HK1026232A1 - Human bikunin - Google Patents

Description

Human bicucullin peptide

Technical Field

The compositions of the present invention relate to the field of proteins that inhibit serine protease activity. The invention also relates to the field of nucleic acid constructs, vectors, and host cells for producing serine protease inhibitory proteins, pharmaceutical formulations comprising such proteins, and methods of use thereof.

Background

Blood loss is a serious complication of major surgical procedures, such as open heart surgery and other complex procedures. Cardiac surgery patients constitute a large segment of recipients. Blood transfusion is at risk of infectious disease and side effects. In addition, blood donations are expensive and often short-lived. Pharmacological approaches to reduce blood loss and the consequent need for blood transfusion have been reported (reviewed by Scott et al, annual book of thoracic surgery (Ann. Thorac. Surg.) 50: 843-.

Protein serine protease inhibitors

The Kunitz family of bovine serine protease inhibitors, aprotinin (aprotinin), is the agent Trasylol^The active substance of (1). Aprotinin (Trasylol) has been reported^) Can effectively reduce blood loss in the operative period (Royston et al, scalpel (Lancet) ii: 1289-1291, 1987; dietrrich et al, thoracic cardiovascular surgery (Thorac. Cardiovasc. Surg.37: 92-98, 1989; Fraedrich et al, thoracic cardiovascular surgery (Thorac. Cardiovasc. Surg.37: 89-91, 1989); W.VanOeveren et al (1987), Ann Thorac. Surg.44, 640-page 645; Bistrup et al, (1988) scalpel I (Lancet I), 366-type), but also reports of adverse reactions, including hypotension and flushing (Bohrer et al, Anesthesia (Anesthesia) 45: 853-type 854, 1990) and allergic reactions (Dietrrich et al, Supra). The use of aprotinin is not recommended for patients who have previously been exposed to aprotinin (Dietrich et al, Supra). Trasylol^Have been used in the treatment of hyperfibrinolytic (hyper fibrinolytic) hemorrhage and traumatic hemorrhagic shock.

Aprotinin is known to inhibit several serine proteases, including trypsin, chymotrypsin, plasmin and kallikrein, and is used in the treatment of acute pancreatitis, shock syndrome in different states, hyperfibrinolytic (hyper-fibrinolytic) hemorrhage and myocardial infarction (Trapnell et al, (1974) J.UK.surgically 61: 177; J.McMichan et al (1982) circulatory shock (circulatory shock) 9: 107; Auer et al (1979) Acta neurovir.49: 207; Sher (1977) J.Obstet.Gynecol.129: 164; Schneider (1976), Artzneim. First.26: 1606.) generally accepted as Trandol^Blood loss is reduced in vivo by inhibiting kallikrein and plasmin. Aprotinin (3-58, Arg15, Ala17, Ser42) was found to exhibit improved plasma kallikrein inhibition compared to the native aprotinin itself (WO 89/10374).

Problems with aprotinin

Since aprotinin is of bovine origin, there is a certain risk of allergic reactions after the patient has been exposed to the drug again. Therefore, human functionally equivalent aprotinin would be most useful and desirable due to its lower risk of anaphylaxis.

Aprotinin also induces nephrotoxicity in rodents and dogs when administered repeatedly at high doses (Bayer, Trasylol)^Protease inhibitors (Inhibitor of protease; glass et al, Verhandlenegen der Deutschen Gesellschaft fur Innere Medizin, 78.Kongress ", Bergmann, Munchen, 1972, p. 1612-1614). There is a hypothesis that this effect is due to the accumulation of aprotinin in the negatively charged tubules in the vicinity of the kidney due to the high net positive charge (WO 93/14120).

Therefore, it is an object of the present invention to find human proteins having a functional activity similar to aprotinin. It is also an object of the present invention to find proteases which may be less charged, yet exhibit the same, highly similar or improved specificity of proteases found on aprotinin, especially for plasmin and kallikrein. Such inhibitors may be repeatedly used as a medicament for human patients with reduced risk of side-effects and reduced renal toxicity.

Brief summary of the invention

The serine protease inhibitors provided by the present invention specifically inhibit kallikrein, which is isolated from human placental tissue by affinity chromatography.

The present invention provides a newly discovered human protein, herein referred to as human placental dikonin (bikunin), which comprises two serine protease inhibitor domains of the Kunitz class. In a specific embodiment, the invention implements a protein having the following amino acid sequence:

ADRERSIHDF CLVSKVVGRC RASMPRWWYN VTDGSCQLFV YGGCDGNSNN 50

YLTKEECLKK CATVTENATG DLATSRNAAD SSVPSAPRRQ DSEDHSSDMF 100

NYEEYCTANA VTGPCRASFP RWYFDVERNS CNNFIYGGCR GNKNSYRSEE 150

ACMLRCFRQQ ENPPLPLGSK VVVLAGAVS 179

(SEQ ID NO: 1)

In a preferred embodiment of the present invention, there is provided a human placental bicurin peptide protein having the amino acid sequence:

ADRERSIHDF CLVSKVVGRC RASMPRWWYN VTDGSCQLFV YGGCDGNSNN 50

YLTKEECLKK CATVTENATG DLATSRNAAD SSVPSAPRRQ DSEDHSSDMF 100

NYEEYCTANA VTGPCRASFP RWYFDVERNS CNNFIYGGCR GNKNSYRSEE 150

ACMLRCFRQQ ENPPLPLGSK 170

(SEQ ID NO: 52)

In one aspect, the biological activity of the protein of the invention is that it binds to and significantly inhibits the biological activity of trypsin, human plasma and tissue kallikrein, human plasmin and factor VIIa. In a preferred embodiment, the present invention provides native human placental bicurin peptide protein in glycosylated form. In a further embodiment, the invention comprises a native human bicining peptide protein formulated to contain at least one cysteine-cysteine disulfide bond. In a preferred embodiment, the protein comprises at least one intra-chain cysteine-cysteine disulfide bond selected from the group of cysteine pairs comprising CYS11-CYS61, CYS20-CYS44, CYS36-CYS57, CYS106-CYS156, CYS115-CYS139, and CYS131-CYS152, wherein the numbering of the cysteines is according to the amino acid sequence of native human placental biconin peptide. One of ordinary skill will recognize that the proteins of the invention can be folded into a suitable three-dimensional structure to retain the biological activity of native human bicining peptide, which may not occur, or may occur with one or more, or all, of the native intrachain cysteine-cysteine disulfide bonds. In a most preferred embodiment, the protein of the invention is suitably folded and formed with all suitable native cysteine-cysteine disulfide bonds.

As the following examples, the active proteins of the present invention may be purified from human tissues, such as placenta, or by synthetic protein chemistry techniques. It is also understood that the protein of the present invention can be obtained by a molecular biological technique in which a self-replicating vector is capable of expressing the protein of the present invention in a transformed cell. Such proteins may be designed into a non-secreted or secreted form of the transformed cell. To facilitate secretion from transformed cells, or to enhance the functional stability of the translated protein, or to facilitate folding of the bicining peptide protein, the NH of the native human bicining peptide protein may be₂The terminal portion is added with a specific signal peptide sequence.

In one embodiment, the invention thus provides a native human bicining peptide protein with at least a portion of the native signal peptide sequence intact. Accordingly, one embodiment of the present invention provides a native bicining peptide having at least a portion of the signal peptide, having the sequence:

AGSFLAWLGSLLLSGVLA -1

ADRERSIHDFCLVSKVVGRCRASMPRWWYNVTDGSCQLFVYGGCDGNSNN 50

YLTKEECLKKCATVTENATGDLATSRNAADSSVPSAPRRQDSEDHSSDMF 100

NYEEYCTANAVTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNSYRSEE 150

ACMLRCFRQQENPPLPLGSKVVVLAGAVS 179

(SEQ ID NO: 2)

In a preferred embodiment, the invention provides a native human placental bicurin peptide protein with a partially complete leader sequence having the sequence number: 52 and having an entire leader segment having the amino acid sequence:

MAQLCGLRRSRAFLALLGSLLLSGVLA -1

(SEQ ID NO: 53)

In another embodiment, the invention provides a partially intact leader sequence bicunning peptide protein having the sequence number: 52 and having the complete leader segment with the amino acid sequence:

MLR AEADGVSRLL GSLLLSGVLA -1

(SEQ ID NO: 54)

In a preferred numbering system for use herein, NH is the amino acid sequence of native human placental bicurin peptide₂The end is designated as amino acid number + 1. One will readily recognize that a functional protein fragment derivable from native human placental dikonin will retain at least a portion of the biological activity of native human placental dikonin and act as a serine protease inhibitor.

In one embodiment, the protein of the invention comprises a fragment of native human placental dicurnine peptide comprising at least one functional Kunitz-like domain having the amino acid sequence of amino acids 7-159 of native human placental dicurnine peptide, hereinafter referred to as "dicurnine peptide (7-159)". The invention thus embodies a protein having the following amino acid sequence:

IHDFCLVSKVVGRCRASMPRWWYNVTDGSCQLFVYGGCDGNSNN 50

YLTKEECLKKCATVTENATGDLATSRNAADSSVPSAPRRQDSEDHSSDMF 100

NYEEYCTANAVTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNSYRSEE 150

ACMLRCFRQ 159

(SEQ ID NO: 3)

Wherein the amino acid numbering corresponds to the amino acid sequence of native human placental dikonin. Another functional variant of this embodiment may be a fragment of native human placental dikonin, which comprises at least one functional Kunitz-like domain and has the amino acid sequence of amino acids 11-156 of native human placental dikonin, the dikonin (11-156).

CLVSKVVVGRCRASMPRWWYNVTDGSCQLFVYGGCDGNSNN 50

YLTKEECLKKCATVTENATGDLATSRNAADSSVPSAPRRQDSEDHSSDMF 100

NYEEYCTANAVTGPCRASFPRWYFDVERNSCNNFIYGGCRGNHNSYRSEE 150

ACMLRC 156

(SEQ ID NO: 50)

It will be appreciated that the individual Kunitz-like domains remain fragments of native placental dicurnine peptide. In particular, the present invention provides a protein having the amino acid sequence of the first Kunitz-like domain consisting of amino acids 7-64 of native human placental bikunin peptide, hereinafter referred to as "bikunin peptide (7-64)". Thus in one embodiment, a protein having at least one Kunitz-like domain is comprised having the amino acid sequence:

IHDFCLVSKVVGRCRASMPRWWYNVTDGSCQLFVYGGCDGNSNN 50

YLTKEECLKKCATV 64

(SEQ ID NO: 4)

Wherein the amino acid numbering corresponds to the amino acid sequence of native human placental dikonin. Another form of the protein of the invention may be the first Kunitz-like domain, which consists of the amino acid sequence of amino acids 11-61 of natural human placental bicurin peptide, having the amino acid sequence "bicurin peptide (11-61)":

CLVSKVVGRCRASMPRWWYNVTDGSCQLFVYGGCDGNSNN 50

YLTKEECLKKC 61

(SEQ ID NO: 5)

The present invention also provides a protein having the amino acid sequence of a Kunitz-like domain consisting of the amino acid sequence of native human placental bicurin amino acids 102-159, hereinafter referred to as "bicurin (102-159)". Thus, one embodiment of the invention includes at least one Kunitz-like domain protein having the amino acid sequence:

YEEYCTANAVTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNSYRSEE 150

ACMLRCFRQ 159

(SEQ ID NO: 6)

Wherein the amino acid numbering corresponds to the amino acid sequence of native human placental dikonin. Another version of this domain may be a Kunitz-like domain consisting of the amino acid sequence of native human placental bicurin amino acids 106-156, the "bicurin peptide (106-156)" having the amino acid sequence:

CTANAVTGPCRASFPRWYFDVERNSCNNFIYGGCRGNKNSYRSEE 150

ACMLRC 156

(SEQ ID NO: 7)

One of ordinary skill will thus recognize that fragments of native human bicining peptide proteins can be designed that retain at least a portion of the biological activity of the native protein. Such fragments can be combined in different orientations or in multiple combinations to provide alternative proteins that retain some, or the same or more, of the biological activity of the native human bicistronic protein.

It will be readily appreciated that the biologically active protein of the invention may comprise one or more of the subject Kunitz-like domains in combination with other Kunitz-like domains from other sources. The biologically active proteins of the invention may comprise one or more Kunitz domains of this class in combination with additional protein domains of other origin having various biological activities. The biological activity of the proteins of the present invention can be combined with other known proteins to provide multifunctional fusion proteins with predictable biological activity. Thus, in one embodiment, the invention includes a composition comprising at least one peptide corresponding to or functionally equivalent to the sequence No.: 5 or sequence number: 7 in a sequence of amino acids.

An open reading frame that stops at the early stop codon can still encode a functional protein. The present invention includes such alternative terminations and in one embodiment provides a protein having the amino acid sequence:

ADRERSIHDFCLVSKVVGRCRASMPRWWYNVTDGSCQLFVYGGCDGNSNN 50

YLTKEECLKKCATVTENATGDLATSRNAADSSVPSAPRRQDS 92

(SEQ ID NO: 8)

In one embodiment, the invention provides a substantially purified, or recombinantly produced, native human bicurin peptide protein having a complete segment of the leader sequence and a portion of the complete native transmembrane region. Thus, one embodiment of the present invention forms a native human bicining peptide having the entire leader sequence, and at least a portion of a transmembrane domain (underlined), having an amino acid sequence selected from:

1)EST MLR AEADGVSRLL GSLLLSGVLA -1

2)PCR MAQLCGL RRSRAFLALL GSLLLSGVLA -1

3)λcDNA MAQLCGL RRSRAFLALL GSLLLSGVLA -1

1)ADRERSIHDF CLVSKVVGRC RASMPRWWYN VTDGSCQLFV YGGCDGNSNN 50

2)ADRERSIHDF CLVSKVVGRC RASMPRWWYN VTDGSCQLFV YGGCDGNSNN 50

3)ADRERSIHDF CLVSKVVGRC RASMPRWWYN VTDGSCQLFV YGGCDGNSNN 50

1)YLTKEECLKK CATVTENATG DLATSRNAAD SSVPSAPRRQ DSEDHSSDMF 100

2)YLTKEECLKK CATVTENATG DLATSRNAAD SSVPSAPRRQ DSEDHSSDMF 100

3)YLTKEECLKK CATVTENATG DLATSRNAAD SSVPSAPRRQ DSEDHSSDMF 100

1)NYEEYCTANA VTGPCRASFP RWYFDVERNS CNNFIYGGCR GNKNSYRSEE 150

2)NYEEYCTANA VTGPCRASFP RWYFDVERNS CNNFIYGGCR GNKNSYRSEE 150

3)NYEEYCTANA VTGPCRASFP RWYFDVERNS CNNFIYGGCR GNKNSYRSEE 150

1)ACMLRCFRQQ ENPPLPLGSK VVVLAGLFVM VLILFLGASM VYLIRVARRN 200

2)ACMLRCFRQQ ENPPLPLGSK VVVLAGLFVM VLILFLGASM VYLIRVARRN 200

3)ACMLRCFRQQ ENPPLPLGSK VVVLAGLFVM VLILFLGASM VYLIRVARRN 200

1)QERALRTVWS SGDDKEQLVK NTYVL 225

2)QERALRTVWS FGD 213

3) QERALRTVWS SGDDKEQLVK NTYVL 225 wherein sequence 1) is derived from the consensus sequence SEQ ID NO: 45 derived ESTs, sequence 2) are PCR cloned SEQ ID NO: 47, SEQ ID NO: 3) is a lambda DNA clone SEQ ID NO: 49. in a preferred embodiment, the protein of the invention comprises the sequence of seq id no: 45, 47 or 49, wherein the protein is cleaved in the region between the tail of the last Kunitz domain and the transmembrane region.

The invention also embodies proteins lacking a signal peptide. The invention therefore provides a cell having the sequence number contiguous with the transmembrane amino acid sequence: 52, transmembrane amino acid sequence:

EST VVVLAGLFVM VLILFLGASM VYLIRVARRN 200

EST QERALRTVWS SGDDKEQLVK NTYVL 225

(SEQ ID NO: 69)

Transmembrane amino acid sequence:

PCR VVVLAGLFVM VLILFLGASM VYLIRVARRN 200

PCR QERALRTVWS FGD 213

(SEQ ID NO: 68)

Or a transmembrane amino acid sequence:

λcDNA VVVLAGLFVM VLILFLGASM VYLIRVARRN 200

λcDNA QERALRTVWS SGDDKEQLVK NTYVL 225

(SEQ ID NO: 67)

The amino acid sequence of the protein of the invention makes it clear to the skilled person that suitable nucleotide sequences can be used in molecular biology techniques to produce the protein of the invention. Thus, one embodiment of the present invention provides a nucleic acid sequence encoding human bicining peptide having the consensus DNA sequence of FIG. 3(SEQ ID NO: 9) which is translated into the amino acid sequence of the native human placental bicining peptide sequence of FIG. 3(SEQ ID NO: 10). In another embodiment, the present invention provides the consensus nucleic acid sequence of FIG. 4C (SEQ ID NO: 51) encoding the amino acid sequence of FIG. 4D (SEQ ID NO: 45).

In a preferred embodiment, the present invention provides a DNA sequence of FIG. 4F (SEQ ID NO: 48) encoding the sequence of SEQ ID NO: 49, and a nucleic acid sequence encoding a native human placental dikonin. In another embodiment, the invention provides a method of encoding a sequence number: 47 (SEQ ID NO: 46).

One will readily recognize that specific allelic mutations, as well as conservative substitutions, may be designed into nucleic acid sequences while still obtaining protein amino acid sequences that fall within the invention. It will be appreciated by those skilled in the art that specific natural allelic mutations of the proteins of the invention, as well as conservative substitutions of the amino acid sequences of the proteins of the invention, will not significantly alter the biological activity of the protein and fall within the scope of the invention.

The present invention also provides pharmaceutical compositions comprising human placental dicuronine peptides and fragments thereof for reducing intraoperative blood loss in a treated patient.

The present invention also provides a method of reducing blood loss during surgery in a surgical patient wherein an effective amount of the disclosed human serine protease inhibitor of the present invention is administered to the patient in a biologically acceptable carrier.

The invention also provides variants of placental dicuronine peptides comprising amino acid substitutions thereby altering the specificity of the protease, and the specific Kunitz structures described aboveA domain. Preferred substitution sites are as indicated below, from Xaa in the amino acid sequence of native placental dikonin¹To Xaa³²The position of (a). From Xaa¹To Xaa¹⁶The substitution is preferred for the variant of the biconinin peptide (7-64), and Xaa¹⁷To Xaa³²Substitutions of the biculinin peptide (variant 102-159) are preferred.

Thus, the proteins embodied by the invention have the amino acid sequence:

(SEQ ID NO: 11)

Ala Asp Arg Glu Arg Ser Ile Xaa¹ Asp Phe 10

Cys Leu Val Ser Lys Val Xaa² Gly Xaa³ Cys 20

Xaa⁴ Xaa⁵ Xaa⁶ Xaa⁷ Xaa⁸ Xaa⁹ Trp Trp Tyr Asn 30

Val Thr Asp Gly Ser Cys Gln Leu Phe Xaa¹⁰ 40

Tyr Xaa¹¹ Gly Cys Xaa¹² Xaa¹³ Xaa¹⁴ Ser Asn Asn 50

Tyr Xaa¹⁵ Thr Lys Glu Glu Cys Leu Lys Lys 60

Cys Ala Thr Xaa¹⁶ Thr Glu Asn Ala Thr Gly 70

Asp Leu Ser Thr Ser Arg Asn Ala Ala Asp 80

Ser Ser Val Pro Ser Ala Pro Arg Arg Gln 90

Asp Ser Glu His Asp Ser Ser Asp Met Phe 100

Asn Tyr Xaa¹⁷ Glu Tyr Cys Thr Ala Asn Ala 110

Val Xaa¹⁸ Gly Xaa¹⁹ Cys Xaa²⁰ Xaa²¹ Xaa²² Xaa²³ Xaa²⁴ 120

Xaa²⁵ Trp Tyr Phe Asp Val Glu Arg Asn Ser 130

Tys Asn Asn Pre Aaa²⁶ Tyr Xaa²⁷ Gly Cys Xaa²⁸ 140

Xaa²⁹ Xaa³⁰ Lys Asn Ser Tyr Xaa³¹ Ser Glu Glu 150

Ala Cys Met Leu Arg Cys Pne Arg Xaa³² Gln 160

Glu Asn Pro Pro Leu Pro Leu Gly Ser Lys 170

Val Val Val Leu Ala Gly Ala Val Ser 179

(SEQ ID NO：11).

Wherein Xaa¹-Xaa³Each independently represents a naturally occurring amino acid residue other than cysteine (Cys), with the proviso that at least Xaa¹-Xaa³²One amino acid residue in the sequence differs from the corresponding amino acid residue in the native sequence.

Herein, the term "naturally occurring amino acid residue" means any of the 20 commonly occurring amino acids, i.e., alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly) histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val).

By substituting one or more amino acids at one or more of the above positions, the inhibitor-specific profile of native placental dicuronine or individual Kunitz-like domains-dicuronine (7-64) or dicuronine (102-159) can be altered to preferentially inhibit other serine proteases, such as but not limited to enzymes of the complement cascade (complement cascade), transfer factor/factor VIIa (TF/FVIIa), factor xa (fxa), thrombin, neutrophil elastase, cathepsin G or protease-3.

Examples of preferred variants of placental digonin peptide include those wherein Xaa¹Is an amino acid residue selected from the group comprising His, Glu, Pro, Ala, Val or Lys, especially wherein Xaa¹Is His or Pro; or wherein Xaa²Is an amino acid residue selected from the group comprising Val, Thr, Asp, Pro, Arg, Tyr, Glu, Ala, Lys, especially wherein Xaa²Val or Thr; or wherein Xaa³Is an amino acid residue selected from the group comprising Arg, Pro, Ile, Leu, Thr, especially wherein Xaa³Is Arg or Pro; or wherein Xaa⁴Is an amino acid residue selected from the group comprising Arg, Lys and Ser, Gln, in particular wherein Xaa⁴Is Arg or Lys; or wherein Xaa⁵Is an amino acid residue selected from the group comprising Ala, Gly, Asp, Thr, especially wherein Xaa⁵Is Ala; or wherein Xaa⁶Is an amino acid residue selected from the group comprising Ser, Ile, Tyr, Asn, Leu, Val, Arg, Phe, especially wherein Xaa⁶Is Ser or Arg; or wherein Xaa⁷Is an amino acid residue selected from the group comprising Met, Phe, Ile, Glu, Thr and Val, especially wherein Xaa⁷Is Met or Ile; or wherein Xaa⁸Is an amino acid residue selected from the group comprising Pro, Lys, Thr, Gln, Asn, Leu, Ser or Ile, especially wherein Xaa⁸Is Pro or Ile; or wherein Xaa⁹Is an amino acid residue selected from the group comprising Arg, Lys or Leu, in particular wherein Xaa⁹Is Arg; or wherein Xaa¹⁰Is an amino acid residue selected from the group comprising Val, Ile, Lys, Ala, Pro, Phe, Trp, Gln, Leu and Thr, especially wherein Xaa¹⁰Val; or wherein Xaa¹¹Is an amino acid residue selected from the group comprising Gly, Ser and Thr, especially wherein Xaa¹¹Is Gly; or wherein Xaa¹²Is an amino acid residue selected from the group comprising Asp, Arg, Glu, Leu, Gln, Gly, especially wherein Xaa¹²Is Asp or Arg; or wherein Xaa¹³Is an amino acid residue selected from the group comprising Gly or Ala; or wherein Xaa¹⁴Is an amino acid residue selected from the group comprising Asn or Lys; or wherein Xaa¹⁵Is an amino acid residue selected from the group comprising Gly, Asp, Leu, Arg, Glu, Thr, Tyr, Val and Lys, especially wherein Xaa¹⁵Is Leu or Lys; or wherein Xaa¹⁶Is an amino acid residue selected from the group comprising Val, Gln, Asp, Gly, Ile, Ala, Met and Val, especially wherein Xaa¹⁶Is Val or Ala; or wherein Xaa¹⁷Is an amino acid residue selected from the group consisting of His, Glu, Pro, Ala, Lys and Val, especially wherein Xaa¹⁷Glu or Pro; or wherein Xaa¹⁸Is an amino acid residue selected from the group comprising Val, Thr, Asp, Pro, Arg, Tyr, Glu, Ala or Lys, especially wherein Xaa¹⁸Is Thr; or wherein Xaa¹⁹Is an amino acid residue selected from the group comprising Arg, Pro, Ile, Leu or Thr, especially wherein Xaa¹⁹Is Pro; or wherein Xaa²⁰Is an amino acid residue selected from the group comprising Arg, Lys, Gln and Ser, especially wherein Xaa²⁰Is Arg or Lys; or wherein Xaa²¹Is an amino acid residue selected from the group of Ala, Asp, Thr or Gly, especially wherein Xaa²¹Is Ala; or wherein Xaa²²Is an amino acid residue selected from the group comprising Ser, Ile, Tyr, Asn, Leu, Val, Arg or Phe, especially wherein Xaa²²Is Ser or Arg; or wherein Xaa²³Is prepared from Met, Phe, Ile, Glu, Leu,Amino acid residue selected from the group of Thr and Val, especially wherein Xaa²³Is Phe or Ile; or wherein Xaa²⁴Is an amino acid residue selected from the group comprising Pro, Lys, Thr, Asn, Leu, Gln, Ser or Ile, especially wherein Xaa²⁴Is Pro or Ile; or wherein Xaa²⁵Is an amino acid residue selected from the group comprising Arg, Lys or Leu, in particular wherein Xaa²⁵Is Arg; or wherein Xaa²⁶Is an amino acid residue selected from the group comprising Val, Ile, Lys, Leu, Ala, Pro, Phe, Gln, Trp and Thr, especially wherein Xaa²⁶Val or Ile; or wherein Xaa²⁷Is an amino acid residue selected from the group comprising Gly, Ser and Thr, especially wherein Xaa²⁷Is Gly; or wherein Xaa²⁸Is an amino acid residue selected from the group comprising Asp, Arg, Glu, Leu, Gly or Gln, especially wherein Xaa²⁸Is Arg; or wherein Xaa²⁹Is an amino acid residue selected from the group comprising Gly and Ala; or wherein Xaa³⁰Is an amino acid residue selected from the group comprising Asn or Lys; or wherein Xaa³¹Is an amino acid residue selected from the group comprising Gly, Asp, Leu, Arg, Glu, Thr, Tyr, Val and Lys, especially wherein Xaa³¹Is Arg or Lys; or wherein Xaa³²Is an amino acid residue selected from the group comprising Val, Gln, Asp, Gly, Ile, Ala, Met and Thr, especially wherein Xaa³²Is Gln or Ala.

Drawings

The following detailed description together with the claims and the accompanying drawings will provide a better understanding of the present invention, in which:

FIG. 1 depicts the nucleic acid sequence of EST R35464(SEQ ID NO: 12) and the translation of this DNA sequence (SEQ ID NO: 13) to generate a DNA sequence in open reading frame with some sequence similarity to aprotinin. The translation product has 5 of 6 cysteines at appropriate intervals that are characteristic of Kunitz-like inhibitor domains (indicated in bold). The position normally occupied by the remaining cysteine (codon 38) is replaced by phenylalanine (indicated by an asterisk).

FIG. 2 depicts the nucleic acid sequence of EST R74593(SEQ ID NO: 14) and the translation of this DNA sequence (SEQ ID NO: 15) which results in an open reading frame homologous to the serine protease inhibitor domain of the Kunitz class. The translation product contains 6 cysteines at appropriate intervals that are characteristic of Kunitz-like inhibitor domains (in bold). However, this reading frame sequence contains stop codons at codon 3 and codon 23.

FIG. 3 depicts a deduced nucleic acid sequence, labeled "consensus sequence" human placental biconin peptide (SEQ ID NO: 9), that pairs with the amino acid sequence of a translated protein (SEQ ID NO: 10), labeled "translated". Also shown as a control are nucleic acid sequences of ESTs H94519 (SEQ ID NO: 16), N39788 (SEQ ID NO: 17), R74593(SEQ ID NO: 14) and R35464(SEQ ID NO: 12). The underlined nucleotides in the consensus sequence correspond to the PCR sites described in the examples. The amino acids underlined in the translated consensus sequence are those residues which were themselves confirmed by amino acid sequencing of purified human placental dikonin. The nucleotide and amino acid codes are standard single letter codes, with "N" in the nucleic acid code referring to the undefined nucleic acid and "x" referring to the stop codon in the amino acid sequence.

FIG. 4A depicts an initial overlap of a series of ESTs having some nucleic acid sequences homologous to those encoding human placental bicinin or portions thereof. The relative positions of the bicining (7-64) and the bicining (102-159) peptides are shown for reference and are referred to as KID 1 and KID 2, respectively.

FIG. 4B depicts a more comprehensive EST overlap after incorporation of additional ESTs. The number at the upper end of the X-axis indicates the base pair length, starting from the first base of the most 5' EST sequence. The length of each bar is proportional to the length in base pairs of the individual EST comprising the gap. The increased number of ESTs is shown to the right of their EST bars, respectively.

FIG. 4C depicts the corresponding arrangement of the corresponding oligonucleotides for each of the overlapping ESTs shown schematically in FIG. 4B. The sequence labeled as bicining peptide above (SEQ ID NO: 51) represents the consensus oligonucleotide sequence derived from overlapping nucleotides at each position. Numbers refer to base pair positions in the EST plots. The underlined oligonucleotides in EST R74593 (positions 994 and 1005 on the figure) are base insertions that are consistently absent in all other overlapping ESTs and appear in R74593.

FIG. 4D depicts amino acid translation of the consensus oligonucleotide sequence (SEQ ID NO: 45) for the bicining peptide depicted in FIG. 4C.

FIG. 4E depicts the nucleotide sequence (SEQ ID NO: 46) and corresponding amino acid translation product (SEQ ID NO: 47) of the human placental biconin peptide coding sequence derived from a human placental cDNA library by PCR-based amplification.

FIG. 4F depicts the nucleotide sequence (SEQ ID NO: 48) and corresponding amino acid translation product (SEQ ID NO: 49) of a native human placental diconin peptide-encoding clone isolated from a human placental-derived cDNA library by clonal hybridization.

FIG. 4G compares the translated amino acid arrangement of the oligonucleotide sequences of placental bicining peptide by EST overlap (SEQ ID NO: 45), PCR-based cloning (SEQ ID NO: 47), and classical lambda clonal hybridization (SEQ ID NO: 49).

FIG. 5 shows a graph of the purification of human placental dicuronine from placental tissue after Superdex 75 Gel Filtration (Superdex 75 Gel-Filtration). The graphs are an overlay of the protein elution curve measured by OD280 nm (solid line), the activity of the eluted protein in the trypsin inhibition assay (percent inhibition is shown as circles), and the activity of the eluted protein in the kallikrein inhibition assay (percent inhibition is shown as boxes).

FIG. 6 shows a graph of the purification of human placental diconin peptide from placental tissue by C18 Reverse Phase Chromatography (C16 Reverse-Phase Chromatography). The graphs are an overlay of the protein elution curve measured by OD 215nm (solid line), the activity of the eluted protein in the trypsin inhibition assay (percent inhibition is shown as circles), and the activity of the eluted protein in the kallikrein inhibition assay (percent inhibition is shown as boxes).

FIG. 7 depicts a silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel of highly purified placental dicuronine (lane two) and a series of protein molecular size standards (lane 1), the size of which is expressed in kilodaltons. The migration is from top to bottom.

FIG. 8 shows the amount of trypsin inhibitory activity that occurs in cell-free fermentation broths derived from the growth of yeast strain SC101 (panel 8A) or WHL341 (panel 8B) stably transformed with plasmid (pS604) directing the expression of placental dikonin (102-159).

FIG. 9 shows SDS-PAGE silver staining (left panel) of cell-free fermentation broth for growth of the yeast strain SC101 (recombinants 2.4 and 2.5) stably transformed with a plasmid directing the expression of bovine aprotinin or placental dikonin (102-159) and Western blotting (right panel) with pAb against placental dikonin (102-159). The migration is from top to bottom.

FIG. 10 is a photograph showing silver staining (lane 2) of highly purified placental dicuronine (102-159) SDS-PAGE and a series of protein molecular size standards whose sizes are expressed in kilodaltons (lane 1). The migration is from top to bottom.

FIG. 11 is a photograph showing the results of Northern blotting of mRNA from different human tissues hybridized to 32P-labeled cDNA probes encoding placental bicurin peptide (102-159) (panel 11A) or placental bicurin peptide (1-213) (panel 11B). Migration is from top to bottom. The number to the right of each blot indicates the size of the adjacent RNA standard reference in kilobases. The mRNA derived organs are shown below each blot lane.

Figure 12 depicts immunohybridization with placental-derived placental digonin peptides to synthetic reduced placental digonin peptides (7-64) (panel a) or 102-159 (panel B) rabbit antisera. In each plate, the contents are: molecular size standards reference (lane 1); native placental digonin peptide isolated from human placenta (lane 2); synthetic placental digonin peptides (7-64) (lane 3) and synthetic placental digonin peptides (102-159) (lane 4). Tricine 10-20% SDS-PAGE gels were blotted and visualized with protein A-purified primary polyclonal antibody (dissolved in 20ml of 0.1% DAS/Tris-buffered salt (pH7.5)), followed by alkaline phosphatase conjugated goat anti-rabbit secondary antibody. Migration is from top to bottom.

FIG. 13 depicts a Coomassie blue stained 10-20% Tricine SDS-PAGE gel of 3 mg of highly purified placental dikonin (1-170) from the baculovirus/Sf 9 expression system (lane 2). Lane 1 contains molecular size standards. The migration is from top to bottom.

FIG. 14 depicts a comparison of the effect of increasing the concentration of Sf 6-derived human placental dikonin (1-170) (filled circles) or synthetic placental dikonin (102-159) (open circles) or aprotinin (open squares) on the time to partial thromboplastin activation in human plasma. Coagulation is initiated by calcium chloride. Protein concentration is plotted against the extension of clotting time. The uninhibited clotting time was 30.8 seconds.

Detailed Description

The present invention includes a newly discovered human protein, referred to herein as human placental dicurnine peptide, which contains two domains of a Kunitz-type serine protease inhibitor. The invention also includes pharmaceutical compositions comprising placental dicurnine and fragments thereof for reducing blood loss during surgery in a patient with surgery or severe trauma.

The present invention also provides a method for reducing blood loss during surgery in a surgical or trauma patient in which an effective amount of a human serine protease inhibitor as disclosed herein is administered to the patient in a biologically acceptable carrier.

The preferred use of placental dicurnine peptides, isolated domains and other variants is for reducing blood loss due to trauma or surgery which may cause significant blood loss. These methods and formulations reduce or eliminate the need for Whole blood donations (Whole donor blood) or blood products, thereby reducing the risk of infection or other adverse side effects, while also reducing the cost of surgery. This method is therefore useful for reducing blood loss in normal patients, i.e., those who do not have a natural or preoperative clotting factor deficiency. Reducing blood loss is reducing blood loss during surgery, or post-surgery blood loss (post surgical repair), or both. Preferred surgical procedures include, but are not limited to, thoracic and abdominal surgery, total or partial hip replacement surgery, and surgery to treat patients with ocular epithelial injuries. Preferred thoracic surgical procedures include, but are not limited to, aortic coronary bypass surgery, cardiac aneurysm and aortic aneurysm resection surgery, esophageal varices surgery, coronary vein bypass surgery. Preferred abdominal surgeries include, but are not limited to, liver transplantation, radical prostatectomy, diverticulum coli surgery, tumor debulking, abdominal artery surgery, and duodenal ulcer surgery, as well as liver or spleen trauma repair. Preferred therapeutic applications for trauma include, but are not limited to, calming severely injured patients who have wounds due to loss of limbs or severe chest/abdominal injuries. In the case of application to reducing surgical blood loss, it is preferred to administer placental bicinin, individual domains or other variants prior to and during surgery; however, for trauma, placental digonin peptide variants, individual domains, or other variants should be administered as soon as possible after injury, and should be included on emergency vehicles that are transported to the wound.

Factor XII (also known as Hageman factor) is a serine protease found in the circulation in zymogen form (80KD) at 29-40. mu.g/ml (see Pixley et al, (1993) methods (meth.in Enz.), 222, 51-64) and is activated by tissue and plasma kallikrein. Once activated, it participates in the internal pathways of activated blood coagulation when blood or plasma contacts "heterogeneous" or anionic surfaces. Once activated, factor XIIa cleaves and activates a number of other plasma proteases, including factor XI, prekallikrein and C1 of the complement system. Thus, the release of bradykinin by activated kallikrein (see Colman, (1984) j. clin. invest, 73, 1249) factor XII can be involved in the induction of hypotensive responses.

Sepsis is a disease caused by endotoxin or Lipopolysaccharide (LPS) of bacterial infection. Exposure of factor XII to LPS results in the activation of factor XII. Patients with sepsis also frequently have symptoms of intravascular coagulation, also due to activation of factor XII by LPS. Septic shock can also be caused by bacterial infection and is associated with fever, low systemic vascular resistance, low arterial blood pressure. It is a common cause of patient death in the U.S. intensive care unit, with seventy-five percent of patients who die from septic shock having sustained hypotension (see Parillo, et al (1989) medical review yearbook (Ann rev. med.)40, 469-.

Adult respiratory distress syndrome is characterized by pulmonary edema, hypoxemia, reduced lung compliance. Although the proteolytic pathways of coagulation and fibrinolysis are believed to play a role in this, the pathogenesis of this disease remains unknown (see Carvalho et al (1988) Proc. laboratory clinical medicine (J. LabClin. Med., 112: 270-)).

The protein of the invention is also a human Kunitz-type inhibitor of kallikrein, a factor XII activator. It is therefore another object of the present invention to provide a method for the prevention or treatment of systemic inflammatory responses such as septic shock, Adult Respiratory Distress Syndrome (ARDS), preeclampsia, multiple organ failure, Disseminated Intravascular Coagulation (DIC). Administration of the peptides of the invention for treatment or prophylaxis will cause a reduction in these inflammations and benefit the patient.

Plasmin plays an important role in extracellular matrix degradation and activation of the matrix-metallo protease (MMP) cascade. Collectively, these proteases mediate the migration of and tissue invasion by endothelial cells and metastatic cancer cells in angiogenesis/neovascularization. New blood vessel formation is essential for tumor growth, and metastasis mediates the process of tumor expansion and is associated with an extremely poor prognosis for the patient.

Some preclinical studies have shown that Kunitz-like serine protease inhibitors with protease specificity similar to aprotinin can be used as cancer drugs. For example, aprotinin reduces the growth and invasion of cancer and also increases tumor necrosis when administered to hamsters with highly invasive fibrosarcoma or mice with malignant-like breast cancer (Latner et al (1974), British cancer journal (Br.J. cancer) 30: 60-67; Latner and Turner (1976), British cancer journal (Br.J. cancer) 33: 535-538). In addition, 200,000KIU of aprotinin, when administered intraperitoneally to C57B1/6 Cr male mice on days 1 and 14 after inoculation with Lewis lung carcinoma cells, reduced lung metastasis by 50%, although there was no effect on the primary tumor mass, Giraldi et al, (1977) european journal of cancer (eur.j. cancer), 13: 1321-1323). Similarly, 10,000KIU of aprotinin, when administered daily to C57BL/6J mice 13-16 days after inoculation with Lewis lung carcinoma cells, reduced 90% of lung metastases while having no effect on primary tumor growth (Uetsuji et al, (1992), J.J.Surg.J.22: 429-422). In the same study, it was also demonstrated that the amount of pulmonary metastasis was increased when plasmin and kallikrein were administered on the same dose schedule. These results led the authors to believe that the possibility of metastasis may be reduced if aprotinin is administered to the patient during surgery. Black and Steger (1976, European Pharmacol., 38: 313-319) found that aprotinin inhibits the transplanted Murphy-Strum lymphosarcoma of the odontoid class in mice, indicating an effect involved in the inhibition of the cytokinin-forming enzyme system. The growth rate of primary tumors was reduced by 90% after intraperitoneal injection of 10,000KIU of aprotinin twice daily to female ddY mice (each of these mice had a single self-squamous cell carcinoma from 3-methylcholanthrene treatment) over a seven week period. Tumor regression was observed in some animals. All animals treated with vehicle died over a period of 7 weeks, while the aprotinin-treated group remained alive. The reduction in tumor growth is associated with hyperkeratosis (Ohkoshi, Gann (1980), 71: 246-250).

Clinically, the survival rate of 26 patients in the surgical treatment group receiving intravenous injection of aprotinin was 70% within two years after surgery, and there was no tumor recurrence; while 26 patients in the placebo group had only 38% survival at the same time and the rate of tumor recurrence was quite high (Freeman et al, British gastroenterology Association (Br. Soc. gastroenterol.) (1980) appendix A: 902). In a case study (Guthrie et al, J.Clin.Pract) (1981) 35: 330-. Intraperitoneal injections (bolus) of 500,000KIU of aprotinin were repeated every 8 hours, while intravenous injections of 200,000KIU of aprotinin were continued every 6 hours for a total of seven days per month. Treatment was discontinued after 4 months due to the induction of an allergic reaction to aprotinin. More recent evidence has further corroborated the role of plasmin as a target in the effect of aprotinin on metastasis.

The mechanism of these phenomena can be linked to the fact that aprotinin blocks the invasive capacity of cancer cell lines (Liu G., et al, International journal of cancer (Int J. cancer)1995, 60: 501-. Furthermore, since the proteins of the present invention are also potent inhibitors of plasmin and kallikrein, they can also be designed for use as anti-cancer agents. For example, they may be designed to retard the growth of primary tumors by limiting neovascularization and invasion of primary tumors; and for blocking metastasis by inhibiting tissue penetration. The compounds may be administered locally to the tumor or systemically. In a preferred mode of treatment, postoperative administration for tumor elimination can be performed to minimize the risk of metastasis. In such a method, the blood loss inhibiting properties of the compound have the added advantage of providing a clearer surgical field. Another preferred mode of administration is combination therapy in combination with MMP inhibitors or chemotherapeutic agents. Another preferred mode of administration is the practice of local gene therapy of placental bicurin peptides designed to be selectively expressed in tumor cells or the stroma and vascular beds associated therewith.

Preferred types of cancer to be treated are vascular-dependent solid tumors, such as breast, colon, lung, prostate and ovarian cancers, all of which have the potential for high metastasis, and local delivery of high concentrations of the protein is feasible, e.g., lung cancer by lung transport, colon cancer by liver transport to liver metastasis, skin cancer, e.g., head and neck or melanoma by subcutaneous transport. Since the protein of the present invention is human in origin, it is less relevant to allergic reactions and anaphylaxis observed in Supra as Guthrie et al when used again.

In addition, the proteins of the invention are designed to reduce thromboembolic complications associated with activation of the intrinsic pathway of coagulation. This may include prevention of pulmonary embolism in patients with advanced cancer, a common cause of death (Donati MB., (1994), Haemostasis 24: 128-.

Edema of the brain and spinal cord results from brain trauma and spinal cord injury, stroke, cerebral ischemia, cerebral and subarachnoid hemorrhage, surgery (including open heart surgery); infectious diseases, such as encephalitis or meningitis: granulomatous diseases, such as sarcoid tumors and complications of focal or invasive cancer. And this edema is one of the causes of high morbidity or mortality from these events. Bradykinin has been shown to disrupt the blood-brain barrier (Greenwood J., (1991), radioneurology (neurobiology), 33: 95-100; Whittle et al, (1992), Acta neurohir., 115: 53-59). Injection of bradykinin into the carotid artery in vivo caused cerebral edema in Spontaneously Hypertensive Rats (SHR) with common carotid artery occlusion (Kamiya, (1990), Nippon Ika Daigaku Zasshi.57: 180-191). In a murine spinal cord wound model, increased levels of bradykinin in extracellular fluid following injury were found (Xu et al, (1991), J. neurohem, 57: 975-. Bradykinin is released from high molecular weight kininogens by serine proteases including kallikrein (Coleman (1984) J. Clin invest.73: 1249), and it has been found that serpin can block the aggravation of cerebral edema caused by cerebral ischemia in SHR mice (Kamiya, (1990), Nippon Ika Daigaku Zasshi.57: 180-191; Kamiya et al, (1993), Stroke, 24: 571-575) and rabbits with cerebral cold damage (Unterberg et al, (1986), J. Neurosgurry, 64: 269-276).

These phenomena suggest that cerebral edema is caused by the local proteolytic release of kinins, such as the release of bradykinin from high molecular weight kininogen, followed by the bradykinin-induced increase in blood brain barrier penetration. Thus, placental bikunin peptides and fragments thereof can be designed as agents for preventing edema in patients at risk of edema. Especially those with a high risk of death or brain injury. This includes patients with head and spinal trauma, patients with multiple trauma, patients undergoing surgery on the brain or spinal cord and associated blood vessels, or other conventional procedures including open heart surgery, patients with stroke, bleeding from the brain or subarachnoid hemorrhage, brain infectious diseases, brain granulomatous diseases or diffuse or focal cancers and tumors of the brain or other conditions including multiple sclerosis involving breakdown of the blood-brain barrier, or patients with other brain or spinal cord infections. Patients will receive administration of placental digonin in the form of intravenous or intracranial infusion or bolus (bolus) injection. Additional doses of bicurin peptide may be administered intermittently over the following one to three weeks. Dosage levels will be designed to be elevated above the levels required to neutralize plasma or bradykinin and other vasoactive peptides formed by serine protease action, and circulating concentrations sufficient to reduce edema. Since the protein is of human origin, repeated administration during the course of therapy will not result in the development of an immune response to the protein. Placental diconin peptides and fragments thereof can be designed for monotherapy and also in combination with other agents such as neuropathic and neuroprotective agents for prophylaxis.

Recent evidence (Dola Cadena R.A. et al (1995), FASEB J.9: 446-. Accordingly, the protease of the present invention is designed to be useful as a drug for treating arthritis and anemia in humans based on its ability to inhibit human kallikrein.

The use of aprotinin for treating male non-insulin Diabetic (NIDDM) patients can significantly improve total glucose absorption and reduce metabolic clearance of insulin (Laurenti et al, (1996), diabetes Medicine (diabetes Medicine 13: 642-.

Daily treatment of patients at risk of preterm birth with urinary trypsin inhibitors for two weeks significantly reduces recurrent uterine contractions (Kanayama et al, (1996), european obstetrics and gynecology and biological journal of reproduction (Eur j. obstet. gynecol. & reprod. biol.). accordingly, the human proteins of the invention can be designed to prevent preterm birth.

Aprotinin has been shown to stimulate mouse myoblast differentiation in culture (Wells and Strickland, DeVelopment (DeVelopment), (1994), 120: 3639-. TGFb is present as a non-activated pro-polypeptide and is activated by a defined proteolysis. It has been suggested that the mechanism of action of aprotinin is involved in inhibiting the processing of the TGFb pro-form into the mature active form of the protease. TGFb has been shown to be up-regulated in various fibrotic lesions and has long been considered as a possible target for anti-fibrotic treatments. For example, in model mice with pulmonary fibrosis, TGF-b concentrations paralleled the extent of bleomycin-induced infection. Furthermore, plasmin levels in alveolar macrophages were consistent with mature TGF-b levels, and the addition of the plasmin inhibitor α 2-antiplasmin abolished the posttranslational activation of macrophages to TGFb pro (Khal et al, (1996) am.J.Respir, cell.mol.biol.15: 252-259). The data suggest that plasmin is involved in the formation of the activated form of TGFb by alveolar macrophages and that this process plays a pathological role in bleomycin-induced lung infections.

Based on these circumstances, placental dicurnine peptides and fragments thereof have been designed for use as therapeutic agents for various fibrotic diseases, including fibrosis of the lung, liver, kidney and skin (scleroderma).

Aerosol aprotinin has been shown to control 50% of mice infected with lethal doses of influenza or paramyxovirus (Ovcharenko and Zhirnov, Antiviral research (Antiviral research), (1994), 23: 107-. Inhibition of the development of lethal hemorrhagic bronchopneumonia and normalization of weight gain was also observed following aerosolized aprotinin treatment. Based on these circumstances, placental dicurnine peptides and fragments thereof have been designed for use as therapeutic agents for various respiratory-related influenza-like diseases.

The human placental dikonin, single domain, and other variants of the invention are all designed for use in medical/therapeutic applications that require native aprotinin or aprotinin analogs with other inhibitory properties, especially those that require large doses. This includes diseases treated with human proteins that are used for their property of inhibiting human serine proteases; the serine protease comprises trypsin, plasmin, kallikrein, elastase, cathepsin G and proteinase-3; these diseases include, but are not limited to: acute pancreatitis (pancreatic elastase and trypsin), infection, thrombocytopenia, preservation of platelet function, organ preservation, wound healing, various forms of shock including shock lung, endotoxin shock, and postoperative complications; blood coagulation disorders, such as excessive fibrinolytic hemorrhage (hyperfibrinolytic hemorrhage); acute and chronic infectious reactions, in particular the treatment or prevention of organ damage, such as pancreatitis and radiation-induced enteritis, complex-mediated inflammatory reactions, such as immune vasculitis, glomerulonephritis and various arthritides; collagen diseases, especially rheumatoid arthritis; various arthritis caused by metabolic-related waste products (e.g., gout); degradation of the elastic component of the organ-associated tissue segment, such as atherosclerosis (serum elastase) or emphysema (neutrophil elastase); adult respiratory distress syndrome, intestinal infectious disease, and psoriasis.

An unexpectedly important finding was that the synthetic peptides encoding the bicining peptides (7-64) and (102-159) were able to fold properly into the correct three-dimensional structures (example 2 and example 1, respectively) with the biological activity of active protease inhibitors. Each of these bicining peptide fragments reduced 6 mass units of 3 intrachain disulfide bonds formed by the 6 cysteine residues on each fragment upon folding, and this reduction in mass was consistent for each folding case. Another unexpected finding was that synthetic peptides encoding the bicining peptides (7-64), the bicining peptides (102-159) and the bicining peptides (1-170) were highly inhibitory to plasmin, as well as tissue and plasma kallikrein (examples 4, 3 and 10, respectively). Trasylol^Inhibition of plasmin and kallikrein is believed to involve the use of Trasylol in open heart surgery^The mechanism of blood loss is reduced. This unexpected discovery of the specificity of the Kunitz domain of the present invention makes it a suitable therapeutic agent for use in hemostasis during surgery or trauma where substantial blood loss or other fibrinolysis and/or inhibition of kallikrein is beneficial.

Furthermore, we show in this disclosure (example 10) that placental dicurnine peptide (1-70) is a potent inhibitor of factor XIa, and is a moderate inhibitor of factor Xa. Factor XIa plays a very critical role in the internal pathway of blood coagulation, converting inactive factor IX to active factor IXa. Thus, placental dicuronine inhibits two key enzymes of the internal pathway, kallikrein and factor XIa. Consistent with these, we also show that placental dicurnine peptide (1-170) is a potent inhibitor of activated partial thromboplastin time, as measured by clotting time due to internal pathways. On the other hand, we show that placental dicurin peptide (1-170) is a very weak inhibitor of the tissue factor VIIa complex, indicating that this placental dicurin peptide is not important in the regulation of the extrinsic coagulation cascade. Based on these unexpected findings placental dicuronine peptides were designed as therapeutics for diseases in which activation of the intrinsic pathway of coagulation is a major pathogenesis. Examples of such diseases include posttraumatic shock and disseminated intravascular coagulation.

One major advantage of the Kunitz domains of the invention is that these domains are human proteins, in contrast to Trasylol^Less positively charged and therefore reduces the risk of renal injury in the case of administration of large doses of protein. Since it is of human origin, with an equivalent dose of Trasylol^In contrast to administration, the proteins of the invention can thus be administered to human patients with a significantly reduced unwanted immune response. In addition, it was found that the bicining peptide (102-159), the bicining peptide (7-64) and the bicining peptide (1-170) were in vivo linked to Trasylol^Compared to significantly more potent inhibitors of plasma kallikrein (examples 3, 4 and 10). Therefore, the bicining peptide and the fragment thereof are expected to be more effective in reducing blood loss of patients in vivo.

The serine protease inhibitor should be administered in an amount sufficient to provide a level above normal plasma levels. For use in preventing a decrease in blood volume caused by bleeding during or after coronary artery bypass surgery (CABG), the protein of the present invention should be used in place of Trasylol in consideration of the difference in effectiveness^. Transsylol is described in surgeon's commonly used Reference (Physicians Desk Reference), (1995)^Is briefly described, in appendix A. listing Trasylol^. Briefly, placental dicurin peptide, single domain or other variant at a loading dose (loading dose) is slowly administered to a patient in a supine position for a period of 20 to 30 minutes after anesthesia but prior to a sternotomy. The total dose generally used is about 2X 10 depending on such factors as patient weight and operation time⁶KIU (kallikrein inhibitory unit) to 8X 10⁶KIU is between. After completion of the loading dose, a constant infusion dose (infusion dose) follows until the procedure is completed and the patient leaves the operating room. Preferred delivered doses are in the range of 250,000 to 500,000KIU per hour. An initial volume of Pump (Pump prime dose) is added to the initial fluid of the cardiopulmonary bypass circuit, and the initial fluid in liquid form is replaced before the cardiopulmonary bypass circuit is formed. Preferred initial doses for pumping are in the range of one toIn the total amount of two million KIUs.

The proteins of the invention may be used by formulating pharmaceutical compositions by any means known in the art. Such compositions comprise the active ingredient in admixture with one or more pharmaceutically acceptable carriers, diluents, fillers, binders and other excipients, depending on the mode of administration and the dosage form envisaged. Therapeutically inert inorganic or organic carriers known to those skilled in the art include, but are not limited to, lactose, corn starch or derivatives thereof, talc, vegetable oils, waxes, fats, polyols such as polyethylene glycol, water, sucrose, ethanol, glycerol, and the like. Various preservatives, lubricants, dispersants, flavoring agents, humectants, antioxidants, sweeteners, colorants, stabilizers, salts, buffers and the like may also be added as needed to aid in the stability of the formulation or to aid in the enhancement of the activity or its bioavailability or to produce an acceptable mouthfeel or odor upon oral administration. The inhibitors may be used in such compositions as are their original compounds, or optionally in the form of their pharmaceutically acceptable salts. The proteins of the present invention may be administered alone, or in various combinations, as well as in combination with other therapeutic agents. The compositions so formulated may be administered in any suitable manner known to those skilled in the art, as desired.

Parenteral administration includes intravenous (i.v), subcutaneous (s.c.), intraperitoneal (i.p.), and intramuscular (i.m.). Intravenous administration can be used to obtain rapid adjustment of the peak plasma concentration of the drug as needed. In addition, such drugs may be administered continuously through an intravenous catheter at a desired rate. Suitable excipients include sterile, non-pyrogenic liquid diluents, such as sterile water for injection, sterile buffered solutions or sterile saline. The resulting composition is administered by intravenous injection or infusion prior to or during surgery.

Entrapment of drugs by liposomes may help to increase the half-life of the drug and enhance targeting of the drug to phagosomes such as neutrophils and macrophages involved in inflammation. The selectivity of the liposome target should be enhanced by the introduction of ligands on the exterior of the liposome that bind to macromolecules specific to the target organ/tissue, such as the GI tract and lung. In addition, i.m. or s.c. injection of the precipitate (deposit injection) may be used to achieve a sustained release effect of the drug with or without encapsulation of the drug in digestible microspheres (e.g. Poly-DL-Lactide-Co-glyColide) or protective pharmaceutical agents containing collagen. To improve the convenience of the dosage form, an i.p. implant reservoir and septum such as the secuseal system may be utilized. Convenience and patient compliance may also be improved by using an injection pen (e.g., a Novo Pin or Q-pen) or a needleless jet injector (e.g., from Bioject, Mediject, or Becton Dickinson). Delivery to the destination through a cannula (cannula) can also be precisely controlled using an implantable pump. Examples include subcutaneously implanted osmotic pumps from ALEA such as ALZNET osmotic pumps.

Drug delivery to the nose can be achieved by incorporating the drug into bioadhesive specific carriers (< 200mm) such as those containing cellulose, polyacrylate or polyorthophilic substances (PolyCarbophil) together with appropriate absorption enhancers such as phospholipids or acylcarnitines. Commercially available systems include those developed by Biosys and Scios Nova.

Pulmonary delivery represents a mode of administration of drugs into the circulation without the intestine. The lower airway epithelium is highly permeable to proteins in a large range of molecular sizes up to 20 kDa. Micron-sized dry powder usable with dry powder inhalers such as Inhale^TM、Dura^TM、Fisons(Spinhaler^TM) And Glaxo (Rotahaler)^TM) Or based on Astra (Turbohaler)^TM) Metered dose inhalers for propellant delivery to the distal alveolar surface contain these dry powders with the medicament in a suitable carrier, such as mannitol, sucrose or lactose. Solution formulations with or without liposomes can be delivered using an ultrasonic nebulizer.

Oral delivery can incorporate the drug into tablets, coated tabletsTablets, dragees, hard and soft gelatin capsules, solvents, lubricants, suspensions, or inert coated capsules and the like are designed to release the drug in a dosage form in the colon having low digestive protease activity. Examples of the latter include OROS-CT/Osmet by ALZA^TMSystem, and PULSINCAP from Scherer Drug Delivery Systems^TMProvided is a system. Other systems utilize azo-crosslinked polymers that are degraded by colon-specific bacterial azo hydrolases, or polyacrylic polymers that are activated by elevated pH in the colon. The above systems can be used in combination with a wide range of absorption enhancers. Rectal delivery may be achieved by incorporating the drug into a suppository.

In its preferred pharmaceutical use, the preferred mode of administration of the placental dicurnine peptides of the invention is parenteral, preferably by the intravenous route via the midline, in order to reduce blood loss during surgery.

The amount of the pharmaceutical composition used will depend on the subject and the condition being treated. The necessary amount can be determined without undue experimentation using protocols known to those skilled in the art. In addition, the necessary amount can be calculated based on the measured amount of the target protease, e.g., plasmin or kallikrein, to be inhibited for treatment of the disease condition. Because the active agents contemplated by the present invention are considered non-toxic, the treatment is preferably administered in doses exceeding the optimal required amount of the active agent.

Additionally, placental dikonin, individual domains or other variants can be used to isolate natural substances, e.g., homologous proteases isolated from human material using affinity-based isolation methods, and to induce protease antibodies that can be further used to explore tissue distribution and useful functions of placental dikonin.

Searching human sequence data

The presence of a unique protein functionally homologous to aprotinin was deduced from a unique analysis of the entry sequence of the expressed sequence tag database (hereinafter dbEST) of NCBI (national center for biological information, Md.). By TBlastN derivation (BLAST or basic local homology search tool using the method of Altschul et al (1990) J. mol Biol 215: 403-^Nucleic acid sequences having sequence homology. This search for numerous clones selectively narrows down to two distinct clones that may be capable of encoding a deduced amino acid sequence that may correspond to a human protein that is functionally homologous to aprotinin. The selected amino acid sequences were R35464(SEQ ID NO: 12) and R74593(SEQ ID NO: 14), which were derived from a human placental nucleic acid library. One of the 6 cysteines critical for the formation of the covalent structure of the Kunitz domain was deleted in the translated protein sequence of the longest open reading frame of R35464(SEQ ID NO: 13), which means that the nucleic acid sequence of R35464 did not produce a functional inhibitor. Similarly, the longest translated open reading frame of R74593(SEQ ID NO: 15) contains a stop codon 5' of the coding Kunitz-like sequence, which means that this sequence cannot be translated to produce a functional secreted Kunitz domain. The meaning of these sequences alone is not even clear. It is possible that they represent a) pseudogene products, b) untranslated mRNA regions, or c) products encoding viable mRNAs that are incorrectly sequenced.

Discovery of human biconin peptide

In order to specifically isolate and determine the actual human sequence, primers were designed that hybridized to the 5 'and 3' sequences of the cDNA fragment encoding the Kunitz-like sequence we propose to find in R35464 and R74593. Primers used to amplify the fragment of R74593 encoding a Kunitz-like sequence were: CGAAGCTTCATCTCCGAAGCTCCAGACG (3 'primer with Hind III site; SEQ ID NO: 33) and AGGATCTAGACAATAATTACCTGACCAAGGA (5' primer with Xba site; SEQ ID NO: 34).

These primers were used by PCR (30 cycles) to amplify a DNA sequence from Clontech (MATCHMAKER, Cat # HL4003 AB, Clontech Laboratories,palo Alto, CA) of the 500 base pair product of the human placental cDNA library was subcloned into Bluescript-SK + and passed through T₃Sequence for primers^TMKit version 2.0 sequencing. Surprisingly, the fragments obtained with our primers do not differ from the sequence of R74593 as listed in the dbEST database. In particular, our new sequence contains an additional intervening guanosine 3 'of the putative stop codon, but it is 5' in the region encoding the Kunitz-like sequence (fig. 3). This additional G insertion allows the stop codon to be shifted out of the reading frame of the Kunitz-like domain (in the correct sequence of R74593, G is located at base 114; FIG. 3).

Subsequent queries in dbEST for sequences homologous to the R74593-class Kunitz peptide sequence yielded H94519 and N3978 derived from human retinal libraries. These sequences contain Kunitz-like sequences that are nearly identical to the Kunitz-like domain encoded by R35464, except that they contain all 6 characteristic cysteines. Each nucleotide sequence was overlapped with R74593 (with the insertion G at base pair 114 corrected) and R35464 to obtain a consensus nucleotide sequence of a partially human placental biconin peptide (SEQ ID NO: 9; FIG. 3). Translation of the consensus sequence resulted in an open reading frame from-18 to +179 residues (FIG. 3: full translation of SEQ ID NO: 10) containing two complete Kunitz-like domain sequences located within the regions of amino acid residues 17-64 and 102-159, respectively.

Attempts were made to obtain additional 5 'sequences by querying the dbEST with R35464 and the possible matches resulting from these queries with additional 5' sequences were then used to re-query the dbEST. Under such a form of repeated queries, a series of overlapping 5' sequences were found, including clones H16866, T66058, R34808, R87894, N40851 and N39876 (fig. 4). Alignment of these sequences indicates that the presence of the 5' ATG may serve as a starting site for the synthesis of a consensus translated protein sequence. From this selection information, it is now possible to selectively screen and determine the nucleic acid and polypeptide sequences of human proteins functionally homologous to aprotinin.

A re-query of dbEST reveals many new EST entries, as briefly shown in FIG. 4B. The overlap of these additional ESTs allows us to construct a longer consensus oligonucleotide sequence (FIG. 4C) that extends both 5 'and 3' beyond the original oligonucleotide sequence shown in FIG. 3. In fact, the new sequence, 1.6kb in total length, extends all the way to the 3' poly-A tail. Overlapping ESTs added at each base pair along the sequence can enhance confidence in specific regions, for example, in sequences overlapping with EST R745933' (fig. 3). Several overlapping ESTs within this region confirmed the two key base deletions associated with R74593 (R74593 is located in bold underline in fig. 4C, positions 994 and 1005). Translation of the new consensus sequence (FIG. 4D) within the coding frame of the bicining peptide produced a placental bicining peptide that was larger (248 amino acids) than the mature sequence (179 amino acids) encoded by the original consensus sequence (SEQ ID NO: 1) and was terminated by an in-frame stop codon within the oligonucleotide consensus sequence. This increase in size is due to a frameshift of the 3' coding region caused by the deletion of the insertion of two bases characteristic of EST R74593. Frame-shifting allows the stop codon of the original consensus sequence (FIG. 3) to be shifted out of frame, which allows read-through into a new reading frame that encodes an additional amino acid sequence. The new translation product (FIG. 4D) is identical to the original protein consensus sequence (SEQ ID NO: 1) between residues +1 and +175 (encoding the Kunitz domain), but contains a new extended C-terminus showing a putative transmembrane domain 24 residues long (underlined in FIG. 4D), followed by a cytoplasmic domain of 31 residues. The exact sequence around the start codon methionine and the signal peptide is somewhat speculative due to considerable heterogeneity between overlapping ESTs in this region.

Through Geneworks^TMProtein sequence analysis was performed with the highlighted aspartic acids at positions 30 and 67 as putative N-linked glycosylation consensus sites. Aspartic acid 30 was not observed in N-terminal sequencing of the full-length protein isolated from human placenta, consistent with its glycosylation.

Cloning of human biconin peptide

The presence of human mRNA corresponding to the hypothetical human biculinine nucleotide sequence deduced from the analysis of FIG. 3 was confirmed as follows. Primer hybridizing to Kunitz-encoded cDNA sequence 5' of R35464 (consensus nucleic acid sequence 3-27bp in fig. 3):

GGTCTAGGAGGCCGGGTCGTTTCTCGCCTGGCTGGGA

(5 'primer derived from the R35464 sequence with an XbaI site; SEQ ID NO: 35), nucleic acid primers hybridizing 3' to the Kunitz-coding sequence of R74593 (consensus nucleotide sequence of 680-700bp in FIG. 3) were used to PCR amplify the Clontech human placenta library, expecting a fragment of about 670bp in length encoding the consensus nucleic acid sequence of the placental biculinin cDNA of FIG. 3. (as shown schematically in fig. 4A).

A fragment of the desired length (about 872bp) predicted by EST overlap (as shown schematically in FIG. 4) can be amplified from the Clontech human placental library using a 5 ' primer that hybridizes to the 126bp sequence of R87894 (as shown schematically in FIG. 4A) 5 ' to the putative ATG start site described above, plus a3 ' primer identical to R74593 used above.

Sequencing of the 872bp fragment showed that it contained at its 5 'end a region of nucleotides corresponding to 110 to 218bp of EST R87894 and at its 3' end a region of nucleotides from 310 to 542bp of the consensus sequence of human placental bicinin deduced from EST overlap analysis. The 3' nucleotide sequence contained all Kuntz-like domains encoded by placental dicurnine peptide (102-159).

To obtain a cDNA encoding the entire extracellular region of the protein, the following 5' PCR primers were designed which hybridized to EST R34808:

CACCTGATCGCGAGACCCC (SEQ ID NO: 36) this primer was used with the same EST 74593 3' primer to amplify (30 cycles) an approximately 780 base pair cDNA product from a human placental cDNA library. This product was gel purified and cloned into a TA vector (Invitrogen) for DNA sequencing by dideoxy (Sanger F., et al (1977) Proc. Natl. Acad. Sci. (USA), 74: 5463-:

vector specificity:

GATTTAGGTGACACTATAG(SP6)(SEQ ID NO：37)

TAATACGACTCACTATAGGG(T7)(SEQ ID NO：38)

gene specificity:

TTACCTGACCAAGGAGGAGTGC(SEQ ID NO：39)

AATCCGCTGCATTCCTGCTGGTG(SEQ ID NO：40)

CAGTCACTGGGCCTTGCCGT(SEQ ID NO：41

the resulting cDNA sequence and its translation product are shown in FIG. 4E. At the nucleotide level, this sequence showed only minor differences with the consensus EST sequence (FIG. 4D). The translation product of the sequence contains the coding sequence of the in-frame initiation ATG site, a signal peptide, and mature placental dicuronine and a transmembrane domain. Due to the selectivity of the 3' primer pair for PCR amplification, the translated sequence of the PCR product is deleted in the cytoplasmic domain by at least 12 amino acids. The selectivity of this 3' PCR primer (designed based on the sequence of R74593) also introduced an artificial variation of S to F at amino acid position 211 of the translated PCR-derived sequence. The signal peptide deduced from the translation of the PCR fragment also differed somewhat from the EST consensus sequence.

To obtain full-length placental dicurnine peptide cDNA, PCR-derived products (fig. 4E) were gel-purified and used to isolate non-PCR-based full-length clones representing the dicurnine peptide sequence. The PCR-derived cDNA sequence was run through High Prime (Boehringer Mannheim) to generate a cDNA sequence⁵²P-CTP labeling and use to probe placenta cDNA libraries by clonal hybridization techniques (Strategene, Unizap)^TMLambda library). After 3 rounds of screening and plaque purification, about 2X 10⁶And (4) carrying out plaque culture. Two clones considered to be full length (. about.1.5 kb) were identified by comparison with the EST consensus sequence (see above) and restriction enzyme analysis. Among these clonesThe dideoxy analysis of one of (a) gives the oligonucleotide sequence shown in FIG. 4F. The translation product from this sequence yields a sequence with an in-frame initiation codon methionine, a signal peptide, and mature placental bicurin peptide. The mature placental digonin peptide sequence is identical to the translated mature protein sequence derived from the EST consensus sequence, although the length and sequence of the signal peptide sequence is different. Unlike PCR derivatives, cDNA derived from clonal hybridization contains the entire extracellular domain, transmembrane domain, cytoplasmic domain, and in-frame stop codon. In fact, this clone extends all the way to the poly-A tail. The hydrophobic signal peptide following the start codon methionine is identical to the signal peptide of the PCR-derived clone. We have expressed and purified a soluble fragment of placental dicurnine peptide, dicurnine peptide (1-170), from Sf9 cells and found it to be a functional protease inhibitor (example 10). Furthermore, soluble fragments of placental digonin peptide isolated from human placenta by us are also active protease inhibitors (example 7). Native proteins, as well as protein forms expressed by Sf9 cells, may be glycosylated at the aspartic acid residue at position 30, based on PTH-amino acid recovery (recovers) in N-terminal sequencing (examples 7 and 9).

Based on these phenomena, it appears that the full-length placental diconin peptide has the ability to serve as both a cell membrane surface penetrating protein and a soluble protein. Other membrane penetrating proteins containing Kunitz domains are known to undergo proteolytic processing to produce mixtures with soluble and membrane associated forms. These include two forms of amyloid precursors, known as APP 751(Esch F. et al (1990) Science, 248: 1122-1124) and APP 770(Wang R et al (1991) J.biol Chem, 266: 16960 16964).

Contact activation is a process activated by exposure of the damaged vascular surface to components of the coagulation cascade. Angiogenesis is a process involving the local activation of plasmin at the endothelial surface. The specificity of placental dikonin and its putative ability to anchor to the cell surface suggests that the physiological functions of transmembrane placental dikonin may include modulation of contact activation and angiogenesis.

The amino acid sequences of placental dicurin (7-64), dicurin (102-159) and full-length placental dicurin (FIG. 4F) were found using the genetic Computer group program (Genetics Computer group program) Fast A in the PIR (version 46.0) and Patch X (version 46.0) protein databases and the Geneseq (version 20.00) protein database in patent sequences. The same protein sequence was searched for by the genetic computer group program TFasta (Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85: 2444-. EST and STS subsets of GenBank and EMBL were not included in this series of searches. The best fit from these searches also contained only 50% sequence identity over the full length of the 58 amino acid protein sequence derived from the R74593 and R35464 clones we analyzed.

Isolation of human bicurin peptide

As described above, synthetic peptides corresponding to the bicining (7-64) and the bicining (102-159) peptides as determined by translation of the consensus sequence of the bicining (FIG. 3) can be folded (examples 2 and 1, respectively) to produce activated kallikrein inhibitor proteins (examples 4 and 3, respectively). We investigated this unexpected property to design a purification route to isolate native placental dicurnine peptide from human tissue.

In the first step, highly purified natural functional kallikrein inhibitors are isolated using kallikrein-agarose affinity chromatography as a purification step. The isolated native human biculinin peptide has the same N-terminus (50 amino acid residues sequenced) as the translated amino acid residues +1 to +50 (example 7) of the predicted consensus nucleic acid sequence, confirming for the first time the presence of a novel native kallikrein inhibitor isolated from human placenta.

A list of known Kunitz-like domains is given below. It is believed that the residues that come into contact with the target protease are important for a particular purposeMarked (bold/underlined). These particular residues are designated as position Xaa for specific reference as marked by Xaa as follows^1-16：

Xaa 1 1 111 1 1

1 2 3 456789 0 1 234 5 6

1)IHDFCLVSKVV GRCRASMPRW WYNVTDGSCQ LFVYGGCDGN SNNYLTKEEC LKKCATV

2)YEEYCTANAVT GPCRASFPRW YFDVERNSCN NFIYGGCRGN KNSYRSEEAC MLRCFRQ

3)-HSFCAFKADD GPCKAIMKRF FFNIFTRQCE EFIYGGCEGN QNRFESLEEC KKMCTRD

4)-PDFCFLEEDP GICRGYITRY FYNNQTKQCE RFKYGGCLGN MNNFETLEEC KNICEDG

5)-PSWCLTPADR GLCRANENRF YYNSVIGKCR PFKYSGCGGN ENNFTSKQEC LRACKKG

6)-AEICLLPLDY GPCRALLLRY YYRYRTQSCR QFLYGGCEGN ANNFYTWEAC DDACWRI

7)-PSFCYSPKDE GLCSANVTRY YFNPRYRTCD AFTYTGCGGN DNNFVSREDC KRACAKA

8)-KAVCSQEAMT GPCRAVMPRT TFDLSKGKCV RFITGGCGGN RNNFESEDYC MAVCKAM

9)RPDFCLEPPYT GPCKARIIRY FYNAKAGLCQ TFVYGGCRAK RNNFKSAEDC MRTCGGA

10)----CQLGYSA GPCMGMTSRY FYNGTSMACE TFQYGGCMGN GNNFV EKEC LQTC

11) VAACNLPIVR GPCRAFIQLW AFDAVKGKCV LFPYGGCQGN GNKFYSEKEC REYCGVP

12)-EVCCSEQAET GPCRAMISRW YFDVTEGKCA PFFYGGCGGN RNNFDTEEYC MAVCGSA

13)----CKLPKDE GTCRDFILKW YYDPNTKSCA RFWYGGCGGN ENHFGSQKEC EKVC

14)-PNVCAFPMEK GPCQTYMTRW FFNFETGECE LFAYGGCGGN SNNTLRHEKC EKFCKFT

Wherein SEQ ID NO. 1) is bicining (7-64) (SEQ ID NO: 4) (ii) a Sequence No. 2) is bicining (102-159) (SEQ ID NO: 6) (ii) a Sequence No. 3) is tissue factor pathway inhibitor precursor 1(seq id NO: 18) (ii) a Sequence No. 4) is tissue factor pathway inhibitor precursor 1(SEQ ID NO: 19) (ii) a Sequence No. 5) is a tissue factor pathway inhibitor precursor (SEQ ID NO: 20) (ii) a Sequence 6) is a tissue factor pathway inhibitor precursor 2(SEQ ID NO: 21) (ii) a Sequence 7 is tissue factor pathway inhibitor precursor 2(SEQ ID NO: 22); sequence 8 is an amyloid precursor protein homolog (SEQ ID NO: 23); sequence 9) is aprotinin (SEQ ID NO: 24) (ii) a Sequence 10) is a meta-alpha-trypsin inhibitor precursor (seq id NOs: 25) (ii) a Sequence 11 is a precursor of meta-alpha-trypsin inhibitor (SEQ ID NOs: 26); sequence 12) is an amyloid precursor protein (SEQ ID NO: 27) (ii) a Sequence 13) is a collagen alpha-3 (VI) precursor (SEQ ID NO: 28) (ii) a And sequence 14) was HKI-B9(SEQ ID NO: 29).

It can be seen that placental dicurnine peptides (7-64) and (102-159) both have the same number and spacing of cysteine residues, as seen in members of the Kunitz family of serine protease inhibitors. Cysteine is known to form the precise bond of 3 intrachain disulfide bonds and is invariant to all previously known members of the Kunitz family (Laskowski, M et al, 1980, Ann. Rev. biochem. 49: 593-. Based on this known bonding pattern and the fact that folding of placental dicurnine peptides (7-64) and (102-159) into active protease inhibitors is accompanied by a reduction in mass consistent with three intrachain disulfide bond formation (examples 2 and 1), it is likely that disulfide bond formation within the Kunitz domain of placental dicurnine occurs between the following cysteine residues: c11 and C61; c20 and C44; c36 and C57; c106 and C156; c115 and C139; c131 and C152. Moreover, this disulfide bonding form is more likely in larger placental dicurnine peptides containing two Kunitz domains, because this form of protein is also an activated serine protease inhibitor, and because sequencing the N-terminus of native placental dicurnine peptide through 50 cycles (example 7) results in a sequence that is silent at the position where the cysteine is expected to appear.

The placental diconin peptides, isolated domains or other variants of the invention can be produced by standard solid phase peptide synthesis using t-Boc chemistry described in Merrified R.B. and Barany G. "peptides, assays, synthesis, biology 2", Gross E et al, Academic Press (1980) Chapter I; the F-moc chemistry is described in Carpino L.A. and Han G.Y. (1970) J.Amer Chem Soc.92, 5748-5749 and is described in example 2. Alternatively, expression of DNA encoding placental digonin peptide can be used to produce a recombinant placental digonin peptide variant.

The invention also relates to DNA constructs encoding the placental dicurnine peptide protein variants of the invention. These constructs can be obtained, for example, by Beaucage S.L. and CaruthersM.H. (1981) Tetrahedron Lett, 22, pages 1859-1862; matleucci m.d. and carothers M.H (1981), j.am.chem.soc.103, page 3185, or by screening genomic or cDNA libraries with probes designed to hybridize to DNA sequences encoding placental dicronin. The genomic or cDNA sequence may be modified at one or more positions to obtain a cDNA encoding an amino acid substitution or deletion as described in the disclosure.

The invention also relates to expression vectors containing DNA recombinants encoding the placental digonin peptides, isolated domains or other variants of the invention that can be used to produce recombinant placental digonin peptide variants. The cDNA should be linked to a suitable promoter sequence which shows transcriptional activity in the host cell of choice, with appropriate terminator and polyadenylation signals. The cDNA encoding the placental bicurin peptide variant may be fused to a 5' signal peptide which results in the secreted protein encoded by the cDNA. The signal peptide may be one that is recognized by the host organism. In the case of mammalian host cells, the signal peptide may also be a native signal peptide present in full-length placental dicurin peptide. Methods for preparing such vectors for expressing placental dicurnine peptide are well known in the art and are described, for example, in Molecular Cloning, Sambrook et al: a laboratory Manual, Cold Spring Harbor, New York, (1989).

The invention also relates to transformed cells containing recombinants of DNA encoding the placental digonin peptides, isolated domains or other variants of the invention that can be used to produce recombinant placental digonin peptide variants. There are many combinations of expression vectors and host organisms that can be used to produce placental dicurnine peptide variants. Suitable host cells include Sf6 insect cells infected with baculovirus, mammalian cells such as BHK, CHO, HeLa and C-127, bacteria such as E.Coli and yeasts such as Saccharomyces cerevisiae. Methods of using mammalian, insect and microbial expression systems requiring expression of placental dicurnine are well known in the art and have been described, for example, in Ausubel F.M et al, Current Protocols in Molecular Biology, John Wiley & Sons (1995), Chapter 16. Preferred for placental bicurin peptide fragments containing a single Kunitz inhibitor domain, such as bicurin peptides (7-64) and (102-159), are the yeast and e.coli expression systems, with the yeast expression system being most preferred. Typically, yeast expression can be performed as described for the aprotinin variant of U.S. patent 5,164,482 and adapted to example 5 for placental dikonin (102-159) of this specification. Coli expression can be performed using the method described in us patent 5,032,573. For larger placental digonin peptide variants containing two inhibitor domains, such as variant digonin peptide (7-159), mammalian and yeast systems are most preferred.

DNA encoding a placental diconin peptide variant with substituted amino acids of the native amino sequence can be identified using Kunkel T.A (1985) proc.natl.acad Sci USA 82: 488-492 was prepared as described for the expression of recombinant proteins. Briefly, the DNA to be mutagenized is cloned into a single stranded phage vector such as M13. The oligonucleotide extending to the region to be altered and encoding the substitution is hybridized with the single-stranded DNA to prepare a double strand by a conventional molecular biological method. This DNA was then transformed into an appropriate bacterial host and verified by dideoxy sequencing. The correct DNA was cloned into the expression plasmid. Alternatively, the target DNA can be mutagenized by conventional PCR techniques, sequenced, and then inserted into an appropriate expression plasmid.

The present invention relates to a substantially purified protein having serine protease inhibitory activity, selected from the group consisting of proteins comprising any one of the following amino acid sequences numbered according to the amino acid sequence of the natural human placental dicurnine peptide set forth in fig. 4F, wherein the N-terminal residue resulting from removal of the signal peptide in fig. 4F is designated as residue No. 1:

ADRERSIHDF CLVSKVVGRC RASMPRWWYN VTDGSCQLFV YGGCDGNSNN 50

YLTKEECLKK CATVTENATG DLATSRNAAD SSVPSAPRRQ DSEDHSSDMF 100

NYEEYCTANA VTGPCRASFP RWYFDVERNS CNNFIYGGCR GNKNSYRSEE 150

ACMLRCFRQQ ENPPLPLGSK VVVLAGLFVM VLILFLGASM VYLIRVARRN 200

QERALRTVWS SGDDKEQLVK NTYVL 22S

(SEQ ID NO：70)；

ADRERSIHDF CLVSKVVGRC RASMPRWWYN VTDGSCQLFV YGGCDGNSNN 50

YLTKEECLKK CATVTENATG DLATSRNAAD SSVPSAPRRQ DSEDHSSDMF 100

NYEEYCTANA VTGPCRASFP RWYFDVERNS CNNFIYGGCR GNKNSYRSEE 150

ACMLRCFRQQ ENPPLPLGSK VVVLAGLFVM VLILFLGASM VYLIRVARRN 200

QERALRTVWS FGD 213

(SEQ ID NO：71)；

the protein wherein said protein is glycosylated or contains at least one intrachain cysteine-cysteine disulfide bond, or both glycosylated and contain at least one intrachain cysteine-cysteine disulfide bond.

The invention relates to a pharmaceutical composition for inhibiting serine protease activity, which comprises one of the proteins as described above together with a pharmaceutically acceptable carrier.

The present invention relates to an isolated nucleic acid sequence encoding the above-mentioned protein.

The present invention relates to a self-replicating protein expression vector comprising a nucleic acid sequence encoding and capable of expressing the protein. The invention relates to the use of the protein in the preparation of a medicament for inhibiting serine protease activity.

The invention relates to the use of a protein as described above for the preparation of a medicament for the treatment of disease conditions comprising: cerebral edema, spinal cord edema, multiple sclerosis, ischemia, intraoperative blood loss, sepsis, septic shock, fibrosis, diseases associated with pathological blood clotting or clotting, combined trauma, stroke, cerebral or subarachnoid hemorrhage, brain inflammation, spinal cord inflammation, brain infection, cerebral granuloma, spinal cord infection, spinal cord granuloma, open heart surgery, gastric cancer, cervical cancer, or prevention of metastasis.

The present invention relates to a method for preparing the protein by using recombinant DNA technology.

The following specific examples are provided for the purpose of illustration and are not intended to limit certain aspects and preferred embodiments of the present invention.

Example 1: preparation of synthetic placental dicurnine (102-159)

Materials and methods/reagents used. Fluorogenic substrate Tos-Gly-Pro-Lys-AMC was purchased from Bachem BioScience Inc (King of Prussia, Pa.). PNGB, Pro-Phe-Arg-AMC, Ala-Ala-Pro-Met-AMC, bovine trypsin (type III), human plasma kallikrein, and human plasmin are from Sigma (St. Louis, Mo.).

Recombinant aprotinin (Trasylol)^) From Bayer AG (Wuppertal, Germany). Pre-placed Gln Wang resin was purchased from Novabiochem (La Jolla, Calif.). Thioanisole, ethanedithiol (ethanedithiol) and tert-butyl methyl ether were purchased from Aldrich (Milwaukee, Wis.). Quantification of functional placental dicuronine (7-64) and (102-159)

The amount of trypsin inhibitory activity present in refolded samples at each stage of purification was determined using GPK-AMC as substrate. Bovine trypsin (200 picomolar) was mixed with either biculinin (7-64) or (102-159) from different purification stages in buffer A (50mM, Hepes, pH7.5, 0.1M NaCl, 2mM CaCl)₂And 0.01% triton X-100) at 37 ℃ for 5 minutes. GPK-AMC (20 μ M final concentration) was added and the amount of coumarin produced was determined by measuring the fluorescence (ex 370nm, em 432nm) on a Perikin-Elmer LS-50B fluorometer. Calculating the percent inhibition for each sample tested according to equation 1; wherein R is₀Is the increase in fluorescence in the presence of an inhibitor, R₁Is the ratio determined without the sample. The unit of activity of the inhibitor is defined as the amount required to obtain 50% inhibition in the experiment under the experimental conditions described above.

Percent inhibition of 100 × [1-R × ]₀/R₁] (1)

And (4) synthesizing. Placental dicuronine (102-159) was synthesized on an Applied Biosystems model 420A peptide synthesizer using NMP-HBTU Fmoc chemistry. Peptides were synthesized with an 8-fold excess of amino acids for each coupling on pre-placed Gln resins. Cleavage and deprotection were accomplished by treatment with 84.6% trifluoroacetic acid (TFA), 4.4% thioanisole, 2.2% dithiol, 4.4% liquefied phenol, and 4.4% water for two hours at room temperature. The crude peptide was precipitated, centrifuged and washed twice with tert-butyl methyl ether. Peptides were purified on a Dynamax 60AC18 reverse phase HPLC column with a TFA/acetonitrile gradient. The final product (61.0mg) yielded the correct amino acid combination and molecular weight, and the predicted sequence was determined by Electrospray mass spectrometry (MH + ═ 6836.1; Calcd ═ 6835.5):

YEEYCTANAV TGPCRASFPR WYFDVERNSC NNFIYGGCRG NKNSYRSFEA

CMLRCFRQ(SEQ ID NO.6)

and (5) purifying. Refolding of placental biconin (102-159) was carried out according to the method of Tam et al (J.Am.chem.Soc.1991, 113: 6657-62). A portion of the purified peptide (15.2mg) was dissolved in 4.0ml of 0.1M Tris, pH6.0 and 8M urea. Oxidation of disulfide bonds was accomplished by dropping a solution containing 23% DMSO, and 0.1M Tris, pH6.0, to a final concentration of 0.5mg/ml peptide in 20% DMSO, 0.1M Tris, pH6.0, and 1M urea. The solution was stirred at 25 ℃ for 24 hours and then diluted 1: 10 with a buffer containing 50mM Tris, pH8.0 and 0.1M NaCl. This material was purified by a kallikrein affinity column prepared by covalently binding 30 mg of bovine pancreatic kallikrein (Bayer AG) to 3.5 ml of CNBr-activated Sapharose (pharmacia) according to the manufacturer's recommendations. The refolded material was placed on an affinity column and washed with 50mM Tris, pH8.0, and 0.1M NaCl at a flow rate of 1 ml/min until no absorbance could be detected at 280nm wash. The column was eluted with 3 volumes of 0.2M acetic acid, pH4.0 and 1.7, respectively. The active ingredient (shown below) was aggregated and the pH of the solution was adjusted to 2.5. This material was placed directly on a Vydac C18 reverse phase column (5 μm, 0.46 x 25 cm) equilibrated with 22.5% acetonitrile in 0.1% TFA. The separation was accomplished in a linear gradient from 22.5 to 40% acetonitrile in 1.0 ml/min 0.1% TFA over a period of 40 minutes. The active fractions were aggregated, lyophilized, redissolved in 0.1% TFA and stored at-20 ℃ until use.

And (6) obtaining the result. Synthetic placental diconin (102-159) peptide was refolded as described above using 20% DMSO as the oxidizing agent and purified by the two-step purification method shown below to obtain active trypsin inhibitors (Table 1 below).

TABLE 1

Purification Table for isolation of synthetic placental digonin (102-159)

TABLE 1
	Purification step volume mg/mg unitcSpA yield (ml) ml (U) (U/mg)
8.0M Urea 4.03.75a 15.0 0 0 -
	20％DMSO 32.0 0.47a 15.0 16,162 1,078 100
Kallikrein affinity 9.80.009b 0.09 15,700 170,000 97
	C18 3.0 0.013ab 0.04 11,964 300,000 74

a protein determined by AAA.

b purified protein (1.7X 10) determined by quenching correlation coefficient (inactivation)⁴Liter mole¹Centimeter^-1) OD280 nm determined protein.

c one unit is defined as the material required to inhibit 50% of trypsin activity in routine experiments.

The layer-folded crude refolded material on the immobilized bovine pancreatic kallikrein column selectively separated 6.0% of the protein and exhibited 97% trypsin inhibitory activity. Subsequent reverse phase chromatography using C18 yielded 2 further purifications with an overall recovery of 74%. Reduced and refolded placental digonin peptide (102-159) exhibited elution times of 26.3 and 20.1 minutes, respectively, on RPHPLC. Mass spectrometry analysis of the purified material revealed a molecular weight of 6829.8 with a loss of 6 mass units compared to the starting material. This demonstrates the complete formation of 3 disulfide bonds of the expected peptide sequence.

With preformed Amphiline in combination with PI standard^PAGP plate (pH3.5 to 9.5) was used to determine the isoelectric point of purified refolded synthetic placental dicomin peptide (102-159) by Electrophoresis using Multiphor II Electrophoresis system and focusing for 1.5 hours according to the manufacturer's recommendations. After staining the migration distance from the cathodic edge of the gel to the different protein bands was determined. Each unknown PI is determined by a standard curve generated from the curves resulting from the migration distance standard and its corresponding PI. Using this method, the PI of placental dicurnine peptide (102-159) was determined to be 8.3, which matched the value predicted from the amino acid sequence. This is lower than the value of 10.5 for aprotinin determined by PI (Tenstad et al, 1994, Acta Physiol. Scand.152: 33-50).

Example 2

Preparation of synthetic placental bikunin peptide (7-64)

Placental digoxin (7-64) was synthesized, refolded and purified essentially as described for placental digoxin (102-159), with the following modifications: stirring the synthetic peptide as a solution in 20% DMSO at 25 ℃ for 30 hours while folding; c18 RP-HPLC purification was accomplished with a linear gradient of 25 to 45% acetonitrile in 0.1% TFA over 40 minutes. The active fraction collected from the first C18 was re-loaded onto the column and fractionated with a linear gradient (60 min, 1 ml/min) of 20 to 40% in 0.1% TFA.

And (6) obtaining the result. The finally obtained purified reduced peptide exhibited MH⁺6563, in accordance with the following sequence:

IHDFCLVSKV VGRCRASMPR WWYNVTDGSC QLFVYGGCDG NSNNYLTKEE

CLKKCATV(SEQ ID NO：4)

refolding and purification yielded a functional Kunitz domain with trypsin inhibitor activity (see Table 2 below)

TABLE 2A purification Table of isolated synthetic placental dikonin peptides (7-64)
	Purification step volume mg/mg unit SpA yield (ml) ml (U) (U/mg)
8.0M Urea 8.02.520.000-
	20％DMSO 64.0 0.31 20.0 68,699 3,435 100
Kallikrein (Kall) 11.70.101.1643,33336,11062 affinity ratio pH4.0
	Kallikrein (Kall) 9.00.645.849728577.2 affinity ratio pH1.7
C18-1 4.6 0.14 0.06 21,905 350,143 31.9
	C18-2 1.0 0.08 0.02 7,937 466,882 11.5

The purified refolded protein exhibited MH + 6558, that is 5 ± 1 mass units less than the reduced peptide. This confirms that refolding results in at least one appropriate form of disulfide bond.

The PI of placental dicurin peptide (7-64) was determined using the PI method for determining placental dicurin peptide (102-158). Placental dicurin peptide (7-64) exhibited a PI much higher than expected (PI 7.9). Refolded placental digonin peptide (7-64) migrated to the cathodic edge of the gel (pH9.5), and its PI value could not be accurately determined under these conditions.

Subsequent preparation of synthetic placental dikonin (7-64).

Because the synthetic placental digonin peptides may not undergo complete deprotection prior to purification and refolding, refolding is repeated with the protein identified to be completely deprotected. The synthesis, refolding and purification of placental digoxin (7-64) is essentially identical to that used for placental digoxin (102-159), with the following modifications: in refolding, the synthetic peptide (0.27 mg/ml) in the form of a 20% DMSO solution was stirred at 25 ℃ for 30 hours; c18 RP-HPLC purification was accomplished with a 22.5 to 50% linear gradient of acetonitrile in 0.1% TFA over 40 minutes.

And (6) obtaining the result. The resulting purified reduced peptide exhibited MH⁺6567.5, which is matched with the following sequence:

IHDFCLVSKV VGRCRASMPRW WYNVTDGSC QLFVYGGCDG

NSNNYLTKEE CLKKCATV(SEQ ID NO：4)

folding and purification resulted in a functional Kunitz domain with trypsin inhibitor activity (as shown in table 2B below).

TABLE 2B

Purification Table for isolation of synthetic placental digonin peptide (7-64)

TABLE 2B
	Purification step volume milligram per milligram unit (U) SpA yield (ml) milliliters (U/mg))
8.0M Urea 4.92.110.500-20% DMSO 39.00.2710.5236,00022,500100 kallikrein affinity 14.50.30.43120,000279,07050.9 Rate (pH2) C18 reverse 0.21.20.2470,676294,48330.0

The purified folded protein exhibited MH + ═ 6561.2, that is, it was 6.3 mass units less than the reduced peptide. This indicates that the folding caused the formation of the expected disulfide bonds.

The PI of refolded placental digonin (7-64) was determined using the PI method for determining placental digonin (102-159). Refolded placental dicuronine (7-64) showed a PI of 8.85, slightly higher than predicted (PI 7.9).

Example 3

In vivo specificity of functional placental dicuronine fragment (102-159)

A protease. Quantification of bovine trypsin, human trypsin and bovine pancreatic kallikrein was performed by active site titration (active site titration) using P-nitrophenyl-P' -guanidinobenzoate HCl as previously described (Chase, t. and Shaw, E. (1970) Methods enzmo1., 19: 20-27). Human kallikrein was determined by active site titration using bovine aprotinin as standard, PFR-AMC as substrate and presumably forming a 1: 1 complex. K of Trypsin and plasmin against GPK-AMC under these conditions_m29 μ M and 726 μ M, respectively; k of human plasma kallikrein and bovine pancreatic kallikrein on PFR-AMC_m457. mu.M and 81.5. mu.M, respectively; k of elastase to AAPR-AMC_mIs 1600. mu.M. Quantification of human tissue kallikrein (Bayer, germany) was performed using active site titration with P nitrophenyl P' -guanidinobenzoate HCL as previously described (Chare, t., andShow，E.(1970)Methods Enzmol.19：20-27)。

inhibition kinetics: inhibition of trypsin by placental bicurin (102-159) or aprotinin was determined by incubating 50PM trypsin with placental bicurin (102-159) (0-2nM) or aprotinin (0-3nM) in buffer A in a total volume of 1.0. mu.l. After 5 minutes at 37 ℃ 15. mu.l of 2mM GPK-AMC were added and the change in fluorescence was monitored (as above). Inhibition of human plasmin by placental dicurin (102-159) and aprotinin was determined using plasmin (50pM) in a buffer containing 50mM Tris-Hd (pH7.5), 0.1M NaCl and 0.02% triton X-100, and placental dicurin (102-159) (0-10nM) and aprotinin (0-4 nM). After 5 minutes, incubation was performed at 37 ℃ and 25. mu.l of 20mM GPK-AMC was added and the change in fluorescence was monitored. Inhibition of human plasma kallikrein by either placental dicurin (102-159) or aprotinin was determined using kallikrein (2.5nM) and placental dicurin (102-159) at 0-3nM or aprotinin (2.5nM) in 50mM Tris-Hd (pH8.0), 50mM NaCl and 0.02% triton X-100. After 5 minutes, 15. mu.l of 20mM PFR-AMC were added at 37 ℃ and the change in fluorescence was monitored. Inhibition of bovine pancreatic kallikrein by placental dikonin (102-159) and aprotinin was determined in a manner similar to that of kallikrein (92pM), placental dikonin (102-159) (0-1.6nM) and aprotinin (0-14PM) and at a final substrate concentration of 100. mu.M. The apparent inhibition constant Ki was determined using the nonlinear regression data analysis program, Enzfitter software (Biosoft, Cambridge, uk):

kinetic data for each experiment were analyzed with the equation for tight binding inhibitors:

V_i/V_o＝1-(E_o+I_o+K₁ ^*-[(E_o+I_o+K_i ^*)²-4E_oI_o)]^1/2)/2E_o (2)

wherein V_i/V_oIs the relative enzyme activity (ratio of inhibited to uninhibited), E_oAnd I_oConcentrations of enzyme and inhibitor, respectively, K_iThe value is corrected to the bottom according to the following equationThe product effect is as follows:

K_i＝K_i ^*/(1+[S_o]/K_m) (3)

(Boudier, C. and Bieth, J.G. (1989) Biochim Biophys acta, 995: 36-41)

For inhibition of human neutrophil elastase by placental dikonin (102-159) and aprotinin, elastase (19nM) was incubated with placental dikonin (102-159) (150nM) or aprotinin (0-7.5. mu.M) in a buffer containing 0.1M Tris-HCl (pH8.0) and 0.05% Triton X-100. After 5 minutes at 37 deg.C, AAPM-AMC (500. mu.M or 1000. mu.M) was added and fluorescence was measured over a period of two minutes. At 1/V pairs [ I ] at two different substrate concentrations]Dixon plot (Dixon plot) of (K)_iValues (Dixon et al, 1979).

The inhibition of human tissue kallikrein by aprotinin, placental biculinine fragments (7-64) or placental biculinine fragments (102-159) was determined by incubating human tissue kallikrein at 0.35nM with placental biculinine (7-64) (0-40nM) or placental biculinine (102-159) (0-2.5nM), or aprotinin (0-0.5nM) in a reaction volume of 1 ml containing 50mM Tris-HCl buffer pH9.0, 50mM NaCl and 0.1% TritonX-100. After 5 minutes, 5. mu.l of 2mM PFR-AMC was added at 37 ℃ to reach a final concentration of 10. mu.M and the change in fluorescence was monitored. K of human tissue kallikrein on PFR-AMC under the conditions of this assay_mThe value was 5.7. mu.M. The inhibition of human factor Xa (American Dianositica, Inc, Greenwich, CT) by synthetic placental dicurnine (102-159), recombinant placental dicurnine and aprotinin was determined as follows: human factor Xa in an amount of 0.87nM was incubated with increasing amounts of inhibitor in a buffer containing 20mM Tris (pH7.5), 0.1M NaCl, and 0.1% BSA. After 5 minutes at 37 ℃ 30. mu.l of 20mM LGR-AMC (Sigma) was added and the change in fluorescence was monitored. The inhibition of human urokinase by the Kunitz inhibitor was determined by incubating urokinase (2.7ng) with the inhibitor in a total volume of 1 ml of buffer containing 50mM Tris-HCl (pH8.0), 50mM NaCl and 0.1 TritonX-100. 3After 5 minutes at 7 ℃ 35. mu.l of 20mM GGR-AMC (Sigma) were added and the change in fluorescence was monitored. Inhibition of factor XIa (purchased from Enzyme Research Labs, south ben, IN) was performed by combining factor XIa (0.1nM) with placenta bicurin (7-64) 0 to 800nM, placenta bicurin (102-159)0 to 140nM or aprotinin 0 to 40nM IN a medium containing 50mM Hepes pH7.5, 100mM NaCl, 2mM CaCl₂Incubation was performed in a total volume of 1 ml of buffer with 0.01% TritonX-100 and 1% BSA. After 5 minutes at 37 deg.C, 40mM Boc-Glu (OBzl) -Ala-Arg-AMC (Bachem Biosciences, King of Prussia, Pa.) was added and the change in fluorescence was monitored.

And (6) obtaining the result. A direct comparison of the inhibitory properties of placental dicurin (102-159) and aprotinin was made by determining their inhibition constants for the various proteases under the same conditions. K_iThe values are listed in table 3 below.

TABLE 3

Ks for inhibition of various proteases by the biconin peptide (102-159)_iValue of

TABLE 3
	Protease (concentration) biconin aprotinin substrate (concentration) Km(102-159) enzyme peptide (mM) Ki(nM) Ki(nM)
Trypsin (48.5pM) 0.40.8 GPK-AMC (0.03mM) 0.022
	Chymotrypsin (5nM) 0.240.86 AAPF-pNA (0.08mM) 0.027
Bovine pancreatic kallikrein 0.40.02 PFR-AMC (0.1mM) 0.08(92.0pM)
	Human plasma kallikrein 0.319.0 PFR-AMC (0.3mM) 0.46(2.5nM)
Human plasmin (50pM) 1.81.3 GPK-AMC (0.5mM) 0.73
	Human neutrophilic elastin 323.08500.0 AAPM-AMC (1.0 μ M) 1.6 enzyme (19nM)
Factor XIIa > 300.012,000.0 PFR-AMC (0.2. mu.M) 0.35
	Human tissue kallikrein 0.130.004 PFR-AMC (10. mu.M) 0.0057(0.35nM)
Factor Xa (0.87nM) 274 N.I. LGR-AMC (0.6mM) N.D. at 3. mu.M
	Urokinase 110004500 GGR-AMC (0.7mM) N.D.
Factor XIa (0.1nM) 15288E (OBz) AR-MC (0.4mM) 0.46

Under the conditions used, placental dicurin (102-159) and aprotinin inhibited bovine trypsin and human plasmin to a comparable (similar) extent. K for inhibiting elastase by aprotinin₁Was 8.5. mu.M. Placental digonin peptide (102-159) K inhibiting elastase_iIs 323 nM. K inhibition of bovine pancreatic kallikrein by placental digonin (102-159)_iValue ratio of K to aprotinin_iThe value was 20 times higher. In contrast, placental dikonin (102-159) is a more potent inhibitor of human plasma kallikrein than aprotinin, which binds to the parentThe neutrality is 56 times higher.

Placental dicuronine (102-159) is more potent than Trasylol as a kallikrein inhibitor^More than 50 times higher, because of this, only the specific Trasylol need be used to maintain an effective patient dose of inhibitor KIU^A lesser amount of human placental digoxin or a fragment thereof (i.e., placental digoxin (102-159)). This reduces the cost of each dose of drug and reduces the likelihood of adverse nephrotoxic effects occurring in the patient from re-exposure to the drug. Moreover, since the protein is of human origin, it is less immunogenic in humans than aprotinin from cattle. This also results in a greatly reduced risk of it causing an adverse immunogenic response when the patient is re-exposed to the agent.

Example 4

In vitro specificity of functional placental diconin peptide fragments (7-64).

The in vitro specificity of functional human placental dikonin (7-64) was determined using the materials and methods described in the examples above.

As a result: the table below shows the effectiveness of placental dikonin (7-64) as an inhibitor of various serine proteases in vitro. The data presented are from placental dicurnine (102-159) and aprotinin (Tasylol)^) Data for screening inhibition were compared.

TABLE 4A

Ks for inhibiting various proteases by bikunin peptide (7-64)_iValue of

TABLE 4A
	Proteinase (concentration) bicurin inhibin (102-159) (7-64) Ki(nM) peptide Ki(nM)Ki(nM)
Trypsin (48.5pM) 0.170.80.4
	Bovine pancreatic kallikrein (92.0pM) 0.40.020.4
Human plasma kallikrein (2.5nM) 2.419.00.3
	Human plasmin (50pM) 3.11.31.8
Bovine chymotrypsin (5nM) 0.60.90.2
	Factor XIIa > 30012000 > 300
Elastase > 1008500323

The results show that the amino acid sequence encoding placental dikonin (7-64) refolds into active serine protease inhibitors that are effective against at least the four tryptase serine proteases.

Table 4B below also shows the effectiveness of refolded placental dikonin (7-64) as a variety of serine protease inhibitors in vitro. Refolded placental digonin peptide (7-64) was prepared from proteins determined to be fully deprotected prior to purification and refolding. The data presented are from placental dicurnine (102-159) or aprotinin (Trasylol)^) Data for screening inhibition were compared.

TABLE 4B

K for refolding biculinin (7-64) for inhibition of various proteases_iValue of

TABLE 4B
	Proteinase (concentration) bicurin inhibin (102-159) (7-64) Ki(nM) peptide Ki(nM)Ki(nM)
Trypsin (50pM) 0.20.80.3
	Human plasma kallikrein (0.2nM) 0.719.00.7
Human plasmin (50pM) 3.71.31.8
	Factor XIIa was not done 12,0004,500
Factor XIa (0.1nM) 20028815
	Human tissue kallikrein 2.30.0040.13

Surprisingly, placental dikonin (7-64) was more effective than aprotinin in inhibiting human plasma kallikrein, and was at least similar in effectiveness as a plasmin inhibitor. These data indicate that placental dikonin is at least as effective as aprotinin in vitro experiments, and it is expected that it should have better or similar capabilities in vivo.

Example 5

Expression of placental digonin variant (102-159) in Yeast

The DNA sequence encoding placental digonin 102-159(SEQ ID NO: 6) was generated using synthetic oligonucleotides. The DNA end product (5 'to 3') consisted of 15 nucleotides from the yeast α -mating factor propeptide sequence fused to the in-frame cDNA sequence encoding placental biconin peptide (102-159) followed by an in-frame stop codon. This cDNA, when cloned into the yeast expression vector pS604, was able to direct the expression of a fusion protein containing the N-terminal yeast α -mating factor propeptide fused to the 58 amino acid sequence of placental dikonin (102-159). Processing of the fusion protein at the KEX-2 cleavage site at the junction of the α -mating factor and the Kunitz domain was designed to release the Kunitz domain at its native N-terminus.

A5' sense oligonucleotide containing the following sequence and a HindIII site for cloning was synthesized.

GAA GGG GTA AGC TTG GAT AAA AGA TAT GAA GAA TAC TGCACC GCC AAC GCA GTC ACT GGG CCT TGC CGT GCA TCC TTC CCACGC TGG TAC TTT GAC GTG GAG AGG(SEQ ID NO：42)

A3' antisense oligonucleotide containing a BamHI site and a stop codon for cloning and having the following sequence was synthesized:

CGC GGA TCC CTA CTG GCG GAA GCA GCG GAG CAT GCA GGC CTC

CTC AGA GCG GTA GCT GTT CTT ATT GCC CCG GCA GCC TCC ATA

GAT GAA GTT ATT GCA GGA GTT CCT CTC CAC GTC AAA GTA CCA

GCG

(SEQ ID NO：43)

the oligonucleotides were dissolved in 10mM Tris buffer pH8.0 containing 1mM EDTA, and 12 micrograms of each oligonucleotide, combined with 0.25M NaCl. For hybridization, the oligonucleotides were boiled for 5 minutes to denature and cooled from 65 ℃ to room temperature over a period of 2 hours. The overlapping sequences were extended with the Klenow fragment and digested with HindIII and BamHI. The resulting digested double-stranded fragments were cloned into PUC 19 and verified by sequencing. The clone containing the correct sequence fragment was digested with BamHI/HindIII to release the bicinin peptide containing a fragment with the following + chain sequence:

GAA GGG GTA AGC TTG GAT AAA AGA TAT GAA GAA TAC TGC ACC

GCC AAC GCA GTC ACT GGG CCT TGC CGT GCA TCC TTC CCA CGC

TGG TAC TTT GAC GTG GAG AGG AAC TCC TGC AAT AAC TTC ATC

TAT GGA GGC TGC CGG GGC AAT AAG AAC AGC TAC CGC TCT GAG

GAG GCC TGC ATG CTC CGC TGC TTC CGC CAG TAG GGA TCC(SEQ

ID.：44)

then, it was gel purified and ligated into BamHI/HindIII digested pS 604. The ligation mixture was extracted with phenol/chloroform and purified using an S-200 micro spin column (minispin column). The ligation products were transduced into yeast strains SC101 and WHL341 and plated onto ura selection plates. 12 clones from each strain were restreaked on ura shed (drop out) plates. The single clones were inoculated into 2 ml ura DO medium and grown overnight at 30 ℃. Cells were pelleted at 14000 Xg 2 min and the content of placental digonin peptide (102-159) in the supernatant was assessed.

Detection of expression of placental digonin (102-159) in transformed Yeast

First, the ability of the supernatant (50 microliters per test) to inhibit trypsin activity in vitro was determined using the method described in example 1(1 ml test volume). Negative controls used yeast clones expressing inactive variants of aprotinin and unused media samples. Yeast clones expressing native aprotinin were used as positive controls and presented in comparative form.

A second method for quantifying placental digoxin (102-159) expression developed the use of polyclonal antibodies (pAbs) to synthetic peptides to monitor the accumulation of recombinant peptides using Western blotting. These studies were only performed on strains derived from SC101, since they produced stronger inhibitory activity than recombinants derived from WHL 341.

To generate pAb, two 6-8 week old New Zealand white female rabbits (Hazelton Research Labs, Denuer, Pa.) were immunized daily with 0 to 250 micrograms of purified reduced synthetic placental dikonin (102-159) in the form of complete Freund's adjuvant and then boosted daily with 125 micrograms of the same antigen in the form of incomplete Freund's adjuvant on days 14, 35 and 56 and 77. Antisera used in this study were collected after the third boost using off-the-shelf methods. Polyclonal antibodies were purified using antiserum to protein a.

Clones 2.4 and 2.5 from transformed yeast SC101 (FIG. 8) and control aprotinin were grown overnight at 30 ℃ in 50 ml ura DO medium. The cells were pelleted and the supernatant was concentrated 100-fold using Centriprep 3(Amicon, Beverly, Mass.) concentrate. SDS-PAGE was performed on each sample (30. mu.l) using the manufacturer's method in 10-20% tricin buffer gel (Novex, San Diego, Calif.). The gels were either developed with silver staining kits (interclass Separation Systems, Nantick, Mass.) or transferred to nitrocellulose filters and developed with purified polyclonal antibodies to synthetic dicurnine (102-159). Goat anti-rabbit antibodies conjugated with alkaline phosphatase were used as secondary antibodies according to the manufacturer's recommendations (Kirkegaardand Perry, Gaithersburg, Md.).

Purification of placental dicuronine from SC101 transformed strains (102-159)

The fermentation broth of a1 liter culture from SC101 strain 2.4 was harvested by centrifugation (4000 g.times.30 min), and then applied to a 1.0 ml column of dehydrated chymotrypsin-Sepharose (Takara Biochemical Inc., CA) previously treated with 2mM CaCl containing 0.1M NaCl₂And 0.01% (v/v) triton X-100 in 50mM Hepes buffer. The column was washed with the same buffer but containing 1.0M NaCl until the A280 nm dropped to zero, at which time the column was eluted with 0.1M formic acid at pH 2.5. The eluted fractions were collected and placed on a C18 column (Vydac, 5 μm, 4.6X 250 mm) equilibrated with 0.1% TFA and then with a linear gradient of 20 to 80% acetonitrile in 0.1% TFAAnd (4) eluting. Fractions containing placental dicuronine (102-159) were collected and re-chromatographed on C18 using a 22.5 to 50% acetonitrile gradient in 0.1% TFA.

And (6) obtaining the result. Figure 8 shows the percentage of inhibition of trypsin activity by 12 clones derived from transformation of each SC101 and WHL341 strains. The results showed that all 12 clones of yeast strain SC101 transformed with the trypsin inhibitor placental digoxin (102-159) had the ability to produce a comparable amount of trypsin inhibitory activity compared to two negative controls that did not exhibit trypsin inhibition. This activity was therefore related to the expression of specific inhibitors in cells transformed with the placental dicurnine variant (102-159). The yeast WHL341 sample contained minimal trypsin inhibitory activity. This may be associated with the observed slow growth of the strain under the conditions used.

FIG. 9 shows SDS-PAGE and Western blotting of the supernatant of the yeast SC101 strain. SDS-PAGE of supernatants derived from recombinant yeasts 2.4 and 2.5 expressing placental dicurnin (102-159) and from yeasts expressing aprotinin was silver stained and the resulting protein bands were electrophoresed to a position of approximately 6kDa, coinciding with the size of each recombinant Kunitz domain desired. Western blots showed that the 6kDa bands expressed by strains 2.4 and 2.5 reacted with pAb for placental dikonin (102-159). The same 6kDa band in aprotinin did not react with the same antibody, demonstrating the specificity of this antibody for the placental digonin variant (102-159).

The final product of the C-terminal domain of placental diconin peptide was highly purified by silver-stained SDS-PAGE (fig. 10). The overall recovery of the broth-derived trypsin inhibitory activity in the final product was 31%. N-terminal sequencing of the purified inhibitor showed that 40% of the protein was correctly processed to the correct N-terminus of placental biconin peptide (102-159), whereas 60% of the material contained a portion of yeast α -mating factors. The purified material contains an activated serine protease inhibitor which exhibits K on in vitro inhibition of plasma kallikrein_iIs 0.35 nM.

In general, protease inhibitory activity and accumulation of proteins immunochemically correlated with synthetic dicurnine peptide (102-159) in fermentation broth, and isolation of placental dicurnine peptide (102-159) from a transformed line provided evidence of placental dicurnine peptide expression in recombinant yeast as described herein, demonstrating for the first time the use of yeast for the production of placental dicurnine peptide fragments.

Additional constructs were made in an effort to increase the expression level of the Kunitz domain contained in placental diguanine peptide 102-159, while increasing production of proteins with the correct N-terminus. We hypothesized that the N-terminal residue of placental digonin 102-159 (YEEY- -) might have a cleavage site that is only poorly recognized by the yeast KEX-2 protease, which enzymatically removes the yeast alpha-factor prepro region. Thus, we prepared yeast expression constructs for the production of placental biculinin 103-159(EEY N-terminal.), 101-159 (NYEY N-terminal.) and 98-159 (DMFNYEEY.) to modify the P' secondary site around the KEX-2 cleavage site. In an attempt to increase the expression level of the recombinant protein. We used yeast-preferred codons instead of mammalian-preferred codons to make some of the constructs described below. These constructs were prepared essentially as described above for placental digoxin 102-159 (referred to as construct #1), with the following modifications:

construct #2 placental Dicunin peptide 103-159, Yeast codon usage

5' sense oligonucleotide

GAAGGGGTAA GCTTGGATAA AAGAGAAGAA TACTGTACTG

CTAATGCTGT TACTGGTCCA TGTAGAGCTT CTTTTCCAAG

ATGGTACTTT GATGTTGAAA GA(SEQ ID NO：55)

And 3' antisense oligonucleotides

ACTGGATCCT CATTGGCGAA AACATCTCAA CATACAGGCT

TCTTCAGATC TGTAAGAATT TTTATTACCT CTACAACCAC

CGTAAATAAA ATTATTACAA GAATTTCTTT CAACATCAAA

GTACCATCT(SEQ ID NO：56)

Prepared as described for the production of the expression vector for placental digonin 102-159 (construct #1 above).

Construct #3 placental diconin 101-159, Yeast codon usage

5' sense oligonucleotide

GAAGGGGTAA GCTTGGATAA AAGAAATTAC GAAGAATACT

GTACTGCTAA TGCTGTTACT GGTCCATGTA GAGCTTCTTT

TCCAAGATGG TACTTTGATG TTGAAAGA(SEQ ID NO：57)

The same 3' antisense oligonucleotide as used in construct #2 was prepared using the method for producing an expression vector for the expression of placental biconin 102-159 (construct #2, supra).

Construct #4 placental diconin peptide 98-159, codon usage of Enzymogen

5' sense oligonucleotide

GAAGGGGTAA GCTTGGATAA AAGAGATATG TTTAATTACG

AAGAATACTG TACTGCTAAT GCTGTTACTG GTCCATGTAG

AGCTTCTTTT CCAAGATGGT ACTTTGATGT TGAAAGA(SEQ ID NO：58)

And 3' antisense oligonucleotides identical to those used in construct #2 were prepared using the methods described for the production of expression constructs (construct #1 above).

Yeast strain SC101 (MAT. alpha., ura 3-52, Suc 2) was transformed with a plasmid containing each of the above cDNAs, and the protein was expressed by the method described above for producing placental biculinine 102-159 using human codon usage. Approximately 250 ml of each yeast culture was harvested and the supernatant from centrifugation (15 min. times.3000 RPM) was purified separately using 1mm of kallikrein-Sepharose as described above. The relative amount of trypsin inhibition in applysate, the amount of purified protein recovered and the N-terminal sequence of the purified protein were determined, and the data are shown in Table 7 below.

TABLE 7

Relative production levels of different proteins containing the C-terminal Kunitz domain of placental dicurnine peptide

TABLE 7
	N-terminal inhibition sequencing in construct applysate: relative concentration amounts (pmol) of the preparation of the sequence preparation
No no expression was detected in # 2103-159
	# 3101-15925% inhibition without low expression
# 498-15993% inhibition 910 DMFNYE well expressed the correct product
	# 1102-15982% incorrectly processed protein that inhibits the expression activity of 480 AKEEGV

The results indicate that placental diconin peptide fragments of different lengths containing the C-terminal Kunitz domain have extensive variation in the expression of functional secreted proteins. Constructs expressing the 101-159 and 103-159 fragments produced little or low activity in the supernatant prior to purification, and N-terminal sequencing of 0.05 ml aliquots of each purified fragment produced undetectable amounts of inhibitor. On the other hand, expression of placental digoxin 102-159 or 98-159 resulted in an appreciable amount of protease activity prior to purification. However, N-terminal sequencing showed that the purified protein recovered from 102-159 expression was again mostly incorrectly processed, indicating that its N-terminal was identical to the processing at a site within the yeast α -mating factor pro sequence of most of the preproteins. However, the purified proteins recovered from the expression of placental diconin peptide 98-159 were all processed at the correct site and yielded the correct N-terminus. Moreover, the recovered protein was almost doubled in mass compared with the recovery of placental digonin 102-159. Placental diconin peptide 98-159 therefore exhibits a preferred fragment length for production of the C-terminal Kunitz domain by the α -mating factor prepro sequence/KEX-2 processing system of sarcina cerevisiae (s.

Example 6

Alternative for Yeast expression

The 58 amino acid peptide derived from the R74593 translation product can also be PCR amplified from the R87894-R74593 PCR product cloned in the TA vector TM (Invitrogen, San Diego, Calif.) after DNA sequencing, or from human placenta cDNAPCR. The amplified DNA product consisted of 19 nucleotides from the yeast α -ligand leader sequence, which matched the R74593 sequence encoding YEEY-CFRQ (residue 58) so that the translation product was in frame, to form an α -ligand/Kunitz domain fusion protein. The protein sequence also contains a Kex 2 cleavage that releases the Kunitz domain at its native N-terminus.

The 5' sense oligonucleotide containing a HindIII site for cloning will contain the following sequence:

GCCAAGCTTG GATAAAAGAT ATGAAGAAT ACTGCACCGC CAACGCA

(SEQ ID NO：30)

the 3' antisense oligonucleotide containing a BamHI site and stop codon for cloning has the following sequence:

GGGGATCCTC ACTGCTGGCG GAAGCAGCGG AGCAT(SEQ ID NO：31)

the full-length 206-nucleotide cDNA sequence for cloning into the yeast expression vector had the following sequence:

CCAAGCTTGG ATAAAAGATA TGAAGAATAC TGCACCGCCA ACGCAGTCAC

TGGGCCTTGC CGTGCATCCT TCCCACGCTG GTACTTTGAC GTGGAGAGGA

ACTCCTGCAA TAACTTCATC TATGGAGGCT GCCGGGGCAA TAAGAACAGC

TACCGCTCTG AGGAGGCCTG CATGCTCCGC TGCTTCCGCC AGCAGTGAGG

ATCCCC(SEQ ID NO：32)

after PCR amplification, the DNA will be digested with HindIII, BamHI and cloned into the HindIII and BamHI digested yeast expression vector pMT15 (see U.S. Pat. No. 5,164,482, incorporated herein by reference). The resulting plasmid vector was used to transform the SC 106 strain as described in U.S. Pat. No. 5,164,482. URA 3+ yeast transformants were isolated and cultured under induction conditions. The yield of the recombinant placental digonin peptide variant was determined by the amount of trypsin inhibitory activity that accumulated in the culture supernatant over time using the in vitro assay method described above. The fermentation broth was centrifuged at 9000rpm for 30 minutes. The supernatant was then filtered through 0.4 and then 0.2 micron filter, diluted to 7.5ms conductivity and adjusted to pH3 with citric acid. The sample batch was taken up on 200 ml S-Sepharose fast flow (Pharmacia), in 50mM sodium citrate pH3 and stirred for 60 minutes. The gel was then subjected to a series of washes: 2 liters each of 50mM pH3.0 sodium citrate; 50mM Tris HCl, pH 9.0; 20mM HEPES, pH 6.0. The washed gel was transferred to a suitable column and eluted with a linear gradient of 0 to 1M sodium chloride in 20mM HEPES pH 6.0. The eluted fractions containing in vitro trypsin inhibitory activity were collected and further purified in two ways a) by chromatography on a column (essentially as described in example 2) in which the dehydrated trypsin was immobilised; b) performing chromatography on a column immobilized with bovine kallikrein; or c) combining conventional chromatographic steps including gel filtration and/or anion-exchange chromatography.

Example 7

Isolation and characterization of native human placental diconin peptides from placenta

Biculinin protein was purified from fully frozen placenta (Analytical Biological Services, inc., Wilmington, DE) to apparent homogeneity. The placenta (740 g) was melted to room temperature and cut into pieces of 0.5 to 1.0 cm size, placed on ice and washed with 600 ml of PBS buffer. The washing solution was decanted and 240 ml of placenta fragments were placed on a Waring blender. 300 ml of a buffer containing 0.1M Tris (pH8.0) and 0.1M NaCl was added, and the mixture was stirred at high speed for two minutes, poured into a 750.0 ml centrifuge tube and placed on ice. This process was repeated until all material was processed. The mixed tissue slurry was centrifuged at 4500 Xg for 60 min at 4 ℃. The supernatant was filtered through cheesecloth and the placental diconin peptide was purified using a kallikrein affinity column made according to manufacturer's recommendations with 70 mg of bovine pancreatic kallikrein (bayer ag) covalently bound to 5.0 ml of CNBr-activated sepharose (pharmacia). This material was placed on an affinity column, further washed with 0.1M Tris (pH8.0), 0.5M NaCl at a flow rate of 2.0 ml/min, and then diluted with 3 volumes of pH 4.00.2M acetic acid. Fractions containing kallikrein and trypsin inhibitory activity (as below) were collected, frozen and lyophilized. Placental dicurin peptide was further chromatographed by gel filtration using Superdex 7510/30(Pharmacia) column bound to a Beckman System Gold HPLC System. Briefly, the column was equilibrated at a flow rate of 0.5 ml/min with 0.1M Tris, 0.15M NaCl and 0.1% Triton X-100. The lyophilized samples were reconstituted in 1.0 ml of 0.1M pH8.0Tris and injected onto a gel filtration column as 200. mu.l aliquots. Fractions (0.5 ml) were collected and assayed for trypsin and kallikrein inhibitory activity. The active fractions were collected and the pH of the solution was adjusted to 2.5 by the addition of TFA. This material was placed directly on a Vydac C18 reverse phase column (5 μm, 0.46 × 25 cm) pre-equilibrated with 20% acetonitrile in 0.1% TFA. The separation was performed with a linear gradient of 20 to 80% acetonitrile in 0.1% TFA at 1.0 ml/min for 50 minutes, followed by an initial wash with 20% acetonitrile in 0.1% TFA for 20 minutes prior to separation. Fractions (1 ml) were collected and assayed for trypsin and kallikrein activity. The fractions containing the inhibitory activity were concentrated using a Speed-Vac concentrator (Savant) and analyzed for N-terminal sequence.

Functional analysis of placental bikunin

The ability to inhibit bovine trypsin and human plasma kallikrein was determined to identify functional placental dikonin. Trypsin inhibitory Activity Gly-Pro-Lys-Carbamic coumarin was used as a substrate in 96-well microtiter plates (Perkin Elmer) at room temperature in assay buffer (50mM Hepes, pH7.5, 0.1M NaCl, 2.0mM CaCl₂0.1% Triton X-100). The amount of coumarin produced by trypsin was determined by measuring its fluorescence (ex 370nm, em 432nm) on a Perkin-Elmer LS-50B fluorometer equipped with a plate reader. Trypsin (23. mu.g in 100. mu.l buffer) was mixed with the 20. mu.l sample to be assayed and incubated for 10 minutes at 25 ℃. The reaction was initiated by adding 50. mu.l of the substrate GPK-AMC (final concentration 33. mu.M) to the test buffer. Fluorescence intensity was measured and the percent inhibition of each fraction was determined by the following formula:

percent inhibition of 100 × [1-F × ]₀/F₁]Wherein F₀Is unknown fluorescence, and F₁Is the fluorescence of trypsin in the control. The kallikrein inhibitory activity of the fractions was similarly determined using 7.0nM kallikrein in test buffer (50mM Tris, pH8.0, 50mM NaCl, 0.1% triton X-100) and 66.0. mu.M Pro-Phe-Arg-AMC as substrates.

Determination of in vitro specificity of placental dicurnine

The in vitro specificity of native placental dikonin was determined using the materials and methods described in the preceding examples. Monitoring of unbound trypsin fraction by using GPK-AMC as substrate placental bicinin was quantified by active site titration of a known concentration of trypsin.

Protein sequencing

The volume of 1 ml of fraction (C18-29 Delaria) was reduced to 300 ml on a Speed Vac to reduce the amount of organic solvent. The sample was then placed on a Hewlett-Packard mini biphasic reaction column and washed with 1 ml of 2% trifluoroacetic acid. The samples were sequenced by Edman degradation on a Hewlett-Packard type G1005A protein sequencing system. Version 3.0 of the sequencing method and all reagents by Hewlett-Packard. The sequence was confirmed over 50 cycles.

And (6) obtaining the result. Placental diconin peptide was purified to apparent homogeneity using a series of kallikrein affinities, gel filtration, and reverse phase chromatography (see purification table 5 below):

TABLE 5

Purification Table of native placental diconin peptide (1-179)

TABLE 5
	Step volume OD280 OD280 unitsa(U) Unit/OD 280 (ml) (/ ml)

Placenta supernatant 1800.041.775,0603,000,00040.0
	Kallikrein affinity 20.00.173.3616,0004,880 pH4.0
Kallikrein affinity 10.20.454.5612,0002,630 pH1.7
	Superdex 75 15.0 0.0085 0.13 3,191 24,546

a one unit is defined as the amount required to inhibit 50% of trypsin activity in a standard assay.

Most of the kallikrein and trypsin inhibitory activity was eluted from a kallikrein affinity column eluted at pH 4.0. Subsequent gel-filtration chromatography (FIG. 5) produced peaks of kallikrein and trypsin inhibitory activity with molecular weight values in the range of 10 to 40kDa, as determined by a standard curve generated by standard shifts of molecular weight under equivalent conditions. Reverse phase C18 chromatography (fig. 6) produced 4 peaks of inhibitory activity with the strongest elution at approximately 30% acetonitrile. The activity associated with the first peak eluted at C18 (fraction 29) shows the amino acid sequence starting with the first amino acid from the amino acid sequence of the predicted placental diconin peptide (ADRER.; SQID NO: 1) and is identical to the predicted sequence from 50 cycle sequencing (underlined amino acids in FIG. 3). Cysteine residues in this sequence are silent as expected from sequencing of oxidized proteins. Cysteine residues at amino acid positions 11 and 20 of mature placental digonin were found from subsequent sequencing of S-pyridylethylated proteins, in which PTH-pyridylethyl-cysteine was recovered at cycles 11 and 20.

Interestingly, the aspartic acid at position 30 of the sequence was silent, indicating that this position is susceptible to glycosylation. Fraction 29 yielded a primary sequence corresponding to placental dicurnine peptide starting at residue No. 1 (27 picomoles, in cycle 1) plus a secondary sequence derived from placental dicurnine peptide starting at residue 6 (sihd..). This indicates that the end product sequenced in fraction 29 is highly pure and most likely responsible for the protease inhibitory activity associated with this fraction (FIG. 6).

Thus, the final preparation of placental dicuronine from C18 chromatography was highly purified based on silver-stained SDS-PAGE analysis (fig. 7), the migrating protein on a 10 to 20% acrylamide tricin gel (novex, San Diego, CA) having an apparent molecular weight of 24KDa and calibrated with the following molecular weight standards: insulin (2.9 KDa); bovine trypsin inhibitor (5.8 KDa); lysozyme (14.7 KDa); beta-lactoglobulin (18.4 KDa); carbodehydratase (carbonic anhydrase) (29 KDa); and ovalbumin (43 KDa). The size of the placental diconin peptide on SDS-PAGE as described above was identical to that expected for the full length coding sequence (FIG. 4F).

In agreement with the expectation, the purified protein reacted with the antibody to placental dicurnine peptide (7-64) produced a band with the same molecular weight as the pure preparation detected on the gel with silver staining (fig. 7) according to the above N-terminal sequencing results (fig. 12A). However, when the same preparation was reacted with an antibody to the synthetic placental digoxin peptide (102-159), no band corresponding to the full-length protein was observed. In contrast, a fragment of about 6kDa was observed which co-migrated with synthetic biculinin (102-159). A simple explanation for these results is that the purified protein preparation undergoes degradation after purification to yield an N-terminal fragment containing the N-terminal domain and a C-terminal fragment containing the C-terminal domain C-terminal fragment. Assuming that the fragment active against antisera to placental diconin peptide (7-64) lacks the C-terminal tail of the full-length protein, this size (24kDa) suggests a higher degree of glycosylation.

Table 6 below shows the in vitro inhibitory capacity of placental dicurnine peptides against different serine proteases. Data and data from aprotinin (Traslol)^) Comparison of (a).

TABLE 6

K of placental digonin peptide for inhibiting different proteases_iValue of

TABLE 6
	Protease (concentration) placental biconin peptide Ki(nM) aprotinin Ki(nM)
Trypsin (48.5pM) 0.130.8
	Human plasmin (50pM) 1.91.3

The results indicate that placental dicuronine peptide isolated from a natural source (human placenta) is a potent inhibitor of serine protease of tryptase.

Example 8

Expression pattern of placental dicurnine in different human organs and tissues

Multiple tissue Northern blots were purchased from Clontech, which contained 2 micrograms of Poly A + RNA from human heart, brain, placenta, lung, liver, skeletal muscle, kidney, and pancreas. Two different cDNA probes were used: 1) gel-purified cDNA encoding placental dicuronine (102-159); 2) PCR-derived 780 base pair cDNA released from TA clones was digested with EcoRI and gel-purified. For each probe³²P-dCTP was labeled and randomly labeled with a kit from Boehringer Mannheim biochemicals (Indiana) and then used to hybridize with multi-tissue Northern according to manufacturer's recommendations. Autoradiography was performed with Biomax and Adobe photoshop and Umax Scanner scans at 18 hour exposure time.

And (6) obtaining the result. The tissue expression patterns observed with the placental digoxin (102-159) probe (fig. 11A) or a larger probe containing two Kunitz domains of placental digoxin (fig. 11B) were essentially consistent with what was expected. Placental digonin mRNA is most abundant in the pancreas and placenta. Significant levels were also observed in lung, brain and kidney, whereas levels were lower in heart and liver and the mRNA was not detected in skeletal muscle. In all cases, the transcript length was 1.95kb, which is very similar to that deduced from the overlap of the full-length cDNA clones and ESTs described above.

The widespread distribution of this mRNA in tissues indicates that placental digonin peptide is widely expressed. Since the protein also has a leader sequence, it is widely exposed to the human immune system and must be recognized as a self-protein. Additional evidence for the widespread distribution of placental dicurin peptide mRNA in tissues derives from the fact that some estentries with homology to placental dicurin peptide (fig. 4B) are derived from adult or infant brain, as well as human retina, breast, ovary, olfactory table and placenta. It can therefore be concluded that administration of a natural human protein to a patient will not elicit an immune response.

Interestingly, the expression pattern of placental dikonin was somewhat reminiscent of the high levels of aprotinin found in bovine lungs and pancreas. To further elucidate the expression pattern of placental dicurnine peptide, RT-PCR of total RNA from human cells was determined: unstimulated Human Umbilical Vein Endothelial Cells (HUVECs), HK-2 (derived from the renal proximal tubule system), TF-1 (erythroleukemia line) and Phorbolester (PMA) stimulated human peripheral blood leukocytes. The primers used were:

CACC TGATCGCG AGACCCC (sense; sequence 59);

CTGGCGGAAGCAGCGGAGCATGC (antisense; sequence 60);

designed to amplify a 600b.p. cDNA fragment encoding placental digonin peptide. The comparison can be extrapolated to the general case by amplifying the 800b.p. actin segment by including actin primers. Whereas the 800b.p. fragment detected on ethidium bromide agarose gel was consistent in intensity in all lanes, the placental biculinine fragment of 600b.p was absent from HUVECs but was present in significant amounts in any other cell line. We conclude that placental dicurnine peptide is not expressed in at least some endothelial cells, but is expressed in some red blood cell populations.

Example 9

Purification and characterization of placental dicurin peptide (1-170) highly purified from baculovirus/Sf 9 expression System

Large fragments of placental dicurnine peptide containing two Kunitz domains (placental dicurnine peptide 1-170) were expressed in Sf6 cells as follows. The placental dikonin cDNA from PCR (FIG. 4E) and contained in the TA vector (see examples above) was digested with HindIII and Xbal to generate a fragment flanked by 5 'XbI and 3' HindIII sites. The fragment was gel filtered and cloned into M13mp19 vector (New England Biolabs, BeUerly, Mass.). In vitro mutagenesis (Kunkel T.A., (1985) Proc. NatlAcad. Sci.USA, 82: 488-Asn 492) was used to generate a3 'Pstl site at the 5' XbaI site; and 5' of the sequence coding for the ATG start site, the native placental digonin peptide signal peptide and the mature placental digonin peptide coding sequence. The oligonucleotides used for mutagenesis had the following sequence:

5′CGCGTC TCG GCT GAC CTG GCC CTG CAG ATG GCG CAC GTG TGC

GGG 3′(SEQ ID NO：61)

in a similar manner, stop codons (TAGs) and BgIII/XmaI sites were generated at the 3' end of the cDNA using the following oligonucleotides:

5′CTG CCC CTT GGC TCA AAG TAG GAA GAT CTT CCC CCC GGG GGG

GTG GTT CTG GCG GGG CTG 3′(SEQ ID NO：62)

the stop codon is in frame with the sequence encoding placental dicurnine and results in a termination that occurs immediately following lysine at amino acid residue 170, thus encoding a truncated placental dicurnine fragment that lacks the putative transmembrane domain. The products digested from PstI and BglII were isolated and cloned into the Bac Pac8 vector for expression of a placental diconin peptide fragment containing both Kunitz domains but truncated at the N-terminus immediately adjacent to the putative transmembrane segment (1-170).

Culture medium was harvested 72 hours post-infection and Sf-9 insect cells were optimized for expression of bicining to infection diversity of 1: 1. After harvest, the pH of the baculovirus cell culture supernatant (2 liters) was adjusted to 8.0 by adding Tris-HCl. Using the procedure for the purification of native placental dicurin from placenta as described in example 7 above, the dicurin was purified by chromatography using a 5 ml bovine pancreatic kallikrein affinity column, the eluate was adjusted to pH2.5 with TFA and subjected to chromatography on a C18 reverse phase column (1.0X 25 cm) equilibrated with 10% acetonitrile in 0.1% TFA at a flow rate of 1 ml/min. Bicinin in 0.1% TFAElution was performed with a linear gradient of 10 to 80% acetonitrile. The active fractions were collected, lyophilized, and redissolved in 50mM Hepes (pH7.5), 0.1M NaCl, 2mM CaCl₂And 0.1% triton X-100 and stored at-20 ℃ until use. The concentration of recombinant biculinine peptide was determined by amino acid analysis.

And (6) obtaining the result. Recombinant biculinin peptide was purified from baculovirus cell culture supernatant in the following two-step purification procedure to produce active trypsin inhibitors (table 8 below).

TABLE 8

Purification of recombinant bicining peptides from transformed culture supernatants

TABLE 8
	Purification step volume OD 280/OD 280 units (U) specific activity (ml) total volume in milliliters (units/OD)
Supernatant 2300.09.020,7006,150,00297

Kallikrein affinity 23.00.122.7640,70014,746
	C18 inverse 0.43.841.5411,11172,150

The crude product was subjected to affinity column chromatography on immobilized bovine pancreatic kallikrein, and 0.013% of protein was selectively separated and showed trypsin inhibitory activity of 0.67%. Most of the trypsin inhibitory activity present in the starting supernatant was not bound to immobilized kallikrein, independent of bikunin peptide (results not shown). Subsequent C18 reverse phase chromatography yielded a further 5-fold purification with a 0.2% recovery. The final product, highly purified by SDS-PAGE, had a molecular weight of 21.3kDa and was immunoblotted against rabbit anti-placental dikonin 102-159 (not shown). N-terminal sequencing (26 cycles) resulted in the expected sequence of mature placental bicinin peptide (fig. 4F) starting at residue +1 (adrer..) indicating correct processing of the signal peptide in Sf9 cells.

Placental dicuronine peptide (100 picomoles) purified from Sf9 cells was alkylated with pyridylethyl, digested with CNBr, and then sequenced without resolubilization of the resulting fragments. Sequencing for 20 cycles yielded the following sequence N-terminus:

sequence number placental diconin peptide residues #

LRCFrQQENPP-PLG- - -21 picomolar 154- -

ADRERSIHDFCLVSKVVGRC 20 picomolar 1-20(SEQ ID NO: 64)

FNYeYCTANAVTTGPCRASF 16 picomole 100-

Pr- -Y-V-dGS-Q-F-Y-G6 picomolar 25-43(SEQ ID NO: 66)

Thus, the N-terminus corresponding to each of the four desired fragments was recovered. This confirms that Sf6 expresses a protein containing the entire extracellular domain of placental digonin peptide (1-170). Additional samples of undigested placental digonin peptide (1-170) were sequenced N-terminal (50 cycles) to generate amino acid sequences that lacked any PTH-amino acid for 30 cycles (PTH-aspartate is desired). The sequencing of the native protein from human placenta gave corresponding results (example 7) and the residue was glycosylated as predicted from the amino acid sequence surrounding this aspartic acid residue. Furthermore, cysteine residues in this region are silent, as predicted for disulfide bonds.

Example 10

Inhibitory specificity of purified placental diconin peptide from Sf6 cells

The in vitro specificity of recombinant bicining peptides was determined using the materials and methods described in examples 3, 4 and 7. In addition, inhibition of human tissue kallikrein by bicining peptide was determined by incubating 0.35nM of human tissue kallikrein recombinant bicining peptide in a buffer containing 50mM Tris (pH9.0)50mM NaCl and 0.01% triton X-100. After 5 minutes at 37 ℃ 5. mu.l of 2mM PFR-AMC was added and the change in fluorescence was monitored.

Inhibition of tissue plasminogen activator (tPA) was also determined by the following method: tPA (single stranded form of human melanoma cell culture from Sigma Chemical Co, St Louis, Mo.) was preincubated with inhibitor for 2 hours at room temperature in 20mM Tris buffer pH7.2 containing 150mM NaCl and 0.02% sodium azide. The reaction was then transferred to a reaction system containing the following concentrations of the starting components to initiate the reaction: tPA (7.5nM), inhibitor 0 to 6.6. mu.M, DIle-Lpro-Larg-p-nitroaniline (1mM) in 28mM Tris buffer pH8.5 containing 0.004% (v/v) triton X-100 and 0.005% (v/v) sodium azide. The formation of p-nitroaniline was determined at 37 ℃ after two hours incubation by measurement at A405 nm.

The following table shows the effectiveness of recombinant biculinin peptides as in vitro inhibitors of different serine proteases. The data presented are in comparison to data obtained from screening with recombinant bicining or aprotinin.

TABLE 9

K inhibition of various proteases by recombinant placental dikonin (1-170) or aprotinin_iComparison of values

TABLE 9
	Protease (concentration) recombinant biculinin aprotinin Ki(nM) Ki(nM)
Trypsin (48.5pM) 0.0640.8
	Human plasma kallikrein (2.5nM) 0.1819.0
Human tissue kallikrein (0.35nM) 0.040.004
	Bovine pancreatic kallikrein (100pM) 0.120.02
Human plasmin (50pM) 0.231.3
	Factor Xa (0.87nM) 180 inhibits 5% at 31. mu.M
Factor XIa (0.1nM) 3.0288
	Tissue plasminogen activator (7.5nM) < 60 with no inhibition at 6.6. mu.M
Tissue factor VIIa 800 is not inhibited at 1. mu.M

The results indicate that recombinant biculinin peptides can be expressed in insect cells and produce active protease inhibitors that are inhibitors of at least 5 serine proteases.

Recombinant dicurnine is more effective than aprotinin on human plasma kallikrein, trypsin and plasmin. Surprisingly, recombinant bicining was more efficient than the synthetic bicining fragments (7-64) and (102-159) for all enzymes tested. These data indicate that recombinant biculinin peptide is more effective than aprotinin for in vitro experiments and thus it is expected to be more potent in vivo.

In addition to determining effectiveness against specific proteases, placental dikonin (1-170) was identified for its ability to prolong Activated Partial Thromboplastin Time (APTT) and compared to aprotinin-related activity. The inhibitor was diluted in 20mM Tris buffer pH7.2 containing 150mM NaCl and 0.02% sodium azide and added (0.1 ml) to the mixture in MLA Electrora^800 Automatic coagulation time instrument (Automatic coagulation timer) a small cup in a coagulation instrument (Medical Laboratory Automation, inc. The instrument was placed in APTT shift 300 seconds activation time and repeat mode, and 1 ml of Plasma (Speielly assisted Reference Plasma Lotl-6-5/85, Helena Laboratories, Beaumomt, TX), APTT reagent (Automated APTT-lot102345) available from Oregon Teknika Corp. Durhan. NC) and 25mM CaCl were added₂Automatic dispersion to initiate coagulation and automatic monitoring of coagulation time. The results (FIG. 14) show that doubling of clotting time requires a final concentration of aprotinin of about 2. mu.M, but only 0.3. mu.M Sf 9-derived placental dicurin. These data indicate that placental dikonin is a potent anticoagulant useful as a therapeutic agent for the treatment of diseases involving pathological activation of the coagulation intrinsic pathway.

While specific embodiments of the invention have been described in detail for purposes of illustration, it will be apparent to those skilled in the art that the methods and formulations described may be modified without departing from the spirit and scope of the invention. Accordingly, the invention is not to be restricted except in light of the attached claims.

Claims (8)

1. A purified protein having serine protease inhibitory activity comprising any one of the amino acid sequences selected from the group consisting of:

ADRERSIHDF CLVSKVVGRC RASMPRWWYN VTDGSCQLFV YGGCDGNSNN 50

YLTKEECLKK CATVTENATG DLATSRNAAD SSVPSAPRRQ DSEDHSSDMF 100

NYEEYCTANA VTGPCRASFP RWYFDVERNS CNNFIYGGCR GNKNSYRSEE 150

ACMLRCFRQQ ENPPLPLGSK 170

(SEQ ID NO：52)；

MAQLCGL RRSRAFLALL GSLLLSGVLA -1

ADRERSIHDF CLVSKVVGRC RASMPRWWYN VTDGSCQLFV YGGCDGNSNN 50

YLTKEECLKK CATVTENATG DLATSRNAAD SSVPSAPRRQ DSEDHSSDMF 100

NYEEYCTANA VTGPCRASFP RWYFDVERNS CNNFIYGGCR GNKNSYRSEE 150

ACMLRCFRQQ ENPPLPLGSK VVVLAGLFVM VLILFLGASM VYLIRVARRN 200

QERALRTVWS SGDDKEQLVK NTYVL 225

(SEQ ID NO：49)；

ADRERSIHDF CLVSKVVGRC RASMPRWWYN VTDGSCQLFV YGGCDGNSNN 50

YLTKEECLKK CATVTENATG DLATSRNAAD SSVPSAPRRQ DSEDHSSDMF 100

NYEEYCTANA VTGPCRASFP RWYFDVERNS CNNFIYGGCR GNKNSYRSEE 150

ACMLRCFRQQ ENPPLPLGSK VVVLAGLFVM VLILFLGASM VYLIRVARRN 200

QERALRTVWS SGDDKEQLVK NTYVL 225

(SEQ ID NO：70)；

AGSFLAWL GSLLLSGVLA -1

ADRERSIHDF CLVSKVVGRC RASMPRWWYN VTDGSCQLFV YGGCDGNSNN 50

YLTKEECLKK CATVTENATG DLATSRNAAD SSVPSAPRRQ DSEDHSSDMF 100

NYEEYCTANA VTGPCRASFP RWYFDVERNS CNNFIYGGCR GNKNSYRSEE 150

ACMLRCFRQQ ENPPLPLGSK VVVLAGAVS 179

(SEQ ID NO：2)；

MLR AEADGVSRLL GSLLLSGVLA -1

ADRERSIHDF CLVSKVVGRC RASMPRWWYN VTDGSCQLFV YGGCDGNSNN 50

YLTKEECLKK CATVTENATG DLATSRNAAD SSVPSAPRRQ DSEDHSSDMF 100

NYEEYCTANA VTGPCRASFP RWYFDVERNS CNNFIYGGCR GNKNSYRSEE 150

ACMLRCFRQQ ENPPLPLGSK VVVLAGLFVM VLILFLGASM VYLIRVARRN 200

QERALRTVWS SGDDKEQLVK NTYVL 225

(SEQ ID NO：45)；

MAQLCGL RRSRAFLALL GSLLLSGVLA -1

ADRERSIHDF CLVSKVVGRC RASMPRWWYN VTDGSCQLFV YGGCDGNSNN 50

YLTKEECLKK CATVTENATG DLATSRNAAD SSVPSAPRRQ DSEDHSSDMF 100

NYEEYCTANA VTGPCRASFP RWYFDVERNS CNNFIYGGCR GNKNSYRSEE 150

ACMLRCFRQQ ENPPLPLGSK VVVLAGLFVM VLILFLGASM VYLIRVARFN 200

QERALRTVWS FGD 213

(SEQ ID NO：47)；

ADRERSIHDF CLVSKVVGRC RASMPRWWYN VTDGSCQLFV YGGCDGNSNN 50

YLTKEECLKK CATVTENATG DLATSRNAAD SSVPSAPRRQ DSEDHSSDMF 100

NYEEYCTANA VTGPCRASFP RWYFDVERNS CNNFIYGGCR GNKNSYRSEE 150

ACMLRCFRQQ ENPPLPLGSK VVVLAGLFVM VLILFLGASM VYLIRVARRN 200

QERALRTVWS FGD 213

(SEQ ID NO：71)；

IHDF CLVSKVVGRC RASMPRWWYN VTDGSCQLFV YGGCDGNSNN 50

YLTKEECLKK CATV 64

(SEQ ID NO：4)；

CLVSKVVGRC RASMPRWWYN VTDGSCQLFV YGGCDGNSNN 50

YLTKEECLKK C 61

(SEQ ID NO：5)；

YEEYCTANA VTGPCRASFP RWYFDVERNS CNNFIYGGCR GNKNSYRSEE 150

ACMLRCFRQ 159

(SEQ ID NO：6)；

CTANAVTGPC RASFPRWYFD VERNSCNNFI YGGCRGNKNS YRSEE 150

ACMLRC 156

(SEQ ID NO：7)；

IHDF CLVSKVVGRC RASMPRWWYN VTDGSCQLFV YGGCDGNSNN 50

YLTKEECLKK CATVTENATG DLATSRNAAD SSVPSAPPRQ DSEDHSSDMF 75

NYEEYCTANA VTGPCRASFP RWYFDVERNS CNNFIYGGCR GNKNSYRSEE 125

ACMLRCFRQ 159

(SEQ ID NO：3)；

CLVSKVVGRC RASMPRWWYN VTDGSCQLFV YGGCDGNSNN 50

YLTKEECLKK CATVTENATG DLATSRNAAD SSVPSAPRRQ DSEDHSSDMF 100

NYEEYCTANA VTGPCRASFP RWYFDVERNS CNNFIYGGCR GNKNSYRSEE 150

ACMLRC 156

(SEQ ID NO：50)；

ADRERSIHDF CLVSKVVGRC RASMPRWWYN VTDGSCQLFV YGGCDGNSNN 25

YLTKEECLKK CATVTENATG DLATSRNAAD SSVPSAPRRQ DSEDHSSDMF 75

NYEEYCTANA VTGPCRASFP RWYFDVERNS CNNFIYGGCR GNKNSYRSEE 125

ACMLRCFRQQ ENPPLPLGSK VVVLAGAVS 179

(SEQ ID NO：1)；and

ADRERSIHDF CLVSKVVGRC RASMPRWWYN VTDGSCQLFV YGGCDGNSNN 50

YLTKEECLKK CATVTENATG DLATSRNAAD SSVPSAPRRQ DS 92

(SEQ ID NO：8).

2. a protein according to claim 1, wherein said protein is glycosylated or contains at least one intrachain cysteine-cysteine disulfide bond, or is both glycosylated and contains at least one intrachain cysteine-cysteine disulfide bond.

3. A pharmaceutical composition for inhibiting serine protease activity comprising a protein of claim 1 or claim 2 together with a pharmaceutically acceptable carrier.

4. An isolated nucleic acid sequence encoding the protein of claim 1.

5. A self-replicating protein expression vector comprising a nucleic acid sequence encoding and capable of expressing the protein of claim 1 or claim 2.

6. Use of a protein according to claim 1 or claim 2 in the manufacture of a medicament for inhibiting serine protease activity.

7. Use of a protein according to claim 1 or claim 2 in the manufacture of a medicament for the treatment of disease conditions comprising: cerebral edema, spinal cord edema, multiple sclerosis, ischemia, intraoperative blood loss, sepsis, septic shock, fibrosis, diseases associated with pathological blood clotting or clotting, combined trauma, stroke, cerebral or subarachnoid hemorrhage, brain inflammation, spinal cord inflammation, brain infection, cerebral granuloma, spinal cord infection, spinal cord granuloma, open heart surgery, gastric cancer, cervical cancer, or prevention of metastasis.

8. A method of producing a protein according to claim 1 or claim 2 by recombinant DNA techniques.