WO1993025675A1 - Mutants of the epidermal growth factor domains of human thrombomodulin - Google Patents

Abstract

This invention relates to the identification of critical amino acid residues in the 6-EGF region of thrombomodulin [TM]. Modification of these residues has a predictable effect on the ability of TM to modify cofactor activity, e.g., bind to thrombin and/or to mediate potentiation of thrombin mediated activation of protein C.

Description

MUTANTS OF THE EPIDERMAL GROWTH FACTOR DOMAINS OF HUMAN THROMBOMODULIN

BACKGROUND OF THE INVENTION Native TM is an endothelial cell surface glycoprotein that binds to thrombin with high affinity. Binding of TM to thrombin alters its conformation, leading to accelerated activation of protein C. Activated protein C then catalyzes the proteolytic inactivation of clotting factors Va and Villa in the presence of cofactor protein S, thereby inhibiting the coagulation cascade. Complex formation between thrombin and TM also results in direct inhibition of the procoagulant activities of thrombin, namely, fibrin formation and platelet activation. Thus, TM plays a critical role in maintenance of the anticoagulant surface.

Mature human TM is composed of a single polypeptide chain of 559 residues and consists of 5 domains: an amino- terminal "lectin-like" domain, an "6 EGF domain" comprising six epidermal growth factor (EGF) -like repeats, an 0- glycosylation domain, the transmembrane domain and cytoplasmic domain. Various structure-function studies using proteolytic fragments of rabbit TM or deletion mutants of recombinant human TM have localized its cofactor activity to the last three EGF-like repeats.

In order to understand how TM interacts with thrombin and protein C, an alanine-scanning mutagenesis technique {Science 244 , 1081-1085, 1989) was utilized in the present studies.

A six EGF-like repeat of human TM and seventy seven mutants were expressed in Escherichia coli , and the effects of mutations on cofactor activities were analyzed directly in periplasmic extracts prepared by osmotic shock.

The results have identified a number of critical residues within the last three EGF-like domains of TM involved in thrombin-dependent activation of protein C. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a comparison of the M388L analog, the wild type 6-EGF and p select controls in the cofactor activation assay described herein. Figs. 2a-c compare the relative cofactor activity of the various alanine mutations described herein.

Fig. 3 provides estimates for the K_d of 22 analogs.

SUMMARY OF THE INVENTION This invention provides for a novel family of TM analogs. They are defined by amino acid substitutions primarily in the last 3 EGF repeats. The substitutions are illustrated by alanine substitutions and in general provide sites of modification resulting in controllable and predictable changes in the cofactor activity of the analog. Although the work was done on analogs comprising only the 6-EGF domains

, the cofactor activities are predictable for larger analogs comprising some or all of the other domains of native TM. Unless otherwise stated amino acids are referred to by either their full name, three letter designation or the standard single letter designation.

More particularly, this invention provides for a thrombomodulin analog which has a modified cofactor activity upon binding to thrombin, as compared to TM_E having natural sequence, said analog having an amino acid modification at a position corresponding to natural sequence at: a) 336 (asparagine) ; b) 337 (tyrosine) ; c) 340 (valine) ; d) 341 (aspartic acid) ; e) 365 (glutamic acid) ; f) 369 (leucine) ; g) 388 (methionine) ; h) 447 (isoleucine) ; or i) 454 (leucine) .

The modifications set forth above provide a level of cofactor activity which is less than or equal to about 25% of the wild type activity or and increased cofactor activity. The analogs may comprise one or more of the above modifications. Preferred modifications are where said analog has modifications at both positions 365 and 369. These modifications result in about a 30% increase in cofactor activity. The exemplified modifications are for alanine, however, aliphatic amino acids such as glycine, valine, leucine or isoleucine are also useful.

Generically it is preferred that the above analogs are soluble and/or comprising at least one structural domain which is EGF4, EGF5, and EGF6. Also preferred are additional domains including: a) the lectin domain; b) EGF domains 1, 2, or 3; or c) the 0-linked glycosylation domain.

Where protease resistance is desired the analogs may further comprise modifications at positions 456 or 457 which confer protease resistance to said analog. Preferred substitutions are 456gly or 457gln. In combination a preferred analog has substitutions at positions 365, 369, 388, 456, and 457.

A further preferred analog provides for uniform termini during production of the recombinant protein. Said analogs have terminal sequences of GPQP at the amino terminus, and LTPP at the carboxy terminus.

Nucleic acids encoding the above analogs are also described herein. A second series of analogs is described herein having about 50% or less of the cofactor activity of the control TMgM388L. More particularly said thrombomodulin analog upon binding to thrombin, induces a modified cofactor activity as compared to binding with TM_EM388L of less than or equal to 50%, said analog having an amino acid substitution at one or more positions corresponding to: aa) 349 (aspartic acid) ; ab) 355 (asparagine) ; ac) 357 (glutamic acid) ; ad) 358 (tyrosine) ; ae) 359 (glutamine) ; af) 363 (leucine) ; ag) 371 (valine) ;

second letter represent the relative position of the modification with regard to other residues in the listing. The analogs of this list may have modified modified K_d for binding thrombin, a modified k_cat/K_m, or both. The following analogs are a subset of the above list wherein the analogs have 25% or less of the cofactor activity of the control, TM_EM388L. These analogs are: aa) 349 (aspartic acid) ; ac) 357 (glutamic acid) ; ad) 358 (tyrosine) ; ae) 359 (glutamine) ; ag) 371 (valine) ; ai) 376 (phenylalanine) ; be) 398 (aspartic acid) ; bd) 400 (aspartic acid) ; be) 402 (asparagine) ; bg) 408 (glutamic acid) ; bh) 413 (tyrosine) ; bi) 414 (isoleucine) ; bj) 415 (leucine); bk) 416 (aspartic acid) ; bl) 417 (aspartic acid) ; ca) 423 (aspartic acid) ; cb) 424 (isoleucine) ; cc) 425 (aspartic acid) ; cd) 426 (glutamic acid) ; cf) 429 (asparagine) ; ck) 439 (asparagine) ; en) 444 (phenylalanine) ; or cr) 461 (aspartic acid) .

The modifications set forth above with regard to protease activity, aliphatic substitutions, oxidation resistance and uniform termini are also applicable for the above analogs having less than 50% of the cofactor activity of the control. Preferred are those listed above having less than

30% of the activity of the control. These analogs are represented by mutations in domain 4: aa) 349 (aspartic acid) ; ac) 357 (glutamic acid) ; ad) 358 (tyrosine) ; ae) 359 (glutamine) ; ag) 371 (valine) ; or ai) 376 (phenylalanine) .

There is also described herein analogs having an essentially an unmodified K^ compared to TM_EM388L. EGF5 and EGF6 are known to play an important role in high affinity binding to thrombin. EGF4 with a less critical role in binding is critical for conferring cofactor activity to the TM/thrombin complex. For this reason those analogs having modifications in the latter to EGF repeats can have approximately native cofactor activity but a reduced K_d e.g (S406A) . Analogs having modifications in the latter two EGF repeats which resulted in reduced cofactor activity are listed below: be) 398 (aspartic acid) ; bd) 400 (aspartic acid) ; be) 402 (asparagine) ; bg) 408 (glutamic acid) ; bh) 413 (tyrosine) ; bi) 414 ( isoleucine) ; b j ) 415 (leucine) ; bk) 416 (aspartic acid) ; bl) 417 (aspartic acid); ca) 423 (aspartic acid) ; cb) 424 (isoleucine) ; cc) 425 (aspartic acid) ; cd) 426 (glutamic acid) ; cf) 429 (asparagine) ; ck) 439 (asparagine) ; en) 444 (phenylalanine) ; or cr) 461 (aspartic acid) . Also provided herein are nucleic acids encoding the analogs described above.

The above analogs may also grouped by their respective domains as well as by their respective relative activity, i.e., EGF4, EGF5 or EFG6. For example the analogs of EGF4 having 50% of the control cofactor activity are: aa) 349 (aspartic acid) ; ab) 355 (asparagine) ; ac) 357 (glutamic acid) ; ad) 358 (tyrosine) ; ae) 359 (glutamine) ; af) 363 (leucine) ; ag) 371 (valine) ; ah) 374 (glutamic acid) ; ai) 376 (phenylalanine) ; aj) 384 (histidine); or ak) 385 (arginine) . Those in EGF4 having less than 25% of the cofactor activity of the control are: aa) 349 (aspartic acid) ; ac) 357 (glutamic acid) ; ad) 358 (tyrosine) ; ae) 359 (glutamine) ; ag) 371 (valine) ; or ai) 376 (phenylalanine) . In EGF5, the following modifications resulted in analogs having at least a 50% reduction in cofactor activity: be) 398 (aspartic acid) ; bd) 400 (aspartic acid) ; be) 402 (asparagine) ; bf) 403 (threonine) ; bg) 408 (glutamic acid) ; bh) 413 (tyrosine) ; bi) 414 (isoleucine) ; bj) 415 (leucine); bk) 416 (aspartic acid) ; bl) 417 (aspartic acid) ; or bm) 420 (isoleucine) . Amongst these analogs are those where the analog has essentially an unmodified k_cat/K_m compared to TM_EM388L. In EGF5, the analogs can be further subgrouped according to those modifications resulted in analogs having at least a 75' reduction in cofactor activity: be) 398 (aspartic acid) ; bd) 400 (aspartic acid) ; be) 402 (asparagine) ; bg) 408 (glutamic acid) ; bh) 413 (tyrosine) ; bi) 414 (isoleucine) ; bj) 415 (leucine); bk) 416 (aspartic acid) ; or bl) 417 (aspartic acid) . And again among these analogs are those with essentially unmodified k_cat/K_m compared to TM_EM388L. Nucleic acids encoding the above analogs are also claimed.

With regard to EGF6 the groups are provided below. Those having a cofactor activity of less than 50% of the control are: ca) 423 (aspartic acid) ; cb) 424 (isoleucine) ; cc) 425 (aspartic acid) ; cd) 426 (glutamic acid) ; ce) 428 (glutamic acid) ; cf) 429 (asparagine) ; eg) 432 (phenylalanine) ; eh) 434 (serine) ; ci) 436 (valine) ; cj) 438 (histidine); ck) 439 (asparagine) ; cl) 440 (leucine) ; cm) 443 (threonine) ; en) 444 (phenylalanine) ; co) 445 (glutamic acid) ; cp) 456 (arginine) ; cq) 458 (isoleucine) ; or cr) 461 (aspartic acid) .

Those having a cofactor activity of less than 25% of the control are: ca) 423 (aspartic acid) ; cb) 424 (isoleucine) ; cc) 425 (aspartic acid) ; cd) 426 (glutamic acid) ; cf) 429 (asparagine) ; ck) 439 (asparagine) ; cl) 440 (leucine) ; en) 444 (phenylalanine) ; or cr) 461 (aspartic acid) . Again the preferred analogs are those set forth above with additional modifications for solubility, protease resistance, oxidation resistance as well as uniform terminal ends. The nucleic acid encoding these analogs are also a part of the claimed invention. As with the other groups, these analogs include those wherein said analog has an essentially unmodified k_cat/K_m compared to TM^BBQL .

The analogs can be further subgrouped according to those possessing a modified amino acid at a position, wherein said analog has essentially equivalent K^ for thrombin compared to an analog having at said position the native residue, wherein said position corresponds to: aa) 349 (aspartic acid) ; ab) 355 (asparagine) ; ac) 357 (glutamic acid) ; ad) 358 (tyrosine) ; or ae) 359 (glutamine) .

These analogs may have a modified k_cat/K_m of less than 30% of the control.

The following sites embrace describe analogs having a modified K^ or k_cat/K_m compared to an analog having at said position the native residue, wherein said position corresponds to: af) 363 (leucine) ; ag) 371 (valine) ; ah) 374 (glutamic acid) ; ai) 376 (phenylalanine) ; aj) 384 (histidine); ak) 385 (arginine) ; be) 398 (aspartic acid) ; bd) 400 (aspartic acid) ; or be) 402 (asparagine) .

These further include those analogs having both a modified K_d and k_cat/K_m, especially those having been modified by at least 20%.

The following sites describe analogs having a lower cofactor activity and a K^ or k_cat/K_m that is essentially equivalent when compared to an analog having at said position the native residue, wherein said position corresponds to: lutamic acid) ; yrosine) ; soleucine) ; eucine); spartic acid) ; spartic acid) ; soleucine) ; spartic acid) ; soleucine) ; spartic acid) lutamic acid) lutamic acid) sparagine) ; henylalanine) ; erine) ; aline) ; istidine) ; sparagine) ; eucine) ; threonine) ; henylalanine) ; lutamic acid) ; rginine) ; isoleucine) ; or

aspartic acid) . The following positions describe a subgrouping of those modifications which resulted in at least a 75% reduction in cofactor activity yet essentially little change in k_cat/_K_m: bg) 408 (glutamic acid) ; bh) 413 (tyrosine) ; bi) 414 (isoleucine) ; bj) 415 (leucine) ; bk) 416 (aspartic acid) ; bl) 417 (aspartic acid) . ca) 423 (aspartic acid) ; cb) 424 (isoleucine) ; cc) 425 (aspartic acid) ; cd) 426 (glutamic acid) ; cf) 429 (asparagine) ; ck) 439 (asparagine) ; en) 444 (phenylalanine) ; or cr) 461 (aspartic acid) . A further subgrouping can be made of the above modifications wherein the K_d for thrombin is modified by at least 30%. This invention further provides for methods. More specifically there is described herein a method useful for screening for analogs of thrombomodulin which exhibit a modified K^ for thrombin binding, comprising the steps of: a) making an amino acid substitution at a position: bg) 408 (glutamic acid) ; bh) 413 (tyrosine) ; bi) 414 (isoleucine) ; bj) 415 (leucine); bk) 416 (aspartic acid) ; bl) 417 (aspartic acid) ; b ) 420 (isoleucine) ; ca) 423 (aspartic acid) ; cb) 424 (isoleucine) ; cc) 425 (aspartic acid) ; cd) 426 (glutamic acid) ; ce) 428 (glutamic acid) ; cf) 429 (asparagine) ; eg) 432 (phenylalanine) ; ch) 434 (serine) ; ci) 436 (valine) ; cj) 438 (histidine); ck) 439 (asparagine) ; cl) 440 (leucine) ; cm) 443 (threonine) ; en) 444 (phenylalanine) ; co) 445 (glutamic acid) ; cp) 456 (arginine) ; cq) 458 (isoleucine) ; cr) 461 (aspartic acid) ; and b) comparing the K^ for thrombin to a control molecule. Various embodiments of this invention include those wherein said K_β is modified by at least 33%, or where said modification is an amino acid substitution, or wherein said control molecule is TMJJM388L. A preferred grouping of modifications for use in the method are: bg) 408 (glutamic acid) ; bh) 413 (tyrosine) ; bi) 414 (isoleucine) ; bj) 415 (leucine); bk) 416 (aspartic acid) ; bl) 417 (aspartic acid) . ca) 423 (aspartic acid) ; cb) 424 (isoleucine) ; cc) 425 (aspartic acid) ; cd) 426 (glutamic acid) ; cf) 429 (asparagine) ; ck) 439 (asparagine) ; en) 444 (phenylalanine) ; or cr) 461 (aspartic acid) . An another method is described herein wherein the method is useful for screening for analogs of thrombomodulin which induce a modified cofactor activity upon binding to thrombin, comprising the steps of: a) making an amino acid modification at a position: aa) 349 (aspartic acid) ; ab) 355 (asparagine) ; ac) 357 (glutamic acid) ; ad) 358 (tyrosine) ; ae) 359 (glutamine) ; and b) comparing the rate of cofactor activity upon binding to thrombin with the rate of a control molecule.

The preferred embodiments set forth for the first method are applicable here also.

DEFINITIONS "Cofactor activity" refers to the relative ability of the TM analogs to complex with thrombin and potentiate the ability of thrombin to activate protein C. The assay procedures used to measure cofactor activity are provided in Example 5. Cofactor activity is also referred to as k_cat when thrombin and protein C are at saturation concentrations.

"Essentially equivalent" refers to numbers which are statistically similar at the p=.01 level.

"K^" refers to the relative binding affinity between the TM analog and thrombin. High K^ values represent low binding affinity. The precise assays and means for determining K^ are provided in example 5.

"KJJJ" refers to the Michaelis constant and is derived in the standard way by measuring the rates of catalysis measured at different substrate concentrations. It is equal to the substrate concentration at which the reaction rate is half of its maximal value. In this study, K is determined by keeping thrombin concentrations at a constant level e.g. (1 nM) and using saturation levels of TM (e.g. 100 nM or greater depending on the kd. Reactions are carried out using increasing concentration of protein C (e.g., 1-60 μM) . K^ and k_cat are then determined using Lineweaver-Burke plotting or nonlinear regression analysis. _^ "Modification" or "modified amino acid" refers either a deletion or a substitution of a native amino acid residue for either a peptide bond or a substituent amino acid.

"Natural sequence" refers to the native sequence of thrombomodulin as provided in sequence ID. No. 1.

"Thrombomodulin analog" refers to a molecule having TM-like biological properties with regard to the activation of protein C and/or the binding to thrombin. Said analogs have substantial identity with the primary sequence of the domains of native TM that are responsible for the biological activity of TM. Analogs include deletion mutants, soluble analogs, protease resistant analogs and oxidation resistant analogs. ⁿTM_E" refers to an analog of TM consisting of the six EFG repeats.

DETAILED DESCRIPTION

A DNA sequence encoding the full-length native human thrombomodulin protein was isolated and described in (European Patent Application No. 88870079.6, which is incorporated herein by reference) . The cDNA sequence encodes a 60.3 kDa protein of 575 amino acids, which includes a signal sequence of about 18 amino acids.

The sequences for bovine, mouse and human thrombomodulin exhibit a high degree of homology with one another. By analogy with other proteins, the structure of thrombomodulin can be presumptively divided into domains. The term "domain" refers to a discrete amino acid sequence that can be associated with a particular structure, function or characteristic. The full length thrombomodulin gene encodes a precursor peptide containing the following domains:

Approximate Amino Acid Position Domain

-18--1 Signal sequence 1-226 N-terminal domain (lectin-like)

227-462 6 EGF-like repeat domain

463-497 0-linked Glycosylation

498-521 Transmembrane domain

522-557 Cytoplasmic domain See C. S. Yost et al . , (1983) Cell , 34 : 759 - 166 and D. Wen eϋ al . , (1987) Biochemistry, 26:4350-4357, both incorporated herein by reference.

In comparison to native thrombomodulin, the TM analogs of the present invention are modified in at least one of 22 amino acid residues in the 6-EGF region 227-462. Modifications of the native sequence are indicated by the following formula: Z N Z' wherein Z and Z' are the single letter designation for the natural amino acids and where Z is the native residue and Z' is the substituted residue and wherein N is the number of the residue being modified. For example M388L represents deletion of the native methionine at position 388 of TM and introduction of a leucine residue.

Where the modification is described as changing a parameter (e.g., K_d, k_cat or I^) by at least %, the modification can be plus or minus. It being understood that a particular site of modification is determined as critical and substitutions being available therein to affect the desired change in %.

TM analogs of the present invention may be further modified to embrace the 6 epidermal growth factor [EGF] -like domain plus or minus the 0-linked glycosylation domain. TM analogs preferably include any or all of the following characteristics: i) they are soluble in aqueous solution in the absence of detergents; ii) they retain activity after exposure to oxidants; iii) they are protease resistant through modification of residues 456 and 457; iv) they have uniform amino and carboxy termini; and, v) when bound to thrombin, they potentiate the thrombin-mediated activation of protein C and through the elimination of chondroitin sulfate have a reduced ability to inhibit the direct anti-coagulant activities of thrombin such as the conversion of fibrinogen to fibrin or the activation and aggregation of platelets.

Soluble TM analogs that retain activity after exposure to oxidants are termed "oxidation resistant". Such analogs are described in detail in co-pending co-assigned USSN 506,325 filed April 9, 1990, incorporated herein by reference.

A preferred TM analog are those rendered oxidation resistant by substitution of the methionine at position 388 ,in particular with leucine, M 388 L. As used herein, a "soluble TM analog" is a TM analog which is soluble in an aqueous solution and preferably can be secreted by a cell. For pharmacological administration, the soluble TM analog or an insoluble analog comprising the native cytoplasmic domain, may optionally be combined with phospholipid vesicles, detergents or other similar compounds well known to those skilled in the art of pharmacological formulation. The preferred TM analogs of the present invention are soluble in the blood stream, making the analogs useful in various anticoagulant and other therapies. These modifications do not significantly affect activities of native thrombomodulin such as affinity for thrombin or activity in protein C activation.

A. General Methods For Making TM Analogs. This invention relies upon molecular genetic manipulations that can be achieved in a variety of known ways. The recombinant cells, plasmids, and DNA sequences of the present invention provide means to produce pharmaceutically useful compounds wherein the compound, secreted from recombinant cells, is preferably a soluble derivative of thrombomodulin.

Generally, the definitions of nomenclature and descriptions of general laboratory procedures used in this application can be found in J. Sambrook et al . , Molecular Cloning, A Laboratory Manual , (1989) Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. The manual is hereinafter referred to as Sambrook and is hereby incorporated by reference.

Unless otherwise stated all reagents and equipment are used according to the manuf cturer's instructions.

Oligonucleotides that are not commercially available can be chemically synthesized according to the solid phase phosphoramidite triester method first described by S.L. Beaucage and M.H. Caruthers, (1981) Tetrahedron Letts . , 22(20) :1859-1862 using an automated synthesizer, as described in D.R. Needham-VanDevanter et al . , (1984) Nucleic Acids Res . , 12:6159-6168. Purification of oligonucleotides was by either native acrylamide gel electrophoresis or by anion-exchange

HPLC as described in J.D. Pearson and F.E. Regnier, (1983) J. Chrom. , 255:137-149. Nucleotide sizes are given in either kilobases (kb) or base pairs (bp) . These are estimates derived from agarose or acrylamide gel electrophoresis or from published DNA sequences.

The sequence of the cloned genes and synthetic oligonucleotides can be verified using the chemical degradation method of A.M. Maxam et al . , (1980) Methods in Enzymology, 65:499-560. The sequence can be confirmed after the assembly of the oligonucleotide fragments into the double- stranded DNA sequence using the method of Maxam and Gilbert, supra, or the chain termination method for sequencing double- stranded templates of R.B. Wallace et al., (1981) Gene, 16: 21 - 26. Southern Blot hybridization techniques were carried out according to Southern et al . , (1975) J. Mol . Biol . , 58:503.

Embodiments of this invention involve the creation of novel peptides and genes by in vi tro mutagenesis. Target genes are isolated in intermediate vectors and cloned for amplification in prokaryotes such as E. coli , Bacillus or Streptomyces. Most preferred is E. coli because that organism is easy to culture and more fully understood than other species of prokaryotes. The Sambrook manual contains methodology sufficient to conduct all subsequently described clonings in E. coli . Strain MH-1 is preferred unless otherwise stated. All E. coli strains are grown on Luria broth (LB) with glucose, or M9 medium supplemented with glucose and acid-hydrolyzed casein amino acids. Strains with resistance to antibiotics were maintained at the drug concentrations described in Sambrook. Transformations were performed according to the method described by D.A. Morrison, (1977) J. Bact. , 132:349-351 or by J.E. Clark-Curtiss and R. Curtiss, (1983) Methods in Enzymology, 101:347-362, Eds. R. Wu et al . , Academic Press, New York. Representative vectors include pBR322 and the pUC series which are available from commercial sources.

B. Gene Synthesis The gene encoding native human thrombomodulin has been isolated and sequenced, both in its genomic form and as a cDNA (Zuzuki, K., et al. (1987) Embo. J. 6:1891-1897 which is herein incorporated by reference). Sequence ID. Nos. 1 and 23 provide the amino acid sequence and nucleic acid sequence, respectively according to the standard numbering scheme.

Publication of the full length DNA sequence encoding human thrombomodulin and thrombin facilitates the preparation of genes and is used as a starting point to construct DNA sequences encoding TM peptides. The peptides of the present invention are preferably soluble derivatives which lack the stop transfer sequence of TM in addition to having internal amino acid substitutions. Furthermore, these analogs are secreted from eukaryotic cells which have been transfected or transformed with plasmids containing genes which encode these polypeptides. Methods for making modifications, such as amino acid substitutions, deletions, or the addition of signal sequences to cloned genes are known. Specific methods used herein are described below.

The full length gene for thrombomodulin can be prepared by several methods. Human genomic libraries are commercially available. Oligonucleotide probes, specific to these genes, can be synthesized using the published gene sequence. Methods for screening genomic libraries with oligonucleotide probes are known. The publication of the gene sequence for thrombomodulin demonstrates that there are no introns within the coding region. Thus a genomic clone provides the necessary starting material to construct an expression plasmid for thrombomodulin using known methods. A thrombomodulin encoding DNA fragment can be retrieved by taking advantage of restriction endonuclease sites which have been identified in regions which flank or are internal to the gene. (R.W. Jackman et al., (1987) Proc . Natl . Acad. Sci . USA. , 54:6425-6429) . Alternatively, the full length genes can also be obtained from a cDNA bank. For example, messenger RNA prepared from endothelial cells provides suitable starting material for the preparation of cDNA. A cDNA molecule containing the gene encoding thrombomodulin is identified as described above. Methods for making cDNA banks are well known (See Sambrook, supra) .

Genes encoding TM peptides may be made from wild- type TM genes first constructed using the gene encoding full length thrombomodulin. A preferred method for producing wild- type TM peptide genes for subsequent mutation combines the use of synthetic oligonucleotide primers with polymerase extension on a mRNA or DNA template. This polymerase chain reaction (PCR) method amplifies the desired nucleotide sequence. U.S. Patents 4,683,195 and 4,683,202 describe this method.

Restriction endonuclease sites can be incorporated into the primers. Genes amplified by the PCR reaction can be purified from agarose gels and cloned into an appropriate vector. The emphasis of this invention is on the substitution of native amino acids of the 6-EGF region.

Although the initial determination of the 22 critical residues was via alanine scanning, the invention is not limited to alanine substitutions. Alterations in the natural gene sequence of TM beyond the 43 residues identified can be introduced by the techniques of in vitro mutagenesis which include the single site mutation techniques as described herein, cassette mutagenesis whereby multiple bases are replaced and random mutagenesis where a part of the gene or cDNA is altered by insert of a randomly generated oligomuleotide. One can also use the polymerase chain reaction with primers that have been designed to incorporate appropriate mutations.

The TM peptides described herein are secreted when expressed in eukaryotic cell culture. Secretion may be obtained by the use of the native signal sequence of the thrombomodulin gene. Alternatively, genes encoding the TM peptides of the present invention may be ligated in proper reading frame to a signal sequence other than that corresponding to the native thrombomodulin gene. For example, the signal sequence of t-PA, (see WO 89/00605 incorporated herein by reference) or of hypodermin A or B (see EP 326,419 which is incorporated hereby by reference) can be linked to the polypeptide (See Table 2) . In the preferred embodiment of the present invention, use is made of the signal sequence of t-PA which contains the second intron of the human t-PA gene. The inclusion of the intron enhances the productivity of the adjacent structural gene. With the analogs of this invention, those portions of the gene encoding the transmembrane and cytoplasmic domains of the carboxyl terminal region of the native thrombomodulin gene are typically deleted. Therefore, it is necessary to add a stop codon so that translation will be terminated at the desired position. Alternatively, a stop codon can be provided by the desired expression plasmid. Additionally a polyadenylation sequence is helpful to ensure proper processing of the mRNA in eukaryotic cells encoding the TM analog. Also, it may be necessary to provide an initiation codon, if one is not present, for expression of the TM peptides. Such sequences may be provided from the native gene or by the expression plasmid.

Preferred cloning vectors suitable for replication and integration in prokaryotes or eukaryotes and containing transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of TM peptides are described herein. The vectors are comprised of expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the plasmid in both eukaryotes and prokaryotes, i.e., shuttle vectors, and selection markers for both prokaryotic and eukaryotic systems.

C. Expression of TM Peptides in Prokaryotic Cells In addition to the use of cloning methods in E. coli for amplification of cloned sequences it may be desirable to express TM analogs in prokaryotes. As discussed in greater detail below, the carbohydrate moieties of the mature protein are not essential for activity as a cofactor for the activation of protein C but do have an effect on the direct anticoagulant properties of the TM analogs as well as the molecule's half life in circulation. Expression of thrombomodulin analogs in E. coli has provided a useful tool for analysis of this issue. It is possible to recover a therapeutically functional protein from E. coli transformed with an expression plasmid encoding a soluble TM analog. Methods for the expression of cloned genes in bacteria are well known. To obtain high level expression of a cloned gene in a prokaryotic system, it is essential to construct expression vectors which contain, at the minimum, a strong promoter to direct mRNA transcription termination. Examples of regulatory regions suitable for this purpose are the promoter and operator region of the E. coli β- galactosidase gene, the E. coli tryptophane biosynthetic pathway, or the leftward promoter from the phage lambda. The inclusion of selection markers in DΝA vectors transformed in E. coli are useful. Examples of such markers include the genes specifying resistance to ampicillin, tetracycline, or chloramphenicol.

See Sambrook for details concerning selection markers and promoters for use in E. coli . In the described embodiment of this invention p select is used as a vector for the subcloning and amplification of desired gene sequences.

D. Expression of TM Peptides in Eukaryotic Cells

It is- expected that those of skill in the art are knowledgeable in the expression systems chosen for expression of the desired TM peptides and no attempt to describe in detail the various methods known for the expression of proteins in eukaryotes will be made.

The DΝA sequence encoding a soluble TM analog can be ligated to various expression vectors for use in transforming host cell cultures. The vectors typically contain marker genes and gene sequences to initiate transcription and translation of the heterologous gene. The vectors preferably contain a marker gene to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase, metallothionein, hygromycin, or neomycin phosphotransferase. The nuclear polyhedral viral protein from Autographa calif ornica is useful to screen transfected insect cell lines from Spodoptera frugiperda and Bombyx mori to identify recombinants. For yeast, Leu-2, Ura-3, Trp-1, and His-3 are known selectable markers (Gene (1979) 8:17-24) . There are numerous other markers, both known and unknown, which embody the above scientific principles, all of which would be useful as markers to detect those eukaryotic cells transfected with the vectors embraced by this invention.

Of the higher eukaryotic cell systems useful for the expression of TM analogs, there are numerous cell systems to select from. Illustrative examples of mammalian cell lines include RPMI 7932, VERO and HeLa cells, Chinese hamster ovary (CHO) cell lines, WI38, BHK, COS-7, C127 or MDCK cell lines. A preferred mammalian cell line is CHL-l. When CHL-l is used hygromycin is included as a eukaryotic selection marker. CHL-l cells are derived from RPMI 7932 melanoma cells, a readily available human cell line. The CHL-l cell line has been deposited with the ATCC according to the conditions of the Budapest Treaty and has been assigned #CRL 9446, deposited June 18, 1987. Cells suitable for use in this invention are commercially available from the American Type Culture Collection. Illustrative insect cell lines include Spodoptera frugiperda (fall Armyworm) and Bombyx mori (silkworm) .

As indicated above, the expression vector, e.g., plasmid, which is used to transform the host cell, preferably contains gene sequences to initiate the transcription and sequences to control the translation of the TM peptide gene sequence. These sequences are referred to as expression control sequences. When the host cell is of insect or mammalian origin, illustrative expression control sequences include but are not limited to the following: the retroviral long terminal repeat promoters (Nature, 257:479-483, 1982), SV40 promoter (Science, 222:524-527, 1983); thymidine kinase promoter; ( Cell , 27:299-308, 1982), or the beta-globin promoter, ( Cell , 33:705-716, 1983) . The recipient vector nucleic acid containing the expression control sequences is cleaved using restriction enzymes and adjusted in size as necessary or desirable. This segment is ligated to a DNA sequence encoding at the TM peptide by means well known in the art.

When higher animal host cells are employed, polyadenylation or transcription termination sequences need to be incorporated into the vector. An example of a polyadenylation sequence is the polyadenylation sequence from SV40, which may also function as a transcription terminator.

Genes incorporated into the appropriate vectors can be used to direct synthesis of proteins in either transient expression systems or in stable clones. In the former case yields are low, but the experiments are quick. In the latter case it takes more time to isolate high producing clones. Different vectors may be used for the two different types of experiments. In particular, in the case of transient expression, sequences may be included within the plasmid that allow the plasmid to replicate to a high copy number within the cell. These sequences may be derived from virus such as SV40 (e.g. C. Doyle et al . , (1985) J. Cell Biol . , 100:704-714) or from chromosomal replicating sequences such as murine autonomous replicating sequences (Weidle et al . , (1988) Gene, 73:427-437) . The vector for use in transient expression should also contain a strong promoter such as the SV40 early promoter (e.g., A. van Zonnenfeld et al . , (1987) Proc. Natl . Acad. Sci . USA. , 83:4670-4674) to control transcription of the gene of interest. While transient expression provides a rapid method for assay of gene products, the plasmid DNA is not incorporated into the host cell chromosome. Thus, use of transient expression vectors does not provide stable transfected cell lines. A description of a plasmid suitable for transient expression is provided by A. Aruffo & B. Seed, (1987) Proc. Natl . Acad. Sci . USA . , 84:8573-8577.

TM analogs may alternatively be produced in the insect cell lines described above using the baculovirus system. This system has been described by V.A. Luckow and M.D. Summers (1988) Bio/Technology, 6:47-55. Generally, this expression system provides for a level of expression higher than that provided by most mammalian systems. The baculovirus infects the host insect cells, replicates its genome through numerous cycles, and then produces large amounts of polyhedron crystals. The polyhedron gene can be replaced with a TM peptide gene. The polyhedron promoter will then make large amounts of analog protein following infection of the culture host cell and replication of the baculovirus genome. The non- secreted gene product is harvested from the host 3-7 days post infection. Alternatively, the TM peptide may be secreted from the cells if appropriate signal sequences are present on the protein. The host cells are competent or rendered competent for transfection by various means. There are several well- known methods of introducing DNA into animal cells. These include: calcium phosphate precipitation, DEAE-dextran technique, fusion of the recipient cells with bacterial protoplasts containing the DNA, treatment of the recipient cells with liposomes containing the DNA, electroporation and microinjection of the DNA directly into the cells. See, B. Perbal, "Practical Guide to Molecular Cloning, ^~ 2nd edition, John Wiley & Sons, New York and Wigler, et al . , (1987) Cell , 16:777-785.

E. Culturing Cells

It is preferred that the host cell is capable of rapid cell culture and able to appropriately glycosylate expressed gene products. Cells known to be suitable for dense growth in tissue culture are particularly desirable and a variety of invertebrate or vertebrate cells have been employed in the art, both normal and transformed cell lines.

The transfected cells are grown up by means well known in the art. For examples, see Biochemical Methods in

Cell Culture and Virology, Kuchler, R. J. , Dowden, Hutchinson and Ross, Inc. (1977) . The expression products are harvested from the cell medium in those systems where the protein is secreted from the host cell or from the cell suspension after disruption of the host cell system by, e.g., mechanical or' enzymatic means, which are well known in the art.

F. Purification of TM Analogs

It is preferred that the TM peptides of this invention be secreted by cultured recombinant eukaryotic cells. The TM analogs are produced in serum-free or serum supplemented media and are secreted intact. If prokaryotic cells are used, the TM analogs may be deposited intracellularly. The peptides may be fully or partially glycosylated or non-glycosylated. Following the growth of the recombinant cells and concomitant secretion of TM analogs into the culture media, this "conditioned media" is harvested. The conditioned media is then clarified by centrifugation or filtration to remove cells and cell debris. The proteins contained in the clarified media are concentrated by adsorption to any suitable resin such as, for example, Q Sepharose or metal chelators, or by use of ammonium sulfate fractionation, polyethylene glycol precipitation, or by ultrafiltration. Other means known in the art may be equally suitable. Further purification of the TM analogs can be accomplished in the manner described in Galvin, J. B., et al . , (1987) J. Biol . Chem. , 262:2199-2205 and Salem, H.H. et al . , (1984) J. Biol . Chem. , 255:12246-12251 and in the manner described in the embodiment disclosed herein. The purification of TM analogs secreted by cultured cells may require the additional use of, for example, affinity chromatography, ion exchange chromatography, sizing chromatography or other protein purification techniques.

Recombinant TM analogs may be produced in multiple conformational forms which are detectable under nonreducing chromatographic conditions. Removal of those species having a low specific activity is desirable and is achieved by a variety of chromatographic techniques including anion exchange or size exclusion chromatography. Recombinant TM analogs may be concentrated by pressure dialysis and buffer exchanged directly into volatile buffers (e.g., N-ethylmorpholine (NEM) , ammonium bicarbonate, ammonium acetate, and pyridine acetate) . In addition, samples can be directly freeze-dried from such volatile buffers resulting in a stable protein powder devoid of salt and detergents. In addition, freeze-dried samples of recombinant analogs can be efficiently resolubilized before use in buffers compatible with infusion (e.g., phosphate buffered saline) . Other suitable buffers might include hydrochloride, hydrobromide, sulphate acetate, benzoate, malate, citrate, glycine, glutamate, and aspartate.

G. Oxidation and Protease Resistant TM analogs.

Native thrombomodulin is susceptible to oxidation and when oxidized loses its ability to promote the activation of protein C. Many of the disease conditions requiring anticoagulation are also associated with high levels of toxic oxygen radicals, which can inactivate biomolecules and cause significant tissue damage. Examples of these conditions are reperfusion injury associated with myocardial infarction, DIC associated with septicemia, and alveolar fibrosis associated with adult respiratory distress syndrome. In addition, any wound, such as occurring in surgical procedures, involves the influx of activated monocytes, polymorphonuclear leukocytes, etc. which can create toxic oxygen species as well as releasing a host of proteolytic enzymes, such as elastase. The connection between endothelial cell damage, inflammation and thrombosis has long been recognized (See The Molecular and Cellular Biology of Wound Repair, ed. Clark, R.A.F. and P.M. Henson 1988, for example) . Thrombomodulin is subject to inactivation by exposure to toxic oxygen species and that this is expected to have a significant role in many pathogenic states.

Methods for rendering amino acids, specifically methionines, resistant to oxidation are well known in the art. It is possible to chemically modify thiol groups with iodoacetic acid, for example, to form oxidation resistant sulphonium (Gundlach, H.G. , et al., (1959) J^". Biol . Chem. 234:1754) . A preferred method is by removing the susceptible amino acid or replacing it with one or more different amino acids that will not react with oxidants. The amino acids leucine, alanine and glutamine would be particularly preferred amino acids because of their size and neutral character. Two methionines of thrombomodulin subject to oxidation are those located at residue 291 and 388. If only one methionine is to be blocked or eliminated, it is preferred that it be the residue at position 388.

To determine the resistance to loss of thrombomodulin activity due to oxidation, the test material (100 - 250 μg/ml) is first incubated with an oxidant such as, for example, chloramine-T, hydrogen peroxide at 5-lOmM chloramine-T or 200-1000 mM hydrogen peroxide in a buffer of 0.2% N-ethylmorpholine and 0.008% Tween 80 at pH 7.0 for 20 minutes at room temperature. After such oxidant exposure, the test material is evaluated using one of the bioactivity assays described below, specifically for the ability to act as a cofactor for the activation of protein C. Those mutant TM analogs that retain at least 60%, and preferably 90%, of activity they had prior to exposure to oxidants are considered to be oxidation resistant as compared to wild-type (non- mutant) TM analog or native thrombomodulin. Protease activity has been a problem with recombinant production of TM. The resulting TM has two chains. Protease resistant species of TM are those designed to be resistant to protease cleavage at amino acid residues, 456/457. In the native numbering system, these residues are arginine and histidine. These residues are preferably altered to glycine and glutamine, although other substitutions are possible. This modification will result in the preparation of single chain TM. Such modifications can be introduced using the methods described herein or by following the methods described in U.S. Pat. application Ser. No. 07/830,577 filed on February 5, 1992 entitled Protease-Resistant Thrombomodulin Analogs. H. Production of TM analogs having unique amino and carboxy termini.

Heterogeneity of the termini of recombinant TM is a further problem during production. To provide a uniform amino terminus, the amino terminus should be modified so that the processing enzyme of the host cell will generate a single N-terminus in the mature protein. In the preferred cell system of this invention, deleting the first three amino acids and beginning expression with the fourth amino acid (glu) provides for a fully functional analog having a homogeneous amino terminus.

Heterogeneity of the carboxy terminus is also a problem when producing TM from recombinantly altered cells. Ending the molecule at the pro-pro residues at positions 489 and 490, seven amino acids from the native TM carboxy terminus provides a preferred TM analog. This carboxy-terminus is particularly resistant to c-terminal exonucleases and provides a fully functional soluble TM.

I. Laboratory Assays for Measuring TM Activity.

A number of laboratory assays for measuring TM activity are available. Protein C cofactor activity can be measured in the assay described by Salem, et al . , (1984) J. Biol . Chem. 255(19) :12246-12251 and Galvin, et al., (1987) J. Biol . Chem. 262(5) :2199-2205. In brief, this assay consists of two steps. The first is the incubation of the test TM analog with thrombin and protein C under defined conditions (see Examples below) . In the second step, the thrombin is inactivated with hirudin or antithrombin III and heparin, and the activity of the newly activated protein C is determined by the use of a chromogenic substrate, whereby the chromophore is released by the proteolytic activity of activated protein C. This assay is carried out with purified reagents.

Alternatively the effect of a TM analog can be measured using plasma in clotting time assays such as the activated partial thromboplastin time (APTT) , thrombin clotting time (TCT) and/or prothrombin time (PT) . These assays distinguish between different mechanisms of coagulation inhibition, and involve the activation of protein C. Prolongation of the clotting time in any one of these assays demonstrates that the molecule can inhibit coagulation in plasma. The above assays are used to identify soluble TM analogs that are able to bind thrombin and to activate protein C in both purified systems and in a plasma milieu. Further assays are then used to evaluate other activities of native thrombomodulin such as inhibition of thrombin catalyzed formation of fibrin from fibrinogen (Jakubowski, et al . , (1986) J. Biol . Chem. 261 ( B ) :3876-3882) , inhibition of thrombin activation of Factor V (Esmon, et al . , (1982) J. Biol . Chem. 257:7944-7947) , accelerated inhibition of thrombin by antithrombin III and heparin cofactor II (Esmon, et al . , (1983) J. Biol . Chem. 258:12238-12242), inhibition of thrombin activation of Factor XIII (Polgar, et al . , (1987) Thromb . Haemostas . 58: 140 ) , inhibition of thrombin mediated inactivation of protein S (Thompson and Salem, (1986) J. Clin. Inv. 78(1):13-17) and inhibition of thrombin mediated platelet activation and aggregation (Esmon, et al . , (1983) J^". Biol . Chem. 258:12238-12242) . In the present invention, the TM analogs do not have all activities equal to that of native thrombomodulin.

J. Methods for Altering the Glycosylation of TM Analogs.

Carbohydrate substituents on proteins can affect both biological"activity and circulating half-life. In order to make many of the TM analogs of the present invention, O-linked glycosaminoglycan carbohydrate such as is found in the native thrombomodulin protein is eliminated. There are numerous ways for accomplishing this objective, including the treatment of the 0-linked carbohydrate containing protein with a glycanase known to specifically degrade sulfated glycosaminoglycans, such as chondroitinase ABC or hyaluronidase. This method is described in Bourin, M, et al . , (1988) J^". Biol . Chem. 263(17) :8044-8052, which is herein incorporated by reference. A second method for eliminating the 0-linked carbohydrate is by introducing site directed mutations into the protein. The attachment of glycosaminoglycans is often directed by the consensus recognition sequence of amino acids X-serine-glycine-X-glycine-X (Bourdon, M.A. , et al . , (1987) PNAS, U. S.A . 84:3194-3198) where X is any amino acid. The recognition sequence for other types of 0-linked sugars is threonine/serine-X-X-proline. The 0-linked domain of thrombomodulin has one potential glycosaminoglycan addition site (aa 472) and three other potential 0-linked carbohydrate addition sites (aa 474, 480 and 486) . Any change introduced into the nucleotide sequence that removes or changes the identity of any one or more of the amino acids in this recognition sequence will eliminate the potential 0-linked carbohydrate attachment site. Methods of introducing site directed mutations into a nucleotide sequence are described above.

A preferred method of eliminating 0-linked carbohydrate from a TM analog is by making an analog peptide that does not include the amino acids that are considered to be the O-linked domain, i.e., amino acids 468 through 485 of the native thrombomodulin gene sequence as shown in Table l. Methods of accomplishing this are well known in the art and have been described above. The circulating half-life of a protein can be altered by the amount and composition of carbohydrate attached to it. The TM analogs cf the present invention contain both 0-linked and N-linked carbohydrate. In addition to the potential glycosylation sites discussed above there are potential N-linked sites at amino acids 364, 391 and 393 and potential 0-linked sites at amino acids 319, 393 and 396. Methods of altering carbohydrate composition in addition to those described above are: l) expression of the TM analog gene in bacteria such E. coli , which does not have the cellular mechanisms necessary to glycosylate mammalian proteins, 2) expression of the TM analog gene in various eukaryotic cells, as each has its own characteristic enzymes that are responsible for the addition of characteristic sugar residues, and 3) treatment with chemicals such as hydrofluoric acid. Hydrofluoric acid, for example, chemically digests acid and neutral pH sugars while leaving intact basic sugars such as N- acetyl glucosamines and, under certain conditions, galactosamines.

K. Formulation and Use of Thrombomodulin Analogs While soluble forms of TM are preferred, the following methods can be altered by the addition of surfactants or liposomes to permit use of nonsoluble TM. The TM analogs described herein may be prepared in a lyophilized or liquid formulation. The material is to be provided in a concentration suitable for pharmaceutical use as either an injectable or intravenous preparation, preferably in single dose for mutations.

TM can be administered alone or as mixtures with other physiologically acceptable active materials, such as antibiotics, other anti coagulants, one-chain t-PA, or inactive materials, or with suitable carriers such as, for example, water or normal saline. The analogs can be administered parenterally, for example, by injection. Injection can be subcutaneous, intravenous or intramuscular.

These compounds are administered in pharmaceutically effective amounts and often as pharmaceutically acceptable salts, such as acid addition salts. Such salts can include, e.g., hydrochloride, hydrobromide, phosphate, sulphate, acetate, benzoate, malate, citrate, glycine, glutamate, and aspartate, among others. The analogs described herein may display enhanced in vivo activity by incorporation into micelles. Methods for incorporation into ionic detergent micelles or phospholipid micelles are known.

An antithrombotic agent can be prepared using the soluble TM analogs described herein and can consist of a completely purified analog alone or in combination with a thrombolytic agent as described above. Compounds of the present invention which are shown to have the above recited physiological effects can find use in numerous therapeutic applications such as, for example, the inhibition of blood clot formation. Thus, these compounds can find use as therapeutic agents in the treatment of thrombotic disease 'and of various circulatory disorders, such as, for example, coronary or pulmonary embolism, strokes, as well as the prevention of reocclusion following thrombolytic therapy and enhancement of thrombolytic therapies. These compounds also have utility in the cessation of further enlargement of a clot during an infarction incident. Further, the compounds disclosed can be useful for treatment of systemic coagulation disorders such as disseminated intravascular coagulation (DIC) , which is often associated with septicemia, certain cancers and toxemia of pregnancy.

Some of the analogs disclosed herein exhibit low rates of activation of protein C or higher K_d for thrombin (having lower affinity) . These analogs are low action forms of TM. They are useful for patients with low levels of protein C or low fibrinogen levels where less potent anticoagulants are indicated for management of bleeding complications. These compounds of this invention can be administered to mammals for veterinary use, such as with domestic animals, and for clinical use in humans in a manner similar to other therapeutic agents, that is, in a physiologically acceptable carrier. In general, the administration dosage for the TM analog will range from about 0.0002 to 5000 μg/kg, and more usually 0.02 to 500 μg/kg of the host body weight. These dosages can be administered by constant infusion over an extended period of time, until a desired circulating level has been attained, or preferably as a bolus injection.

The following examples are provided by way of illustration and not by way of limitation. Those of skill will recognize that the methods described below can be varied in noncritical aspects.

Example 1. Production of TM mutants.

Thrombomodulin mutants can be recombinantly produced by isolation of the critical EGF domain of human TM (amino acid 227 to 462) using polymerase chain reaction of human genomic DNA. The following primers can be used: (Sequence ID No. 21 5'CCGGGATCCTCAACAGTCGGTGCCAATGTGGCG3* and Seq. ID. No. 22 5'CCGGGATCCTGCAGCGTGGAGAACGGCGGCTGC3' . This fragment, through a series of intermediate constructs, is then placed under the control of β-lactamase promoter and signal sequence in pKT279 purchased from Strategene, La Jolla, CA.. An EcoR5- Bgl2 fragment of the resultant plasmid and a Scal-Sacl fragment of pGEM-3Zf DNA containing the fl origin of replication were then inserted into pSELECT-1 vector at EcoR5- BamHl and Scal-Sacl sites,respectively, to construct an E.coli expression vector designated pTHR211. Plasmids, pSELECT-1 vector, and pGEM-3Zf were purchased from Promega Corporation, Madison, WI. The bacterial host is E. coli strain DH5 alpha from

GIBCO BRL, Grand Island, NY.

Example 2. Si te -directed mutagenesis

Plasmids coding for TM mutants were constructed using a site-directed mutagenesis procedure described by Kunkel et.al., (1987) Methods in Enzymology 154 , 367-382. Briefly, a single-stranded uracil DNA prepared from E. coli strain CJ236 with R408 helper phage was used as a template for the synthesis of the mutagenic strand in the presence of specific oligonucleotides using T4 DNA polymerase and T4 DNA ligase.

The T4 DNA polymerase and E. coli strain CJ236 (dut^" ung^") were from BioRad Laboratories, Richmond, CA. A restriction enzyme recognition sequence in oligonucleotides was incorporated without changing the amino acid sequence, and resulting DH5 alpha tranformants were characterized by restriction digests of isolated plasmid DNAs. Three independent positive clones were isolated for each mutant except Y368A, E411A, I414A and E408A mutants, for which only 2 positive clones were obtained. In cases where there were large discrepancies between cofactor activities of triplicates, plasmids were further characterized by dideoxy sequencing. Example 3. Production of E. coli shockates containing TM mutants

DH5 alpha cells expressing TM mutants are grown overnight in 1.5 ml of Luria broth containing ampicillin (50μg/ml) at 37°C. Cells are harvested by centrifuging at

14,000 rpm in a microfuge for 25 seconds, washed once with 0.5 ml of 100 mM Tris, pH8.0 / 50mM NaCl, and suspended in 0.5 ml of 300 mM Tris, pH8.0/20% sucrose/l mM EDTA/0.5 mM MgCl₂.

After a 10 minute incubation at room temperature, cells are spun as before, and resuspended in 150 μl of 0.5 mM MgCl .

Following 10 minute incubation at 4°C, shockates are obtained by centrifuging 5 minutes at full speed.

The shockates are assayed immediately or less preferably stored at -70°C until ready for assaying (no longer than 30 days) .

Example 4. Assays for thrombin -dependent activation of protein C by TM mutants

The ability of TM mutants to act as cofactor for thrombin-mediated activation of protein C was assayed directly in the shockates. Recombinant human protein C was from Dr. John McPherson, Genzyme Corp., Framingham, MA., and was purified as described (BioTechnology 8:655-661, 1990) . Twenty five μl of each shockate was mixed with equal volumes of recombinant human protein C (final concentration of 0.3 μM) and human alpha thrombin (Sigma Chemicals, St. Louis, MO, at a final concentration of InM) in a microtiter plate. All reagents used were diluted in 20 mM Tris, pH7.4/100 mM NaCl/3.75 mM CaCl₂/0.1 % NaN₃ (w/V) containing 5 mg/ml bovine serum albumin.

Mixtures were incubated for 1 hr at 37°C and the reaction was terminated by addition of 25 μl of hirudin at 800 units/ml (Sigma Chemicals, St. Louis, MO) . The amount of activated protein C was determined by addition of 100 μl of chromogenic substrate D-valyl-L-leucyl-L-arginine-p- nitroanilide (S-2266) (lmM) . The change is measured by the absorbance at 405 nm with time using a plate reader. Data is recorded as milliOD unit/min and determined for each sample by measuring the absorbance every 10 seconds for 15 minutes using a Molecular Devices plate reader. All assays contained triplicate shockate samples each of DH5 alpha cells transfected with either pSELECT-1 vector (no TM) , pTHR211 (wild type) or pMJM57 (pTHR211 with methionine at 388 altered to leucine) , as internal controls. Cofactor activities of TM mutants were expressed as mean % of that obtained for pMJM57 and are provided in Fig. 1. Statistical Analysis Each mutant was assayed for activity at least twice

(three times for those mutants for which only two positive clones were isolated) , and all the data were included in the determination of the significance of difference using Student t-Test. Coefficient of variation between plates was 16.7% (n=18) . The results are provided in Fig. 2A-C. Western blot analysis

E. coli shockates were run in 10% Tris-tricine SDS PAGE under reduced conditions according to the manufacture's specifications (Novex Inc., San Diego, CA) . Reduced and alkylated samples were prepared by boiling shockates in sample buffer (62.5 mM Tris, pH6.8/2% SDS/10% glycerol/0.0025% bromophenol blue) containing 10 mM dithiothreitol for 10 minutes, followed by incubation with 50 mM iodoacetamide.

Proteins were transferred to nitrocellulose filter in transfer buffer (192 mM glycine/25 mM Tris,pH8.3/20 % methanol) at 4°C. The nitrocellulose filter was blocked with a blocking buffer (1% bovine serum albumin in 10 mM Tris,ph7.5/0.9 % NaCl/0.05 % NaN₃) , and then incubated with mouse polyclonal antiserum (raised against reduced and alkylated EGF domain of human thrombomodulin) in the blocking buffer. After washing with a washing buffer (10 mM Tris,pH7.5/0.9 % NaCl/0.05 % NaN₃/0.05 % Tween 20), the filter was incubated with biotinylated goat anti-mouse IgG antibodies in the blocking buffer containing 0.05% Tween 20. Proteins were detected using the Vectastain ABC solution (Vector Laboratories, Burlingame, CA) and ECL detection system (Amersham Corporation, Arlington Heights, IL) according to the manufacture's specifications. Example 5. Assays for determining the K_d of the TM/ Thrombin Complex

The activity of selected alanine mutants was assayed as described above in Example D except that the final concentration of thrombin in the reaction mixture was varied from 1 to 60 nM and the reaction was terminated by the addition of 25 μl of 8000 units/ml hirudin. Cofactor activity at each thrombin concentration was determined from the mOD/min in the presence of mutant minus the mOD/min in the absence of mutant (corrected mOD/min) . The reciprocal of the corrected mOD/min was then plotted against the reciprocal of the thrombin concentration. Double reciprocal plots gave straight lines and linear regression analysis was used to determine K^ (nM) from the intercept on the 1 / thrombin concentration axis (-l/K_jj) . K^ values for 22 mutants are provided in Fig. 3.

Example 6. .Results of alanine mutation experiments of the EGF domain of TM.

Using the methods described in the examples set forth above, it was established that the EGF domain of human thrombomodulin produced from E. coli is active in thrombin- dependent activation of protein C. Using this system, native human soluble 6EGF had cofactor activity of 5.7±1.9 mOD/min (n=18) . Assays were performed at final Ca⁺² concentration of 2.5 mM to reduce the activation of protein C by thrombin alone, as shown by a negligible amount of activated protein C produced in the absence of 6EGF (pSELECT control had 0.2% activity of native 6EGF) .

In earlier studies, it was observed that methionine residue at 388 was susceptible to oxidation, which led to the reduced cofactor activity. Substitution of methionine 388 with leucine in the purified 6EGF or extracellular domain of thrombomodulin resulted in 2-fold higher specific activity. In the current system, soluble M388L mutant of 6EGF in shockates had cofactor activity of 12.8±3.3 mOD/min (n=18) ,

2.3-fold higher than wild type 6EGF. Since expression levels of wild type 6EGF and M388L mutant in shockates were similar judging from Western blot analyses, the relative activity of wild type 6EGF to M388L mutant measured directly in shockates should reflect that of purified proteins.

The alanine scanning mutagenesis of EGF domain of human thrombomodulin identified critical residues for its activity. Various studies reported so far using deletion mutants have localized cofactor activity of thrombomodulin to the 4th, 5th and 6th repeats of EGF domain, while thrombin- binding is more specifically localized to the 5th and 6th repeats (Kurosawa, S., et al., 1988, J^". Biol . Chem. 263:5993- 5996) . In order to identify the critical residues involved for activity in this region of thrombomodulin, each residue between amino acid 333 to 462 (except Ala,Cys,Gly and Pro) was systematically replaced with alanine by site-directed mutagenesis, and the effect of substitutions on activity was determined directly in shockate samples.

Seventy seven mutants were constructed using M388L mutant as a template to increase the basal level of activity. Three independent clones were isolated for each mutant and assayed at least twice (or three assays for 4 mutants with 2 clones) . The results are expressed as mean percentages of

M388L mutant with standard deviations in error bars (Fig. 2) . The significance of difference for each mutant, calculated as described under materials and methods, is also included in the figure. Of 77 mutations, 22 mutations resulted in cofactor activity below 25 % of M388L mutant. They were D349A, E357A, Y358A, F376A, D398A, D400A, N402A, E408A, Y413A, I414A, L415A, D416A, D417A, D423A, I424A, D425A, E426A, N429A, N439A, L440A, F444A and D461A. Other mutations produced proteins with activity ranging between 25 to 100 % of the control.

Two mutants, Q365A and L369A, were found to have 10 to 15% higher activity than the M388L mutant. This effect was additive in a double mutant of Q365A/L369A. In order to demonstrate that these changes in the activity were not due to altered levels of expression of mutant proteins, shockate samples were analysed by Western blots using mouse polyclonal antibodies raised against EGF domain of human thrombomodulin. The data indicated that decreases in the activity of 22 mutants could not be accounted for by the differences in their expression levels.

Of 77 mutations assayed, 21 mutations involved negatively-charged residue to alanine conversions, while 7 involved positively-charged residue to alanine conversion. Twenty two critical residues identified here included 11 negatively-charged residues:

Asp349, Glu357, Asp398, Asp400, Glu408, Asp416, Asp417, Asp423, Asp425, Glu426, and Asp461 but no positively-charged residue. Six of these residues (Glu408, Asp4l6,Asp417,

Asp423,Asp425 and Glu426) were found within a region spanning the third loop of the 5th repeat to the interdomain, the region known to play a key role in thrombin-binding.

While overall negative charges were presumably important for the binding to the anionic exosite of thrombin, substitutions of hydrophobic residues in this region also resulted in the loss of cofactor activity (Y413A, I414A, L415A, and I424A) . Decreased cofactor activity of D461A mutant might also be due, at least partially, to the decreased affinity for thrombin. In earlier studies using purified deletion mutants, it was shown that the deletion of residues 447-462 in the 6th repeat of EGF domain of human thrombomodulin increased K^ for thrombin about 6-fold without affecting k_cat/K_m (Parkinson, J.S., et al . , Biochem. Biophys . Res . Comm. (in press, 1992)). In the same studies, the deletion of residues 333-350 caused 90% reduction in the k_j-_g^/K_jjj value without altering K^ for thrombin. Asp349 might be responsible for this loss of activity. Similar finding has been reported on Asp349 by Suzuki et.al. (Zushi, M. , et al., J^". Biol . Chem. 260:19886-19889, 1991).

Asn439 and Phe444 were part of the proposed consensus sequence -C-X-D/N-X-X-X-X-F/Y-X-C-X-C- for the 3-hydroxylation of Asp or Asn residue (Stemflow, J. , et al., PNAS 84:368-372, 1987) . While 3-hydroxylation of Asp or Asn has not shown to be obligatory for Ca²⁺ binding, it could contribute to the higher affinity for Ca²⁺ (Hanford, P.A., et al . , Nature 351:161-167, 1991). It is noteworthy that Asp423, Asp425 and Glu426, critical residues identified within the thrombin- binding fragment of 6EGF, may also constitute a part of Ca²⁺binding site, as demonstrated for EGF-like domain of human protein S and Factor IX. (See Hanford, supra . )

Thrombomodulin peptides according to this invention, e.g., D349A, E357A, Q365A, L369A, D425A and N439A, have been expressed in mamalian cells, i.e., in HEK293 (human embryonic kidney) cells, and the resultant peptides demonstrated essentially the same alterations in specific activity when compared with wild-type and M388L as was found in those peptides expressed in E. coli .

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Nagashima, Mariko Morser, Michael J. Parkinson, John F.

(ii) TITLE OF INVENTION: Mutants of the Epidermal

Growth Factor Domain of Human Thrombomodulin

(iii) NUMBER OF SEQUENCES: 23

(iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Kenneth A. Weber

(B) STREET: One Market Plaza, Steuart Tower,

Suite 2000

(C) CITY: San Francisco

(D) STATE: California (E) COUNTRY: USA

(F) ZIP: 94105

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US

(B) FILING DATE: (C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Weber, Kenneth A.

(B) REGISTRATION NUMBER: 31,677

(C) REFERENCE/DOCKET NUMBER: 14618-47 (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 415-543-9600

(B) TELEFAX: 415-543-5043

SUBSTITUTE SHEET (2) INFORMATION FOR SEQ ID N0:1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 462 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(ix) FEATURE:

(A) NAME/KEY: Protein (B) LOCATION: 1..462

(D) OTHER INFORMATION: /note= "Native Human Thrombomodulin"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

Ala Pro Ala Glu Pro Gin Pro Gly Gly Ser Gin Cys Val Glu His Asp 1 5 10 15

Cys Phe Ala Leu Tyr Pro Gly Pro Ala Thr Phe Leu Asn Ala Ser Gin 20 25 30 lie Cys Asp Gly Leu Arg Gly His Leu Met Thr Val Arg Ser Ser Val 35 40 45 Ala Ala Asp Val lie Ser Leu Leu Leu Asn Gly Asp Gly Gly Val Gly 50 55 60

Arg Arg Arg Leu Trp He Gly Leu Gin Leu Pro Pro Gly Cys Gly Asp 65 70 75 80

Pro Lys Arg Leu Gly Pro Leu Arg Gly Phe Gin Trp Val Thr Gly Asp 85 90 95

Asn Asn Thr Ser Tyr Ser Arg Trp Ala Arg Leu Asp Leu Asn Gly Ala 100 105 110

Pro Leu Cys Gly Pro Leu Cys Val Ala Val Ser Ala Ala Glu Ala Thr 115 120 125 Val Pro Ser Glu Pro He Trp Glu Glu Gin Gin Cys Glu Val Lys Ala 130 135 140

Asp Gly Phe Leu Cys Glu Phe His Phe Pro Ala Thr Cys Arg Pro Leu 145 150 155 160

Ala Val Glu Pro Gly Ala Ala Ala Ala Ala Val Ser He Thr Tyr Gly 165 170 175

Thr Pro Phe Ala Ala Arg Gly Ala Asp Phe Gin Ala Leu Pro Val Gly 180 185 190

Ser Ser Ala Ala Val Ala Pro Leu Gly Leu Gin Leu Met Cys Thr Ala 195 200 205 Pro Pro Gly Ala Val Gin Gly His Trp Ala Arg Glu Ala Pro Gly Ala 210 215 220

SUBSTITUTESHEET Trp Asp Cys Ser Val Glu Asn Gly Gly Cys Glu His Ala Cys Asn Ala 225 230 235 240

He Pro Gly Ala Pro Arg Cys Gin Cys Pro Ala Gly Ala Ala Leu Gin 245 250 255 Ala Asp Gly Arg Ser Cys Thr Ala Ser Ala Thr Gin Ser Cys Asn Asp

260 265 270

Leu Cys Glu His Phe Cys Val Pro Asn Pro Asp Gin Pro Gly Ser Tyr 275 280 285

Ser Cys Met Cys Glu Thr Gly Tyr Arg Leu Ala Ala Asp Gin His Art 290 295 300

Cys Glu Asp Val Asp Asp Cys He Leu Glu Pro Ser Pro Cys Pro Gin 305 310 315 320

Arg Cys Val Asn Thr Gin Gly Gly Phe Glu Pro Ser Pro Cys Pro Gin 325 330 335 Tyr Asp Leu Val Asp Gly Glu Cys Val Glu Pro Val Asp Pro Cys Phe

340 345 350

Arg Ala Asn Cys Glu Tyr Gin Cys Gin Pro Leu Asn Gin Thr Ser Tyr 355 360 365

Leu Cys Val Cys Ala Glu Gly Phe Ala Pro He Pro His Glu Pro His 370 375 380

Arg Cys Gin Met Phe Cys Asn Gin Thr Ala Cys Pro Ala Asp Cys Asp 385 390 395 400

Pro Asn Thr Gin Ala Ser Cys Glu Cys Pro Glu Gly Tyr He Leu Asp 405 410 415 Asp Gly Phe He Cys Thr Asp He Asp Glu Cys Glu Asn Gly Gly Phe

420 425 430

Cys Ser Gly Val Cys His Asn Leu Pro Gly Thr Phe Glu Cys He Cys 435 440 445

Gly Pro Asp Ser Ala Leu Ala Arg His He Gly Thr Asp Cys 450 455 460

SUBSTITUTE SHEET (2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 462 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(ix) FEATURE:

(A) NAME/KEY: Modified-site (B) LOCATION: 336..454

(D) OTHER INFORMATION: /note= "Xaa is the natural amino acid for that position as specified in SEQ ID NO 1 or an aliphatic amino acid of the group comprising Gly, Ala, Val, Leu and He".

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Ala Pro Ala Glu Pro Gin Pro Gly Gly Ser Gin Cys Val Glu His Asp 1 5 10 15

Cys Phe Ala Leu Tyr Pro Gly Pro Ala Thr Phe Leu Asn Ala Ser Gin 20 25 30

He Cys Asp Gly Leu Arg Gly His Leu Met Thr Val Arg Ser Ser Val 35 40 45

Ala Ala Asp Val He Ser Leu Leu Leu Asn Gly Asp Gly Gly Val Gly 50 55 60 Arg Arg Arg Leu Trp He Gly Leu Gin Leu Pro Pro Gly Cys Gly Asp 65 70 75 80

Pro Lys Arg Leu Gly Pro Leu Arg Gly Phe Gin Trp Val Thr Gly Asp 85 90 95

Asn Asn Thr Ser Tyr Ser Arg Trp Ala Arg Leu Asp Leu Asn Gly Ala 100 105 110

Pro Leu Cys Gly Pro Leu Cys Val Ala Val Ser Ala Ala Glu Ala Thr 115 120 125

Val Pro Ser Glu Pro He Trp Glu Glu Gin Gin Cys Glu Val Lys Ala 130 135 140 Asp Gly Phe Leu Cys Glu Phe His Phe Pro Ala Thr Cys Arg Pro Leu 145 150 155 160

Ala Val Glu Pro Gly Ala Ala Ala Ala Ala Val Ser He Thr Tyr Gly 165 170 175

Thr Pro Phe Ala Ala Arg Gly Ala Asp Phe Gin Ala Leu Pro Val Gly 180 185 190

Ser Ser Ala Ala Val Ala Pro Leu Gly Leu Gin Leu Met Cys Thr Ala 195 200 205

SUBSTITUTE SHEET

SUBSTITUTE SHEET (2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 462 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(ix) FEATURE:

(A) NAME/KEY: Modified-site (B) LOCATION: 365..369

(D) OTHER INFORMATION: /note= "Xaa is the natural amino acid for that position as specified in SEQ ID NO 1 or an aliphatic amino acid of the group comprising Gly, Ala, Val, Leu and He".

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

Ala Pro Ala Glu Pro Gin Pro Gly Gly Ser Gin Cys Val Glu His Asp 1 5 10 15

Cys Phe Ala Leu Tyr Pro Gly Pro Ala Thr Phe Leu Asn Ala Ser Gin 20 25 30

He Cys Asp Gly Leu Arg Gly His Leu Met Thr Val Arg Ser Ser Val 35 40 45

Ala Ala Asp Val He Ser Leu Leu Leu Asn Gly Asp Gly Gly Val Gly 50 55 60 Arg Arg Arg Leu Trp He Gly Leu Gin Leu Pro Pro Gly Cys Gly Asp 65 70 75 80

Pro Lys Arg Leu Gly Pro Leu Arg Gly Phe Gin Trp Val Thr Gly Asp 85 90 95

Asn Asn Thr Ser Tyr Ser Arg Trp Ala Arg Leu Asp Leu Asn Gly Ala 100 105 110

Pro Leu Cys Gly Pro Leu Cys Val Ala Val Ser Ala Ala Glu Ala Thr 115 120 125

Val Pro Ser Glu Pro He Trp Glu Glu Gin Gin Cys Glu Val Lys Ala 130 135 140 Asp Gly Phe Leu Cys Glu Phe His Phe Pro Ala Thr Cys Arg Pro Leu 145 150 155 160

Ala Val Glu Pro Gly Ala Ala Ala Ala Ala Val Ser He Thr Tyr Gly 165 170 175

Thr Pro Phe Ala Ala Arg Gly Ala Asp Phe Gin Ala Leu Pro Val Gly 180 185 190

Ser Ser Ala Ala Val Ala Pro Leu Gly Leu Gin Leu Met Cys Thr Ala 195 200 205

SUBSTITUTE SHEET Pro Pro Gly Ala Val Gin Gly His Trp Ala Arg Glu Ala Pro Gly Ala ' 210 215 220

Trp Asp Cys Ser Val Glu Asn Gly Gly Cys Glu His Ala Cys Asn Ala 225 230 235 240 He Pro Gly Ala Pro Arg Cys Gin Cys Pro Ala Gly Ala Ala Leu Gin

245 250 255

Ala Asp Gly Arg Ser Cys Thr Ala Ser Ala Thr Gin Ser Cys Asn Asp 260 265 270

Leu Cys Glu His Phe Cys Val Pro Asn Pro Asp Gin Pro Gly Ser Tyr 275 280 285

Ser Cys Met Cys Glu Thr Gly Tyr Arg Leu Ala Ala Asp Gin His Arg 290 295 300

Cys Glu Asp Val Asp Asp Cys He Leu Glu Pro Ser Pro Cys Pro Gin 305 310 315 320 Arg Cys Val Asn Thr Gin Gly Gly Phe Glu Cys His Cys Tyr Pro Asn

325 330 335

Tyr Asp Leu Val Asp Gly Glu Cys Val Glu Pro Val Asp Pro Cys Phe 340 345 350

Arg Ala Asn Cys Glu Tyr Gin Cys Gin Pro Leu Asn Xaa Thr Ser Tyr 355 360 365

Xaa Cys Val Cys Ala Glu Gly Phe Ala Pro He Pro His Glu Pro His 370 375 380

Arg Cys Gin Met Phe Cys Asn Gin Thr Ala Cys Pro Ala Asp Cys Asp 385 390 395 400 Pro Asn Thr Gin Ala Ser Cys Glu Cys Pro Glu Gly Tyr He Leu Asp

405 410 415

Asp Gly Phe He Cys Thr Asp He Asp Glu Cys Glu Asn Gly Gly Phe 420 425 430

Cys Ser Gly Val Cys His Asn Leu Pro Gly Thr Phe Glu Cys He Cys 435 440 445

Gly Pro Asp Ser Ala Leu Ala Arg His He Gly Thr Asp Cys 450 455 460

SUBSTITUTE SHEET (2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 462 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(ix) FEATURE:

(A) NAME/KEY: Modified-site (B) LOCATION: 456..457

(D) OTHER INFORMATION: /note= "Xaa is the natural amino acid for that position as specified in SEQ ID NO 1 or an aliphatic amino acid of the group comprising Gly, Ala, Val, Leu and He".

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 456..457

(D) OTHER INFORMATION: /note= "protease resistence".

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Ala Pro Ala Glu Pro Gin Pro Gly Gly Ser Gin Cys Val Glu His Asp 1 5 10 15

Cys Phe Ala Leu Tyr Pro Gly Pro Ala Thr Phe Leu Asn Ala Ser Gin 20 25 30

He Cys Asp Gly Leu Arg Gly His Leu Met Thr Val Arg Ser Ser Val 35 40 45

Ala Ala Asp Val He Ser Leu Leu Leu Asn Gly Asp Gly Gly Val Gly 50 55 60 Arg Arg Arg Leu Trp He Gly Leu Gin Leu Pro Pro Gly Cys Gly Asp 65 70 75 80

Pro Lys Arg Leu Gly Pro Leu Arg Gly Phe Gin Trp Val Thr Gly Asp 85 90 95

Asn Asn Thr Ser Tyr Ser Arg Trp Ala Arg Leu Asp Leu Asn Gly Ala 100 105 110

Pro Leu Cys Gly Pro Leu Cys Val Ala Val Ser Ala Ala Glu Ala Thr 115 120 125

Val Pro Ser Glu Pro He Trp Glu Glu Gin Gin Cys Glu Val Lys Ala 130 135 140 Asp Gly Phe Leu Cys Glu Phe His Phe Pro Ala Thr Cys Arg Pro Leu 145 150 155 160

Ala Val Glu Pro Gly Ala Ala Ala Ala Ala Val Ser He Thr Tyr Gly 165 170 175

SUBSTITUTE SHEET Thr Pro Phe Ala Ala Arg Gly Ala Asp Phe Gin Ala Leu Pro Val Gly 180 185 190

Ser Ser Ala Ala Val Ala Pro Leu Gly Leu Gin Leu Met Cys Thr Ala 195 200 205 Pro Pro Gly Ala Val Gin Gly His Trp Ala Arg Glu Ala Pro Gly Ala 210 215 220

Trp Asp Cys Ser Val Glu Asn Gly Gly Cys Glu His Ala Cys Asn Ala 225 230 235 240

He Pro Gly Ala Pro Arg Cys Gin Cys Pro Ala Gly Ala Ala Leu Gin 245 250 255

Ala Asp Gly Arg Ser Cys Thr Ala Ser Ala Thr Gin Ser Cys Asn Asp 260 265 270

Leu Cys Glu His Phe Cys Val Pro Asn Pro Asp Gin Pro Gly Ser Tyr 275 280 285 Ser Cys Met Cys Glu Thr Gly Tyr Arg Leu Ala Ala Asp Gin His Arg 290 295 300

Cys Glu Asp Val Asp Asp Cys He Leu Glu Pro Ser Pro Cys Pro Gin 305 310 315 320

Arg Cys Val Asn Thr Gin Gly Gly Phe Glu Cys His Cys Tyr Pro Asn 325 330 335

Tyr Asp Leu Val Asp Gly Glu Cys Val Glu Pro Val Asp Pro Cys Phe 340 345 350

Arg Ala Asn Cys Glu Tyr Gin Cys Gin Pro Leu Asn Gin Thr Ser Tyr 355 360 365 Leu Cys Val Cys Ala Glu Gly Phe Ala Pro He Pro His Glu Pro His 370 375 380

Arg Cys Gin Met Phe Cys Asn Gin Thr Ala Cys Pro Ala Asp Cys Asp 385 390 395 400

Pro Asn Thr Gin Ala Ser Cys Glu Cys Pro Glu Gly Tyr He Leu Asp 405 410 415

Asp Gly Phe He Cys Thr Asp He Asp Glu Cys Glu Asn Gly Gly Phe 420 425 430

Cys Ser Gly Val Cys His Asn Leu Pro Gly Thr Phe Glu Cys He Cys 435 440 445 Gly Pro Asp Ser Ala Leu Ala Xaa Xaa He Gly Thr Asp Cys 450 455 460

SUBSTITUTE SHEET (2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 462 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(ix) FEATURE:

(A) NAME/KEY: Modified-site (B) LOCATION: 456..457

(D) OTHER INFORMATION: /note= "Xaa is the natural amino acid for that position as specified in SEQ ID NO 1 or an aliphatic amino acid of the group comprising Gly, Ala, Val, Leu and He".

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 456..457

(D) OTHER INFORMATION: /note= "Xaa is Gly at position 456 or Xaa is Gin at position

457".

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 456..457 (D) OTHER INFORMATION : /note= "protease resistence" .

(xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 5 :

Ala Pro Ala Glu Pro Gin Pro Gly Gly Ser Gin Cys Val Glu His Asp 1 5 10 15 Cys Phe Ala Leu Tyr Pro Gly Pro Ala Thr Phe Leu Asn Ala Ser Gin

20 25 30

He Cys Asp Gly Leu Arg Gly His Leu Met Thr Val Arg Ser Ser Val 35 40 45

Ala Ala Asp Val He Ser Leu Leu Leu Asn Gly Asp Gly Gly Val Gly 50 55 60

Arg Arg Arg Leu Trp He Gly Leu Gin Leu Pro Pro Gly Cys Gly Asp 65 70 75 80

Pro Lys Arg Leu Gly Pro Leu Arg Gly Phe Gin Trp Val Thr Gly Asp 85 90 95 Asn Asn Thr Ser Tyr Ser Arg Trp Ala Arg Leu Asp Leu Asn Gly Ala

100 105 110

Pro Leu Cys Gly Pro Leu Cys Val Ala Val Ser Ala Ala Glu Ala Thr 115 120 125

SUBSTITUTE SHEET Val Pro Ser Glu Pro He Trp Glu Glu Gin Gin Cys Glu Val Lys Ala 130 135 140

Asp Gly Phe Leu Cys Glu Phe His Phe Pro Ala Thr Cys Arg Pro Leu 145 150 155 160

Ala Val Glu Pro Gly Ala Ala Ala Ala Ala Val Ser He Thr Tyr Gly 165 170 175

Thr Pro Phe Ala Ala Arg Gly Ala Asp Phe Gin Ala Leu Pro Val Gly 180 185 190

Ser Ser Ala Ala Val Ala Pro Leu Gly Leu Gin Leu Met Cys Thr Ala 195 200 205

Pro Pro Gly Ala Val Gin Gly His Trp Ala Arg Glu Ala Pro Gly Ala 210 215 220

Trp Asp Cys Ser Val Glu Asn Gly Gly Cys Glu His Ala Cys Asn Ala 225 230 235 240 He Pro Gly Ala Pro Arg Cys Gin Cys Pro Ala Gly Ala Ala Leu Gin

245 250 255

Ala Asp Gly Arg Ser Cys Thr Ala Ser Ala Thr Gin Ser Cys Asn Asp 260 265 270

Leu Cys Glu His Phe Cys Val Pro Asn Pro Asp Gin Pro Gly Ser Tyr 275 280 285

Ser Cys Met Cys Glu Thr Gly Tyr Arg Leu Ala Ala Asp Gin His Arg 290 295 300

Cys Glu Asp Val Asp Asp Cys He Leu Glu Pro Ser Pro Cys Pro Gin 305 310 315 320 Arg Cys Val Asn Thr Gin Gly Gly Phe Glu Cys His Cys Tyr Pro Asn

325 330 335

Tyr Asp Leu Val Asp Gly Glu Cys Val Glu Pro Val Asp Pro Cys Phe 340 345 350

Arg Ala Asn Cys Glu Tyr Gin Cys Gin Pro Leu Asn Gin Thr Ser Tyr 355 360 365

Leu Cys Val Cys Ala Glu Gly Phe Ala Pro He Pro His Glu Pro His 370 375 380

Arg Cys Gin Met Phe Cys Asn Gin Thr Ala Cys Pro Ala Asp Cys Asp 385 390 395 400 Pro Asn Thr Gin Ala Ser Cys Glu Cys Pro Glu Gly Tyr He Leu Asp

405 410 415

Asp Gly Phe He Cys Thr Asp He Asp Glu Cys Glu Asn Gly Gly Phe 420 425 430

Cys Ser Gly Val Cys His Asn Leu Pro Gly Thr Phe Glu Cys He Cys 435 440 445

Gly Pro Asp Ser Ala Leu Ala Xaa Xaa He Gly Thr Asp Cys 450 455 460

SUBSTITUTE SHEET (2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 462 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(ix) FEATURE:

(A) NAME/KEY: Modified-site (B) LOCATION: 365..457

(D) OTHER INFORMATION: /note= "Xaa is the natural amino acid for that position as specified in SEQ ID NO 1 or an aliphatic amino acid of the group comprising Gly, Ala, Val, Leu and He".

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

Ala Pro Ala Glu Pro Gin Pro Gly Gly Ser Gin Cys Val Glu His Asp 1 5 10 15

Cys Phe Ala Leu Tyr Pro Gly Pro Ala Thr Phe Leu Asn Ala Ser Gin 20 25 30

He Cys Asp Gly Leu Arg Gly His Leu Met Thr Val Arg Ser Ser Val 35 40 45

Ala Ala Asp Val He Ser Leu Leu Leu Asn Gly Asp Gly Gly Val Gly 50 55 60 Arg Arg Arg Leu Trp He Gly Leu Gin Leu Pro Pro Gly Cys Gly Asp 65 70 75 80

Pro Lys Arg Leu Gly Pro Leu Arg Gly Phe Gin Trp Val Thr Gly Asp 85 90 95

Asn Asn Thr Ser Tyr Ser Arg Trp Ala Arg Leu Asp Leu Asn Gly Ala 100 105 110

Pro Leu Cys Gly Pro Leu Cys Val Ala Val Ser Ala Ala Glu Ala Thr 115 120 125

Val Pro Ser Glu Pro He Trp Glu Glu Gin Gin Cys Glu Val Lys Ala 130 135 140 Asp Gly Phe Leu Cys Glu Phe His Phe Pro Ala Thr Cys Arg Pro Leu 145 150 155 160

Ala Val Glu Pro Gly Ala Ala Ala Ala Ala Val Ser He Thr Tyr Gly 165 170 175

Thr Pro Phe Ala Ala Arg Gly Ala Asp Phe Gin Ala Leu Pro Val Gly 180 185 190

Ser Ser Ala Ala Val Ala Pro Leu Gly Leu Gin Leu Met Cys Thr Ala 195 200 205

SUBSTITUTE SHEET Pro Pro Gly Ala Val Gin Gly His Trp Ala Arg Glu Ala Pro Gly Ala 210 215 220

Trp Asp Cys Ser Val Glu Asn Gly Gly Cys Glu His Ala Cys Asn Ala 225 230 235 240 He Pro Gly Ala Pro Arg Cys Gin Cys Pro Ala Gly Ala Ala Leu Gin

245 250 255

Ala Asp Gly Arg Ser Cys Thr Ala Ser Ala Thr Gin Ser Cys Asn Asp 260 265 270

Leu Cys Glu His Phe Cys Val Pro Asn Pro Asp Gin Pro Gly Ser Tyr 275 280 285

Ser Cys Met Cys Glu Thr Gly Tyr Arg Leu Ala Ala Asp Gin His Arg 290 295 300

Cys Glu Asp Val Asp Asp Cys He Leu Glu Pro Ser Pro Cys Pro Gin 305 310 315 320 Arg Cys Val Asn Thr Gin Gly Gly Phe Glu Cys His Cys Tyr Pro Asn

325 330 335

Tyr Asp Leu Val Asp Gly Glu Cys Val Glu Pro Val Asp Pro Cys Phe 340 345 350

Arg Ala Asn Cys Glu Tyr Gin Cys Gin Pro Leu Asn Xaa Thr Ser Tyr 355 360 365

Xaa Cys Val Cys Ala Glu Gly Phe Ala Pro He Pro His Glu Pro His 370 375 380

Arg Cys Gin Xaa Phe Cys Asn Gin Thr Ala Cys Pro Ala Asp Cys Asp 385 390 395 400 Pro Asn Thr Gin Ala Ser Cys Glu Cys Pro Glu Gly Tyr He Leu Asp

405 410 415

Asp Gly Phe He Cys Thr Asp He Asp Glu Cys Glu Asn Gly Gly Phe 420 425 430

Cys Ser Gly Val Cys His Asn Leu Pro Gly Thr Phe Glu Cys He Cys 435 440 445

Gly Pro Asp Ser Ala Leu Ala Xaa Xaa He Gly Thr Asp Cys 450 455 460

SUBSTITUTE SHEET - 53 -

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 462 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(ix) FEATURE:

(A) NAME/KEY: Modified-site (B) LOCATION: 336..461

(D) OTHER INFORMATION: /note= "Xaa is the natural amino acid for that position as specified in SEQ ID NO 1 or an aliphatic amino acid of the group comprising Gly, Ala, Val, Leu and He".

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: amino terminal

(D) OTHER INFORMATION: /note= "Gly-Pro-Gln-Pro is an amino terminal modification" .

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: carboxy terminal

(D) OTHER INFORMATION: /note= "Leu-Thr-Pro-Pro is a carboxy terminal modification" .

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

Ala Pro Ala Glu Pro Gin Pro Gly Gly Ser Gin Cys Val Glu His Asp 1 5 10 15

Cys Phe Ala Leu Tyr Pro Gly Pro Ala Thr Phe Leu Asn Ala Ser Gin 20 25 30

He Cys Asp Gly Leu Arg Gly His Leu Met Thr Val Arg Ser Ser Val 35 40 45

Ala Ala Asp Val He Ser Leu Leu Leu Asn Gly Asp Gly Gly Val Gly 50 55 60 Arg Arg Arg Leu Trp He Gly Leu Gin Leu Pro Pro Gly Cys Gly Asp 65 70 75 80

Pro Lys Arg Leu Gly Pro Leu Arg Gly Phe Gin Trp Val Thr Gly Asp 85 90 95

Asn Asn Thr Ser Tyr Ser Arg Trp Ala Arg Leu Asp Leu Asn Gly Ala 100 105 110

Pro Leu Cys Gly Pro Leu Cys Val Ala Val Ser Ala Ala Glu Ala Thr 115 120 125

Val Pro Ser Glu Pro He Trp Glu Glu Gin Gin Cys Glu Val Lys Ala 130 135 140

SUBSTITUTE SHEET Asp Gly Phe Leu Cys Glu Phe His Phe Pro Ala Thr Cys Arg Pro Leu 145 150 155 160

Ala Val Glu Pro Gly Ala Ala Ala Ala Ala Val Ser He Thr Tyr Gly 165 170 175

Thr Pro Phe Ala Ala Arg Gly Ala Asp Phe Gin Ala Leu Pro Val Gly 180 185 190

Ser Ser Ala Ala Val Ala Pro Leu Gly Leu Gin Leu Met Cys Thr Ala 195 200 205

Pro Pro Gly Ala Val Gin Gly His Trp Ala Arg Glu Ala Pro Gly Ala 210 215 220

Trp Asp Cys Ser Val Glu Asn Gly Gly Cys Glu His Ala Cys Asn Ala 225 230 235 240

He Pro Gly Ala Pro Arg Cys Gin Cys Pro Ala Gly Ala Ala Leu Gin 245 250 255 Ala Asp Gly Arg Ser Cys Thr Ala Ser Ala Thr Gin Ser Cys Asn Asp

260 265 270

Leu Cys Glu His Phe Cys Val Pro Asn Pro Asp Gin Pro Gly Ser Tyr 275 280 285

Ser Cys Met Cys Glu Thr Gly Tyr Arg Leu Ala Ala Asp Gin His Arg 290 295 300

Cys Glu Asp Val Asp Asp Cys He Leu Glu Pro Ser Pro Cys Pro Gin 305 310 315 320

Arg Cys Val Asn Thr Gin Gly Gly Phe Glu Cys His Cys Tyr Pro Xaa 325 330 335 Xaa Asp Leu Xaa Xaa Gly Glu Cys Val Glu Pro Val Xaa Pro Cys Phe

340 345 350

Arg Ala Xaa Cys Xaa Xaa Xaa Cys Gin Pro Xaa Asn Xaa Thr Ser Tyr 355 360 365

Xaa Cys Xaa Cys Ala Xaa Gly Xaa Ala Pro He Pro His Glu Pro Xaa 370 375 380

Xaa Cys Xaa Xaa Xaa Cys Asn Gin Thr Ala Cys Pro Ala Xaa Cys Xaa 385 390 395 400

Pro Xaa Xaa Gin Ala Ser Cys Xaa Cys Pro Glu Gly Xaa Xaa Xaa Xaa 405 410 415 Xaa Gly Phe Xaa Cys Thr Xaa Xaa Xaa Xaa Cys Xaa Xaa Gly Gly Xaa

420 425 430

Cys Xaa Gly Xaa Cys Xaa Xaa Xaa Pro Gly Xaa Xaa Xaa Cys Xaa Cys 435 440 445

Gly Pro Asp Ser Ala Xaa Ala Xaa Xaa Xaa Gly Thr Xaa Cys 450 455 460

SUBSTITUTE SHEET (2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 462 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(ix) FEATURE:

(A) NAME/KEY: Modified-site (B) LOCATION: 349..461

(D) OTHER INFORMATION: /note= "Xaa is the natural amino acid for that position as specified in SEQ ID NO 1 or an aliphatic amino acid of the group comprising Gly, Ala, Val, Leu and He".

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

Ala Pro Ala Glu Pro Gin Pro Gly Gly Ser Gin Cys Val Glu His Asp 1 5 10 15

Cys Phe Ala Leu Tyr Pro Gly Pro Ala Thr Phe Leu Asn Ala Ser Gin 20 25 30

He Cys Asp Gly Leu Arg Gly His Leu Met Thr Val Arg Ser Ser Val 35 40 45

Ala Ala Asp Val He Ser Leu Leu Leu Asn Gly Asp Gly Gly Val Gly 50 55 60 Arg Arg Arg Leu Trp He Gly Leu Gin Leu Pro Pro Gly Cys Gly Asp 65 70 75 80

Pro Lys Arg Leu Gly Pro Leu Arg Gly Phe Gin Trp Val Thr Gly Asp 85 90 95

Asn Asn Thr Ser Tyr Ser Arg Trp Ala Arg Leu Asp Leu Asn Gly Ala 100 105 110

Pro Leu Cys Gly Pro Leu Cys Val Ala Val Ser Ala Ala Glu Ala Thr 115 120 125

Val Pro Ser Glu Pro He Trp Glu Glu Gin Gin Cys Glu Val Lys Ala 130 135 140 Asp Gly Phe Leu Cys Glu Phe His Phe Pro Ala Thr Cys Arg Pro Leu 145 150 155 160

Ala Val Glu Pro Gly Ala Ala Ala Ala Ala Val Ser He Thr Tyr Gly 165 170 175

Thr Pro Phe Ala Ala Arg Gly Ala Asp Phe Gin Ala Leu Pro Val Gly 180 185 190

Ser Ser Ala Ala Val Ala Pro Leu Gly Leu Gin Leu Met Cys Thr Ala 195 200 205

SUBSTITUTE SHEET Pro Pro Gly Ala Val Gin Gly His Trp Ala Arg Glu Ala Pro Gly Ala 210 215 220

Trp Asp Cys Ser Val Glu Asn Gly Gly Cys Glu His Ala Cys Asn Ala 225 230 235 240

He Pro Gly Ala Pro Arg Cys Gin Cys Pro Ala Gly Ala Ala Leu Gin 245 250 255

Ala Asp Gly Arg Ser Cys Thr Ala Ser Ala Thr Gin Ser Cys Asn Asp 260 265 270

Leu Cys Glu His Phe Cys Val Pro Asn Pro Asp Gin Pro Gly Ser Tyr 275 280 285

Ser Cys Met Cys Glu Thr Gly Tyr Arg Leu Ala Ala Asp Gin His Arg 290 295 300

Cys Glu Asp Val Asp Asp Cys He Leu Glu Pro Ser Pro Cys Pro Gin 305 310 315 320 Arg Cys Val Asn Thr Gin Gly Gly Phe Glu Cys His Cys Tyr Pro Asn

325 330 335

Tyr Asp Leu Val Asp Gly Glu Cys Val Glu Pro Val Xaa Pro Cys Phe 340 345 350

Arg Ala Xaa Cys Xaa Xaa Xaa Cys Gin Pro Xaa Asn Gin Thr Ser Tyr 355 360 365

Leu Cys Xaa Cys Ala Xaa Gly Phe Ala Pro He Pro His Glu Pro Xaa 370 375 380

Xaa Cys Xaa Met Xaa Cys Asn Gin Thr Ala Cys Pro Ala Xaa Cys Xaa 385 390 395 400 Pro Xaa Xaa Gin Ala Ser Cys Xaa Cys Pro Glu Gly Xaa Xaa Xaa Xaa

405 410 415

Xaa Gly Phe Xaa Cys Thr Xaa Xaa Xaa Xaa Cys Xaa Xaa Gly Gly Xaa 420 425 430

Cys Xaa Gly Xaa Cys Xaa Xaa Xaa Pro Gly Xaa Xaa Xaa Cys He Cys 435 440 445

Gly Pro Asp Ser Ala Leu Ala Xaa His Xaa Gly Thr Xaa Cys 450 455 460

SUBSTITUTE SHEET (2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 462 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(ix) FEATURE:

(A) NAME/KEY: Modified-site (B) LOCATION: 349..461

(D) OTHER INFORMATION: /note= "Xaa is the natural amino acid for that position as specified in SEQ ID NO 1 or an aliphatic amino acid of the group comprising Gly, Ala, Val, Leu and He".

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

Ala Pro Ala Glu Pro Gin Pro Gly Gly Ser Gin Cys Val Glu His Asp 1 5 10 15

Cys Phe Ala Leu Tyr Pro Gly Pro Ala Thr Phe Leu Asn Ala Ser Gin 20 25 30

He Cys Asp Gly Leu Arg Gly His Leu Met Thr Val Arg Ser Ser Val 35 40 45

Ala Ala Asp Val He Ser Leu Leu Leu Asn Gly Asp Gly Gly Val Gly 50 55 60 Arg Arg Arg Leu Trp He Gly Leu Gin Leu Pro Pro Gly Cys Gly Asp 65 70 75 80

Pro Lys Arg Leu Gly Pro Leu Arg Gly Phe Gin Trp Val Thr Gly Asp 85 90 95

Asn Asn Thr Ser Tyr Ser Arg Trp Ala Arg Leu Asp Leu Asn Gly Ala 100 105 110

Pro Leu Cys Gly Pro Leu Cys Val Ala Val Ser Ala Ala Glu Ala Thr 115 120 125

Val Pro Ser Glu Pro He Trp Glu Glu Gin Gin Cys Glu Val Lys Ala 130 135 140 Asp Gly Phe Leu Cys Glu Phe His Phe Pro Ala Thr Cys Arg Pro Leu 145 150 155 160

Ala Val Glu Pro Gly Ala Ala Ala Ala Ala Val Ser He Thr Tyr Gly 165 170 175

Thr Pro Phe Ala Ala Arg Gly Ala Asp Phe Gin Ala Leu Pro Val Gly 180 185 190

Ser Ser Ala Ala Val Ala Pro Leu Gly Leu Gin Leu Met Cys Thr Ala 195 200 205

SUBSTITUTE SHEET

SUBSTITUTE SHEET (2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 462 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(ix) FEATURE:

(A) NAME/KEY: Modified-site (B) LOCATION: 349..376

(D) OTHER INFORMATION: /note= "Xaa is the natural amino acid for that position as specified in SEQ ID NO 1 or an aliphatic amino acid of the group comprising Gly, Ala, Val, Leu and He".

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

Ala Pro Ala Glu Pro Gin Pro Gly Gly Ser Gin Cys Val Glu His Asp 1 5 10 15

Cys Phe Ala Leu Tyr Pro Gly Pro Ala Thr Phe Leu Asn Ala Ser Gin 20 25 30

He Cys Asp Gly Leu Arg Gly His Leu Met Thr Val Arg Ser Ser Val 35 40 45

Ala Ala Asp Val He Ser Leu Leu Leu Asn Gly Asp Gly Gly Val Gly 50 55 60 Arg Arg Arg Leu Trp He Gly Leu Gin Leu Pro Pro Gly Cys Gly Asp 65 70 75 80

Pro Lys Arg Leu Gly Pro Leu Arg Gly Phe Gin Trp Val Thr Gly Asp 85 90 95

Asn Asn Thr Ser Tyr Ser Arg Trp Ala Arg Leu Asp Leu Asn Gly Ala 100 105 110

Pro Leu Cys Gly Pro Leu Cys Val Ala Val Ser Ala Ala Glu Ala Thr 115 120 125

Val Pro Ser Glu Pro He Trp Glu Glu Gin Gin Cys Glu Val Lys Ala 130 135 140 Asp Gly Phe Leu Cys Glu Phe His Phe Pro Ala Thr Cys Arg Pro Leu 145 150 155 160

Ala Val Glu Pro Gly Ala Ala Ala Ala Ala Val Ser He Thr Tyr Gly 165 170 175

Thr Pro Phe Ala Ala Arg Gly Ala Asp Phe Gin Ala Leu Pro Val Gly 180 185 190

Ser Ser Ala Ala Val Ala Pro Leu Gly Leu Gin Leu Met Cys Thr Ala 195 200 205

SUBSTITUTE SHEET Pro Pro Gly Ala Val Gin Gly His Trp Ala Arg Glu Ala Pro Gly Ala ' 210 215 220

Trp Asp Cys Ser Val Glu Asn Gly Gly Cys Glu His Ala Cys Asn Ala 225 230 235 240 He Pro Gly Ala Pro Arg Cys Gin Cys Pro Ala Gly Ala Ala Leu Gin

245 250 255

Ala Asp Gly Arg Ser Cys Thr Ala Ser Ala Thr Gin Ser Cys Asn Asp 260 265 270

Leu Cys Glu His Phe Cys Val Pro Asn Pro Asp Gin Pro Gly Ser Tyr 275 280 285

Ser Cys Met Cys Glu Thr Gly Tyr Arg Leu Ala Ala Asp Gin His Arg 290 295 300

Cys Glu Asp Val Asp Asp Cys He Leu Glu Pro Ser Pro Cys Pro Gin 305 310 315 320 Arg Cys Val Asn Thr Gin Gly Gly Phe Glu Cys His Cys Tyr Pro Asn

325 330 335

Tyr Asp Leu Val Asp Gly Glu Cys Val Glu Fro Val Xaa Pro Cys Phe 340 345 350

Arg Ala Asn Cys Xaa Xaa Xaa Cys Gin Pro Leu Asn Gin Thr Ser Tyr 355 360 365

Leu Cys Xaa Cys Ala Glu Gly Xaa Ala Pro He Pro His Glu Pro His 370 375 380

Arg Cys Gin Met Phe Cys Asn Gin Thr Ala Cys Pro Ala Asp Cys Asp 385 390 395 400 Pro Asn Thr Gin Ala Ser Cys Glu Cys Pro Glu Gly Tyr He Leu Asp

405 410 415

Asp Gly Phe He Cys Thr Asp He Asp Glu Cys Glu Asn Gly Gly Phe 420 425 430

Cys Ser Gly Val Cys His Asn Leu Pro Gly Thr Phe Glu Cys He Cys 435 440 445

Gly Pro Asp Ser Ala Leu Ala Arg His He Gly Thr Asp Cys 450 455 460

SUBSTITUTESHEET (2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 462 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(ix) FEATURE:

(A) NAME/KEY: Modified-site (B) LOCATION: 398..461

(D) OTHER INFORMATION: /note= "Xaa is the natural amino acid for that position as specified in SEQ ID NO 1 or an aliphatic amino acid of the group comprising Gly, Ala, Val, Leu and He".

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:

Ala Pro Ala Glu Pro Gin Pro Gly Gly Ser Gin Cys Val Glu His Asp 1 5 10 15

Cys Phe Ala Leu Tyr Pro Gly Pro Ala Thr Phe Leu Asn Ala Ser Gin 20 25 30

He Cys Asp Gly Leu Arg Gly His Leu Met Thr Val Arg Ser Ser Val 35 40 45

Ala Ala Asp Val He Ser Leu Leu Leu Asn Gly Asp Gly Gly Val Gly 50 55 60 Arg Arg Arg Leu Trp He Gly Leu Gin Leu Pro Pro Gly Cys Gly Asp 65 70 75 80

Pro Lys Arg Leu Gly Pro Leu Arg Gly Phe Gin Trp Val Thr Gly Asp 85 90 95

Asn Asn Thr Ser Tyr Ser Arg Trp Ala Arg Leu Asp Leu Asn Gly Ala 100 105 110

Pro Leu Cys Gly Pro Leu Cys Val Ala Val Ser Ala Ala Glu Ala Thr 115 120 125

Val Pro Ser Glu Pro He Trp Glu Glu Gin Gin Cys Glu Val Lys Ala 130 135 140 Asp Gly Phe Leu Cys Glu Phe His Phe Pro Ala Thr Cys Arg Pro Leu 145 150 155 160

Ala Val Glu Pro Gly Ala Ala Ala Ala Ala Val Ser He Thr Tyr Gly 165 170 175

Thr Pro Phe Ala Ala Arg Gly Ala Asp Phe Gin Ala Leu Pro Val Gly 180 185 190

Ser Ser Ala Ala Val Ala Pro Leu Gly Leu Gin Leu Met Cys Thr Ala 195 200 205

SUBSTITUTE SHEET Pro Pro Gly Ala Val Gin Gly His Trp Ala Arg Glu Ala Pro Gly Ala 210 215 220

Trp Asp Cys Ser Val Glu Asn Gly Gly Cys Glu His Ala Cys Asn Ala 225 230 235 240

He Pro Gly Ala Pro Arg Cys Gin Cys Pro Ala Gly Ala Ala Leu Gin 245 250 255

Ala Asp Gly Arg Ser Cys Thr Ala Ser Ala Thr Gin Ser Cys Asn Asp 260 265 270

Leu Cys Glu His Phe Cys Val Pro Asn Pro Asp Gin Pro Gly Ser Tyr 275 280 285

Ser Cys Met Cys Glu Thr Gly Tyr Arg Leu Ala Ala Asp Gin His Arg 290 295 300

Cys Glu Asp Val Asp Asp Cys He Leu Glu Pro Ser Pro Cys Pro Gin 305 310 315 320 Arg Cys Val Asn Thr Gin Gly Gly Phe Glu Cys His Cys Tyr Pro Asn

325 330 335

Tyr Asp Leu Val Asp Gly Glu Cys Val Glu Pro Val Asp Pro Cys Phe 340 345 350

Arg Ala Asn Cys Glu Tyr Gin Cys Gin Pro Leu Asn Gin Thr Ser Tyr 355 360 365

Leu Cys Val Cys Ala Glu Gly Phe Ala Pro He Pro His Glu Pro His 370 375 380

Arg Cys Gin Met Phe Cys Asn Gin Thr Ala Cys Pro Ala Xaa Cys Xaa 385 390 395 400 Pro Xaa Thr Gin Ala Ser Cys Xaa Cys Pro Glu Gly Xaa Xaa Xaa Xaa

405 410 415

Xaa Gly Phe He Cys Thr Xaa Xaa Xaa Xaa Cys Glu Xaa Gly Gly Phe 420 425 430

Cys Ser Gly Val Cys His Xaa Leu Pro Gly Thr Xaa Glu Cys He Cys 435 440 445

Gly Pro Asp Ser Ala Leu Ala Arg His He Gly Thr Xaa Cys 450 455 460

SUBSTITUTE SHEET (2) INFORMATION FOR SEQ ID NO:12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 462 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(ix) FEATURE:

(A) NAME/KEY: Modified-site (B) LOCATION: 349..376

(D) OTHER INFORMATION: /note= "Xaa is the natural amino acid for that position as specified in SEQ ID NO 1 or an aliphatic amino acid of the group comprising Gly, Ala, Val, Leu and He".

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:

Ala Pro Ala Glu Pro Gin Pro Gly Gly Ser Gin Cys Val Glu His Asp 1 5 10 15

Cys Phe Ala Leu Tyr Pro Gly Pro Ala Thr Phe Leu Asn Ala Ser Gin 20 25 30

He Cys Asp Gly Leu Arg Gly His Leu Met Thr Val Arg Ser Ser Val 35 40 45

Ala Ala Asp Val He Ser Leu Leu Leu Asn Gly Asp Gly Gly Val Gly 50 55 60 Arg Arg Arg Leu Trp He Gly Leu Gin Leu Pro Pro Gly Cys Gly Asp 65 70 75 80

Pro Lys Arg Leu Gly Pro Leu Arg Gly Phe Gin Trp Val Thr Gly Asp 85 90 95

Asn Asn Thr Ser Tyr Ser Arg Trp Ala Arg Leu Asp Leu Asn Gly Ala 100 105 110

Pro Leu Cys Gly Pro Leu Cys Val Ala Val Ser Ala Ala Glu Ala Thr 115 - 120 125

Val Pro Ser Glu Pro He Trp Glu Glu Gin Gin Cys Glu Val Lys Ala 130 135 140 Asp Gly Phe Leu Cys Glu Phe His Phe Pro Ala Thr Cys Arg Pro Leu 145 150 155 160

Ala Val Glu Pro Gly Ala Ala Ala Ala Ala Val Ser He Thr Tyr Gly 165 170 175

Thr Pro Phe Ala Ala Arg Gly Ala Asp Phe Gin Ala Leu Pro Val Gly 180 185 190

Ser Ser Ala Ala Val Ala Pro Leu Gly Leu Gin Leu Met Cys Thr Ala 195 200 205

SUBSTITUTESHEET

SUBSTITUTE SHEET (2) INFORMATION FOR SEQ ID NO:13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 462 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(ix) FEATURE:

(A) NAME/KEY: Modified-site (B) LOCATION: 398..420

(D) OTHER INFORMATION: /note= "Xaa is the natural amino acid for that position as specified in SEQ ID NO 1 or an aliphatic amino acid of the group comprising Gly, Ala, Val, Leu and He".

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

Ala Pro Ala Glu Pro Gin Pro Gly Gly Ser Gin Cys Val Glu His Asp 1 5 10 15

Cys Phe Ala Leu Tyr Pro Gly Pro Ala Thr Phe Leu Asn Ala Ser Gin 20 25 30

He Cys Asp Gly Leu Arg Gly His Leu Met Thr Val Arg Ser Ser Val 35 40 45

Ala Ala Asp Val He Ser Leu Leu Leu Asn Gly Asp Gly Gly Val Gly 50 55 60 Arg Arg Arg Leu Trp He Gly Leu Gin Leu Pro Pro Gly Cys Gly Asp 65 70 75 80

Pro Lys Arg Leu Gly Pro Leu Arg Gly Phe Gin Trp Val Thr Gly Asp 85 90 95

Asn Asn Thr Ser Tyr Ser Arg Trp Ala Arg Leu Asp Leu Asn Gly Ala 100 105 110

Pro Leu Cys Gly Pro Leu Cys Val Ala Val Ser Ala Ala Glu Ala Thr 115 120 125

Val Pro Ser Giu Pro He Trp Glu Glu Gin Gin Cys Glu Val Lys Ala 130 135 140 Asp Gly Phe Leu Cys Glu Phe His Phe Pro Ala Thr Cys Arg Pro Leu 145 150 155 160

Ala Val Glu Pro Gly Ala Ala Ala Ala Ala Val Ser He Thr Tyr Gly 165 170 175

Thr Pro Phe Ala Ala Arg Gly Ala Asp Phe Gin Ala Leu Pro Val Gly 180 185 190

Ser Ser Ala Ala Val Ala Pro Leu Gly Leu Gin Leu Met Cys Thr Ala 195 200 205

SUBSTITUTE SHEET Pro Pro Gly Ala Val Gin Gly His Trp Ala Arg Glu Ala Pro Gly Ala 210 215 220

Trp Asp Cys Ser Val Glu Asn Gly Gly Cys Glu His Ala Cys Asn Ala 225 230 235 240

He Pro Gly Ala Pro Arg Cys Gin Cys Pro Ala Gly Ala Ala Leu Gin 245 250 255

Ala Asp Gly Arg Ser Cys Thr Ala Ser Ala Thr Gin Ser Cys Asn Asp 260 265 270

Leu Cys Glu His Phe Cys Val Pro Asn Pro Asp Gin Pro Gly Ser Tyr 275 280 285

Ser Cys Met Cys Glu Thr Gly Tyr Arg Leu Ala Ala Asp Gin His Arg 290 295 300

Cys Glu Asp Val Asp Asp Cys He Leu Glu Pro Ser Pro Cys Pro Gin 305 310 315 320 Arg Cys Val Asn Thr Gin Gly Gly Phe Glu Cys His Cys Tyr Pro Asn

325 330 335

Tyr Asp Leu Val Asp Gly Glu Cys Val Glu Pro Val Asp Pro Cys Phe 340 345 350

Arg Ala Asn Cys Glu Tyr Gin Cys Gin Pro Leu Asn Gin Thr Ser Tyr 355 360 365

Leu Cys Val Cys Ala Glu Gly Phe Ala Pro He Pro His Glu Pro His 370 375 380

Arg Cys Gin Met Phe Cys Asn Gin Thr Ala Cys Pro Ala Xaa Cys Xaa 385 390 395 400 Pro Xaa Xaa Gin Ala Ser Cys Xaa Cys Pro Glu Gly Xaa Xaa Xaa Xaa

405 410 415

Xaa Gly Phe Xaa Cys Thr Asp He Asp Glu Cys Glu Asn Gly Gly Phe 420 425 430

Cys Ser Gly Val Cys His Asn Leu Pro Gly Thr Phe Glu Cys He Cys 435 440 445

Gly Pro Asp Ser Ala Leu Ala Arg His He Gly Thr Asp Cys 450 455 460

SUBSTITUTE SHEET (2) INFORMATION FOR SEQ ID NO:14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 462 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(ix) FEATURE:

(A) NAME/KEY: Modified-site (B) LOCATION: 398.. 17

(D) OTHER INFORMATION: /note= "Xaa is the natural amino acid for that position as specified in SEQ ID NO 1 or an aliphatic amino acid of the group comprising Gly, Ala, Val, Leu and He".

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

Ala Pro Ala Glu Pro Gin Pro Gly Gly Ser Gin Cys Val Glu His Asp 1 5 10 15

Cys Phe Ala Leu Tyr Pro Gly Pro Ala Thr Phe Leu Asn Ala Ser Gin 20 25 30

He Cys Asp Gly Leu Arg Gly His Leu Met Thr Val Arg Ser Ser Val 35 40 45

Ala Ala Asp Val He Ser Leu Leu Leu Asn Gly Asp Gly Gly Val Gly 50 55 60 Arg Arg Arg Leu Trp He Gly Leu Gin Leu Pro Pro Gly Cys Gly Asp 65 70 75 80

Pro Lys Arg Leu Gly Pro Leu Arg Gly Phe Gin Trp Val Thr Gly Asp 85 90 95

Asn Asn Thr Ser Tyr Ser Arg Trp Ala Arg Leu Asp Leu Asn Gly Ala 100 105 110

Pro Leu Cys Gly Pro Leu Cys Val Ala Val Ser Ala Ala Glu Ala Thr 115 120 125

Val Pro Ser Glu Pro He Trp Glu Glu Gin Gin Cys Glu Val Lys Ala 130 135 140 Asp Gly Phe Leu Cys Glu Phe His Phe Pro Ala Thr Cys Arg Pro Leu 145 150 155 160

Ala Val Glu Pro Gly Ala Ala Ala Ala Ala Val Ser He Thr Tyr Gly 165 170 175

Thr Pro Phe Ala Ala Arg Gly Ala Asp Phe Gin Ala Leu Pro Val Gly 180 185 190

Ser Ser Ala Ala Val Ala Pro Leu Gly Leu Gin Leu Met Cys Thr Ala 195 200 205

SUBSTITUTE SHEET Pro Pro Gly Ala Val Gin Gly His Trp Ala Arg Glu Ala Pro Gly Ala 210 215 220

Trp Asp Cys Ser Val Glu Asn Gly Gly Cys Glu His Ala Cys Asn Ala 225 230 235 240

He Pro Gly Ala Pro Arg Cys Gin Cys Pro Ala Gly Ala Ala Leu Gin 245 250 255

Ala Asp Gly Arg Ser Cys Thr Ala Ser Ala Thr Gin Ser Cys Asn Asp 260 265 270

Leu Cys Glu His Phe Cys Val Pro Asn Pro Asp Gin Pro Gly Ser Tyr 275 280 285

Ser Cys Met Cys Glu Thr Gly Tyr Arg Leu Ala Ala Asp Gin His Arg 290 295 300

Cys Glu Asp Val Asp Asp Cys He Leu Glu Pro Ser Pro Cys Pro Gin 305 310 315 320 Arg Cys Val Asn Thr Gin Gly Gly Phe Glu Cys His Cys Tyr Pro Asn

325 330 335

Tyr Asp Leu Val Asp Gly Glu Cys Val Glu Pro Val Asp Pro Cys Phe 340 345 350

Arg Ala Asn Cys Glu Tyr Gin Cys Gin Pro Leu Asn Gin Thr Ser Tyr 355 360 365

Leu Cys Val Cys Ala Glu Gly Phe Ala Pro He Pro His Glu Pro His 370 375 380

Arg Cys Gin Met Phe Cys Asn Gin Thr Ala Cys Pro Ala Xaa Cys Xaa 385 390 395 400 Pro Xaa Xaa Gin Ala Ser Cys Xaa Cys Pro Glu Gly Xaa Xaa Xaa Xaa

405 410 415

Xaa Gly Phe He Cys Thr Asp He Asp Glu Cys Glu Asn Gly Gly Phe 420 425 430

Cys Ser Gly Val Cys His Asn Leu Pro Gly Thr Phe Glu Cys He Cys 435 440 445

Gly Pro Asp Ser Ala Leu Ala Arg His He Gly Thr Asp Cys 450 455 460

SUBSTITUTE SHEET (2) INFORMATION FOR SEQ ID NO:15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 462 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(ix) FEATURE:

(A) NAME/KEY: Modified-site (B) LOCATION: 423..461

(D) OTHER INFORMATION: /note= "Xaa is the natural amino acid for that position as specified in SEQ ID NO 1 or an aliphatic amino acid of the group comprising Gly, Ala, Val, Leu and He".

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:

Ala Pro Ala Glu Pro Gin Pro Gly Gly Ser Gin Cys Val Glu His Asp 1 5 10 15

Cys Phe Ala Leu Tyr Pro Gly Pro Ala Thr Phe Leu Asn Ala Ser Gin 20 25 30

He Cys Asp Gly Leu Arg Gly His Leu Met Thr Val Arg Ser Ser Val 35 40 45

Ala Ala Asp Val He Ser Leu Leu Leu Asn Gly Asp Gly Gly Val Gly 50 55 60 Arg Arg Arg Leu Trp He Gly Leu Gin Leu Pro Pro Gly Cys Gly Asp 65 70 75 80

Pro Lys Arg Leu Gly Pro Leu Arg Gly Phe Gin Trp Val Thr Gly Asp 85 90 95

Asn Asn Thr Ser Tyr Ser Arg Trp Ala Arg Leu Asp Leu Asn Gly Ala 100 105 110

Pro Leu Cys Gly Pro Leu Cys Val Ala Val Ser Ala Ala Glu Ala Thr 115 120 125

Val Pro Ser Glu Pro He Trp Glu Glu Gin Gin Cys Glu Val Lys Ala 130 135 140 Asp Gly Phe Leu Cys Glu Phe His Phe Pro Ala Thr Cys Arg Pro Leu 145 150 155 160

Ala Val Glu Pro Gly Ala Ala Ala Ala Ala Val Ser He Thr Tyr Gly 165 170 175

Thr Pro Phe Ala Ala Arg Gly Ala Asp Phe Gin Ala Leu Pro Val Gly 180 185 190

Ser Ser Ala Ala Val Ala Pro Leu Gly Leu Gin Leu Met Cys Thr Ala 195 200 205

SUBSTITUTE SHEET Pro Pro Gly Ala Val Gin Gly His Trp Ala Arg Glu Ala Pro Gly Ala 210 215 220

Trp Asp Cys Ser Val Glu Asn Gly Gly Cys Glu His Ala Cys Asn Ala 225 230 235 240 He Pro Gly Ala Pro Arg Cys Gin Cys Pro Ala Gly Ala Ala Leu Gin

245 250 255

Ala Asp Gly Arg Ser Cys Thr Ala Ser Ala Thr Gin Ser Cys Asn Asp 260 265 270

Leu Cys Glu His Phe Cys Val Pro Asn Pro Asp Gin Pro Gly Ser Tyr 275 280 285

Ser Cys Met Cys Glu Thr Gly Tyr Arg Leu Ala Ala Asp Gin His Arg 290 295 300

Cys Glu Asp Val Asp Asp Cys He Leu Glu Pro Ser Pro Cys Pro Gin 305 310 315 320 Arg Cys Val Asn Thr Gin Gly Gly Phe Glu Cys His Cys Tyr Pro Asn

325 330 335

Tyr Asp Leu Val Asp Gly Glu Cys Val Glu Pro Val Asp Pro Cys Phe 340 345 350

Arg Ala Asn Cys Glu Tyr Gin Cys Gin Pro Leu Asn Gin Thr Ser Tyr 355 360 365

Leu Cys Val Cys Ala Glu Gly Phe Ala Pro He Pro His Glu Pro His 370 375 380

Arg Cys Gin Met Phe Cys Asn Gin Thr Ala Cys Pro Ala Asp Cys Asp 385 390 395 400 Pro Asn Thr Gin Ala Ser Cys Glu Cys Pro Glu Gly Tyr He Leu Asp

405 410 415

Asp Gly Phe He Cys Thr Xaa Xaa Xaa Xaa Cys Xaa Xaa Gly Gly Xaa 420 425 430

Cys Xaa Gly Xaa Cys Xaa Xaa Xaa Pro Gly Xaa Xaa Xaa Cys He Cys 435 440 445

Gly Pro Asp Ser Ala Leu Ala Xaa His Xaa Gly Thr Xaa Cys 450 455 460

SUBSTITUTE SHEET (2) INFORMATION FOR SEQ ID NO:16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 462 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(ix) FEATURE:

(A) NAME/KEY: Modified-site (B) LOCATION: 423..461

(D) OTHER INFORMATION: /note= "Xaa is the natural amino acid for that position as specified in SEQ ID NO 1 or an aliphatic amino acid of the group comprising Gly, Ala, Val, Leu and He".

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:

Ala Pro Ala Glu Pro Gin Pro Gly Gly Ser Gin Cys Val Glu His Asp 1 5 10 15

Cys Phe Ala Leu Tyr Pro Gly Pro Ala Thr Phe Leu Asn Ala Ser Gin 20 25 30

He Cys Asp Gly Leu Arg Gly His Leu Met Thr Val Arg Ser Ser Val 35 40 45

Ala Ala Asp Val He Ser Leu Leu Leu Asn Gly Asp Gly Gly Val Gly 50 55 60 Arg Arg Arg Leu Trp He Gly Leu Gin Leu Pro Pro Gly Cys Gly Asp 65 70 75 80

Pro Lys Arg Leu Gly Pro Leu Arg Gly Phe Gin Trp Val Thr Gly Asp 85 90 95

Asn Asn Thr Ser Tyr Ser Arg Trp Ala Arg Leu Asp Leu Asn Gly Ala 100 105 110

Pro Leu Cys Gly Pro Leu Cys Val Ala Val Ser Ala Ala Glu Ala Thr 115 120 125

Val Pro Ser Glu Pro He Trp Glu Glu Gin Gin Cys Glu Val Lys Ala 130 135 140 Asp Gly Phe Leu Cys Glu Phe His Phe Pro Ala Thr Cys Arg Pro Leu 145 150 155 160

Ala Val Glu Pro Gly Ala Ala Ala Ala Ala Val Ser He Thr Tyr Gly 165 170 175

Thr Pro Phe Ala Ala Arg Gly Ala Asp Phe Gin Ala Leu Pro Val Gly 180 185 190

Ser Ser Ala Ala Val Ala Pro Leu Gly Leu Gin Leu Met Cys Thr Ala 195 200 205

SUBSTITUTE SHEET

SUBSTITUTE SHEET (2) INFORMATION FOR SEQ ID NO:17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 462 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(ix) FEATURE:

(A) NAME/KEY: Modified-site (B) LOCATION: 349..359

(D) OTHER INFORMATION: /note= "Xaa is the natural amino acid for that position as specified in SEQ ID NO 1 or an aliphatic amino acid of the group comprising Gly, Ala, Val, Leu and He".

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:

Ala Pro Ala Glu Pro Gin Pro Gly Gly Ser Gin Cys Val Glu His Asp 1 5 10 15

Cys Phe Ala Leu Tyr Pro Gly Pro Ala Thr Phe Leu Asn Ala Ser Gin 20 25 30

He Cys Asp Gly Leu Arg Gly His Leu Met Thr Val Arg Ser Ser Val 35 40 45

Ala Ala Asp Val He Ser Leu Leu Leu Asn Gly Asp Gly Gly Val Gly 50 55 60 Arg Arg Arg Leu Trp He Gly Leu Gin Leu Pro Pro Gly Cys Gly Asp 65 70 75 80

Pro Lys Arg Leu Gly Pro Leu Arg Gly Phe Gin Trp Val Thr Gly Asp 85 90 95

Asn Asn Thr Ser Tyr Ser Arg Trp Ala Arg Leu Asp Leu Asn Gly Ala 100 105 110

Pro Leu Cys Gly Pro Leu Cys Val Ala Val Ser Ala Ala Glu Ala Thr 115 120 125

Val Pro Ser Glu Pro He Trp Glu Glu Gin Gin Cys Glu Val Lys Ala 130 135 140 Asp Gly Phe Leu Cys Glu Phe His Phe Pro Ala Thr Cys Arg Pro Leu 145 150 155 160

Ala Val Glu Pro Gly Ala Ala Ala Ala Ala Val Ser He Thr Tyr Gly 165 170 175

Thr Pro Phe Ala Ala Arg Gly Ala Asp Phe Gin Ala Leu Pro Val Gly 180 185 190

Ser Ser Ala Ala Val Ala Pro Leu Gly Leu Gin Leu Met Cys Thr Ala 195 200 205

SUBSTITUTE SHEET

SUBSTITUTE SHEET (2) INFORMATION FOR SEQ ID NO:18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 462 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(ix) FEATURE:

(A) NAME/KEY: Modified-site (B) LOCATION: 363..402

(D) OTHER INFORMATION: /note= "Xaa is the natural amino acid for that position as specified in SEQ ID NO 1 or an aliphatic amino acid of the group comprising Gly, Ala, Val, Leu and He".

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:

Ala Pro Ala Glu Pro Gin Pro Gly Gly Ser Gin Cys Val Glu His Asp 1 5 10 15

Cys Phe Ala Leu Tyr Pro Gly Pro Ala Thr Phe Leu Asn Ala Ser Gin 20 25 30

He Cys Asp Gly Leu Arg Gly His Leu Met Thr Val Arg Ser Ser Val 35 40 45

Ala Ala Asp Val He Ser Leu Leu Leu Asn Gly Asp Gly Gly Val Gly 50 55 60 Arg Arg Arg Leu Trp He Gly Leu Gin Leu Pro Pro Gly Cys Gly Asp 65 70 75 80

Pro Lys Arg Leu Gly Pro Leu Arg Gly Phe Gin Trp Val Thr Gly Asp 85 90 95

Asn Asn Thr Ser Tyr Ser Arg Trp Ala Arg Leu Asp Leu Asn Gly Ala 100 105 110

Pro Leu Cys Gly Pro Leu Cys Val Ala Val Ser Ala Ala Glu Ala Thr 115 120 125

Val Pro Ser Glu Pro He Trp Glu Glu Gin Gin Cys Glu Val Lys Ala 130 135 140 Asp Gly Phe Leu Cys Glu Phe His Phe Pro Ala Thr Cys Arg Pro Leu 145 150 155 160

Ala Val Glu Pro Gly Ala Ala Ala Ala Ala Val Ser He Thr Tyr Gly 165 170 175

Thr Pro Phe Ala Ala Arg Gly Ala Asp Phe Gin Ala Leu Pro Val Gly 180 185 190

Ser Ser Ala Ala Val Ala Pro Leu Gly Leu Gin Leu Met Cys Thr Ala 195 200 205

SUBSTITUTE SHEET

SUBSTITUTE SHEET (2) INFORMATION FOR SEQ ID NO:19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 462 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

( ix) FEATURE :

(A) NAME/KEY : Modif ied- site (B ) LOCATION : 408 . . 461

(D) OTHER INFORMATION : /note= "Xaa is the natural amino acid for that position as specified in SEQ ID NO 1 or an aliphatic amino acid of the group comprising Gly, Ala , Val , Leu and He " .

(xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 19 :

Ala Pro Ala Glu Pro Gin Pro Gly Gly Ser Gin Cys Val Glu His Asp 1 5 10 15

Cys Phe Ala Leu Tyr Pro Gly Pro Ala Thr Phe Leu Asn Ala Ser Gin 20 25 30

He Cys Asp Gly Leu Arg Gly His Leu Met Thr Val Arg Ser Ser Val 35 40 45

Ala Ala Asp Val He Ser Leu Leu Leu Asn Gly Asp Gly Gly Val Gly 50 55 60 Arg Arg Arg Leu Trp He Gly Leu Gin Leu Pro Pro Gly Cys Gly Asp

65 70 75 80

Pro Lys Arg Leu Gly Pro Leu Arg Gly Phe Gin Trp Val Thr Gly Asp 85 90 95

Asn Asn Thr Ser Tyr Ser Arg Trp Ala Arg Leu Asp Leu Asn Gly Ala 100 105 110

Pro Leu Cys Gly Pro Leu Cys Val Ala Val Ser Ala Ala Glu Ala Thr 115 120 125

Val Pro Ser Glu Pro He Trp Glu Glu Gin Gin Cys Glu Val Lys Ala 130 135 140 Asp Gly Phe Leu Cys Glu Phe His Phe Pro Ala Thr Cys Arg Pro Leu 145 150 155 160

Ala Val Glu Pro Gly Ala Ala Ala Ala Ala Val Ser He Thr Tyr Gly 165 170 175

Thr Pro Phe Ala Ala Arg Gly Ala Asp Phe Gin Ala Leu Pro Val Gly 180 185 190

Ser Ser Ala Ala Val Ala Pro Leu Gly Leu Gin Leu Met Cys Thr Ala 195 200 205

SUBSTITUTESHEET Pro Pro Gly Ala Val Gin Gly His Trp Ala Arg Glu Ala Pro Gly Ala 210 215 220

Trp Asp Cys Ser Val Glu Asn Gly Gly Cys Glu His Ala Cys Asn Ala 225 230 235 240 He Pro Gly Ala Pro Arg Cys Gin Cys Pro Ala Gly Ala Ala Leu Gin

245 250 255

Ala Asp Gly Arg Ser Cys Thr Ala Ser Ala Thr Gin Ser Cys Asn Asp 260 265 270

Leu Cys Glu His Phe Cys Val Pro Asn Pro Asp Gin Pro Gly Ser Tyr 275 280 285

Ser Cys Met Cys Glu Thr Gly Tyr Arg Leu Ala Ala Asp Gin His Arg 290 295 300

Cys Glu Asp Val Asp Asp Cys He Leu Glu Pro Ser Pro Cys Pro Gin 305 310 315 320 Arg Cys Val Asn Thr Gin Gly Gly Phe Glu Cys His Cys Tyr Pro Asn

325 330 335

Tyr Asp Leu Val Asp Gly Glu Cys Val Glu Pro Val Asp Pro Cys Phe 340 345 350

Arg Ala Asn Cys Glu Tyr Gin Cys Gin Pro Leu Asn Gin Thr Ser Tyr 355 360 365

Leu Cys Val Cys Ala Glu Gly Phe Ala Pro He Pro His Glu Pro His 370 375 380

Arg Cys Gin Met Phe Cys Asn Gin Thr Ala Cys Pro Ala Asp Cys Asp 385 390 395 400 Pro Asn Thr Gin Ala Ser Cys Xaa Cys Pro Glu Gly Xaa Xaa Xaa Xaa

405 410 415

Xaa Gly Phe Xaa Cys Thr Xaa Xaa Xaa Xaa Cys Xaa Xaa Gly Gly Xaa 420 425 430

Cys Xaa Gly Xaa Cys Xaa Xaa Xaa Pro Gly Xaa Xaa Xaa Cys He Cys 435 440 445

Gly Pro Asp Ser Ala Leu Ala Xaa His Xaa Gly Thr Xaa Cys 450 455 460

SUBSTITUTE SHEET (2) INFORMATION FOR SEQ ID NO:20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 462 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(ix) FEATURE:

(A) NAME/KEY: Modified-site (B) LOCATION: 408..461

(D) OTHER INFORMATION: /note= "Xaa is the natural amino acid for that position as specified in SEQ ID NO 1 or an aliphatic amino acid of the group comprising Gly, Ala, Val, Leu and He".

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:

Ala Pro Ala Glu Pro Gin Pro Gly Gly Ser Gin Cys Val Glu His Asp 1 5 10 15

Cys Phe Ala Leu Tyr Pro Gly Pro Ala Thr Phe Leu Asn Ala Ser Gin 20 25 30

He Cys Asp Gly Leu Arg Gly His Leu Met Thr Val Arg Ser Ser Val 35 40 45

Ala Ala Asp Val He Ser Leu Leu Leu Asn Gly Asp Gly Gly Val Gly 50 55 60 Arg Arg Arg Leu Trp He Gly Leu Gin Leu Pro Pro Gly Cys Gly Asp 65 70 75 80

Pro Lys Arg Leu Gly Pro Leu Arg Gly Phe Gin Trp Val Thr Gly Asp 85 90 95

Asn Asn Thr Ser Tyr Ser Arg Trp Ala Arg Leu Asp Leu Asn Gly Ala 100 105 110

Pro Leu Cys Gly Pro Leu Cys Val Ala Val Ser Ala Ala Glu Ala Thr 115 120 125

Val Pro Ser Glu Pro He Trp Glu Glu Gin Gin Cys Glu Val Lys Ala 130 135 140 Asp Gly Phe Leu Cys Glu Phe His Phe Pro Ala Thr Cys Arg Pro Leu 145 150 155 160

Ala Val Glu Pro Gly Ala Ala Ala Ala Ala Val Ser He Thr Tyr Gly 165 170 175

Thr Pro Phe Ala Ala Arg Gly Ala Asp Phe Gin Ala Leu Pro Val Gly 180 185 190

Ser Ser Ala Ala Val Ala Pro Leu Gly Leu Gin Leu Met Cys Thr Ala 195 200 205

SUBSTITUTE SHEET

SUBSTITUTE SHEET (2) INFORMATION FOR SEQ ID NO:21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: CCGGGATCCT CAACAGTCGG TGCCAATGTG GCG 33

SUBSTITUTE SHEET (2) INFORMATION FOR SEQ ID NO:22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: CCGGGATCCT GCAGCGTGGA GAACGGCGGC TGC 33

SUBSTITUTE SHEET (2) INFORMATION FOR SEQ ID NO:23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1674 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: misc_feature (B) LOCATION: 1..1674

(D) OTHER INFORMATION: /note= "Gene sequence does not include the first 54 bases coding for the 18 amino acid signal peptide. " (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:

GCACCCGCAG AGCCGCAGCC GGGTGGCAGC CAGTGCGTCG AGCACGACTG CTTCGCGCTC 60

TACCCGGGCC CCGCGACCTT CCTCAATGCC AGTCAGATCT GCGACGGACT GCGGGGCCAC 120

CTAATGACAG TGCGCTCCTC GGTGGCTGCC GATGTCATTT CCTTGCTACT GAACGGCGAC 180

GGCGGCGTTG GCCGCCGGCG CCTCTGGATC GGCCTGCAGC TGCCACCCGG CTGCGGCGAC 240 CCCAAGCGCC TCGGGCCCCT GCGCGGCTTC CAGTGGGTTA CGGGAGACAA CAACACCAGC 300

TATAGCAGGT GGGCACGGCT CGACCTCAAT GGGGCTCCCC TCTGCGGCCC GTTGTGCGTC 360

GCTGTCTCCG CTGCTGAGGC CACTGTGCCC AGCGAGCCGA TCTGGGAGGA GCAGCAGTGC 420

GAAGTGAAGG CCGATGGCTT CCTCTGCGAG TTCCACTTCC CAGCCACCTG CAGGCCACTG 480

GCTGTGGAGC CCGGCGCCGC GGCTGCCGCC GTCTCGATCA CCTACGGCAC CCCGTTCGCG 5 0 GCCCGCGGAG CGGACTTCCA GGCGCTGCCG GTGGGCAGCT CCGCCGCGGT GGCTCCCCTC 600

GGCTTACAGC TAATGTGCAC CGCGCCGCCC GGAGCGGTCC AGGGGCACTG GGCCAGGGAG 660

GCGCCGGGCG CTTGGGACTG CAGCGTGGAG AACGGCGGCT GCGAGCACGC GTGCAATGCG 720

ATCCCTGGGG CTCCCCGCTG CCAGTGCCCA GCCGGCGCCG CCCTGCAGGC AGACGGGCGC 780

TCCTGCACCG CATCCGCGAC GCAGTCCTGC AACGACCTCT GCGAGCACTT CTGCGTTCCC 840 AACCCCGACC AGCCGGGCTC CTACTCGTGC ATGTGCGAGA CCGGCTACCG GCTGGCGGCC 900

GACCAACACC GGTGCGAGGA CGTGGATGAC TGCATACTGG AGCCCAGTCC GTGTCCGCAG 960

CGCTGTGTCA ACACACAGGG TGGCTTCGAG TGCCACTGCT ACCCTAACTA CGACCTGGTG 1020

GACGGCGAGT GTGTGGAGCC CGTGGACCCG TGCTTCAGAG CCAACTGCGA GTACCAGTGC 1080

CAGCCCCTGA ACCAAACTAG CTACCTCTGC GTCTGCGCCG AGGGCTTCGC GCCCATTCCC 1140 CACGAGCCGC ACAGGTGCCA GATGTTTTGC AACCAGACTG CCTGTCCAGC CGACTGCGAC 1200

CCCAACACCC AGGCTAGCTG TGAGTGCCCT GAAGGCTACA TCCTGGACGA CGGTTTCATC 1260

TGCACGGACA TCGACGAGTG CGAAAACGGC GGCTTCTGCT CCGGGGTGTG CCACAACCTC 1320

CCCGGTACCT TCGAGTGCAT CTGCGGGCCC GACTCGGCCC TTGCCCGCCA CATTGGCACC 1380

SUBSTITUTE SHEET GACTGTGACT CCGGCAAGGT GGACGGTGGC GACAGCGGCT CTGGCGAGCC CCCGCCCAGC 1440

CCGACGCCCG GCTCCACCTT GACTCCTCCG GCCGTGGGGC TCGTGCATTC GGGCTTGCTC 1500

ATAGGCATCT CCATCGCGAG CCTGTGCCTG GTGGTGGCGC TTTTGGCGCT CCTCTGCCAC 1560

CTGCGCAAGA AGCAGGGCGC CGCCAGGGCC AAGATGGAGT ACAAGTGCGC GGCCCCTTCC 1620

AAGGAGGTAG TGCTGCAGCA CGTGCGGACC GAGCGGACGC CGCAGAGACT CTGA 1674

SUBSTITUTE SHEET

Claims

WHAT IS CLAIMED IS:

1. A thrombomodulin analog which induces a modified cofactor activity upon binding to thrombin, as compared to TM_E having natural sequence, said analog having an amino acid modification at a position corresponding to natural sequence at (SEQ ID NO 2) : a) 336 (asparagine) ; b) 337 (tyrosine) ; c) 340 (valine) ; d) 341 (aspartic acid) ; e) 365 (glutamic acid) ; f) 369 (leucine) ; h) 447 (isoleucine) ; or i) 454 (leucine) .

2. An analog of Claim 1, wherein said modified cofactor activity is at least 25% different compared to said TM_E.

3. An analog of Claim 1 having oxidation resistance conferred through modification of the methionine at position 388 (SEQ ID NO. 3) .

4. An analog of Claim 3, wherein said analog has modifications at both positions 365 and 369. (SEQ ID NO 3) .

5. An analog of Claim 4, wherein said modifications at positions 365 and 369 include at least one substitution with an aliphatic amino acid. (SEQ ID NO 3) .

6. An analog of Claim 1, wherein at least one of said substitutions is with an aliphatic amino acid.

7. An analog of Claim 6, wherein said aliphatic amino acid is glycine, alanine, valine, leucine, or isoleucine.

SUBSTITUTE SHEET

8. An analog of Claim 1, which is a soluble analog.

9. An analog of Claim 8, wherein said soluble analog comprises at least one structural domain is EGF4, EGF5 and EGF 6.

10. An analog of Claim 9, further comprising a domain selected from: a) the lectin domain; b) EGF domains 1, 2, or 3; or c) the O-linked glycosylation domain.

11. An analog of Claim 1, further comprising modifications at positions 456 or 457 which confer protease resistance to said analog. (SEQ ID NO 4) .

12. An analog of Claim 11, wherein one of said modifications is 456gly or 457gln. (SEQ ID NO 5) .

13. An analog of Claim 1, having substitutions at positions 365, 369, 388, 456, and 457. (SEQ ID NO 6).

14. An analog of Claim 13, further comprising a terminal sequence which provides a homogeneous amino or carboxy terminus.

15. An analog of Claim 14, wherein said terminal sequence is GPQP at the amino terminus, or LTPP at the carboxy terminus. (SEQ ID NO 7) .

16. An analog of Claim 13, wherein at least one amino acid substitution at 365 or 369 is with an aliphatic amino acid. (SEQ ID NO 3).

17. An analog of Claim 16, wherein said aliphatic amino acid is glycine, alanine, valine, leucine, or isoleucine.

SUBSTITUTE SHEET

18. A nucleic acid encoding an analog of Claim 1.

19. A nucleic acid encoding an analog of Claim 8.

20. A thrombomodulin analog which, upon binding to thrombin, induces a modified cofactor activity as compared to binding with TM_EM388L, said analog having an amino acid substitution at one or more positions corresponding to (SEQ ID NO 8) : spartic acid) ; sparagine) ; lutamic acid) ; yrosine) ; lutamine) ; eucine) ; aline) ; lutamic acid) ; henylalanine) ; istidine) ; rginine) ; lutamine) ; henylalanine) ; spartic acid) ; spartic acid) ; sparagine) ; hreonine) ; lutamic acid) ; yrosine) ; soleucine) ; eucine) ; spartic acid) ; spartic acid) ; isoleucine) ; aspartic acid) ; isoleucine) ; aspartic acid) ; glutamic acid) ;

glutamic acid) ;

SUBSTITUTE SHEET

21. An analog of Claim 20, wherein said modified cofactor activity is lower than that of TM_EM388L.

22. An analog of Claim 20, having a modified Kj or binding thrombin, a modified k_cat/K_n, or both.

23. An analog of Claim 20, wherein said position corresponds to (SEQ ID NO 9) :

SUBSTITUTE SHEET cb) 424 (isoleucine) ; cc) 425 (aspartic acid) ; cd) 426 (glutamic acid) ; cf) 429 (asparagine) ; ck) 439 (asparagine) ; en) 444 (phenylalanine) ; or cr) 461 (aspartic acid) .

24. An analog of Claim 23, further comprising modifications at positions 456 or 457 which confer protease resistance to said analog. (SEQ ID NO 4) .

25. An analog of Claim 24, wherein one of said modifications is 456gly or 457gln. (SEQ ID NO 5) .

26. An analog of Claim 23, having substitutions at positions 365, 369, 388, 456, and 457. (SEQ ID NO 6) .

27. An analog of Claim 26, further comprising a terminal sequence which provides a homogeneous amino or carboxy terminus.

28. An analog of Claim 27, wherein said terminal sequence is GPQP at the amino terminus, or LTPP at the carboxy terminus. (SEQ ID NO 7).

29. An analog of Claim 26, wherein at least one amino acid substitution at 365 or 369 is with an aliphatic amino acid. (SEQ ID NO 3) .

30. An analog of Claim 29, wherein said aliphatic amino acid is glycine, alanine, valine, leucine, or isoleucine.

31. An analog of Claim 23, wherein said modified activity is changed by at least 30%.

SUBSTITUTE SHEET

32. An analog of Claim 20, wherein said position corresponds to (SEQ ID NO 10) : aa) 349 (aspartic acid) ; ac) 357 (glutamic acid) ; ad) 358 (tyrosine) ; ae) 359 (glutamine) ; ag) 371 (valine) ; or ai) 376 (phenylalanine) .

33. An analog of Claim 32 having an essentially unmodified K_d compared to TM_EM388L.

or

35. An analog of Claim 34 having an essentially unmodified l-_c__t/^~~__> upon binding to thrombin compared to

TM_EM388L.

36 . A nucleic acid encoding an analog of Claim 32.

SUBSTITUTE SHEET

37. A nucleic acid encoding an analog of Claim 34.

38. An analog of Claim 20, wherein said position corresponds to (SEQ ID NO 12) : aa) 349 (aspartic acid) ; ab) 355 (asparagine) ; ac) 357 (glutamic acid) ; ad) 358 (tyrosine) ; ae) 359 (glutamine) ; af) 363 (leucine) ; ag) 371 (valine) ; ah) 374 (glutamic acid) ; ai) 376 (phenylalanine) ; aj) 384 (histidine); or ak) 385 (arginine) .

39. An analog of Claim 38, wherein said analog has an essentially unmodified K_d compared to TM_EM388L.

40. An analog of Claim 38, wherein said position corresponds to (SEQ ID NO 10) : aa) 349 (aspartic acid) ; ac) 357 (glutamic acid) ; ad) 358 (tyrosine) ; ae) 359 (glutamine) ; ag) 371 (valine) ; or ai) 376 (phenylalanine) .

41. A. nucleic acid encoding an analog of Claim 40.

42. An analog of Claim 38, wherein said analog has an essentially unmodified K_<ι compared to TM_EM388L.

43. An analog of Claim 20, wherein said position corresponds to (SEQ ID NO 13) : be) 398 (aspartic acid) ; bd) 400 (aspartic acid) ; be) 402 (asparagine) ;

SUBSTITUTE SHEET bf) 403 (threonine) ; bg) 408 (glutamic acid) ; bh) 413 (tyrosine) ; bi) 414 (isoleucine) ; bj) 415 (leucine); bk) 416 (aspartic acid) ; bl) 417 (aspartic acid) ; or bm) 420 (isoleucine) .

44. An analog of Claim 43, wherein said analog has an essentially unmodified k_cat/K,„ compared to TM_EM388L.

45. An analog of Claim 43, wherein said position corresponds to (SEQ ID NO 14) : be) 398 (aspartic acid) ; bd) 400 (aspartic acid) ; be) 402 (asparagine) ; bg) 408 (glutamic acid) ; bh) 413 (tyrosine) ; bi) 414 (isoleucine) ; bj) 415 (leucine); bk) 416 (aspartic acid) ; or bl) 417 (aspartic acid) .

46. An analog of Claim 45, wherein said analog has an essentially unmodified _._CΛt/Y--_ compared to TM_EM388L.

47. A nucleic acid encoding an analog of Claim 45.

48. An analog of Claim 20, wherein said position corresponds to (SEQ ID NO 15) : ca) 423 (aspartic acid) ; cb) 424 (isoleucine) ; cc) 425 (aspartic acid) cd) 426 (glutamic acid) ce) 428 (glutamic acid) cf) 429 (asparagine) ; eg) 432 (phenylalanine) ;

SUBSTITUTE SHEET ch) 434 (serine) ; ci) 436 (valine) ; cj) 438 (histidine); ck) 439 (asparagine) ; cl) 440 (leucine) ; cm) 443 (threonine) ; en) 444 (phenylalanine) ; co) 445 (glutamic acid) ; cp) 456 (arginine) ; cq) 458 (isoleucine) ; or cr) 461 (aspartic acid) .

49. An analog of Claim 48, wherein said analog has an essentially unmodified ^ K_^, compared to TM_EM388L.

50. An analog of Claim 48, wherein said position corresponds to (SEQ ID NO 16) : ca) 423 (aspartic acid) ; cb) 424 (isoleucine) ; cc) 425 (aspartic acid) ; cd) 426 (glutamic acid) ; cf) 429 (asparagine) ; ck) 439 (asparagine) ; cl) 440 (leucine) ; en) 444 (phenylalanine) ; or cr) 461 (aspartic acid) .

51. A nucleic acid encoding an analog of Claim 50.

52. An analog of Claim 50, wherein said analog has an essentially unmodified k_eat/K„ compared to TM_EM388L.

53. An analog of Claim 20, having substitutions at positions 365, 369, 388, 456, and 457. (SEQ ID NO 6).

54. An analog of Claim 53, further comprising a terminal sequence which provides a homogeneous amino or carboxy terminus.

SUBSTITUTE SHEET

55. A thrombomodulin analog possessing a modified amino acid at a position, wherein said analog has essentially equivalent K_d for thrombin compared to an analog having at said position the native residue, wherein said position corresponds to (SEQ ID NO 17) : aa) 349 (aspartic acid) ; ab) 355 (asparagine) ; ac) 357 (glutamic acid) ; ad) 358 (tyrosine) ; or ae) 359 (glutamine) .

56. An analog of Claim 55, having a modified ^~.__._Λt/'K-. of at least 30%.

57. A thrombomodulin analog possessing a modified amino acid at a position, wherein said analog has a modified K_d or k_cat/K,,, compared to an analog having at said position the native residue, wherein said position corresponds to (SEQ ID NO 18) : af) 363 (leucine) ; ag) 371 (valine) ; ah) 374 (glutamic acid) ; ai) 376 (phenylalanine) ; aj) 384 (histidine); ak) 385 (arginine) ; be) 398 (aspartic acid) ; bd) 400 (aspartic acid) ; or be) 402 (asparagine) .

58. An analog of Claim 57, having both a modified K_- and k_cat/K„.

59. An analog of Claim 57, wherein said modified K_d or ]._CΛt/Km, is modified by at least 20%.

60. An analog of Claim 57, wherein both modified K_d and _ _ca /_K_m are modified by at least 20%.

SUBSTITUTE SHEET

61. A thrombomodulin analog which upon binding to thrombin exhibits essentially equivalent k^/K_m compared to TM_EM388L, and wherein said analog has a modification at a position corresponding to (SEQ ID NO 19) : lutamic acid) ; yrosine) ; soleucine) ; eucine) ; spartic acid) ; spartic acid) ; soleucine) ; spartic acid) ; soleucine) ; spartic acid) ; lutamic acid) ; lutamic acid) ; sparagine) ; henylalanine) ; serine) ; aline) ; istidine) ; sparagine) ; leucine) ; hreonine) ; henylalanine) ; lutamic acid) ; arginine) ; isoleucine) ; or

aspartic acid) .

62. An analog of Claim 61, wherein said position corresponds to (SEQ ID NO 20) : bg) 408 (glutamic acid) ; bh) 413 (tyrosine) ; bi) 414 (isoleucine) ; bj) 415 (leucine); bk) 416 (aspartic acid) ; bl) 417 (aspartic acid) ;

SUBSTITUTE SHEET ca) 423 (aspartic acid) cb) 424 (isoleucine) ; cc) 425 (aspartic acid) cd) 426 (glutamic acid) cf) 429 (asparagine) ; ck) 439 (asparagine) ; en) 444 (phenylalanine) or cr) 461 (aspartic acid)

63. An analog of Claim 61, wherein the K_d for thrombin is modified by at least 30%.

64. An analog of Claim 61, wherein said modification is an amino acid substitution.

65. A method useful for screening for analogs of thrombomodulin which exhibit a modified K,ι for thrombin binding, comprising the steps of: a) making an amino acid substitution at a position (SEQ ID NO 19) : lutamic acid) ; tyrosine) ; isoleucine) ; leucine) ; spartic acid) ; spartic acid) ; isoleucine) ; spartic acid) ; isoleucine) ; spartic acid) ; lutamic acid) ; lutamic acid) ; asparagine) ; henylalanine) ; serine) ; aline) ; istidine) ;

sparagine) ;

SUBSTITUTE SHEET cl) 440 (leucine) ; cm) 443 (threonine) ; en) 444 (phenylalanine) ; co) 445 (glutamic acid) ; cp) 456 (arginine) ; cq) 458 (isoleucine) ; or cr) 461 (aspartic acid) ; and b) comparing the K_d for thrombin to a control molecule.

66. A method of Claim 65, wherein said K_d is modified by at least 33%.

67. A method of claim 65, wherein said modification is an amino acid substitution.

68. A method of Claim 65, wherein said control molecule is TM_EM388L.

69. A method of Claim 65, wherein said position corresponds to (SEQ ID NO 20) : bg) 408 (glutamic acid) ; bh) 413 (tyrosine) ; bi) 414 (isoleucine) ; bj) 415 (leucine); bk) 416 (aspartic acid) ; bl) 417 (aspartic acid) ; ca) 423 (aspartic acid) ; cb) 424 (isoleucine) ; cc) 425 (aspartic acid) ; cd) 426 (glutamic acid) ; cf) 429 (asparagine) ; ck) 439 (asparagine) ; en) 444 (phenylalanine) ; or cr) 461 (aspartic acid) .

SUBSTITUTE SHEET

70. A method useful for screening analogs of thrombomodulin which induce a modified cofactor activity upon binding to thrombin, comprising the steps of: a) making an amino acid modification at a position (SEQ ID NO 17) : aa) 349 (aspartic acid) ; ab) 355 (asparagine) ; ac) 357 (glutamic acid) ; ad) 358 (tyrosine) ; or ae) 359 (glutamine) ; and b) comparing the range of cofactor activity upon binding to thrombin with the rate of a control molecule.

71. A method of Claim 70, wherein said ._CΛt/K-- is modified by at least 33%.

72. A method of Claim 70, wherein said modification is an amino acid substitution.

73. A method of Claim 70, wherein said control is TM_RM388L.

SUBSTITUTESHEET