MXPA98002722A - Novedosa plate activation protein - Google Patents

Abstract

Substantially pure platelet activation polypeptide including a sequence of at least 70% identical to SEQ IN No: 1 and a DNA encoding said polypeptide

Description

NOVEDOSA PROTEIN PLATELET ACTIVATION CROSS REFERENCE TO RELATED APPLICATIONS This application claims priority from the application of US Patent Serial No. 60 / 005,074, filed on October 6, 1995. A DECLARATION REGARDING THE SEARCH SUPPORTED WITH FEDERAL FUNDS NORTH AMERICANS This invention was made with government support under concession No. HL-02348 granted by the National Institutes of Health of North America.

BACKGROUND OF THE INVENTION The invention relates to platelet activation proteins. The normal hemostatic system regulates bleeding and thrombosis through a series of complex interactions or between the components of the blood basal wall, circulating blood platelets, and plasma proteins. Because vascular damage causes rapid loss of the protein, fluid and cellular components of the blood, animals have developed rapid responses to fix the spleen and start repairing it. These rapid responses are initiated by the platelet, a highly specialized cell that reacts to vascular damage. Normally, platelets circulate in the blood as immobile and nonadherent cells, inspecting the integrity of the blood spleen. In response to vascular damage, platelets adhere to the desendothelialized areas, and become activated. The activation of platelets induces deep morphological and functional changes in the cell. Platelets change shape, aggregate with other platelets, and stick to other cells. Upon complete activation, platelets secrete the contents of their lisomals, alpha, and dense granules, thus expressing addition molecules, growth factors, coagulation enzymes, and other specialized molecules. The molecules expressed by the activated platelets execute many of the complex cellular and biochemical processes that stop the loss of blood and begin the process of vascular repair. The cellular and biochemical processes initiated by platelets in response to vascular damage can be salvage, but in the absence of this damage these same processes can become harmful. For example, unregulated arterial platelet thrombosis can clog the blood supply to the organs and cause heart attacks, and limb necrosis.

SUMMARY OF THE INVENTION We have now discovered a new polypeptide, designated activated platelet protein-2 (APP-2), which is expressed preferentially in cultured human platelets, but not in platelets at rest. The invention includes a substantially pure DNA encoding a platelet activation polypeptide, having a molecular weight of 25 kilodaltons (kDa). Under non-reducing conditions, it can be found naturally in covalent association with two other proteins in the 145 kDa complex. The 25 kDa polypeptide as expressed in human platelets contains at least 2 putative phosphorylation sites. The protein of the invention can be characterized as containing epitope that binds to the monoclonal antibody (MAb) 3B2. Preferably, the encoded polypeptide is human APP-2, which includes at least 95% of the amino acid sequence of SEQ ID NO: 4, (eg, the protein encoded by SEQ ID NO: 1). A preferred example of this DNA will contain the nucleotide sequence of SEQ ID NO: 3, or any degenerate variant of SEQ ID NO: 3.

More preferably, the DNA includes the nucleotide sequence of SEQ ID NO: 2, or any degenerate variant of SEQ ID NO: 2. Substantially pure DNA containing a strand of at least 12 nucleotides, eg, a probe Hybridization of at least 20 nucleotides, 50 nucleotides, 100 nucleotides or more, which hybridizes at high stringency to a DNA having the sequence of SEQ ID NO: 2, or the complement thereof, is also within the invention. The expression of APP-2 in a cell can be detected by (a) contacting the mRNA obtained from the cell with a labeled hybridization probe comprising, for example, a single strand segment of the isolated DNA encoding a fragment of APP-2; and (b) detect hybridization of the shadow with the mRNA. By "high severity" is meant the following conditions of hybridization and DNA washing: hybridization to 42 ° C in the presence of 50% formamide; a first wash at 65 ° C with 2 x SSC containing 1% SDS; followed by a second wash 65 ° C with 0.1 x SSC. By "substantially pure DNA" is meant DNA that is gene free, which, in the naturally occurring genome of the organism from which the DNA of the invention is derived, flanks the DNA sequence of interest. Thus, the term includes, for example, a recombinant DNA that is incorporated into a vector, a plasmid virus that replicates autonomously, or a genomic DNA from a prokaryotic or eukaryotic cell; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by the polymerase chain reaction (PCR) or restriction endunoclease digestion) independent of other sequences . It also includes a recombinant DNA that (a) is part of a hybrid gene encoding the additional polypeptide sequence, eg, a fusion protein, or (b) has a sequence that is not a nucleotide sequence that is presented natural way (for example, a degenerate variant of a natural sequence, or a sequence that contains mutations that do not occur naturally). Also included is a recombinant DNA which includes a portion of SEQ ID NO: 2, and which codes for an alternative binding variant of APP-2, eg, a polypeptide, the amino terminus of which differs from the amino terminus of the SEQ. ID NO: 1. The DNA must have at least about 50% identity to the termination sequence of SEQ ID NO: 1 or 3, and preferably at least 70% (eg, 80%, 90%, or 95%). %). The identity between the two nucleic acid sequences or polypeptides is a function of the number of equalization or identical positions. For example, when a subunit position in both of the two sequences is occupied by the same monomeric subunit, for example, if a position in each of the two DNA molecules is occupied by an adenine, then they are identical in that position. For example, if the half, for example, 5 positions in a sequence of 10 nucleotides in length, are identical, then the sequences have 50% sequence identity. The length of the comparison sequences will generally be at least 50 nucleotides, preferably at least 60 nucleotides, preferably at least 75 nucleotides, and most preferably 100 nucleotides. Sequence identity is typically measured using sequence analysis computer programs (eg, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wl 53705). For purposes of calculating% sequence identity, separations are considered to be unequal. The invention also includes a vector that contains a DNA encoding a polypeptide that includes the amino acid sequence of SEQ ID NO: 1, for example, a construct in which the coding sequence is operably linked to a promoter or other regulatory sequences for the expression of the polypeptide, a cell that contains this vector. The cell can be prokaryotic or eukaryotic (e.g., a mammalian cell such as a human cell) and preferably expresses the recombinant polypeptide encoded by SEQ ID NO: 2. The invention also includes a substantially pure platelet activation polypeptide as described above. By "platelet activation polypeptide" is meant a polypeptide having the amino acid sequence of a protein that is preferentially expressed naturally by activated platelets as compared to platelets at rest of an animal. Preferably, the animal is invertebrate, for example, a mammal such as a primate, including a human; alternatively the mammal is a rat, mouse, rabbit, guinea, hamster, cow, pig, horse, goat, sheep, dog, or cat. Preferably, the polypeptide contains the amino acid sequence of human APP-2 (SEQ ID NO: 1), for example, in the form of a Flag-APP-2 fusion protein. By "polypeptide" is meant any chain of amino acids, without considering post-transductional length or modification (eg, glycosylation or phosphorylation).

The amino acid sequence of the polypeptide differs only from SEQ ID NO: 1 by conservative amino acid substitutions, for example, the substitution of one amino acid for another of the same class (eg, valine for glycine, arginine for lysine, etc.). ) or by one or more non-conservative substitutions, deletions, or insertions located at positions in the amino acid sequence that do not destroy the function of the protein (for example, its Mab 3B2 binding or its covalent association in the 145 kDa complex). Preferably, the amino acid sequence of the platelet activation polypeptide is at least 50%, more preferably 70%, in the even more preferred 85% or 90%, most preferably 95% identical to SEQ ID NO: 1. By a "substantially pure polypeptide" is meant a polypeptide that has been separated from the components that naturally accompany it. Typically, the polypeptide is substantially pure when it is at least 60%, by weight, free of proteins and other organic molecules that occur naturally, with which it is naturally associated. Preferably, the purity of the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight. A substantially pure APP-2 polypeptide can be obtained, for example, by extraction from a natural source (e.g., mammalian platelets); by the expression of a recombinant nucleic acid encoding an APP-2 polypeptide, in cells or in a cell-free system; or by the chemical synthesis of the protein. Purity can be measured by any suitable method, for example, column chromatography such as immunoaffinity chromatography using Mab 3B2, polyacrylamide gel electrophoresis, or HPLC analysis. A protein is substantially free of the naturally associated compounds when it is separated from those contaminants that accompany it in its natural state. In this way, a protein that is either chemically synthesized or produced in a cellular system different from the cell from which it originates naturally, will be substantially free of its naturally associated components. Accordingly, substantially pure polypeptides include those derived from eukaryotic organisms, but synthesized in E. coli or other eukaryotic cells. In addition to polypeptides of substantially complete length, the invention also includes fragments of these polypeptides. As used herein, "fragments" when a polypeptide is applied, will ordinarily be at least 10 residues, more typically at least 20 residues, and preferably at least 60 residues in length. Fragments of the APP-2 polypeptide can be generated by methods known to those skilled in the art. The ability of a candidate fragment to exhibit a characteristic of APP-2 (e.g., Mab 3B2 binding or any other anti-APP-2 antibody) can be assessed by those methods described herein. Also included in the invention are the APP-2 polypeptides which are encoded by the portions of SEQ ID NO: 2, for example, products of alternative mRNA binding or alternative protein processing cases, or in which a Section of the APP-2 sequence, such as the transmembrane domain and / or the intracellular domain, have been deleted. The transmembrane domain sequence is routinely mastered by identifying a segment consisting of predominantly hydrophobic residues characteristic of a transmembrane domain. The invention also includes a polypeptide that includes at least 20 amino acids of APP-2. Preferably, the polypeptide includes at least 50%, more preferably at least 100%, more preferably at least 200, and most preferably at least 300 amino acids of APP-2. Preferentially, the polypeptide is an antigenic fragment of APP-2 or a soluble fragment of APP-2 lacking the transmembrane membrane of APP-2. APP-2 has been found in covalent association with a complex (APCOM) that migrates at an apparent molecular weight of 145 kDa under non-reducing conditions. This complex contains at least two proteins in addition to APP-2. One of approximately 45 kD and another of approximately 15 kD. These proteins and APCOM are all within the invention, by virtue of their characteristic association with activated platelets, as opposed to deactivated platelets, APCOM and its constituent proteins can be used to generate antibodies (such as MAb 3B2) of diagnosis for activated platelets and for thrombi. The invention also includes a polyclonal or monoclonal antibody that specifically binds to the platelet activation polypeptide of the invention. Preferably, the antibody is Mab 3B2 or binds to the same epitope as MAb 3B2. The invention encompasses not only an intact monoclonal antibody, but also an immunologically active antibody fragment, eg, a Fab or (Fab) 2 fragment.; an individual chain Fv molecule, engineered, or a chimeric molecule, for example, an antibody that contains the binding specificity of an antibody, for example, of murine origin, and the remaining portions of another antibody, for example, of human origin. In preferred embodiments, the antibody can be linked to a detectable label, for example, a radioactive label, a fluorescent label, a magnetic mark, or a colorimetric label. Also within the invention is a method of detecting an activated platelet in a biological sample, which includes the steps of contacting the sample with the labeled antibody, for example, radiolabelled MAb 3B2, and determining whether the antibody binds to a component of the sample. The binding of the antibody indicates that the sample contains an APP.-2 polypeptide, and consequently, an activated platelet. The labeled antibody can also be used in diagnostic form. For example, a platelet thrombus can be located in an animal, for example, a human patient suspected of having undesirable blood clots, by administering to the animal the labeled antibody, e.g., MAb 3B2, and determining where it is located in the animal marks it. Detection of the tag at a given site on the animal indicates the existence of a platelet thrombus at that site.

The antibody of the invention can also be used therapeutically, for example, in a method for selecting a compound to an activated platelet in an animal, which includes the steps of administering to an animal a composition containing the compound bound to an antibody. anti-APP-2, for example, MAb 3B2. Preferably, the compound is a thrombolytic agent such as urokinase, prourokinase, streptokinase, tissue-type plasminogen activator, staphylokinase, or bat tissue plasminogen activator, to dissolve the thrombi; an anti-thrombotic agent such as heparin, hirudin, or factor Xa or factor 5a inhibitors, to inhibit thrombus formation; an anti-proliferative agent such as inhibitors of platelet-derived growth factor or heparin-binding growth factor to inhibit cell proliferation, eg, proliferation of the smooth muscle cell, in a thrombus site, or a anti-migration agent such as inhibitors of migration of smooth muscle cells, an antibody or other specific inhibitor of the function of urokinase or integrin, to prevent or inhibit the migration of cells that contribute to the blockage of a blood spleen, in a thrombus site.

The therapeutic agents can be linked to an anti-APP-2 MAb, for example, MAb 3B2, using a covalent bond, such as a disulfide bond or a covalent crosslinking agent. The MAb and the therapeutic agent can also be reproduced recombinantly, with the two components of the compound linked by a peptide bond. Other characteristics and advantages of the invention will become apparent from the following detailed description and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS Figure la is a photomicrograph showing the results of an immunofluorescence labeling experiment in which activated platelets were incubated with a control antibody, anti-digoxin. Figure Ib is a photomicrograph showing the results of an immunofluorescence labeling experiment in which platelets activated with MAb 3B2 were incubated. Figure 2 is a bar graph showing a comparison of APP-2 expression before or after cell activation as detected by MAb 3B2 and the expression of p-selectin (a platelet activation molecule selectively expressed in the activated platelets) before and after cell activation as detected by an anti-p-selectin antibody. Figure 3 is a photomicrograph showing the immunodetection of APP-2 in a human thrombus using MAb 3B2. Figure 4 is a representation of the nucleotide sequence of SEQ ID NO: 1. Figure 5 is a representation of the amino acid sequence of SEQ ID NO: 2. Figure 6 is a representation of the nucleotide sequence of SEQ ID NO: 3. Figure 7 is a representation of the amino acid sequence of SEQ ID NO: 4.

DETAILED DESCRIPTION CHARACTERIZATION OF APP-2 APP-2 was initially identified and isolated using two-dimensional gel electro-focusing. APP-2 migrates at an apparent molecular weight of 145 and 25 kDa under non-reduced and reduced conditions, respectively. APP-2 can be detected on the surface of activated platelets by virtue of their binding to MAb 3B2. Platelet binding studies showed that anti-APP-2 binds preferentially to activated platelets and that this binding is saturable. The Scatchard analysis indicates an individual class of binding site with a binding constant of 4.19 nM and the presence of approximately 5000 binding sites per platelet. Studies of the association of APP-2 with the platelet membrane suggest that APP-2 is an integral membrane protein. The cloning of APP-2 revealed that APP-2 is a new protein since its sequence does not share homology with any known protein sequence. The following experimental procedures were used to clone and characterize human APP-2.

PREPARATION AND BIKTINILATION OF PLATELETS Obsolete units of human plasma rich in platelets were collected from blood banks. The platelets were isolated by differential centrifugation and then washed twice using a solution of 1 mM EDTA in buffer A (3 mM Hepes, 5.5 M glucose, 137 mM NaCl, 2.7 mM KCl, 3 mM NaH2P04). The platelets were divided into two different portions and counted. The portion to be activated was washed in buffer A and a concentration of 1010 cells / ml was again dispersed in buffer B (buffer A with 2 M CaCl 2 and 1 mM MgCl 2). The platelets were activated by adding bovine thrombin (2 U / ml, Parke-Davis, Morris Plains, NJ) and allowed to incubate at 37 ° C for 30 minutes. The second portion (at rest) of the platelets was incubated in parallel in the absence of thrombin reel. After centrifugation (2500 rpm, 20 minutes) the supernatant was collected and cooled to -80 ° C. The platelet pellet was washed twice in buffer A. After washing, the platelets were dispersed in buffer A. For labeling, fresh NHS-LC-Biotin (Pierce, IL, 40 mg / ml) was added to the platelets ( at rest or activated) at a concentration of 10 μg / ml redispersed platelets and allowed to rotate for 2 hours at 21 ° C. After labeling, the platelets were centrifuged, washed twice in buffer A, redispersed in 20 ml of buffer A, and stored at -80 ° C.

ELECTROMYPHORESIS IN BIDIMENSIQNAL GEL (2 ~ P) PREPARATIVE For 2-D electrophoresis, activated, biotinylated resting platelets (prepared from 20 units of obsolete plasma as described above) were diluted in the sample buffer (Tris-Base 0.125 M, 20% glycerol, 2% sodium dodecyl sulfate (SDS)) at a concentration of 1: 1 (v / v), boiled for 5 minutes, and centrifuged (3000 rpm, 30 minutes). The proteins and the supernatant were precipitated with acetone (1: 4 v / v) at 21 ° C for 10 minutes and centrifuged (14,000 rpm, 20 minutes). The pellets were then redispersed in 10 m of electro-focusing buffer [9.9 M urea, 4% NP-40, 2.2% ampholytes (BIO-RAD Laboratories, Hercules, CA, pH 3-10), 100 mM diotothreitol]. The preparative isoelectric focusing was performed on a Rotofor® electro-focusing device (BIO-RAD Laboratories). The pre-focusing was performed with 45 ml of electro-focusing buffer for 1 hour at 6 W. The mixture was added and the electro-focusing was carried out for 4 h at 12 W. Fractions were collected in 65 x 12 mm plastic test tubes. The pH in each tube was measured to determine the pH gradient generated during the experiment and to facilitate comparison of platelet fractions at rest and activated platelets. Under the conditions described, the isoelectric focusing reproducibly created a pH gradient of 3-10. Isoelectric fractions with similar pH from resting and activated platelets were precipitated with acetone and separated on 12% SDS-polyacrylamide gels. The proteins were electrotransferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA) and blocked with dry milk without 5% fat for 1 hour. More biotinilide proteins were detected when incubated with estrapvidin-alkaline phosphatase (Pierce, Rockford, IL, 1 μg / ml) were diluted in 0.1% BSA in alkaline phosphatase buffer (0.1 M NaCl, 0.1 M Tris-Base, 0.005 M MgCl2 , pH 9.5) for 30 minutes. After washing, the membranes were washed three times in 0.1% Tween-20 (Sigma, St. Louis, MO) in alkaline phosphatase buffer, the tetrazolium transfers of nitro-blue (330 μg / ml) and phosphate were revealed of 5-bromo-4-chloro-3-indolyl (165 μg / ml) in alkaline phosphatase buffer. Transfers from resting and activated platelets were compared to identify protein bands expressed only by activated platelets. After the identification of a protein band of interest, which separated the samples in duplicate by SDS-PAGE. A portion of the gel with a set of samples was stained with a reversible negative stain (Diversified Biotech, MA) while routine inspection was performed on the duplicate set of samples as described. The negatively stained spots matching the biotinylated protein bands were cut from the gel and electroeluted (25 mM Tris-base, 192 mM glycine, 0.1% SDS) overnight at 4 ° C, 10 mAmp / tube. The electrolyzed proteins were precipitated with acetone and redispersed in phosphate buffered saline (PBS). The concentration of the protein sample was determined by staining with Coomassie blue and staining with plaque.

PRODUCTION AND PURIFICATION OF MONOCLONAL ANTIBODIES Female Balb / C mice (Charles River, MA) were immunized subcutaneously with the electroeluted, purified proteins. When the titers of the immunized mice were detectable at a dilution greater than 1/1250, the splenocytes of a mouse were fused with a companion cell line, normal fusion, eg, sp2 / 0 myeloma cell line, to generate cells of hybridoma using methods known in the art. Then, the hybridoma cells were tested for their production of antibodies to platelets activated by radioimmunoassay. The supernatants of the culture medium (25 μl) were incubated with 107 thrombin-activated platelets 100 μl of buffer A and allowed to incubate at 21 ° C for 1 hour. The platelets were washed with 2 ml of cold buffer A, followed by centrifugation at 3000 rpm for 15 minutes. Then goat anti-mouse 125 I antibody (50,000 cpm) was added to each platelet pellet and centrifuged for 1 hour at 21 ° C. The platelets were washed again, centrifuged and the supernatant was removed. The platelet elements were counted in a gamma-counter. The selected hybridomas were cloned by limiting the dilution. The isotype of the monoclonal antibody was determined using commercially available reagents (Zymed, CA). Hybridoma cells, for example, those that produce MAb 3B2, were extended and inoculated into mice to generate the ascites fluid. The MAbs were purified from the ascitic fluid using known methods. Purified APP-2 polypeptides (including antigenic fragments of APP-2) can be used as antigens to produce other MAbs capable of distinguishing activated platelets and platelets at rest. To identify MAbs that bind to the same or similar epitope as MAb 3B2, the purified APP-2 polypeptide (or activated platelets) was incubated either simultaneously or sequentially with a test MAb, eg, a supernatant of the culture of hybridoma tissue or acetic fluid of a MAb of unspecified specificity, and MAb 3B2 that was labeled with a detectable marker. The binding of MAb 3B2 to the activated platelets or purified APP-2 was then measured. A decrease in the binding of 3B2 antibodies in the presence of the test antibody will indicate that the test antibody competes for the same or similar binding epitope in APP-2 as 3B2. RADIOMARCADE OF ANTIBODY The purified MAb 3B2 was radioiodinated at an activity of approximately 15,000 cpm / ng antibody. IMMUNOFLUQRESRESENCE Platelets were obtained from blood coded by gel filtration using a column of SEPHAROSER 2B agarose beads. The platelets were diluted in buffer B, and 107 platelets were added to the slide chambers (Nunc, Naperville, IL, 8 wells / slides). The cells were allowed to absorb for 2 hours at 21 ° C. The wells were washed three times in PBS, fixed for 5 minutes in formaldehyde, and then washed 4 times in PBS. The primary antibodies (at a concentration of 1 μg / ml in PBS with 1% BSA) were incubated for 90 minutes at 21 ° C. The wells were washed three times in PBS. After incubation for 1 hour at 37 ° C with the secondary antibody (goat anti-mouse, FITC 1: 500, Boehringer, Germany), the wells were washed three more times with PBS and the slides were mounted with the anti-mouse solution. - stain (90% glycerol, 10% PBS, 1, 4-diazobicyclo [2.2.2] octane (25 g / 1). The slides were examined at 200X magnification with a Nikon inverted microscope equipped with a mounted camera.

IMMUNQDETECTION OF APP-2 IN HUMAN THROMBLES The human aortic thrombus was fixed in formaldehyde and embedded in paraffin. Serial paraffin sections were tested with a control MAb (anti-digoxin), an anti-p-selectin MAb, or MAb 3B2. The bound MAb was detected by goat anti-mouse antibody coupled to peroxidase. After the development, the sections were visualized by light microscopy. DETECTION OF PLATELET ACTIVATION The monoclonal antibodies of the invention allow the reliable and precise detection of platelet activation. To detect activation of platelets in a biological sample, such as blood from the patient, the sample is incubated with specific platelet activation antibody, for example, MAb 3B2. The unbound antibody is washed and the bound antibody is detected using any standard label or method for labeling antibodies known in the art, for example, enzymes, radioisotopes, fluorescent compounds and metal chelates. For example, the antibodies of the invention can be used in an enzyme linked immunosuppressant assay (ELISA). UNION OF MONOCLONAL ANTIBODIES TO PLATELETS: COMPARATIVE UNION TESTS Platelets were isolated from rabbit blood, cited and gel filtered as described above. Platelets (90 μl, 107 cells) were distributed to 65 x 12 mm plastic tubes. A portion of the platelets was activated with thrombin (10 μl, 1.5 U / ml) and allowed to incubate at 37 ° C for 10 minutes. Another portion (at rest) of the platelets was incubated in parallel in the absence of thrombin. The platelets were fixed in 0.4% freshly prepared formaldehyde in phosphate buffer (pH 7.2, 10 μl) for 30 minutes. After the addition of 10 μl of the neutralization solution (20 mM NH4C1, 0.15 M NaCl, 0.3 M Tris-base, pH 7. 2), the platelets were washed in 2 ml of buffer A.

After centrifugation (3000 rpm, 10 min) and removed from the supernatant, the platelets were incubated for 30 minutes with the anti-APP-2 antibody labeled with 125 I (100,000 cpm / tube). After washing, bound antibodies were recovered in the pellet after centrifugation (3000 rpm, 10 min.) Then the pellets were found in a range-counter. SATURATION UNION TESTS Platelets were spun from fresh human blood by gel filtration. The platelets were diluted to 2.5 x 107 ml in buffer B, and 50 μl was added to each tube. The cells were activated with 50 μl of thrombin (0.3 U / ml) in buffer B for 15 minutes at 37 ° C. Increasing amounts of the radiolabelled antibody (50 μl, 6260-1,400,000 cpm) were then added to each tube in duplicate in the presence or absence of the unlabeled antibody (0.5 μg) as an inhibitor. After incubation at 21 ° C for 45 minutes, cold buffer A (2 ml) is added to each tube and the tubes are centrifuged for 15 minutes at 370 rpm. After removal of the supernatant, radioactivity bound to platelets was counted in a range-counter. IMMUNCTRANSFERENCE Platelets, smooth muscle cells, lymphoblastic leukemia cells CCRF-CEM (ATCC CCL 119), megakaryoblastic cells DAMI (Greenberg et al., 1988, Blood 72: 1968-1977), and red blood cells (between 1 and 5 μg were redispersed in sample buffer and boiled for 5 minutes (β-mercaptoethanol was added for reduced samples) after SDS-PAGE, the proteins were electrotransferred to PVDF membranes and blocked. incubated with primary antibody for 90 minutes at 21 ° C overnight at 4 ° C. The blots were washed with TBS and incubated with the secondary antibody (goat anti-mouse, alkaline phosphatase 1: 2000 [KPL, Gaithersburg, MD ]) for 1 hour After washing three times the 0.1% Tween-20 in alkaline phosphatase buffer, bound antibody was detected colorimetrically as described above.

ASSOCIATION OF APP-2 WITH PLATELET MEMBRANES Platelets in platelet-rich plasma (PRP) (4 x 108, 1 ml) were activated in the presence of 2 μM calcium ionophore, A 23187 (Sigma) at 37 ° C for 1 hour. After centrifugation (2000 rpm, 20 min). The platelets were redispersed and washed twice in 1 ml of buffer A. The platelets were redispersed in 80 μl of one of the following solutions: buffer A; 2 mM EDTA in buffer A; Urea 1 M; 1 M NaCl; glycine 0.1 M, pH 2.8; glycine 0.1 M, pH 11; or 0.1% triton-X in buffer A. After incubation at 21 ° C for 1 hour, the samples were centrifuged for 20 minutes at 2000 rpm. Without dislodging the sediment from the platelets, 50 μl of the supernatant was recovered, concentrated by precipitation with acetone, redispersed in the sample buffer, electrophoresed in 7.5% SDS gels. The presence of APP-2 or, as a control, CD63 in the eluent was determined by immunoblotting with the anti-CD63 monoclonal antibody (1 μg / ml, Biodesing, Kennebunkport, ME) and anti-APP-2. EFFECTS OF ANTI -APP-2 IN PLATELET AGREEMENT. PLATELET PLATELETS were added to 450 μL of buffer 450 of an instrument to study the aggregates of blood platelets contained in a stir bar.

MAb 3B2 (50 μl, final concentration of 5 or 50 μg / ml) was added to the specimen and incubated for several minutes while the changes in light scattering were recorded (instrument for measuring aggregates of blood platelets, Chrono -Log) Then, 5 μl of A23187 (Sigma, 2 μM final concentration) was added to the test piece and aggregation to the platelets was measured using standard methods known in the art. FOSFORILATION EXPERIMENTS The freshly isolated platelet-rich human plasma (approximately ~ 3 X 108 cells in 3.5 ml) was mixed with ~950 μCi of H332P04 and incubated at 37 ° C for 1 hour. Then the calcium ionophore A23187 (2 μM) was added during 15 minutes at 37 ° C. The platelets were isolated by centrifugation at 3000 rpm for 10 minutes at 4 ° C were rapidly used in 0.5% SDS, 50 mM Tris HCl, (pH 8.0) and 1 mM dithiothreitol, and boiled for 15 minutes. The lysate was diluted with immunoassay buffer and microfuge at 13,000 rpm for 60 minutes. The supernatant was preclarified by incubation with 64C5 MAb coupled to safarose for 2 hours. The supernatant was then divided in half and 1 ml was incubated with 200 μl of 3B2 MAb or 64C5 MAb coupled to safarose for 90 minutes at 4 ° C. The supernatant was removed and the MAb-safarose was achieved twice with cold RIPA buffer. After centrifugation, 150 μl of the sample buffer was added with 5% β-mercaptoethanol and the beads were boiled for 5 minutes. Samples were loaded on 12.5% SDS-polyacrylamide gels for electrophoresis. The state of human platelets was loaded onto another pathway as a control after electroblotting to polyvinylidene difluoride membranes, the platelet lysate was immunoblotted with MAb 3B2 and an immunoprecipitate was exposed on a phospho-imaging (Molecular Devices, Sunnyvale, CA). MOLECULAR CLONING OF APP-2 After infecting Y1090 cells of E. coli, the phage from human bone marrow gtll lambda (5 'plus extension, cDNA library, Clonetech, Palo Alto, CA) were cultured until they were formed plates visible at 42 ° C in LB ampicillin plates. The dry nitrocellulose filters, pre-heated in isopropylthio-β-galactosidase (IPTG, 10 mM), were coated in a bacterial cheesecloth and left at 37 ° C for 2.5 hours. After several washes in TBS, the filters were blocked in 5% milk, and left overnight to be incubated with the monoclonal antibody. Then, the filters were processed as described above for immunoblotting. A total of 980,000 phage were selected using this procedure. The phage expressing a protein recognized by the anti-APP-2 antibody was purified to homogeneity by several selection runs. DNA hybridization methods were used to isolate the clones containing additional cDNA sequences from well-known methods, for example, Sambrook et al., 1989. The cDNA probes were labeled by random tilt (Boehringer Mannheim, Indianapolis, IN) using known methods. The radiolabelled probe was added to the prehybridized filters (2 hours at 42 ° C) and incubated overnight at 42 ° C. The filters were then rinsed in a series of washes at 37 ° C, 42 ° C, 48 ° C and 65 ° C in 2 x SSC solution containing 0.1% SDS. The filters were then exposed to X-ray films at -80 ° C. The phage cDNA isolated either by antibody selection or cDNA hybridization was subcloned into the pUC18 vector. Both strands of the DNA clones were sequenced according to known methods using a combination of the primers specific for the internal sequence of the cDNA. The sequences were analyzed with the help of the MacVector program (IBI, New Haven, CT) and the NCBI database (NIH, MD). Using reagents derived from APP-2 cDNA clones that contain some or all of SEQ ID NO: 2, isolation of a full length APP-2 cDNA from any vertebrate species is well within the scope of the invention. the experience of those experts in the molecular biology technique. For example, radiolabelled cDNA probes made from known cDNA inserts can be used to identify and isolate platelet cDNA libraries, cDNAs containing ions with sequence homology to these cDNAs, library selection cDNA with radiolabeled cDNA probes is routine in the molecular biology technique (see Sambrook et al., 1989, Molecular Cloning: a Laboratory Manual, second edition, Cold Spring Harbor Press, Cold Spring Harbor, NY). The cDNA can be isolated and subcloned into a plasmid vector, and the DNA and plasmid is purified by standard techniques. The cDNA insert is sequenced using the dideoxy chain termination method well known in the art (Sambrook et al., supra). The oligonucleotide primers corresponding to the regions of the border vector as well as primers prepared from the previously isolated cDNA clones can be used to determine the sequence of the complete gene. DNA containing a sequence encoding part or all of the amino acid sequence of an activated platelet protein homologous to human APP-2 can then be re-cloned into an expression vector, eg, the pFlag vector system described below , using a variety of methods known in the art. For example, a recombinant polypeptide can be expressed as a Flag-fusion protein produced in E. coli antibodies (or fragments thereof) that bind to an APP-2 epitope, then can be used to detect the expression of the APP-2 in the cDNA clones. BACTERIAL EXPRESSION OF APP ~ 2 The cDNA encoding APP-2 was cloned into the pFlag vector (IBI, New Haven, CT) and used to transform E. coli DH5 cells (Gibco, Grand Island, NY). The clones were cultured in the selection medium (LB + ampicillin). The cultures were diluted 1/100 in fresh medium in sterile flasks and cultured with shaking at 37 ° C until A600 = 0.5. IPTG (0.3 mM) was added to the cell suspension to include protein expression, and the cells were cultured for another 2 hours at 37 ° C. The cells were centrifuged at 3000 rpm for 30 minutes, and the platelets were redispersed in a sample buffer with a volume of 1/10 with 5% β-mercaptoethanol before analysis on an electrophoretic gel.

DEPOSIT Under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the purpose of Patent Procedure, the 3B2 hybridoma and the APP-2 plasmid (in the pFlag vector) were deposited with the American Species Crop Collection (ATCC, by its abbreviations in English) of Rockville MD, USA, the 6 of October of 1995. The two deposits have been given the designations of CRL-11986 and 97314, respectively. The applicant's transferee, President and Members of the Harvard College, symbolizes that the ATCC is a depository agency that grants the permanence of deposits in the accessibility prepared to it by the public if a patent is granted. All restrictions on public availability and material deposited in this way will be irrevocably removed in the granting of a patent. The material will be available during the pendency of the patent application to a determined by the commissioner who will be entitled to it under 37 CFR 1.14 and 35 U.S.C. § 1212. The material deposited will remain with all necessary care to keep it viable and uncontaminated for a period of at least five years after the most recent request for the presentation of a sample of the deposited plasmid, and in any case, for a period at least thirty (30) years after the date of deposit or for the life of the patient, even though the period is longer. The applicant's assignee acknowledges its duty to replace the deposit if the depositing authority is unable to present a sample when requested due to the connection of the deposit. IDENTIFICATION OF APP-2 BY 2-D TECHNIQUES Analytical 2-D electrophoresis revealed several differences in the pattern of the platelet cell activation protein between the resource and activated platelets. Although analytical 2-D electrophoresis offers tremendous resolution, its limitation lies in the resolution of only μg quantities of protein. To overcome this limiting factor and preserve the resolution of the 2-D system, the separation of platelet proteins by preparative 2-D electrophoresis was achieved. This technique allows the cooling of sufficient antigen, for example, APP-2, to produce antibodies against it. APP-2 was routinely purified from 20 units of platelets. This represents approximately 10,000 the amount that was separated using an analytical 2-D system. To identify the proteins specifically expressed by the activated platelets, the outer membrane proteins of resting and activated platelets were radioiodinated and subjected to 2-D gel analysis. These studies revealed two major proteins that were expressed only by activated platelets. A similar strategy was employed to identify those activated proteins or platelets using a preparative 2-D gel electrophoresis method. The biotinylated surface proteins of the resting and activated platelets were subjected to the preparative isoelectric focusing followed by SDS-PAGE, electrotransfer and the detection of esteptavidin-alkaline phosphatase. Many of the biotinylated proteins of the same molecular weight can be identified in the isoelectric reactions both from resting and activated platelet fractions. In addition, a biotinylated protein of approximately 25 kDa was identified to activated platelets that was not present in the corresponding isoelectric fraction of platelets at rest. This protein was called APP-2. After electro-focusing, APP-2 was electroeluted and used as an antigen to produce the MAbs. After somatic cell fusion, 170 of the 1960 wells of the microtiter plates showed growth of the hybridoma. Of these, 11% produced antibodies that bound activated platelets. Based on its apparent avidity, an anti-APP-2 antibody was selected and cloned. The anti-APP-2 MAb was found to have an IgGl coat isotype.

DETECTION OF APP-2 IN PLATELETS AND OTHER CELLS Immunoblotting experiments were performed to analyze the platelet lysate proteins. Under reducing conditions, the anti-APP-2 MAb detected a protein that migrated with the apparent molecular weight of 25 kDa, corresponding to the weight of APP-2 as identified by two-dimensional techniques. Under non-reducing conditions, APP-2 migrated with an apparent molecular weight of 145 kDa. When purified from a Tx-100 platelet lysate by immunoaffinity chromatography with a column of anti-APP-2 antibodies, molecular weight was also 145 kDa under non-reducing conditions. Immunoblotting experiments suggest that APP-2 was linked via disulfide bonds to other peptides in an activated platelet protein complex (APCOM, for its acronym in English). To identify the other members of APCOM peptides, APP-2 was purified by immunoaffinity chromatography and SDS-PAGE. The complex was then reduced and subjected to repeated SDS-PAGE, and the component polypeptides were detected by silver staining. Analysis after reduction of APCOM indicates that it is composed of three different polypeptides of 45 kDa (APP-45), 25 kDa (APP-2) and 15 kDa (APP-15) respectively. Immunoblotting studies were also performed with smooth muscle cells, red blood cells, and DAMI cells, a megaloplast cell line. APP-2 was detected in the lysates from the platelets and from the DAMI cells. EXPRESSION OF APP-2 BY ACTIVATED PLATELETS Immunofluorescence studies were performed with the purified anti-APP-2 antibody to determine whether APP-2 (and by extension, APCOM containing APP-2) was detectable on the surface of the activated platelets. In comparison to a control monoclonal antibody (anti-digoxins) of the same isotype, only anti-APP-2 showed specific binding to activated platelets (Figure la and Figure lb). Experiments were performed to determine if the APP-2 was expressed preferentially by resting or activated platelets. Figure 2 shows a separation of the expression of APP-2 and p-selectin (a molecule activating platelets selectively expressed in activated platelets) before and after cell activation, as detected by the specific MAbs to each protein. Compared to the level of expression of p-selectin, there is minimal expression of platelet APP-2 at rest.

After cell activation, there is a significant increase in the expression of APP-2 compared to platelets at rest. The expression of p-selectin was also increased after cell activation. The magnitude of the increase in binding of the anti-p-selectin MAb to the activated platelets was significantly greater than that seen for the anti-APP-2 MAb, suggesting that there are p-selectin molecules expressed by the activated platelet. Saturation binding studies were performed with the anti-APP-2 monoclonal antibody to determine the number of MAb 3B2 binding sites in activated platelets. The binding of anti-APP-2 to the activated platelets was saturable and was inhibited by an excess of unlabelled anti-APP-2 MAb. Analysis of the binding data indicated an individual class of binding sites with 4683 ± 748 molecules per activated platelet, representing approximately 40-50% of the number of p-selectin molecules per activated platelet. The anti-APP-2 antibody was bound to the platelets with a constant dissociation of 4.19 nM. These data indicate that MAb 3B2 specifically binds preferentially to activated platelets. Their binding was saturable and could be inhibited by competition with the unlabeled antibody. The Scatchard analysis indicated that there is only one individual class of antigen with approximately 5,000 molecules per platelet.

THE NATURE OF THE APP-2 ASSOCIATION WITH THE PLATELET MEMBRANE. The experiments were performed to determine if APP-2 was physically associated with the outer surface of platelets as an integral or peripheral membrane protein. APP-2 was eluted from the platelet membrane by Triton X-100, but no significant amounts were removed by the following solutions: buffer A; 2 mM EDTA; 1 M urea; 1 M NaCl; : glycine 0.1 M, pH 2.8; or 0.1 M glycine pH 11. The elution profile was similar to that obtained for CD 63, which is an integral membrane protein. These results suggest that APP-2, (and, by extension, APCOM) is associated with the surface of platelets as an integral platelet membrane protein. EFFECTS OF MAB ANTI-APP-2 ON PLAQUETTING AGGREGATION Anti-APP-2 MAb was added to platelet-rich plasma before and after stimulation of platelets with A 23187. MAb 3B2 (at concentrations of 5 or 50) μg ml) only did not induce platelet aggregation. The anti-APP-2 MAb also had no apparent effect on the magnitude of aggregation rate after the platelets were stimulated with A23187. These data indicate that the addition of MAb 3B2 to platelets at rest did not induce platelet aggregation and did not prevent them from adding the platelet activating agent, A23187, after the addition. MOLECULAR CLONING OF THE cDNA CODING FOR APP-2 To isolate the cDNA sequence encoding APP-2, a human bone marrow, gtll lambda expression library, was selected with anti-APP-2 MAb. Approximately 970,000 phage plaques were selected, and a positive clone was isolated. This clone was purified to homogeneity by repetitive subcloning. To confirm that the isolated cDNA encoded for APP-2, it was ligated to the vector pFlag for expression in batteries. Bacterial lysates containing the Flag-APP-2 fusion protein, or a Flag-fusion protein, of negative control, were analyzed by immunoblotting. The anti-APP-2 monoclonal antibody bound specifically to the Flag-APP-2 fusion protein, induced, but not to the Flag-fusion protein control. In addition, a monoclonal antibody of its control and its type as the anti-APP-2 MAb did not bind to the Flag-APP-2 fusion protein. The Flag-APP-2 fusion protein had a molecular weight of 47 kDa under reduced conditions, which was consistent with the open reading structure predicted by DNA sequencing. The APP-2 polypeptide can be enzymatically cleaved from the enterokinase fusion protein (IBI, New Haven, CT). The products of the cleavage can then be subjected to additional chromatography to purify the APP-2 polypeptide apart from the Flag portion of the fusion protein. For example, the Flag cleavage product can be removed from the mixture using commercially available reagents that bind to the Flag polypeptide (IBI, New Haven, CT). Alternatively, MAb 3B2 can be used to purify the cleavage product of APP-2 from the mixture using immunoaffinity chromatography. HOMOLOGIES AND PORTIONS OF THE APP-2 SEQUENCE The sequencing of the isolated cDNA with specific internal primers in both directions yielded several partial sequences that were aligned (MacVector Assembyling, IBI, New Haven, CT). The consensus sequence was analyzed against all the sequences reported in the NIH Genbnak. Although the partial sequences showed homology to the APP-2 sequence, no sequence characterized from a known, known gene matched the APP-2 sequence. Sequence analysis revealed that APP-2 is a new protein. Its amino acid sequence is not matched by any other reported protein sequence. A full-length DNA sequence encoding APP-2 was obtained by selecting the same library with the isolated cDNA as a probe. Immunoblot analysis confirmed that the recombinant polypeptide expressed the epitope recognized by MAb 3B2. FOSFORILATION OF APP-2 IN ACTIVATED PLATELETS Immunoprecipitation studies were performed to determine if the potential phosphorylation sites in APP-2 were functional in activated platelets. In activated platelets incubated with 32P04 and activated, a 25 kDa band was specifically immunuprecipitated by the MAb antibody 3B2 anti-APP, but not by a negative control MAb (anti-fibrin). The band identified by MAb 3B2 co-migrated with APP-2 as detected by immunoblotting on platelets in the same gel. Due to experimental conditions for this assay that typically triggered platelet activation, it was unable to determine whether APP-2 was phosphorylated at platelets at rest under these conditions. IMMUNODETECTION OF APP-2 IN HUMAN THROMBOS To determine whether MAb 3B2 could specifically detect APP-2 in thrombi, serial sections of the fixed aortic thrombus were probed with either MAb 3B2, an anti-p-selectin MAb, or anti-MAb. digoxin Figure 3 shows that MAb 3B2 and anti-p-selectin both detect the human thrombus, while the anti-digoxin MAb does not. This suggests that the MAb 3B2 antibody, similar P-selectin can specifically recognize human thrombi. USES OF DIAGNOSTICS AND THERAPEUTICS For administration to human patients, MAb's, for example, MAb 3B2, can be humanized by methods known in the art, for example, MAbs with a desired binding specificity can be humanized commercially (Scotgene, Scotland; Oxford Molecular, Palo Alto, CA). The monoclonal antibodies can be purified using known methods, such as absorption on immobilized protein A or immunoaffinity chromatography. After purification, the MAbs of the invention or the immunologically active fragments thereof, for example, Fab, (Fab) 2, or Fv, can be administered to patients in a pharmaceutically acceptable excipient such as physiological saline. MAbs and / or compounds based on antibodies of the invention, for example, MAbs linked to detectable labels or therapeutic agents, can be administered by any normal route including the intraperitoneal, intramuscular, subcutaneous or intravenous form. The preferred route of administration is expected to be intravenous. These compounds can be administered systematically to the bloodstream as well as locally within the blood vessel at the site of clot formation.

As is well known in medical techniques, the dosage for any patient depends on many factors, depending on the general health of the patients, sex, size, surface area of the body, age, as well as the particular compound to be administered, time and route of administration, and other drugs that are administered concurrently. Doses for the compounds of the invention will vary, but a preferred dose for intravenous administration is from about 1 μg to 500 μg / ml / volume of blood. The determination of the correct dose for a given application is well within the capabilities of a person skilled in the pharmacology art. The compounds of the invention, for example, MAbs or MAbs linked to other therapeutic agents such as antithrombotic agents, thrombolytic agents, anti-proliferative agents, or anti-aging agents, can also be administered simultaneously or sequentially with these agents . Preferred thrombolic agents include plasminogen activators, for example, urokinase, prourokinase, streptokinase, tissue-type plasminogen activator, staphylokinase or plasminogen activator from bat tissue, as well as physiologically active fragments thereof, for example, the plasminogen activator. of individual chain urokinase (scu-PA) in hybrids. Thrombolytic agents are expected to be administered intravenously at about 0.1 to 2.0 kg per body weight. The optimal dose can be adjusted according to the patient's condition and the patient's response to therapy. Other modalities are within the following claims.

LIST OF SEQUENCES (1) GENERAL INFORMATION (i) APPLICANT: President and Members of Harvard College (ii) TITLE OF THE INVENTION: NEW PLATELET ACTIVATION PROTEIN (iii) SEQUENCE NUMBER: 4 (iv) CORRESPONDENCE ADDRESS: (A) RECIPIENT: Fish & Richardson P.C. (B) STREET: 225 Franklin Street (C) CITY: Boston (D) STATE: MA (E) COUNTRY: USA (F) POSTAL CODE: 02110-2804 (v) COMPUTER LEGIBLE FORM: (A) TYPE OF MEANS: Flexible disk (B) COMPUTER: compatible with IBM PC (C) OPERATING SYSTEM: DOS (D) PROGRAM: FastSEC Version 2.0 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: 10 (B) SUBMISSION DATE: 04-OCT-1996 (C) CLASSIFICATION: (vii) DATA FROM THE PREVIOUS APPLICATION: (A) APPLICATION NUMBER: 60 / 005,074 15 (B) DATE OF SUBMISSION: 06-OCT-1995 (viii) INFORMATION OF THE AGENT / LAWYER (A) NAME: Fraser, Janis K (B) REGISTRATION NUMBER: 34,819 20 (C) ORDER NUMBER / REFERENCE: 05433 / 020WO1 (ix) TELECOMMUNICATION INFORMATION (A) TELEPHONE: 617-542-5070 (B) TELEFAX: 617-542-8906 25 (C) TELEX: 200154) INFORMATION FOR SEQ ID NO: l: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1373 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: double D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (ix) CHARACTERISTIC: (A) NAME / KEY: Sequence coding (B) LOCATION: 44 ... 1321 (C) OTHER INFORMATION: (xi) DESCRIPTION FOR SEQ. ID No: 1 CONTINUED GRAPHICS LEAVES

Claims (21)

1. A substantially pure DNA encoding a naturally occurring platelet activation polypeptide, this polypeptide comprises a sequence at least 70% identical to SEQ ID NO: 1.

2. Substantially pure DNA encoding a polypeptide that comprises an amino acid sequence identical at least 95% to SEQ ID NO: 4.

3. The DNA according to claim 2, wherein the DNA comprises the sequence of SEQ ID NO: 3.

4. Substantially pure DNA comprising a strand of at least 20 nucleotides that hybridizes at high stringency to a DNA complementary to the coding sequence of SEQ ID NO: 3.

5. The DNA according to claim 2, wherein the DNA hybridizes at high stringency to a DNA probe consisting of a sequence of 50 nucleotides complementary to the coding sequence of SEQ ID NO: 3.

6. A vector comprising DNA according to claim 2.

The DNA according to claim 2, wherein the DNA is operably linked to regulatory sequences for the expression of the polypeptide, these regulatory sequences comprise a promoter.

8. A cell comprising the DNA of claim 7.

9. A substantially pure polypeptide comprising an identical sequence at least 95% to SEQ ID NO: 4.

10. The polypeptide according to claim 9, wherein the polypeptide comprises the sequence of amino acids encoded by SEQ ID NO: 3.

11. An antibody that specifically binds to the polypeptide of claim 9.

12. The antibody according to claim 11, wherein the antibody binds to the same epitope as MAb 3B2.

The antibody according to claim 11, wherein the antibody is linked to a detectable label.

14. A method for detecting an activated platelet in a biological sample, comprising contacting the sample with the antibody of claim 11 and determining whether the antibody binds to a component of the sample, this binding is an indication that the sample contains an activated platelet.

A method for locating a platelet thrombus in an animal, which comprises administering to the animal the antibody according to claim 13, and determining in the animal where the tag is located, where the detection is marked in a site of the animal indicates the existence of a platelet thrombus on this site.

16. A method of targeting a compound to an activated platelet in an animal, comprising administering to this animal a composition comprising the compound bound to the antibody of claim 11.

17. The method according to claim 16, wherein the The compound is an anti-thrombotic agent, an anti-proliferative agent, or an anti-migration agent.

18. A polypeptide comprising an antigenic fragment of the polypeptide of claim 9.

19. A substantially pure polypeptide having the sequence of the naturally occurring platelet activation polypeptide comprising an epitope that binds to MAb 3B2.

A method for detecting a protein complex of activated platelets in a biological sample, comprising contacting the sample with the antibody of claim 11 and determining whether this antibody binds to a component of the sample having a molecular weight of approximately 145 kDa under non-reducing conditions.

21. An activated platelet protein complex (APCOM) comprising a polypeptide that binds to the antibody of claim 1.