WO1993009137A1

WO1993009137A1 - Inhibitors of collagen-induced platelet aggregation

Info

Publication number: WO1993009137A1
Application number: PCT/US1991/007958
Authority: WO
Inventors: J. Bryan Smith; Carol A. Dangelmaier
Original assignee: Temple University-Of The Commonwealth System Of
Priority date: 1991-10-28
Filing date: 1991-10-28
Publication date: 1993-05-13

Abstract

Polypeptides isolated from snake venom inhibit platelet aggregation by inhibiting platelet adhesion to collagen. Monoclonal antibodies specific for the anti-adhesive polypeptides are described, which may be used to detect and purify those agents from liquids.

Description

INHIBITORS OF COLLAGEN-INDUCED

PLATELET AGGREGATION

Field of the Invention

The invention relates to inhibition of platelet aggregation, by novel polypeptides which interfere with the platelet's adhesion to fibrillar collagen. Reference to Government Grant

The invention described herein was supported in part by National Institutes of Health Grant HL 36579. The government has certain rights in the invention.

Background of the Invention

It is generally regarded that the very first stage in hemostasis and thrombosis involves the adhesion of circulating blood platelets to newly exposed fibrillar collagen (Baumgartner, Thromb. Haemostas., 37, 1-16 (1977)). Following damage to a vessel wall, circulating blood platelets initially adhere to collagen. Fibrillar collagen has been identified as the most thrombogenic macromolecular component of the blood vessel wall (Id.). Platelets which adhere to the thus-exposed collagen release the contents of their dense, nucleotide and amine storage granules, including adenosine diphosphate (ADP) (Zucker, Amer. J. Physiol., 206, 1267-1274 (1964); Holmsen, J. Clin. Lab. Invest., 17, 239-246 (1965)). In addition, arachidonic acid is freed from membrane phospholipids and converted to thromboxane A₂ (TxA₂) (Smith et al., J. Clin. Invest., 53, 1468-1472 (1976); Hamberg et al., Proc. Natl. Acad. Sci. USA, 72, 2994-2998 (1975)). Collagen thus acts both as a solid phase matrix for platelet adhesion and as a primary agonist to cause release of ADP and TxA₂. The latter two soluble agents cause platelet aggregation and further release of dense granule constituents (Kinlough-Rathbone et al., J. Lab. Med., 90, 707-712 (1977)). The result is the formation of a hemostatic plug or thrombus.

It is currently accepted that Arg-Gly-Asp- containing peptides (Plow et al., Proc. Natl. Acad. Sci. USA, 82, 8057-8061 (1985)), including snake venom proteins such as trigramin (Huang et al., J. Biol. Chem., 262,antihesins 16157-16163 (1987)), inhibit platelet aggregation by competitively and specifically inhibiting fibrinogen binding to the receptors associated with the GPIIb/GPIIIa complex on activated platelets. Until now, no specific inhibitors of platelet adhesion to collagen have been described.

What is needed is an inhibitor of platelet aggregation which works against the initial step in thrombosis and hemostasis, that is, against the adhesion of platelets to exposed fibrillar collagen.

Summary of the Invention

Substantially purified polypeptides corresponding to polypeptides obtainable from snake venoms are provided, which polypeptides inhibit adhesion of platelets to collagen. Such platelet adhesion-inhibiting polypeptides (hereinafter collectively referred to as "antihesins") contain an antigenic determinant which is recognized by monoclonal antibody from hybridoma ATCC HB-10904. By "recognized" is meant that the monoclonal an tibody specifically binds to the antihesin antigenic determinant. Also provided are pharmaceutical compositions comprising one or more antihesins and a pharmaceutically acceptable carrier, useful for inhibiting platelet adhesion and aggregation.

The invention also relates to hybridomas, such as ATCC HB10904, which have been prepared providing cell lines producing monoclonal antibodies which recognize an antigenic determinant shared by the antihesins. The hybridomas comprise cell hybrids formed by fusion of cells from a myeloma line and spleen cells from a donor previously immunized with an immunogen containing an antihesin, preferably substantially purified antihesin. ATCC HB-10904 is one such hybridoma. It was deposited on October 17, 1991 in the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD.

The hybridomas may be cultured in vitro or in vivo to secrete antibodies. The invention also relates to the monoclonal antibodies so produced.

The hybrid cell lines may be prepared by first immunizing a splenocyte donor with immunogen comprising antihesin. The spleen cells are fused with myeloma cells in the presence of a fusion promotor. The fused cells are diluted and cultured in separate wells in a medium which will not support the unfused myeloma cells. The supernatant of each well is assayed for the presence of antibody to antihesin by an assay, such as an enzyme-linked immunosorbent assay ("ELISA"). Hybridomas secreting antibody which bind to antihesin are selected and cloned.

The hybridomas are cultured in a suitable medium and the antibody is recovered from the supernatant. Alternatively, the clones are transferred intraperitoneally into a suitable host, e.g. mice, and the resulting malignant ascites and/or serum containing the desired antibody are harvested. According to another embodiment, the invention provides a method for purifying antihesins from a liquid, such as snake venom. The liquid is contacted with an immobilized antibody, preferably a monoclonal antibody, which recognizes an antigenic determinant of antihesin to absorb that polypeptide from the liquid. The antihesin is thereafter eluted from the immobilized monoclonal antibody.

According to another antihesin purification method, lyophylized venom is dissolved in a solvent. The venom is fractionated to separate the polypeptides contained therein. The venom fractions are then assayed for activity in inhibiting the adhesion of platelets to collagen, or, alternatively, for polypeptide which is recognized by monoclonal antibody from hybridoma ATCC HB- 10904. Polypeptide which inhibits platelet adhesion to platelets and/or is recognized by the aforesaid antibody is then purified from the active fractions.

The invention yet further provides a method for inhibiting adhesion of platelets to fibrillar collagen in a mammal comprising administering antihesin to the mammal. According to another embodiment, a method of inhibiting collagen-induced aggregation of mammalian platelets is provided comprising administering antihesin to a mammal to inhibit the occurrence of platelet aggregation in the bloodstream of the mammal.

As used herein, the term "antibody", inclusive of both monoclonal and polyclonal antibodies, shall include not only the intact antibody, but also fragments thereof capable of binding antigen, including, but not limited to, Fab and F(ab)₂ fragments.

As used herein, the expression "corresponds to" or "corresponding to" with regard to the relationship between a substantially purified polypeptide and the native snake polypeptide means that the purified polypeptide is identical to the native polypeptide, or is a synthetic or genetically engineered polypeptide or fragment of the native polypeptide, which has an amino acid sequence substantially the same as that of the native polypeptide, or at least sufficiently duplicative of the native sequence to retain the biological activity of the native polypeptide.

It is accordingly one object of the invention to provide pharmaceutically useful polypeptides which specifically inhibit adhesion of blood platelets to exposed fibrillar collagen.

It is another object to provide polypeptides which may be administered to inhibit platelet aggregation by interfering with the first step of thrombus formation, that is, the adhesion of blood platelets to fibrillar collagen.

It is also an object to provide a method for inhibiting adhesion of blood platelets to collagen in order to inhibit their aggregation.

It is an object of the invention to prevent formation of thrombi.

It is a further object of the invention to provide hybridomas which produce antibodies against the aforesaid novel anti-adhesive polypeptides.

It is yet another object of the invention to provide antibodies, particularly essentially homogeneous antibodies, against the anti-adhesive polypeptides.

It is a further object of the invention to provide a method for purifying from biological materials novel polypeptides which inhibit platelet adhesion to fibrillar collagen.

Description of the Figures

Fig. 1 shows the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (12%, non-reduced) of B. atrox antihesin at various stages of purification. Lane 1, molecular weight standards; lane 2, crude B. atrox venom; lane 3, B. atrox venom after SEPHADEX G-100 column chromatography; lane 4, 50 kDa B. atrox antihesin after high performance liquid chromatography (HPLC).

Fig.2 shows the SDS-PAGE (12%, non-reduced) of c. atrox antihesin at various stages of purification. Lane 1, molecular weight standards; lane 2, crude C. atrox venom; lane 3, C. atrox venom after SEPHADEX G-100 column chromatography; lane 4, C. atrox 13 kDa antihesin after HPLC; lane 5, C. atrox 50 kDa antihesin after HPLC.

Fig. 3 is a Western blot of crude snake venoms with ATCC HB-10904 monoclonal antibody following SDS-PAGE (12%, non-reduced). Lane 1, C. basiliscus (20 micrograms); lane 2, B. atrox (200 ng); lane 3, B. jararaca (200 ng); lane 4, A. halys blomohoffii (200 ng). Molecular weight standards are shown on the right.

Fig. 4 is a concentration-response curve for inhibition of platelet adhesion to collagen (25 micrograms/ml) by the purified antihesin polypeptide from B. atrox (SVP).

Figs. 5A through. 5E are a series of aggregometer tracings showing the effect of the purified antihesin from B. atrox (indicated by + sign) on aggregation in human citrated platelet-rich plasma induced by (5A) collagen (1 microgram/ml); (5B) ADP (5 micromolar); (5C) epinephrine (5 micromolar); (5D) platelet-activating factor (0.25 micromolar); and (5E) the thromboxane mimetic SQ 26655 (0.2 micromolar).

Detailed Description of the Invention

Exposure of platelets to collagen, the most thrombogenic of the vascular wall components, triggers their activation through the release of ADP and TxA₂.

Adhesion of platelets to collagen at 37°C is a very rapid process, which is essentially complete by 1 minute. The factor limiting the extent of adhesion is the amount of collagen to which the platelets are exposed. The polypeptides of the present invention inhibit platelet aggregation in plasma by interfering with this initial step in hemostasis/thrombosis. This is in contrast to the so-called "disintegrin" class of antithrombotic agents which inhibit platelet aggregation by interfering with platelet binding to fibrinogen, a subsequent step in the pathway leading to thrombus formation. The disintegrins, which contain the tetrapeptide sequence Arg-Gly-Asp-Ser, bind to the fibrinogen receptor of platelet glycoprotein Ilb/IIIa, and thereby prevent build-up of platelet aggregates. The disintegrins thus inhibit platelet aggregation induced by ADP, thrombin, epinephrine, and sodium arachidonate, as well as collagen.

The anti-adhesive polypeptides of the invention have no effect on platelet aggregation induced by platelet agonists other than collagen. They do not act on the fibrinogen receptor on platelet glycoprotein Ilb/IIIa. Rather, they bind to distinct receptors on the collagen molecule. The antihesins inhibit platelet aggregation by preventing initial platelet attachment via collagen to the site of vascular injury.

Biological materials such as snake venoms may be screened for antihesins by an assay for platelet adhesion to collagen in the absence of platelet aggregation. We previously described such a method: Smith, J. and Dangelmaier, C., Anal. Biochem., 187, 173-178 (1990). Briefly, suspensions of human platelets are incubated with or without the biological source material, in the presence of an ADP-removing system and antagonists to TxA₂, platelet activating factor (PAF), serotonin, and fibrinogen, which comprise the platelet agonists responsible for aggregation. Suitable antagonists of TxA₂,

PAF, serotonin and fibrinogen are known to those skilled in the art. Collagen is then added to the system and adherence of platelets thereto is determined. The inhibition of the occurrence of platelet adhesion is indicative of the presence of an antihesin polypeptide in the source material.

Once a biological material is identified in this manner as a source of antihesin, the polypeptide may be isolated from the material in one of two ways. According to a first method, the antihesin is purified by a combination of gel filtration and high performance liquid chromatography. According to a second method, immobilized antibody which binds antihesin, preferably monoclonal antibody, is contacted with the biological material. Antihesin is absorbed from the material and thereafter eluted from the immobilized antibody.

The activity of the antihesins is such that they produce greater than 50% inhibition of platelet adhesion to collagen at an antihesin concentration of 100 μg/ml (about 2μM, based upon an antihesin molecular weight of about 50kDa). Antihesins have been isolated with greater than 95 weight percent purity based upon the presence of a single band upon Coomassie blue or silver stained SDS-PAGE. By "substantially purified", as the expression is used herein, is meant a purity of at least about 50 weight percent. While a purity level of 95 weight percent is generally utilized for clinical use, lower purity material may be satisfactory for in vitro applications.

The monoclonal antibodies which may be employed in immunoaffinity purification of antihesins recognize an antigenic determinant shared by such polypeptides, regardless of the source of the polypeptide. We have found that, upon screening numerous snake venoms for antihesin, there is complete correlation between the presence of antihesin activity and immunoreactivity to monoclonal antibody from hybridoma ATCC HB-10904. While the polypeptides recognized by the aforesaid monocloal antibody are beIieved to be closely related, their amino acid compositions are not identical, as is apparent from Table 1, below. Thus, the antibody recognizes an antigenie determinant which is apparently shared by all antihesins, notwithstanding differences in the primary structure of the antihesins. Antibodies which recognize an antigenic determinant shared by all antihesin polypeptides comprises a valuable tool for isolating antihesins from their respective parent biological materials.

Anti-antihesin antibodies may be prepared by immunizing an appropriate host with antihesin, purified as hereinafter described from the venom of any of the following species: Bothrops atrox, Bothrops jararaca, Bothrops mooqeni, Akistrodon halys blomhoffii, Crotalus atrox or Crotalus basiliscus.

According to one method, mice are immunized with purified antihesin. BALB/c AnSkh mice are preferred, although other strains may be used. The immunization schedule and concentration of immunogen administered should be such as to produce useful quantities of suitably primed splenocytes.

Upon completion of the immunization regimen, more fully described below, the mice are sacrificed and their spleens removed. A suspension of splenocytes in a suitable medium is prepared. Approximately 2.5-5 ml of medium per spleen is sufficient. The protocols for in vitro suspension are well established.

The spleen cells are fused with mouse myeloma cells by means of a fusion promotor. The preferred fusion promotor is polyethylene glycol (PEG), molecular weight 1,300-1,600. The mouse myeloma cell line is preferably one of the drug-resistant types, to facilitate selection of hybrids. The most frequently utilized class of myelomas are the 8-azaguanine-resistant cell lines, which are widely known and available. These cell lines lack the enzyme hypoxanthine guanine phosphoribosyl transferase. They do not therefore survive in "HAT"

(hypoxanthine-aminopterin-thymidine) medium. The use of myeloma cells with different genetic deficiencies (e.g., other enzyme deficiencies, drug sensitivities, etc.) that can be selected against in a medium supporting the growth of genotypically competent hybrids is also possible.

Additionally, it is suggested that the myeloma cell line should not itself produce any antibody, although in some circumstances secreting myeloma cell lines may be used.

While the preferred fusion promotor is PEG of average molecular weight 1,300-1,600, other known fusion promotors may be used.

Fusion of cells may be carried out in an adherent monolayer, such as according to the method described by T. J. McKearn, "Fusion of Cells in an Adherent Monolayer" in Monoclonal Antibodies: Hybridomas: A New Dimension in Biological Analysis (Kennett, R. H., McKearn, T. J., and Bechtol, K.B., eds., Plenum Press, New York and London, 368-369, 1980). Other fusion techniques may be employed. A cell ratio of 2-5:1 spleen cells per myeloma cell may be used. This ratio may be varied depending on the source of spleen or myeloma cells.

A mixture of unfused myeloma cells, unfused spleen cells and fused cells are distributed for culturing in separate compartments (e.g., the wells of microtiter plates) in a selective medium in which the unfused myeloma cells will not survive. Distribution of the cells may be by resuspension in a volume of diluent which is statistically calculated to isolate a desired number of cells per compartment. See, for example, McKearn, "Cloning of Hybridoma Cell Lines by Limiting Dilution in Fluid Phase" in Monoclonal Antibodies, p. 374.

When HAT is used as the medium, unfused 8-azaguanine-resistant myeloma cells will not grow. Unfused spleen cells will normally die after a few days, since they are non-malignant. Culturing proceeds for a time sufficient to allow their death. Fused cells continue to reproduce and grow in the selective medium.

The supernatant in each container or compartment having hybrid cell growth is screened and evaluated for the presence of antibody against antihesin. Any suitable antibody-binding detection method may be used, e.g., enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, etc.

After selection and cloning, monoclonal antibody to antihesin may be produced by in vitro culturing of the hybridomas or by in vivo peritoneal exudate in mice. The first method will yield monoclonal antibody of higher purity. The antibody is recovered from the supernatant essentially free of undesired immunoglobulin. Antibody concentrations of 25-50 micrograms/ml are possible by this method. In growth media containing serum (such as fetal calf serum) a small amount of other immunoglobulin is present.

When concentrations of antibody larger than those obtained by in vitro culturing are required, the subject hybridomas may be injected into the peritoneal cavity of syngeneic or semisyngeneic mice. After a suitable period of incubation, the hybridomas cause formation of antibody-secreting tumors, which will produce 4-10 mg of antibody per ml of peritoneal exudate of the injected mouse. Since mice normally have antibodies in their blood and ascites, a contamination of about 5% from the host mouse is inevitable. Purification of ascites monoclonal antibody may remove these contaminants. The resultant antibody is of high titer.

The antihesins may be administered in any situation where inhibition of mammalian (inclusive of human) collagen-induced platelet aggregation is desired. In particular, it is believed that collagen exposure and platelet adhesion plays a thrombotic role in patients with unstable angina and those recovering from myocardial infarction. The antihesins are believed useful in inhibiting platelet aggregation in these conditions and to clear obstructed coronary arteries.

Platelet adhesion and aggregation is also a consequence of invasive medical techniques which cause epithelical damage and exposure of fibrillar collagen. Thus, the polypeptides may find utility in surgery on peripheral arteries, coronary by-pass and other openheart surgical techniques, angioplasty, or other procedures where damage to the endothelial wall of blood vessels may result in collagen exposure, triggering platelet adhesion and aggregation. Antihesins may be administered to patients undergoing these procedures to prevent platelet aggregation and formation of thrombii. The antihesins are thus believed useful in inhibiting thrombosis and reocclusion during and after such procedures. It is believed that the antihesins are particularly useful in preventing reocclusion after angioplasty.

The antihesins may be administered by any convenient route which will result in delivery to the blood stream in an amount effective for inhibiting platelet adhesion to exposed collagen. The antihesins are most effectively administered parenterally, preferably intravenously or intraarterially. For intravenous/intraarterial administration, they may be dissolved in any appropriate intravenous delivery vehicle containing physiologically compatible substances, such as sodium chloride, glycine, and the like, having a buffered pH compatible with physiologic conditions. Such intravenous delivery vehicles are known to those skilled in the art.

The most suitable vehicle is a sterile saline solution.

The antihesins may be administered in conjunction with other antithrombotic agents, such as tissue plasminogen activator or streptokinase in order to inhibit platelet aggregation.

The amount of antihesin administered will depend on the individual clinical circumstances. For example, for clearance of obstructed coronary arteries, the antihesin may be given at a concentration ranging from about 1 to about 10 mg/ml in a solution further containing a clot lysing agent such as tissue plasminogen activator.

For other applications, dosages may advantageously range from about 0.1 to 100 mg/kg. More or less antihesin may be administered as needed.

The practice of the invention is illustrated by the following non-limiting examples.

Example 1

Purification of Bothrops Atrox Antihesin

Lyophilized Bothrops atrox venom (Sigma Chemical Co., St. Louis, MO) was reconstituted in 0.9% NaCl at 100 mg/ml. Bothrops atrox is a South American viper, family Viperidae. subfamily Crotalinae. 0.5 ml of the aqueous solution was applied to a 2 cm × 45 cm SEPHADEX G-100 column and eluted with 0.9% NaCl at a flow rate of 6 ml per hour. Fractions of 2 ml were collected and examined spectroscopically for protein content at 280 nm. The fractions were also examined for ability to inhibit platelet adhesion to collagen according to the following adhesion assay which we have previously described (Smith, J.B. and Dangelmaier, C., Anal. Biochem. 187, 173-178 (1990)).

Human blood from drug-free healthy volunteers was anticoagulated with citric acid-citrate-dextrose according to the procedure of Aster and Jandl, J. Clin. Invest. 43, 834-855 (1964). The platelet-rich plasma

(PRP), obtained by centrifugation at 180gr for 20 min. at room temperature, was centrifuged at 800g for 15 min. at room temperature. The platelet pellet was resuspended in 0.2 volume of autologous plasma and incubated for 1 hour with 1 microCi/ml [9,10-³H(N)]oleic acid (8.9 Ci/nmol) at 37ºC. Following the incubation, the platelets were separated from unincorporated radiolabel by gel filtration on a SEPHAROSE 2B column using a calcium-free Tyrode's buffer containing 0.2% fatty acid-free bovine serum albumin (Sigma Chem. Co.) and 5 mM glucose. The gel-filtered platelets were adjusted to 2.5 × 10⁸ cells/ml and 1-ml samples were stirred at 800 rpm at 37ºC for 1 min. before addition of the following (i) the fibrinogen/fibronectin inhibitor, Arg-Gly-Asp-Ser (220 micromolar); (ii) the TxA₂ receptor agonist, (SQ 29,548) [1S-[1<a,2<b(5Z)3<b,4<a]]-7-[3-[[2-[phenyl-amino) carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]- 5-heptenoic acid (6 micromolar), Bristol-Myers Squibb Co., Princeton, NJ); (iii) the ADP-removing system, creatine phosphate (20 mM) and creatine phosphokinase (50 U/ml); (iv) the platelet-activating factor antagonist, ginkgolide B (100 micromolar), and (v) the serotonin antagonist, cyproheptadine (1 micromolar). (The chemical structure of ginkgolide B is set forth in U.S. Patent 5,002,965.) Snake venom protein fractions were added to the platelet suspensions. One minute later, collagen (5-100 microgram/ml) was added and incubated for 3 min. at 37ºC. Following incubation, the platelet suspensions were decanted into a manifold containing a 10-micron nylon disk under vacuum. The disks containing the platelets that had adhered to collagen were rinsed and then the radioactivity determined.

An about 50 kDa (SDS-PAGE) anti-adhesive polypeptide was present in approximately 15 ml of gel filtration eluate from the SEPHADEX G-100 column.

The partially purified 50 kDa polypeptide was concentrated from approximately 15 ml to 3-5 ml using a CENTRICON PREP apparatus. The concentrate was applied to a reverse phase HPLC column (semi-preparative, 1 × 25 cm, VYDAC C4). The column was eluted over 60 min. using a linear gradient consisting of increasing amounts of acetonitrile in 0.1% trifluoroacetic acid. Elution of protein was followed by monitoring the absorbance at 220 nm. The peak of protein eluting at 70% acetonitrile in 0.1% trifluoroacetic acid was collected, lyophilized and reexamined for its activity in the adhesion assay.

The purification of the B. atrox polypeptide was monitored by SDS-PAGE, as shown in Fig. 1 (Lane 1, molecular weight standards; lane 2, crude venom; lane 3, venom after SEPHADEX G-100 column chromatography; lane 4, purified 50 kDa polypeptide after HPLC). The purified polypeptide migrated as a single band with an apparent molecular weight of 50 kDa in both reduced and nonreduced gels, suggesting that the inhibitory protein is composed of a single polypeptide chain. However, after storage at -20°C for a week or more, larger aggregates of the venom protein were visible, apparently reflecting disulfide interchange.

A partial amino acid sequence of the B. atrox antihesin was determined as follows. The purified polypeptide was pyridethylated in 8M guanidine-HCl with dithiothriotol at pH 8.5, followed by HPLC on a VYDAC C4 column to remove salts. The sample was then cleaved with CNBr in 5M guanidine-HCl/70% formic acid using an overnight incubation at room temperature. The CNBr digest was washed, lyophilized and injected into HPLC. Of the four fragments obtained, a 13 kDa fragment was selected for limited N-terminal sequencing. The sequence determined is as follows: Val-Leu-Pro-Gly-Thr-Xaa-Xaa-Ala-Asp-Gly-Xaa-Val-Val-Ser (SEQUENCE ID N0:1), wherein each Xaa represents an undetermined amino acid.

The amino acid composition of the B. atrox 50 kDa antihesin was determined by hydrolyzing samples in constant boiling 5.7 M HCl in vacuo at 107°C for 24-, 48- and 96-hour periods, followed by amino acid analysis on a Beckman Model 121 M amino acid analyzer using physiologic methodology. The number of cysteine/half-cystine residues was determined as cysteic acid after hydrolysis with dimethylsulfoxide/HCl (Spencer and Wold, Anal. Biochem., 32, 185-190 (1969)). Tryptophan was determined after hydrolysis with mercaptoethanesulfonic acid (Penke et al., Anal. Biochem., 60, 45-50 (1974)). The number of threonine and serine residues was extrapolated to zero time of hydrolysis. The resulting amino acid composition, indicating a 452-residue polypeptide, is set forth in Table 1, below. Examples 2-6

50 kDa Antihesin From Bothrops Jararaca, Bothrops

Moogenii, Crotalus Atrox, Crotalus Basiliscus

and Akistrodon Halys Bromhoffii Following the procedure of Example 1, the venom of the following snakes was screened for the presence of antihesin. Each venom (obtained in lyophilized form from Sigma Chemical Co., St. Louis, MO) was reconstituted in 0.9% NaCl at 10 mg/ml and screened at a final concentration of 10 microgram protein/ml: Agkistrodon acutus, A. bilineatus, A. contortrix contortrix, A. contortrix laticinctus, A. contortrix mokason, A. halys blomhoffii,

A. piscivorus leuostoma, A. piscivorous piscivorous, A. rhodostoma, Austrelaps superba, Bitis arietans, Bothrops asper, B. jararaca, B. moogeni, B. neuwiedi, B. nummifer,

B. schlegeli, Bungarus fasciatus, Bungarus multicinctus, Crotalus adamanteus, C. atrox, C. basiliscus, C. durissus terrificus, Dendroaspsis jamesonii, Naja melanoleuca, Naja naja atra, Ophiophagus hannah, Pseudechis australis, Sepedon hemachatus, Sisturus miliarius barbouri, Trimeresurus gramineus and Vipers ruselli. Of these, the crude venoms of five members of the Viperidae family, Crotalinae subfamily, showed strong inhibitory activity in an assay of platelet adhesion to collagen: Bothrops jararaca, Bothrops moogeni, Akistrodon halys blomhoffii. Crotalus basiliscus and Crotalus atrox. The first three species are South American vipers. The last-mentioned two species are North American rattlesnakes. Each antihesin migrated on SDS-PAGE as a single band with an apparent molecular weight of 50 kDa in both reduced and non-reduced gels, suggesting that the inhibitory protein, like that of B. atrox, is composed of a single polypeptide chain. After storage at -20°C, larger aggregates became visible, again apparently reflecting disulfide interchange.

The amino acid composition of four of the 50 kDa snake venom antihesins is set forth in Table 1. The values in parentheses are the numbers of each amino acid residue. The total number of residues is believed to be accurate within about + 3 residues. The residue numbers are based on a molecular weight of 50 kDa.

TABLE 1

Amino Acid Oomposition of Snake Venom Anti hesins

Residues/mol protein

Amino Acid B. atrox B. jararaca C. basiliscus A. halvs bromhoffii

Aspartic acid/Asparagine 63.6 ± 0.7 (64) 68.5 ± 0.2 (69) 64.4 ± 1.8 (64) 63.2 ± 1.1 (63)

Threonine 17.9 (18) 18.3 (18) 22.5 (23) 19.1 (19)

Serine 27.7 (28) 23.2 (23) 30.2 (30) 25.3 (25)

Glutamiσ acid/Glutamine 43.9 ± 0.6 (44) 47.4 ± 0 (47) 44.8 ± 0.1 (45) 42.7 ± 0 (43)

Proline 27.1 ± 1.2 (27) 24.7 ± 0.4 (25) 18.7 ± 1.1 (19) 23.3 ± 0.4 (23)

Glycine 36.6 ± 0.3 (37) 38.7 ± 0.6 (39) 32.9 ± 0.7 (33) 36.3 ± 0.5 (36)

Alanine 24.7 ± 0.1 (25) 23.3 ± 0 (23) 22.2 ± 0.3 (22) 26.0 ± 0.6 (26)

Valine 20.9 ± 1.1 (21) 21.9 ± 1.1 (21) 21.0 ± 0.5 (21) 24.2 ± 1.0 (24) Cysteine/half-Cystine 27.0 ± 0.9 (27) 35.5 ± 1.5 (36) 30.7 ± 1.1 (31) 28.7 ± 0.7 (29)

Methionine 13.0 ± 0.8 (13) 10.3 ± 0.8 (10) 5.02 ± 0.3 (5) 8.66 + 0.1 (9)

Isoleucine 22.1 ± 1.1 (22) 21.0 ± 1.2 (21) 18.9 ± 0.6 (19) 21.8 ± 1.2 (22)

Leucine 25.7 ± 0.8 (26) 22.6 ± 1.2 (23) 31.7 ± 0.6 (32) 26.3 ± 0.6 (26)

Tyrosine 17.4 ± 0.7 (17) 20.8 + 0.3 (21) 18.5 ± 0.2 (19) 23.5 ± 1.0 (24)

Phenylalanine 13.3 ± 3.2 (13) 13.0 ± 0.7 (13) 16.1 ± 0.3 (16) 12.9 ± 1.3 (13)

Lysine 26.6 ± 3.2 (27) 25.6 ± 1.4 (26) 18.9 ± 0.8 (19) 24.7 ± 0.5 (25)

Histidine 15.5 ± 0.2 (16) 16.5 ± 0.2 (17) 16.1 ± 0.3 (16) 16.5 ± 0.3 (17)

Arginine 11.6 ± 0.2 (12) 12.0 ± 0.3 (12) 21.0 ± 0.3 (21) 10.5 ± 0.3 (11)

Tryptophan 9.02 ± 0.2 (9) 5.87 ± 0.6 (6) 9.10 ± 0.6 (9) 7.44 + 0.3 (7)

Glucosamine 0 (0) 0 (0) 0 (0) 0 (0)

Galactosamine 0 (0) 0 (0) 0 (0) 0 (0)

Total residues 452 451 444 449

Example 7

50 kDa and 13 kDa Crotalus Atrox Antihesins

Venom was harvested from C. atrox glands (Biotoxins Inc., St. Cloud, FL) by standard techniques. The venom was fractionated by SEPHADEX G-100 chromatography and the fractions analyzed for antihesin activity according to Example 1. Partially purified 50 kDa and 13 kDa active polypeptides were further purified by reverse phase HPLC chromatography as in Exmaple 1. The purification was monitored by SDS-PAGE, as shown in Fig. 2 (Lane 1, molecular weight standards; lane 2, crude venom; lane 3, venom after SEPHADEX G-100 column chromatography; lane 4, 13 kDa antihesin after HPLC; lane 5, 50 kDa antihesin after HPLC). The 13 kDa C. atrox antihesin, unlike the 50 kDa polypeptides we have isolated, does not form aggregates after storage. The 50 kDa antihesin was subjected to CNBr digestion and HPLC as in Example 1. In excess of ten cleavage fragments were obtained. An 8 kDa fragment included the amino acid sequence, SEQUENCE ID NO:2, below. A 13 kDa CNBr digest fragment included SEQUENCE ID NO: 3, wherein Xaa represents an undetermined amino acid.

SEQUENCE ID NO: 2

Tyr Ile His Val Ala Leu Val Gly Leu Glu Ile Trp Ser Asn Glu

5 10 15

Asp Lys Ile Thr Val Lys Pro Glu Ala Gly Tyr Thr Leu Asn Ala

20 25 30

Phe Gly Glu Trp Arg Lys Thr Asp Leu Leu

35 40

SEQUENCE ID NO: 3

Tyr His Pro Xaa Thr Lys Asp Ala Asp Ala Lys Asp Tyr Ser Asn

5 10 15

Gly His Ser Val The purified polypeptide from three of the South American vipers (B. atrox, B. iararaca and A. halys blomhoffii) exhibited a strikingly high degree of similarity in amino acid composition, while less similarity was found with C. basiliscus. The South American venom proteins contained 13 methionine residues per molecule, while the protein from C. basiliscus contained only five. Comparison of the 50 kDa anti-adhesive polypeptides with other proteins revealed that the snake venom molecules are unusual in the large number of cysteine residues they posses (an average of 30 residues per molecule, or 6.5 mol%). The B. moogeni and C. atrox antihesins were not analyzed for amino acid composition.

Platelet Adhesion Assay

The purified polypeptides inhibit platelet adhesion to collagen in a competitive fashion as exemplified by the B. atrox antihesin in Fig. 4. The concentration required for 50% inhibition of platelet adhesion was approximately 10 microgram/ml, or 0.2 micromolar, assuming a molecular weight of 50 kDa. Increasing the preincubation time with the snake venom protein beyond one minute did not increase the extent of inhibition (data not shown).

The following experiment demonstrates that the antihesins function by binding to receptors on collagen, thereby blocking adhesion of platelets.

Example 8

Inhibition of Platelet Adhesion by Collagen

Binding of 50 kDa Croalus Atrox Antihesin

Microtiter plate wells were coated with collagen

(2 μg in 50 μl) for 2 hr at room temperature. The coating solution was removed and unreacted binding sites in the wells were blocked by incubation for 2 hr with Tyrodes buffer containing albumin. The buffer was removed and rows of 9 wells were incubated with trifluoracetic acid (vehicle) or 50 μl of different concentrations of 50 kDa C. atrox antihesin from 0.2 to 2 mg/ml. The first 3 wells in each row were incubated with antihesin for 5 min., the next 3 for 10 min. and the next 3 for 20 min. This was removed and the wells were incubated with Tyrodes buffer overnight. The next day the buffer was removed and 50 μl of radiolabeled platelets were added. After allowing the platelets to adhere for 1 hr at room temperature, free platelets were removed, the wells were washed and the adhered platelets were subjected to liquid scintillation counting. The results are set forth in Table 2.

TABLE 2

Platelet Count (cpm)

Antihesin

(μg/well) Collagen- 5 min. 10 min. 20 min.

Antihesin

incubation time

0 1696±69 1740±140 1765±10

10 1339±132 1646±110 1676±98

20 1140±110 1514±158 1593±223

50 754±28 1136±164 1248±19

100 437±100 847±90 754±177

The antihesin inhibited adhesion of platelets to collagen in a concentration-related fashion, with amounts of antihesin from 10 to 100 μg per well. When collagen was treated with antihesin for only five minutes, the inhibitor was most effective in preventing adhesion of subsequently added platelets.

Platelet Aggregation Assay

Purified polypeptide obtained after HPLC was tested for ability to inhibit platelet aggregation in citrated plate- let-rich plasma. At 5 microgram/ml, the B. atrox protein totally abolished platelet aggregation induced by 1 microgram/ml collagen but had no effect on the rate of platelet aggregation induced by ADP, epinephrine, platelet-activating factor or the thromboxane mimetic [1S-[1<a,2<b(5Z)3<a- (1E,3R*),4<a]]-7-[3-(3-hydroxy-1-octenyl)7-oxabicyclo- [2.2.1]hept-2-yl]-5-heptenoic acid (SQ 26655). See Fig. 5A (collagen, 1 microgram/ml), 5B (ADP, 5 micromolar), 5C (epinephrine, 5 micromolar), 5D (platelet-activating factor, 0.25 micromolar), and 5E (SQ 26655, 0.2 micromolar).

In addition, the purified polypeptides inhibited both calcium mobilization and release of [¹⁴C]serotonin in fura 2-loaded platelets stimulated by collagen in the presence of the feedback pathway antagonists (data not shown).

The preparation of an illustrative monoclonal antibody specific for antihesins is described in Example 9.

Example 9

Monoclonal Antibody to Bothrops Atrox Antihesin

A. Immunization

Three female BALB/c AnSkh mice 10 weeks old were immunized subcutaneously with 80 microgram purified B. atrox antihesin/mouse in RIBI ADJUVANT SYSTEM (RIBI, Hamilton, MT) on day 0. The latter consists of 0.5 mg monophosphoryl lipid A from S. minnesota, and 0.5 mg trehalose dimycolate from M. phlei lyophilized in 40 microliters of SQUALENE and 0.2 % TWEEN 80. The adjuvant, which enhances the immune response, was reconstituted in 1.0 ml sterile phosphate-buffered saline. Immediately before immunizations, a 1:1 emulsion of RIBI adjuvant and antigen was prepared. Immunizations were repeated at 3, 6, and 9 weeks. Ten days after each of these injections, blood was removed from the retro-orbital plexis of each mouse, and the mouse displaying the highest antibody titer was selected as the spleen donor. At week 12 the selected donor mouse was immunized by the intraperitoneal route with the same preparation previously used. Four days later the spleen of this mouse was aseptically removed and placed in a sterile plastic petri dish (15 × 60 mm) containing glucose/potassium/sodium solution (GKN). The latter consists of 137 mM NaCl, 110 mM glucose, 11 mM Na₂HPO₄, 5 mM NaH₂PO₄·H₂O and 5 mM KCl. The spleen was teased apart with sterile forceps and then transferred into a centrifuge tube which was placed on ice for two minutes to allow clumps to settle. The cell-suspension was transferred into another centrifuge tube and spun for ten minutes at 1200 rpm. After discarding the supernatant, the cells were resuspended in 5-10 ml of 0.17 M NH₂Cl (ice cold) and placed in ice for 5 minutes with occasional mixing in order to lyse red blood cells. The cell suspension was gently underlain into 10 ml of a 1:1 dilution of GKN:normal serum and centrifuged at 1200 rpm for ten minutes. Fetal bovine serum (FCS) was used as the normal serum. The cells were then washed thrice in Dulbeco's Modified Eagle's Medium (DME, GIBCO, Grand Island, NY). The number and viability of the cells were then determined.

SP2/O-Agl4 myeloma cells used in the hybridization procedure were washed in the same way as the unlysed splenocytes.

B. Hybridization

Fusion was carried out as follows. 1.5 ml of immune splenocytes and 1.5 ml of SP2/O-Agl4 cells were pipeted onto a concanavalin A-coated plate. The cell concentration of each cell type was adjusted so that the ratio of splenocytes to SP2/O-Agl4 cells was 2-3:1, with a total of 7-10 × 10⁷ cells/plate. The plates were then incubated in 5% CO₂ at 37°C for 45-60 minutes to allow for attachment of the cells to concanavalin A. Fusion was performed by adding 1 ml of PEG 1500 (Boehringer Mannheim Biochemicals, Indianapolis, IN) dropwise to each plate. One minute after the addition of the first drop, the PEG solution was removed. The cells were then washed twice with 5 ml of DME. Following addition of 5 ml of DME plus 20% FCS, per plate, the cells were incubated overnight.

C. Selection and Growth of Hybridomas Following overnight incubation, the cells from the above hybridization procedure were transferred into centrifuge tubes and spun at 1500 rpm for 15 minutes. The supernatants were discarded. The cells from each tube were resuspended in 50 ml of DME + HAT + 20% FBS + 50 microliters concentrated supernatant from a lipopolysaccharide-induced RAW 264.7 cell culture (ATCC TIB 71, Rockville, MD). The cell suspensions were transferred into 96-well plates (200 microliters/well). The plates were cultured at 37ºC in a humid 5% CO₂ atmosphere. Seven days later, approximately one-half of the medium was removed from each well and replaced with 100 microliters of DME + HAT + 10% FBS. On day 14 following the fusion and twice each following week, approximately one half of the medium was removed from each well and replaced with 100 microliters of DME + HAT + FBS. Beginning on day 14 following the fusion, all cultures were examined microscopically for the appearance of hybridomas. When hybridomas achieved 50% confluence, supernatant fluids were removed and tested for the presence of specific antibody.

D. Purification of Monoclonal Antibody

Antibody from ascites fluid prepared above may be purified by Protein-A affinity chromatography using a commercially available kit (AFFI-GEL PROTEIN-A, BioRad

Corp., Richmond, CA). Ascites fluid was diluted 2:1 with

1,1,2-trichloro-l,2,2-trifluoroethane (LIPOCLEAN, Behring

Diagnostics, LaJolla, CA), nutated 10 min. at ambient temperature and centrifuged 10 min. at 2200g at 4ºC. The upper aqueous layer was carefully removed and diluted 1:1 in a binding buffer. The crude material was applied to a Protein-A column, and the column was washed with binding buffer such that the absorbance at 280 ran was less than 0.025. The antibody was then eluted with an acidic buffer. The peak of elution was pH neutralized, pooled, dialyzed against 150 mM NaCl, 10 mM Hepes, pH 7.4, concentrated and supplemented with 0.5% w/v sodium azide. The concentration of the antibody was determined by reading the absorbance at 280 nm and calculating the mg/ml by using a 1% extinction coefficient of 10.0. The final antibody concentration was in the range of 2-10 mg/ml.

E. Characterization of Monoclonal Antibody

The sub-class of the monoclonal antibody was determined with a mouse immunoglobulin subtype identification kit (Boehringer Mannheim). Monoclonal antibody 2C3-1 (ATCC HB-10904) was identified as sub-class IgG₁, kappa light chain. The quality of the final antibody preparation was determined by SDS-PAGE according to a modified procedure of Laemmli, Nature 227, 680-685 (1970). Under non-reducing conditions, the antibody was observed to migrate as a single band of approximately 200 kDa. Upon reduction, the purified antibody resulted in bands at 50 kDa and 28 kDa, representing the heavy and light chains of the IgG immunoglobulins.

A combined Western blot/ELISA technique (Towbin et al., Proc. Natl. Acad. Sci. USA 76, 4350 (1979)) was performed using either crude snake venoms or purified snake venom proteins to confirm whether other venom proteins possess the epitope recognized by the monoclonal antibody directed against the 50 kDa antihesin from B. atrox. The procedure, in brief, is as follows. Purified snake venom proteins or crude snake venoms were subjected to SDS-PAGE using a 12% acrylamide running gel and a 4% acrylamide stacking gel, under non-reducing condition. The SDS-PAGE gel was transferred to an electroeluation apparatus which electrically transferred the protein bands onto a poly vinylidene fluoride membrane (IMMOBILON, Millipore Corp., Bedford, MA). After transfer, the membrane was then blocked for 2 hours by incubation with Bovine Lacto Transfer Optimizer (BLOTTO) Johnson et al., Gene Anal. Tech. 1, 3 (1984)). The blocked membrane containing the proteins was then incubated for two hours at room temp, in BLOTTO containing 2 micrograms/ml of the purified mouse monoclonal antibody from ATCC HB-10904. The membrane was washed thrice with BLOTTO containing 0.1% TWEEN 20 detergent. The membrane was then incubated in BLOTTO containing an alkaline phosphataseconjugated antimouse IgG polyclonal antibody (Sigma Chem. Co.) for two hours at room temperature. The membrane was then washed thrice with BLOTTO containing 0.1% TWEEN 20, and developed in an alkaline phosphate substrate comprising nitro-blue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate (both from Sigma), which left a colored precipitate where antigen-monoclonal antibody-(alkaline phosphatase-polyclonal antibody)-complex was present.

The results are shown in Fig. 3. The monoclonal antibody raised against B. atrox antihesin recognized a single homogeneous protein of approximately 50 kDa in each of four crude venoms: Lane 1, C. basiliscus (20 micrograms); lane 2, B. atrox (200 ng); lane 3, B. jararaca (200 ng); lane 4, A. halys blomhoffii (200 ng). Molecular weight standards are shown to the right in Fig. 3. Based upon the strength of the immunoblotting signal, it is believed that the monoclonal antibody binds antihesin with an affinity of about 10⁸ mol^-1.

In the case of C. basiliscus, larger amounts of the venom were required for detection by immunoblotting as compared to the other snake venoms, indicating that the inhibitory protein in this venom was less immunoreactive.

Similar 50 kDa polypeptides which react with monoclonal antibody ATCC HB-10904, and have anti-adhesive properties, are present in the venom of Bothrops moogeni and Crotalus atrox. A single immunoreactive band at about 50 kDa was observed in these venoms.

Monoclonal antibody against antihesin of one snake venom may be used to purify antihesin from the same or different snake venoms. Example 10 describes the construction and operation of a representative immunoaffinity column for this purpose. Example 10

Immunoaffinity Column for Antihesin Purification

Monoclonal antibody such as ATCC HB-10904 is immobilized to form an immunoaffinity resin using commercially available agarose beads coupled to an activated hydroxysuccinimide ester (AFFIGEL 100, Bio-Rad Laboratories, Richmond, CA). Methods are well-established for coupling antibody to resins via hydroxysuccinimide esters. Covalent coupling of the antibody occurs through epsilon-amino groups of lysine in the protein. Coupling is performed using 2-20 mg/ml antibody in a phosphate or sodium bicarbonate buffer system, and is typically conducted overnight at 4°C. Residual reactive groups in the gel matrix are inactivated using 1 M ethanolamine-HCl, pH 8, for two hours at ambient temperature. The resulting immunoaffinity resin is then washed with a storage buffer supplemented with 0.5% sodium azide and stored at 4°C until required. When required, the immunoaffinity resin is packed into a suitable column and the snake venom or other biological source material to be purified is loaded onto the column. Proteins possessing the epitope recognized by the monoclonal antibody are retained in the column and all other nonadherent proteins are eluted using a wash buffer composed of 10 mM phosphate, pH 6.8. Specifically bound proteins are eluted from the column using 100 mM glycine, pH 2.5, thereby regenerating the immunoaffinity matrix. The column resin is reequilibrated using the wash buffer. The column is then ready for another cycle of use.

Purification of the antihesins to chemical homogeneity has permitted partial amino acid sequencing. It is contemplated that the antihesins may be prepared through genetic engineering techniques, utilizing either partial or complete amino acid sequence information. It is thus understood that the scope of the invention is not iimited to polypeptides isolated by following the chromatographic procedures disclosed herein, but also includes antihesin polypeptides as they may be prepared by genetic engineering techniques.

Based upon a complete amino acid sequence, one may prepare a synthetic gene corresponding to the sequence and introduce the gene into an appropriate host by cloning vectors. Alternatively, it is contemplated that antihesin may be obtained by recombination and cloning of the appropriate native gene obtained from venom producing cells.

Based upon a partial amino acid sequence, such as the partial sequences disclosed herein for the Crotalus atrox and Bothrops atrox 50 kDa antihesins, one may prepare suitably labeled synthetic oligonucleotide probes for screening a cDNA library prepared from snake venom glands. An appropriate cDNA library may be prepared according to any of the known techniques for preparing such libraries, such as the techniques described in Chapter 8 of Molecular Cloning: A Laboratory Manual, Second Edition, 1989 (J. Sambrook, E.F. Fritsch and T. Maniatis, editors). According to one methodology, a cDNA library is prepared from polyA⁺ mRNA using a snake venom gland. The library is constructed using, for example, the insertion vector λZAPII (Stratagene, La Jolla, CA) which is equipped with multiple cloning sites within plasmid sequences that can be excised in vivo and converted to a plasmid vector, Bluescript SK (M13-). λZAPII carries a polycloning site downstream from the E. coli lacZ promotor. See the map of λZAP/R in Molecular Cloning, supra. at page 2.52. λZAPII is equivalent to λZAP except that the Sam100 mutation has been removed to allow better growth of the bacteriophage, which, in turn, causes the plaques to become blue much sooner. cDNAs up to 10kb in length may be inserted into the λZAPII polycloning site and expressed in either infected bacteria or induced lysogens. The Bluescript SK(M13-) plasmid carrying the cloned DNA is excised in the presence of f1 or M13 helper bacteriophages, e.g., f1 R408 (Russel et al., Gene 45:333 (1986)).

According to one embodiment, λZAPII containing a cDNA library generated from snake venom gland genetic material is mixed and incubated with a plating bacteria, e.g. NM522, suitable for propagation of the λZAPII bacteriophage, and grown on agar plates. The plaques are transferred to nitrocellulose filters when they reach a diameter of approximately 1.5 mm. The plaques are lysed, washed and fixed to the nitrocellulose filters and hybridized overnight at 42°C using appropriate ³²P-labeled probes for antihesin genes. The probes may take the form of oligonucleotides synthesized on the basis of the least degenerate portions of CNBr cleavage fragments of the purified 13 kDa or 50 kDa antihesins. The oligonucleotides are end-labeled to high specific activity with ³²P using T4 polynucleotide kinase and gamma³²P-ATP. The nitrocellulose filters are dried following hybridization with oligonucleotide probe, and then autoradiographed to identify positive clones.

Plaques containing positive clones are recovered from the agar plates and treated with chloroform to release the λ-particles. Suitable host bacteria are coinfected with the release λ-particles and a helper phage, e.g., R408. Following incubation, the mixture is heated to kill the bacteria and inactivate the parent λZAPII, but not packaged Bluescript phage particles containing single stranded DNA (ssDNA) which are present in the supernatant following centrifugation. The ssDNA is isolated by standard methods well-known to those skilled in the art and analyzed by electrophoresis on agarose gels following EcoR1 digestion of the DNA to determine the size of the selected cloned inserts.

The identity of clones corresponding to antihesin mRNA is verified by excising ssDNA from the selected clones, as described above. The ssDNAs are ³²P-labeled and used as hybridization probes under high stringency conditions in

Northern blot analysis of mRNA purified from the appropriate snake venom gland, e.g., C. atrox gland. Detection of mRNA transcripts of approximately 0.5 kb and 1.4 kb in the

Northern blot indicates transcript sizes similar to those of the 13 kDa and 50 kDa antihesins, respectively. The mRNA identified by Northern blotting is recovered, solubilized and translated in vitro according to known techniques. The translated protein is then analyzed by SDS-PAGE, and then

Western blotted and tested for biological activity as described elsewhere herein.

DNA from the final selected clone(s) is sequenced using the Sanger dideoxy-mediated chain termination method utilizing oligonucleotide primers according to standard published methodologies (Sambrook, et al. Molecular Cloning,

(1989)). Following DNA sequencing, correspondence between portions of the deduced amino acid sequence obtained from the nucleotide sequence of the cDNA and the amino acid sequence of CNBr peptides derived from authentic snake venom protein are determined.

All references cited with respect to synthetic, preparative and analytical procedures are incorporated herein by reference.

The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof and, accordingly, reference should be made to the appended claims, rather than to the foregoing specification, as indicating the scope of the invention. SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANTS: Smith, J. Bryan

Dangelmaier, Carol

(ii) TITLE OF INVENTION: Inhibitors of CollagenInduced Platelet Aggregation (iii) NUMBER OF SEQUENCES: 3

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Temple University - of the

Commonwealth System of Higher Education

(B) STREET: 406 University

Services Building

(C) CITY: Philadelphia

(D) STATE: Pennsylvania

(E) COUNTRY: U.S.A.

(F) ZIP: 19122

(V) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Diskette, 3.50 inch, 720 Kb

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(D) SOFTWARE: WordPerfect 5.1 (vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Monaco, Daniel A.

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(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (215) 568-8383

(B) TELEFAX: (215) 568-5549

(C) TELEX: None (2) INFORMATION FOR SEQ ID NO:1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 amino acids

(B) TYPE: amino acid

(C) TOPOLOGY: linear

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

Val Leu Pro Gly Thr Xaa Xaa Ala Asp Gly Xaa Val Val Ser

5 10 15

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 40 amino acids

(B) TYPE: amino acid

(C) TOPOLOGY: linear

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

Tyr Ile His Val Ala Leu Val Gly Leu Glu Ile Trp Ser Asn Glu

5 10 15

Asp Lys Ile Thr Val Lys Pro Glu Ala Gly Tyr Thr Leu Asn Ala

20 25 30

Phe Gly Glu Trp Arg Lys Thr Asp Leu Leu

35 40

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 amino acids

(B) TYPE: amino acid

(C) TOPOLOGY: linear

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

Tyr His Pro Xaa Thr Lys Asp Ala Asp Ala Lys Asp Tyr Ser Asn

5 10 15

Gly His Ser Val

Claims

1. A substantially purified polypeptide corresponding to a polypeptide from snake venom, which polypeptide inhibits adhesion of platelets to collagen.

2. A polypeptide according to claim 1 comprising the following amino acid sequence wherein each Xaa, either the same or different, is any amino acid: SEQ ID NO:1.

3. A polypeptide according to claim 1 containing the following amino acid sequence: SEQ ID NO: 2.

4. A polypeptide according to claim 1 containing the following amino acid sequence: SEQ ID NO: 3.

5. A polypeptide according to claim 1 comprising the following amino acid residues determined by acid hydrolysis of the intact polypeptide, wherein each number following an amino acid residue designation comprises the number of such amino acid residues contained in the polypeptide, based upon a polypeptide weight of about 50 kDa:

combined aspartic acid and asparagine - 64, threonine - 18,

serine - 28,

combined glutamic acid and glutamine - 44, proline - 27,

glycine - 37,

alanine - 25,

valine - 21,

combined cysteine and half-cystine - 27, methionine - 13,

isoleucine - 22,

leucine - 26,

tyrosine - 17, phenylalanine - 13,

lysine - 27,

histidine - 16,

arginine - 12 and

tryptophan - 9.

6. A polypeptide according to claim 1 comprising the following amino acid residues determined by acid hydrolysis of the intact polypeptide, wherein each number following an amino acid residue designation comprises the number of such amino acid residues contained in the polypeptide, based upon a polypeptide weight of about 50 kDa:

combined aspartic acid and asparagine - 69, threonine - 18,

serine - 23,

combined glutamic acid and glutamine - 47, proline - 25,

glycine - 39,

alanine - 23,

valine - 21,

combined cysteine and half-cystine - 36, methionine - 10,

isoleucine - 21,

leucine - 23,

tyrosine - 21,

phenylalanine - 13,

lysine - 26,

histidine - 17,

arginine - 12 and

tryptophan - 6.

7. A polypeptide according to claim 1 comprising the following amino acid residues determined by acid hydrolysis of the intact polypeptide, wherein each number following an amino acid residue designation comprises the number of such amino acid residues contained in the polypeptide, based upon a polypeptide weight of about 50 kDa:

combined aspartic acid and asparagine - 64, threonine - 23,

serine - 30,

combined glutamic acid and glutamine - 45, proline - 19,

glycine - 33,

alanine - 22,

valine - 21,

combined cysteine and half-cystine - 31, methionine - 5,

isoleucine - 19,

leucine - 32,

tyrosine - 19,

phenylalanine - 16,

lysine - 19,

histidine - 16,

arginine - 21 and

tryptophan - 9.

8. A polypeptide according to claim 1 comprising the following amino acid residues determined by acid hydrolysis of the intact polypeptide, wherein each number following an amino acid residue designation comprises the number of such amino acid residues contained in the polypeptide, based upon a polypeptide weight of about 50 kDa:

combined aspartic acid and asparagine - 63, threonine - 19,

serine - 25,

combined glutamic acid and glutamine - 43, proline - 23,

glycine - 36,

alanine - 26,

valine - 24,

combined cysteine and half-cystine - 29, methionine - 9,

isoleucine - 22,

leucine - 26,

tyrosine - 24,

phenylalanine - 13,

lysine - 25,

histidine - 17,

arginine - 11 and

tryptophan - 7.

9. A polypeptide according to claim 1 which corresponds to a polypeptide from Bothrops atrox.

10. A polypeptide according to claim 1 which corresponds to a polypeptide from Bothrops jararaca.

11. A polypeptide according to claim 1 which corresponds to a polypeptide from Crotalus basiliscus.

12. A polypeptide according to claim 1 which corresponds to a polypeptide from Akistrodon halys bromhoffii.

13. A polypeptide according to claim 1 which corresponds to a polypeptide from Crotalus atrox.

14. A polypeptide according to claim 1 which corresponds to a polypeptide from Bothrops moogeni.

15. A substantially pure polypeptide which inhibits adhesion of platelets to collagen, which polypeptide contains an antigenic determinant which is recognized by monoclonal antibody from hybridoma ATCC HB-10904.

16. A monoclonal antibody which recognizes an antigenic determinant of a snake venom polypeptide, which polypeptide inhibits adhesion of platelets to collagen.

17. A monoclonal antibody according to claim 16 formed by fusion of cells from a myeloma line and spleen cells from a donor previously immunized with snake venom polypeptide from Bothrops atrox.

18. A monoclonal antibody according to claim 16 which recognizes an antigenic determinant of a venom polypeptide from one or more of the following species: Bothrops atrox. Bothrops jararaca, Bothrops moogeni, Akistrodon halys blomhoffii, Crotalus atrox and Crotalus basiliscus.

19. A monoclonal antibody according to claim 16 formed by fusion of cells from a myeloma line and spleen cells from a donor previously immunized with snake venom polypeptide which inhibits adhesion of platelets to collagen.

20. A monoclonal antibody according to claim 19 whwerein the myeloma line and spleen cells are murine.

21. A monoclonal antibody according to claim 20 wherein the hybridoma is formed by fusion of SP2/O-Ag14 myeloma cells and spleen cells from a BALB/c Anskh mouse.

22. A monoclonal antibody according to claim 21 produced by hybridoma ATCC HB-10904.

23. A method for producing a monoclonal antibody which binds to an antigenic determinant of snake venom polypeptide, which polypeptide inhibits adhesion of platelets to collagen, which method comprises the steps of

(a) immunizing a splenocyte donor with an immunogen containing snake venom polypeptide;

(b) removing the spleen from said donor and making a suspension of the spleen cells;

(c) fusing said spleen cells with myeloma cells; (d) diluting and culturing the fused cells in separate wells in a medium which will not support the unfused myeloma cells;

(e) assaying the supernatant in each well containing a hybridoma for the presence of antibody to snake venom polypeptide which inhibits adhesion of platelets to collagen; and

(f) selecting and cloning a hybridoma secreting antibody which binds to an antigenic determinant of said snake venom polypeptide.

24. A method according to claim 23 wherein the donor cells and the myeloma cells are murine.

25. A method according to claim 24 comprising the further steps of transferring said clones into mice and harvesting the malignant ascites or serum from said mice, said ascites or serum containing the desired antibody.

26. A method according to claim 24 comprising the further steps of culturing the hybridoma in a suitable medium and recovering the antibody from the culture supernatant.

27. A method for producing a monoclonal antibody which recognizes an antigenic determinant of a snake venom protein which inhibits adhesion of platelets to collagen, which method comprises culturing hybridoma ATCC HB-10904, and recovering the secreted monoclonal antibodies from the culture medium.

28. A method for producing a monoclonal antibody which recognizes an antigenic determinant of a snake venom protein which inhibits adhesion of platelets to collagen, which method comprises culturing hybridoma ATCC HB-10904, and recovering the secreted monoclonal antibodies from the mouse asσitic fluid or serum.

29. A composition comprising a continuous cell line producing monoclonal antibody, comprising a cell hybrid of a myeloma fused to a spleen cell from a donor previously immunized with an immunogen containing snake venom polypeptide which inhibits adhesion of platelets to collagen, and a culture medium for said hybrid.

30. The composition according to claim 29 wherein the spleen cell and myeloma are murine.

31. The composition according to claim 30 wherein the myeloma is SP2/0-Agl4.

32. The cell line ATCC HB-10904.

33. A method of inhibiting adhesion of platelets to exposed fibrillar collagen in a mammal comprising administering to the mammal a polypeptide according to claim 1.

34. A method of inhibiting adhesion of platelets to exposed fibrillar collagen in a mammal comprising administering to the mammal a polypeptide according to claim 15.

35. A method of inhibiting collagen-induced aggregation of mammalian platelets comprising incubating the platelets with a polypeptide according to claim 1.

36. A method of inhibiting collagen-induced aggregation of mammalian platelets comprising incubating the platelets with a polypeptide according to claim 15.

37. A method of inhibiting collagen-induced aggregation of platelets in a mammal comprising administering a polypeptide according to claim 1 to inhibit the occurence of platelet aggregation in the bloodstream of the mammal.

38. A method of inhibiting collagen-induced aggregation of platelets in a mammal comprising administering a polypeptide according to claim 15 to inhibit the occurrence of platelet aggregation in the bloodstream of the mammal.

39. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a polypeptide according to claim 1.

40. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a polypeptide according to claim 14.

41. A method for purifying from a liquid a polypeptide which inhibits the adhesion of platelets to collagen comprising contacting the liquid with an immobilized antibody which recognizes an antigenic determinant of the polypeptide to absorb the polypeptide from the liquid, and thereafter eluting the polypeptide from the immobilized antibody.

42. A method according to claim 41 wherein the antibody comprises monoclonal antibody.

43. A method according to claim 42 wherein the antibody comprises monoclonal antibody which recognizes an antigenic determinant of a venom polypeptide from one or more of the following species: Bothrops atrox, Bothrops jararaca, Bothrops moogeni, Akistrodon halys blomhoffii, Crotalus atrox and Crotalus basiliscus.

44. A method according to claim 42 wherein the monoclonal antibody is from hybridoma ATCC HB-10904.

45. A method for purifying from venom a polypeptide which inhibits the adhesion of platelets to collagen comprising (a) fractionating the venom to separate the polypeptides contained therein;

(b) assaying the venom fractions for activity in inhibiting the adhesion of platelets to collagen; and

(c) purifying from the active fractions a polypeptide which inhibits the adhesion of platelets to collagen.

46. A method for purifying from venom a polypeptide which inhibits the adhesion of platelets to collagen comprising

(a) fractionating the venom to separate the polypeptides contained therein;

(b) assaying the fractions for polypeptide which is recognized by monoclonal antibody from hybridoma ATCC HB-10904; and

(c) purifying from the active fractions a polypeptide which is recognized by said monoclonal antibody.