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1. WO1992008472 - AGENTS ANTITHROMBOSE

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[ EN ]

ANTITHROMBOSIS AGENTS

Technical Field
The invention relates to inhibitors of platelet adhesion. More specifically, it concerns proteins and peptides which inhibit the binding of von Willebrand

Factor (vWF) to the platelet glycoprotein GPIb-IX complex thus preventing platelet-vessel wall adherence. Some of these peptides are present in snake venom.

Background Art
Thrombosis within the cardiovascular system is believed to be the principal mechanism for vasoocclusive disorders, which are responsible for much morbidity and mortality in Western societies. Thus, antithrombotic drugs have been used extensively for treating many of these disorders (see, for example, Stein et al . ,
Circulation (1989) £0.: 1501-1513) . As with any group of drugs, however, none is entirely satisfactory for all individuals, and additions to the repertoire of possible therapeutic agents are always welcome.
The mechanism for the formation of thromboses is complex, but partially understood. An initial trauma, such as that initiated by rupture of atherosclerotic plaques or following mechanical removal of plaques during angioplasty, results in adhesion of platelets to the damaged vessel wall through platelet-to-nonplatelet interactions, and subsequent platelet aggregation
(platelet-to-platelet interactions) , in combination with deposition of fibrin. This sequence of events is controlled by the interaction of plasma proteins with specific platelet surface glycoprotein receptors. Since platelet adhesion is thought to be the initial response to injury, it is an especially desired target for
inhibition in order to prevent or ameliorate thrombosis and/or restenosis that may be mediated by adherent platelets.
Unstimulated circulating platelets contain receptors for several adhesive proteins. Among these are laminin, which binds to VLA 2 and VLA 6, and collagen, which binds to VLA 2, GPIV, and others. The initial attachment of platelets to the subendothelium is believed to be mediated by the binding of the GPIb-IX complex residing on the platelet surface to von Willebrand Factor (vWF) , which is immobilized in the vessel wall,
particularly under the high shear rate conditions found at the sites of arteriovascular occlusion. ' The platelet GPIb-IX complex, while functional generally on resting platelets, normally does not bind plasma-borne vWF.
Under normal circumstances, the arterial surface does not provide adhesive protein ligands (vWF) for platelet adherence; thus, platelet adhesion is limited to the vWF bound at the sites of vascular damage.
When the presence of bound vWF supports the adhesion of the platelets to the endothelium, the
platelets become activated and are then capable of forming platelet aggregates, associated with the binding of fibrinogen (Fg) and plasma-borne vWF, through the now- activated GPIIb-IIIa receptor. It follows that
substances which specifically inhibit the adhesion of the unactivated platelets through the GPIb-IX-bound vWF interaction would interrupt the formation of thromboses, especially in vessels wherein high shear stress is caused by stenoses.
The GPIb-IX complex, formed from the lb surface membrane heterodimer (Ibα and Ibβ) noncovalently
complexed with GPIX, is present at a density of
approximately 25,000 copies/platelet surface. The absence of this complex has been shown to be responsible for Bernard-Soulier Syndrome, a rare congenital bleeding disorder characterized by the absence of GPIb-IX complex displayed on the platelet surface and defective arterial adhesion of platelets. Defects in von Willebrand factor, such as those characterizing von Willebrand Disease also have been shown to lead to defective arterial adhesion of platelets.
Substances capable of interfering with
GPIb-IX/vWF interaction are known. Kirby et al, Thromb Haemostasis (1975) 3_4:770, reports that Evans' blue dye inhibits the ristocetin- induced binding of vWF to
formaldehyde-fixed platelets in vitro. Geratz et al,
Thromb Haemostasis (1978) , 3.9:411, showed the same effect by aromatic amidino compounds. Phillips et al, Blood
(1988) 22.:1898-1903, showed that ristocetin-induced platelet agglutination as well as shear-induced platelet aggregation in platelet-rich plasma was effectively inhibited by the triphenylmethyl compound aurin
tricarboxylic acid (ATA) at concentrations tenfold lower than those of other compounds previously described. ATA has also been demonstrated to be an effective inhibitor of coronary artery thrombosis in vivo (Strony et al,
Circulation (1989) 8J3: 11-23 (Abstract) ; PCT Application WO 89/04166.
Binding of vWF to GPIb-IX complex has also been inhibited by monoclonal antibodies immunoreactive with the GPIb-IX complex as disclosed by Ruan et al, Brit J Hae otol (1981) 49:511; and Coller et al, Blood (1983), £1:69) . The antibodies inhibit ristocetin-induced binding of vWF to platelets. Becker et al, Blood (1989) 24:690-694, showed that one of these antibodies or its immunoreactive fragments block GPIb function in guinea pigs in vivo, although they exert no effect on platelet aggregation induced by ADP, collagen, or thrombin.
Monoclonal antibodies immunoreactive with human vWF block platelet adhesion to collagen at high shear rates (Fressinaud et al, J Lab Clin Med (1988) 112:58-67, and Cadroy et al., Circulation (1989) 80:Suppl. II-24) . Murine monoclonal antibodies against porcine von Willebrand factor induce an antithrombotic state in normal pigs without effecting intrinsic platelet function (Bellinger et al., Proc Natl Acad Sci (USA) (1987)
£4:8100-8104) .
A proteolytic 45 kd fragment of glycocalicin (a GPIb fragment) and its derivatives inhibit binding of vWF to platelets, and these peptides can be used as
antithrombotic agents, as shown in EPO Publication No. 317278. Fragments of vWF also inhibit this binding, as described in Australian Patent Application AU87/73715. Vincente et al., J Biol Chem (1990) 265:274-280.
discloses peptides derived from GPIb which block
ristocetin- and botrocetin-induced binding of vWF to platelets.
The peptides of the present invention provide alternative approaches to antithrombosis by specifically inhibiting the binding of vWF with the GPIb-IX platelet- borne complex. These are effective agents in
antithrombotic therapies .

Disclosure of the Invention
The invention is directed to peptide inhibitors of platelet adhesion to the subendothelium which are
useful as antithrombotic agents. It has been found that a number of such proteins are present in snake venom, and methods to purify these proteins are provided. Methods for identification of those venoms which contain the
desired inhibitors are also described.
10 Thus, in one aspect, the invention is directed to antithrombotic agents which are inhibitors of
vWF/GPIb-IX interaction. More specifically these
peptides appear to bind to GPIb-IX and block the binding of vWF. These antithrombotic agents are collectively

15 called platelet antiadhesives. The platelet
antiadhesives found in snake venom, as described herein, are 24-28 kd peptides consisting of two nonidentical
disulfide-linked subunits of 12-14 kd each. Significant sequence homology exists between the two subunits of each

20 of the peptide anti-adhesives.
In another aspect, the invention is directed to methods to identify biological fluids, including snake venoms, which provide the platelet antiadhesives of the invention.
„.. In still other aspects, the invention is
directed to methods to prevent or ameliorate thrombosis by the use of the platelet antiadhesives of the
invention, and to pharmaceutical compositions containing these antiadhesives.
30
Brief Description of the Drawings
Figure 1 shows the chromatogram of CHH GPIb
inhibitor purified on CM-sepharose using NH40Ac gradient elution, monitored by absorbance at 254 nm.

Figure 2 shows active fractions from ion-exchange purification of CHH GPIb inhibitor analyzed on RPLC (C4, acetonitrile/TFA gradient elution) monitored at 214 nm.
Figure 3 shows results of SDS-PAGE performed on the active fractions of Figure 2.
Figure 4 shows the RPLC chromatogram of
purified CHH-B on a C4 column.
Figure 5 shows the RPLC chromatogram of
purified CHH-A on a C . column.
Figure 6 shows the amino acid sequences of the C. h. horridus GPIb inhibitor α and β chains.
Figure 7 shows the dose dependent inhibition of

12 t
I-von Willebrand factor binding to fixed-washed platelets induced by botrocetin by CHH-B GPIb inhibitor.
Figure 8 shows the RPLC chromatogram of
purified VCC GPIb inhibitor from Cerastes cerastes on a C4 column.
Figure 9 shows the dose dependent inhibition of ristocetin-induced platelet agglutination by purified
Crotalus h. horridus antiadhesive in PRP (upper panel) and the dose dependent inhibition of agglutination by
Cerastes cerastes antiadhesive.

Modes of Carrying Out the Invention
The platelet antiadhesives of the invention have certain in vitro properties which relate to their behavior as antithrombotic agents. All of the
antiadhesives of the invention fail to inhibit platelet aggregation induced by ADP, collagen, and thrombin; thus they are not inhibitors of Fg binding to GPIIb-IIIa and do not inhibit platelet aggregation. However, they do prevent the agglutination of unstimulated platelets in standard assays, such as those described by Allain, J.P., et al., J Lab Clin Med (1975) 85.:318, and Read, M.S., et al., J Clin Lab Med (1983) 101:74. They also inhibit the binding of labeled vWF to washed platelets using a standard assay such as that of Ruggeri, CM., et al, J Clin Invest (1983) 72:1-12.
The following are assays in which the platelet antiadhesives of the invention give positive results:

1) Inhibition of Ristocetin or Botrocetin Agglutination of Platelets:
In this assay, formaldehyde fixed-washed platelets prepared as described by Brinkhaus, A.M., et al, Meth Enzymol (1989) .169:149-143, are mixed with purified vWF prepared as described by Thorell, L., et al, Thromb Res (1984) 3_5: 431-450, and agglutination is initiated with either the antibiotic ristocetin or the snake venom agglutinin botrocetin. Increasing
concentrations of substances to be tested for platelet antiadhesive activity are incubated with the fixed platelets in the presence of vWF for 1 minute prior to addition of the agglutination inducer. Agglutination is measured in a commercially available aggregometer such as that supplied by Chronolog Corporation (Havertown, PA) and is a measure of the binding of vWF to the GPIb-IX complex.
(While some samples can be tested in platelet-rich plasma (PRP) instead of the fixed platelet assay, this form of the assay is not preferred for crude
materials such as snake venom, which may contain high concentrations of coagulating enzymes . ) 2) Inhibition of Labeled vWF Binding to
Platelets:
The antiadhesives of the invention also inhibit the ristocetin- or botrocetin-induced binding of
radiolabeled, biotinylated or otherwise labeled vWF to platelets. The platelets can be provided as washed platelets and the assay conducted as described by Ruggeri et al. (J Clin Inves (1983) 72.:1-12, supra), to glyco- calicin bound to ELISA plates, or to platelets or GPIX-Ib in any suitable form.

Thus, in general, the antiadhesives of the invention are positive both in assays which measure ability to inhibit platelet agglutination induced by ristocetin- or botrocetin and which measure ability to inhibit ristocetin- or botrocetin-induced vWF binding to platelets. However, they do not interfere with the binding of fibrinogen to the GPIIb-IIIa receptor.

Identifica ion of Sources of Antiadhesives
Biological fluids, such as snake venoms, which contain the platelet antiadhesives of the invention are effectively identified using the ristocetin/botrocetin agglutination inhibition assay with fixed-washed
platelets, as described above, or an assay which measures inhibition of vWF binding to GPIb-IX platelet-borne
complex.
Successful candidates are subjected to
Q purification procedures to obtain the platelet
antiadhesives in isolated and purified form. While a variety of protein purification techniques can be used, such as size chromatography, ion-exchange chromatography,

5 and reverse-phase HPLC, a typical and successful
procedure is as follows:
About 10-1000 mg of crude venom in lyophilized form is reconstituted in dilute acetic acid (0.5M) and applied to a sizing column, such as Sephadex G-50, and eluted in the same solvent. After lyophilization to remove acetic acid, fractions are assayed for
antiadhesive activity using the fixed-washed platelet botrocetin or ristocetin-induced agglutination assay with purified vWF, as described above.
Active fractions identified from the sizing column are then adsorbed onto carboxymethyl (CM) -sephacyl or diethylaminoethyl (DEAE) -sephacyl columns depending on the ionic charge of the antiadhesive in the snake venom, and eluted from the column using increasing ionic
strength ammonium acetate buffer. Fractions from the column are again lyophilized to remove the volatile salt and assayed using the platelet agglutination assay as described above. (In some cases platelet-rich plasma (PRP) can be substituted for the fixed-washed platelets at this stage if the clotting activity present in many crude snake venoms has been removed from active
fractions . )
Active fractions from the ion-exchange steps can then be purified using preparative reversed-phase liquid chromatography (RPLC) on C4 RPLC columns, such as Vydac, and eluting with gradients containing acetonitrile (2-70% acetonitrile) and 0.1% TFA/H20. The slope of the gradient and flow rate are optimized using routine procedures . Active fractions are determined by the platelet agglutination assay described above, now using PRP as the platelet preparation. The active fractions are then pooled, concentrated and tested for homogeneity using analytical HPLC or SDS-PAGE.
Antiadhesives of the invention, obtainable by the foregoing or other purification methods include those from venoms selected from the group consisting of :
Agkistrodon acutus . Agkistrodon halys blomhoffi.
Agkistrodon contortrix mokasen. Bitis arietans . Bitis caudalis , Bitis gabonica. Bitis g. rhinoceros , Bothrops asper. Bothrops alternata. Bothrops atrox. Bothrops cotiara. Bothrops ~iararac . Bothrops newiedi , Bothrops medusa . Bothrops schlegli. Cerastes cerastes , Cerastes vipera. Crotalus adamanteus . C. atrox. C. basilicus . C. durissus totonatacus . C. h. horridus . C. m. molossus. C. ruber. (C. scutalatus. C. v. cereberus . C. v. helleri . C. y_s_ lutosus . Cs. y_5_ oreganus . Echis carinatus sochurecki , Eristicophis macmahoni . Pseudocerastes persicus .
Sistrurus πu. barbouri. Sistrurus c. tergeminus .
Trimeresurus flavoviridis . Trimeresurus gramineus . Vipera lebetina, Vipera ammondytes . Vipera palastinae, and
Vipera r. russelli.
The purified platelet adhesion inhibitors of the invention are then sequenced using standard
procedures. The entire peptide may be sequenced, generally following reduction and alkylation of the cysteine residues in the native protein, which permits separation of individual subunits. Individual subunits can be proteolytically digested to generate fragments which are separated using RPLC and their protein
sequences determined using automated protein sequenators such as Applied Biosystems 473A Protein Sequenator.
Alternatively, the entire sequence of the protein may be determined by retrieving DNA encoding the antiadhesive from cloned snake tissue DNA libraries. A variety of methods are known to obtain such DNAs, including screening with oligonucleotide probes designed on the basis of partial amino acid sequences for the antiadhesives or expression screening using antibodies prepared to the purified antiadhesives.

Recombinant and other Synthetic Production
The platelet antiadhesives (PAA) of the invention can be produced in a variety of ways, includin using recombinant methods.
The genes encoding native PAA or mutants thereof can then be manipulated and expressed using a variety of recombinant systems. Host systems which do not process the translated protein can be used provided there is proper design of the expression system. For example, the expression system is constructed by placing an ATG start codon immediately preceding the desired N-terminus and a termination codon after the desired C-terminus, with appropriate modification of any adjacent sequences. The desired coding sequence is then ligated in operable linkage to a control system functional in procaryotic or eucaryotic hosts, as desired. A large number of control systems are now known in the ar .
If processing of the PAA is desired, certain eucaryotic systems may be advantageous. Attention shoul be paid to the choice of the recombinant host, which choice will determine the nature of the processing. It is also possible to prevent processing by modification o the gene sequence so as to encode substitute amino acids in positions believed to be susceptible to cleavage or glycosylation by proteolytic or glycosylating enzymes. For example, arginine or lysine could be replaced by threonine residues, thus rendering the resulting peptide non-susceptible to trypsin cleavage at those sites. In the alternative, expression can be effected in hosts which are deficient in enzymes capable of processing these peptides.
As the genes encoding the PAA are made
obtainable by the availability of the probes constructed from PAA-encoding DNA or by the availability of anti PAA antibodies, these genes can be manipulated by replacing the codons for one or more amino acids by site directed mutagenesis, to obtain sequences encoding analogs of these peptides which retain PAA activity.
Construction of expression vectors and
recombinant production of the appropriate DNA sequences are performed by methods known in the art per se.
Expression can be in procaryotic or eucaryotic systems. Procaryotes most frequently are represented by various strains of E. coli. However, other microbial strains may also be used, such as bacilli, for example Bacillus subtilis. various species of Pseudomonas , or other bacterial strains. In such procaryotic systems, plasmid vectors which contain replication sites and control sequences derived from a species compatible with the host are used. For example, E. coli is typically transformed using derivatives of pBR322, a plasmid derived from an E. coli species or pUC series vectors . Commonly used procaryotic control sequences, which are defined herein to include promoters for transcription initiation, optionally with an operator, along with ribosome binding site sequences, including such commonly used promoters as the beta-lactamase (penicillinase) and lactose (lac) promoter systems, the tryptophan (trp) promoter system and the lambda-derived PL promoter system compatible with procaryotes can be used.

The expression systems useful in the eucaryotic hosts comprise promoters derived from appropriate
eucaryotic genes. A class of promoters useful in yeast, for example, include promoters for synthesis of
glycolytic enzymes, including those for 3-phosphoglycerate kinase. Other promoters include those from the enolase gene or the Leu2 gene obtained from YEpl3.
Suitable mammalian promoters include the metallothionein promoter, the early and late promoters from SV40 or other viral promoters such as those derived from polyoma, adenovirus II, bovine papilloma virus or avian sarcoma viruses. Suitable viral and mammalian enhancers can also be used. In the event plant cells are used as an expression system, the nopaline synthesis promoter is appropriate. Insect cells may also be used as hosts in conjunction with the bacculovirus -based expression system.
The expression system is constructed from the foregoing control elements operably linked to the PAA encoding sequences using standard methods, employing standard ligation and restriction techniques which are well understood in the art. Isolated plasmids, DNA sequences, or synthesized oligonucleotides are cleaved, tailored, and religated in the form desired.
The constructed vector is then transformed into a suitable host. Depending on the host cell used, transformation is done using standard techniques
appropriate to such cells. The transformed cells are then cultured under conditions favoring expression of the PAA encoding sequence and the recombinantly produced protein recovered from the culture.

In addition to recombinant production, peptides whose deduced sequences are sufficiently short to make direct peptide synthesis practical can be prepared using 5 standard solid-phase techniques.
Thus, compounds within the scope of the present invention can be synthesized chemically by means well
known in the art such as, e.g., solid-phase peptide
synthesis. The synthesis is commenced from the carboxy- 0 terminal end of the peptide using an alpha-amino
protected amino acid, t-Butyloxycarbonyl (Boc)
protective groups can be used for all amino groups even though other protective groups are suitable. For
example, Boc-Val-OH, Boc-Leu-OH, Boc-Arg-OH or Boc-Tyr- 5 OH (i.e., selected BNP analog carboxy-terminal amino
acids) can be esterified to chloromethylated polystyrene resin supports. The polystyrene resin support is
preferably a copolymer of styrene with about 0.5 to 2% divinyl benzene as a cross-linking agent which causes the _,0 polystyrene polymer to be completely insoluble in certain organic solvents. See Stewart, et al., Solid-Phase
Peptide Synthesis. (1969), W.H. Freemen Co., San
Francisco, and Merrifield, J Am Chem Soc (1963) 85:2149- 2154. These and other methods of peptide synthesis are _,5 also exemplified by U.S. Patent Nos. 3,862,925;
3,842,067; 3,972,859; and 4,105,602.
The synthesis may use manual techniques or
automatically employing, for example, an Applied
Biosysterns 430A or 431A Peptide Synthesizer (Foster _ City, California) following the instructions provided in the instruction manual supplied by the manufacturer.
Of course, since automated synthesis also
permits control of the sequence, the above-mentioned
modifications to the amino acid sequence obtained by 35 modifying the gene as described above are available using this method of synthesis. In addition, it is not
necessary that the substituted amino acid be encoded by a gene. Therefore, the D- forms or beta-amino acids can be substituted for those natively present.

Preparation of Antibodies to the Antiadhesives
The invention platelet antiadhesives can also be utilized in immunization protocols to obtain antisera immunospecific for the invention compounds. The
resulting PAA compounds can then be injected into
suitable mammalian subjects such as mice, rabbits, and the like. Suitable protocols involve repeated injection of the immunogen in the presence of adjuvants according to a schedule which boosts production of antibodies in the serum. The titers of the immune serum can readily be measured using immunoassay procedures, now standard in the art, employing the invention compounds as antigens.
The antisera obtained can be used directly or monoclonal antibodies may be obtained by harvesting the peripheral blood lymphocytes or the spleen of the
immunized animal and immortalizing the antibody-producing cells, followed by identifying the suitable antibody producers using standard immunoassay techniques.
Any of the invention compounds that are
relatively small haptens are advantageously coupled to antigenically neutral carriers such as the conventionally used keyhole limpet hemocyanin (KLH) or serum albumin carriers. Coupling to carrier can be done by methods generally known in the art. Coupling can be done using condensing agents such as dicyclohexyl carbodiimide or other carbodiimide dehydrating agents . Linker compounds can also be used to effect the coupling; both homobifunctional and heterobifunctional linkers are available from Pierce Chemical Company, Rockford, IL.

Administration and Utility
The platelet adhesion inhibitors are useful therapeutically to prevent platelet adherence and
thrombus formation and to prevent restenosis of arteries following invasive procedures such as angioplasty.
Indications appropriate for such treatment include, without limitation, atherosclerosis and arteriosclerosis, acute myocardial infarction, chronic unstable angina, transient ischemic attacks and strokes, peripheral vascular disease, arterial thrombosis, preeclampsia, embolism, restenosis and/or thrombosis following
angioplasty, carotid endarterecto y, anastomosis of vascular grafts, and chronic cardiovascular devices
(e.g., in-dwelling catheters or shunts "extracorporeal circulating devices"). These syndromes represent a variety of stenotic and occlusive vascular disorders thought to be initiated by platelet adherence followed by activation of platelets, release of platelet granule contents which include potent growth factor which may be responsible for vascular smooth muscle proliferation in the neointimal layer of the vessel wall leading to restenosis, and subsequent platelet thrombus formation on damaged arterial vessel walls.
The platelet adhesion inhibitors may be used for prevention or abortion of arterial thrombus
formation, in unstable angina and arterial emboli or thrombosis, as well as treatment or prevention of
myocardial infarction (MI) and mural thrombus formation post-MI. They may be coadministered with thrombolytic agents such as streptokinase or plasminogen activators in order to inhibit platelet adherence and subsequent reocclusion of arteries.
In addition these antithro botics can be used to suppress thrombosis and restenosis resulting from organ transplantations and can thus be used to prevent thrombosis-mediated 'organ rejection and transplant-induced atherosclerosis.
The platelet adhesion inhibitor dosage can range broadly depending upon the desired effects and the therapeutic setting. Typically, dosages will be about 0.001 and 10 mg/kg of the body weight of the individual. Administration is preferably parenteral, such as
intravenous on a daily basis, for up to a week or as much as one or two months or more, which will vary dependent on the therapeutic regimen. If peptide fragments of the platelet adhesion inhibitor are used, other routes of administration can be utilized, such as intranasally, sublingually, or the like.
Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Suitable excipients are, for example, water, saline, dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate, cysteine
hydrochloride or the like. In addition, if desired, the injectable pharmaceutical compositions may contain minor amounts of nontoxic auxiliary substances, such as wetting agents, pH buffering agents, and the like. If desired, absorption-enhancing preparations (e.g., liposomes) may be utilized.
The following examples are intended to
illustrate, but not to limit, the invention.

Example 1
Identification of Platelet
Antiadhesive-Containing Snake Venom
For the assay, purified human vWF was prepared from human plasma cryoprecipitate using the procedure of Thorell et al., (supra) and formaldehyde-fixed washed platelets were prepared and assayed as described by
Brinkhous and Read, (supra) . Ristocetin (1.5 mg/ml final concentration) or botrocetin (lOμg/ml final
concentration) were used to initiate platelet
agglutination.
Seventy-three crude, lyophilized snake venoms obtained from either Sigma Chemical Company (St. Louis, MO) or Miami Serpentarium Labs (Salt Lake City, UT) dissolved in distilled H20 at a concentration of 10 mg/ml were subjected to preparative ultrafiltration using
Centricon-10 and Centricon-30 (YM Membrane) microcon- centrators (Amicon, Danvers, MA) . Both filtrates (10 and 50 μl samples) and retentates (10 and 50 μl samples) were used as test samples in the platelet agglutination assay utilizing the prepared fixed-washed platelets with purified vWF. Inhibitory activity was only found in retentate samples from the Centricon-10 and -30
ultrafiltrations . The results are shown in Table 1.

Table 1
Snake Venoms Screened for Anti-GP lb Activity
(Centricon-30 Retentates)

Elapidae Activity

Bungarus caerulus
Bungarus fasciatus
Dendroaspis jamesoni
Naja naja
Naja melanoleuca
Notechis scutatus scutatus
Ophiophagus hannah
Pseudechis porphyriacus
Pseudonaja textilis textilis
Tropidechis carinatus

Viperinae Activity

Bitis arietans +

Bitis caudalis +

Bitis gabonica +

Bitis g. rhinoceros + Bitis nasicornus
Causus rho beatus
Cerastes cerastes +

Cerastes vipera +

Echis carinatus sochurecki +

Echis carinatus leakyi +

Eristicophis macmahoni +

Hypnale hypnale +

Pseudocerastes persicus +

Vipera ammondytes + Vipera aspis
Vipera berus Table 1 (continued)
Vipera lebetina + Vipera palastinae + Vipera r. russelli +

Crotalinae
Agkistrodon acutus
Agkistrodon bilineatus
Agkistrodon c. contortrix
Agkistrodon c laticinctus
Agkistrodon c mokasen + Agkistrodon h blomhoffi + Agkistrodon p leucostoma
Agkistrodon p piscivorous
Agkistrodon rhodostoma
Bothrops atrox + Bothrops asper + Bothrops alternatus + Bothrops cotiara + Bothrops jararaca +

Bothrops lansbergi
Bothrops medusa
Bothrops nasuta
Bothrops newiedi
Bothrops pradoi
Bothrops schlegli +

Crotalus adamanteus +

Crotalus atrox +

Crotalus basilicus +

Crotalus cerastes +

Crotalus d. durrisus +

Crotalus d. totonatacus +

Crotalus d. terrificus 472

•21-

Table 1 (continued)
Croatlus h. horridus + Crotalus m. molossus + Crotalus r. ruber + Crotalus scutalatus + Crotalus v. cereberus + Crotalus v. concolor
Crotalus v. helleri + Crotalus v. lutosus + Crotalus v. oreganus + Crotalus v. viridis
Sistrurus c. tergeminus + Sistrurus m. barbouri + Trimeresures albolabris + Trimeresurus elegans + Trimeresurus flavoviridis + Trimeresurus gramineus + Trimeresurus purpureomaculatus
Trimeresurus wagleri

The antiadhesion activity is present in some but not all species of Viperinae and Crotalinae, but absent in all species of Elapidae tested.

Example 2
Purification of Platelet Antiadhesive From
Crotalus horridus horridus Venom
A solution of 500 mg of Crotalus horridus horridus venom (Miami Serpentarium Labs, Lot #CH18SZ) in 7.0 ml of 0.5 M acetic acid was applied to a column of Sephadex G-50 (fine) (Pharmacia, 2.5 x 100 cm)
equilibrated and eluted with 0.5 M acetic acid. The column was eluted at a flow rate of 25 ml/hr with 5 ml fractions being collected. Ten μl of each fraction was pooled in groups of 10 fractions (i.e., fractions 1-10,
11-20, 21-30, etc.) and lyophilized for analysis. The freeze-dried fractions were resuspended in 500 μl of
distilled water and aliquots assayed for inhibitory
activity in ristocetin induced platelet agglutination of fixed washed platelets reconstituted with purified vWF.
Active inhibitory fractions in this assay (31-40) were

10 pooled and lyophilized to obtain 95 mg of a white
amorphous powder.
This material was dissolved in 5 ml of 0.01M
NH40Ac, pH 4.5 and applied to a Carboxymethyl-sephacyl column (2.2 x 13 cm) equilibrated in 0.01M to 0.5M
-,5 NH40Ac, to pH 6.5 was run and fractions (12 ml) were
collected. The column was monitored at 254 nm/1.0 AUFS to identify fraction with UV absorbance (Figure 1) . UV absorbing fractions were individually lyophilized (20 μl each) , resuspended in 1 ml of distilled H20 and assayed

20 for their ability to inhibit platelet agglutination.
Fractions 13 through 17 displayed inhibitory activity and were individually lyophilized from H20 three times to
remove excess NH40Ac.
Analysis of the peak inhibitory fractions by

25 SDS-polyacrylamide gel electrophoresis (non-reducing) revealed two major proteins that migrated with Mr=23-28 kd. Analysis of these fractions (lOOμg) using analytical reversed-phase C4 liquid chromatography (Vydac 214TP54,
0.46 X 25 cm, 1.0 ml/min flow, 300A, gradient elution

3_ from 15% acetonitrile/0.1% trifluoroacetic acid (TFA) to 70% acetonitrile in 30 min) revealed two major UV (214 nm) absorbing peaks (Figure 2) , which were collected and dried. Reanalysis of these two peaks by SDS-PAGE
revealed that the earlier HPLC eluting peak, termed CHH- 35 A, (24.9 min) migrated at Mr=23 kd and the later eluting peak, termed CHH-B (25.6 min) migrated at Mr=25 kd
(Figure 3) . Under reducing SDS-PAGE, CHH-A and CHH-B collapsed to two distinct protein chains M =12-15 kd. Native CHH-A and CHH-B were both able to inhibit
botrocetin and ristocetin induced platelet agglutination in the washed system and also in platelet rich plasma to the same degree .
Preparative purification of CHH-A and CHH-B from the ion-exchange fractions was accomplished on a semi-preparative C4 (214TP510, 3.5 ml/min) column using the same gradient elution conditions as the analytical analysis. Figure 4 shows the analytical C4 HPLC
chromatogram of purified CHH-B and Figure 5 shows the chromatogram for CHH-A. (The peak at 34 min in each figure is an artifact) .
A portion of the purified protein (CHH-B) was reduced and alkylated (6M guanidine-HCl , 0.25 M Tris-HC1, 20 mM EDTA, containing 20 mM dithiothreitol (DTT) , pH 7.5, 8 hr 25°C. Excess iodoacetamide was added to the reduced protein for 8 hr at room temperature. The reduced and alkylated chains were separated from each other using C4 reversed-phase HPLC. The earlier eluting subunit (CHH-B-β) and later eluting subunit (CHH-B-α) were individually submitted to N- erminal sequence analysis (Edman degradation) using an Applied Biosystem 473A protein sequenator.
The following N-terminal amino acid sequence was obtained for 37 cycles of Edman degradation for CHH-B-α: Asp-Leu-Glu-Cys-Pro-Ser-Gly-Trp-Ser-Ser-Tyr-Asp-Arg-Tyr-Cys-Tyr-Lys-Pro-Phe-Lys-Gln-Glu-Met-Thr-Trp-Ala-Asp-Ala-Glu-Arg-Phe-Cys-Ser-Glu-Gln-Ala-Lys . The complete amino acid sequence for this chain which
contains 127 amino acids is shown in Figure 6.
The following N-terminal amino acid sequence was obtained for 27 cycles for CHH-B-β: Asp-Cys-Pro- Ser-Asp-Trp-Ser-Ser-Tyr-Glu-Gly-His-Cys-Tyr-Arg-Val-Phe-Gln-Gln-Glu-Met-Trp-Asp-Asp-Ala-Glu-Lys-Phe. The
complete amino acid sequence for this chain in shown in Figure 6.
The purified peptide CHH-B was tested in the vWF-platelet binding assay as performed according to the procedure of Ruggeri, et al. (supra) . The results are shown in Figure 7. Binding inhibition is dose dependent and concentrations of pure CHH-B at less than 200 nM inhibited the binding of vWF to the washed platelets.

Example 3
Purification of Antiadhesive From the Venom
of Cerastes cerastes
A solution of 500 mg of Cerastes cerastes venom (Miami Serpentarium Labs, Lot #CC12SZ in 5.0 ml of 0.5 M acetic acid was applied to a column of Sephadex G-50 (fine) (Pharmacia, 2.5 x 100 cm) equilibrated and eluted with 0.5 M acetic acid. The column run at a flow rate of 40 ml/hr with 8 ml fractions collected into polypropylene tubes. Twenty five μl of each fraction was pooled in groups of 10 fractions (1-10, 11-20, 21-30, etc.) and lyophilized. Freeze-dried fractions were resuspended in 100 μl of distilled water and aliquots assayed for inhibitory activity in ristocetin induced platelet agglutination of fixed-washed platelets reconstituted with purified vWF. Active fraction in this assay
(fractions 21-40) were pooled and lyophilized to obtain 198 mg of a white powder.

This material was dissolved in 10 ml of 0.01M
NH40Ac, pH 4.5 and applied to a CM-sepharose column (2.2 x 13 cm) . A pH and salt gradient running from 0.01M to 5 0.5M NH40Ac, to pH 6.5 was run and fractions (10 ml)
collected into polypropylene tubes. The column effluent was monitored at 254 nm/1.0 AUFS to identify fractions with UV absorbance. Fractions were again pooled with 25 μl from each fraction into groups of 10 fractions and _ lyophilized. The fractions were redissolved in 100 μl of distilled H20 and assayed for their ability to inhibit platelet agglutination. Fractions 81-100 displayed
inhibitory activity which were pooled and lyophilized
from H20 three times to remove excess NH40Ac.
5 Analysis of the inhibitory fractions by SDS- PAGE (non-reducing) revealed one major protein band which stained that migrated with Mr=23-28 kd. Under reducing
SDS-PAGE, the native protein collapsed to two distinct protein chains Mr=12-15 kd. Analysis of the fractions on Q analytical C4 reversed-phase liquid chromatography (Vydac 214TP54, 0.46 x 25 cm, 1.0 ml/min flow, 300A) gradient elution from 30% acetonitrile/0.1% TFA to 70%
acetonitrile in 30 min revealed one major UV (214 nm)
absorbing peak as shown in Figure 8 (the peaks at 27 and 31 min are artifacts) .
A portion of the purified protein (CO was
reduced and alkylated with iodoacetamide as in Example l. The carboxyamidomethylated chains were separated using a reversed-phase Vydac-Phenyl column. The earlier eluting 0 subunit (CC-β) and later eluting subunit (CC-o)were
individually submitted to N-terminal sequence analysis using the Applied Biosystems 473A protein sequenator.
The following N-terminal amino acid sequence was obtained for 25 cycles of Edman degradation for CC- β: Leu-Asp-Cys-Pro-Leu-Asp-Ser-Ser-Xaa-His-Glu-Glu-Lys- Cys-Tyr-Lys-Val-Phe-Phe-Leu-Leu-Xaa-Thr- rp-Glu.
The following N-terminal amino acid sequence was obtained for 20 cycles for CC-x : Asp-Gin-Asp-Cys- Leu-Pro-Gly-Trp-Ser-Tyr-Tyr-Glu-Lys-Tyr-Cys-Tyr-Lys-Val- Phe-Glu.

Example 4
10 Purification of Antiadhesive From the Venom
of Pseudocerastes persicus
A solution of 1000 mg of Pseudocerastes
persicus venom (Miami Serpentarium Labs, Lot #PS8SZ) in
7.0 ml of 0.5M acetic acid was applied to a column of *5 Sephadex G-50f (Pharmacia, 2.5 x 100 cm), eluted and
assayed as described in Examples 2 and 3. Fractions
which were active (Fractions 31-50) were pooled and
lyophilized.
Approximately 160 mg of this material was
20 adsorbed and gradient-eluted from a CM-Sepharose column
(2.2 x 13 cm) using an NH40Ac buffer running from 0.01M to 0.5M NH40Ac. Active fractions (6 ml each, Fractions
64-72) were found to be active in the fixed-platelet
assay, pooled and lyophilized from water several times to

25 remove the volatile salt.
Final purification was effected by
semipreparative C4 reversed-phase liquid chromatography employing a gradient of acetonitrile in 0.1% TFA which ran from 15-70% acetonitrile in 30 minutes. A portion of

30 the material (300 μg) was reduced and
carboxyamidomethylated as described in Example 2. The carboxyamidomethylated chains were separated on C4 RPLC as described and the individual chains submitted to N- terminal sequence analysis.
35 The earlier eluting subunit, PP-β, was
subjected to Edman degradation for 50 cycles to obtain the following sequence: Asp-Cys-Pro-Ser-Asp-Trp-Ser-Ser-His-Glu-Gly-His-Cys-Tyr-Lys-Val-Phe-Asn-Leu-Tyr-Lys-Thr-Trp-Glu-Asp-Ala-Glu-Lys-Phe-Cys-Thr-Glu-Gln-Ala-Asn-Gly-Gly-His-Leu-Val-Ser-Ile-Asp-Ser-Lys-Lys-Glu-Ala-Asn-Phe.
The following amino acid sequence was
determined for 31 cycles of the PP-α chain: Ala-Leu-Asn-Cys-Ala-Ser-Gly-Trp-Ser-Ala-Tyr-Asp-Gln-His-Cys-Tyr-Lys-Ala-Phe-Asp-Glu-Pro-Lys-Ser-Trp-Ala-Asp-Asp-Glu-Lys-Phe.

Example 5
Purification of Platelet Antiadhesive
From Vipera r. russelli Venom
Using the methods of Examples 2-4, 1 gram of venom from Vipera r. russelli was purified to yield the GPIb inhibitor RV. A portion of the purified inhibitor was carboxyamidomethylated as described in Examples 2-4, and the subunits separated with . RPLC.
N-terminal sequencing of the later eluting subunit for 22 cycles revealed the following amino acid sequence: Gly-Phe-Ser-Cys-Pro-Asn-Gly-Trp-Ser-Ser-Phe-Gly-Arg-Tyr-Cys-Tyr-Lys-Pro-Ile-Glu-Pro-Leu.

Example 6
Inhibition of Botrocetin/Ristocetin- Induced Agglutination
The purified proteins from Cj. horridus
horridus . as prepared in Example 2, and from C_^ cerastes, as prepared in Example 3, were subjected. o the
botrocetin/ristocetin- induced agglutination inhibition assay using PRP, as described above. The results are shown in Figure 9. Again, a dose dependent effect is shown, concentrations as low as 2-3 μg/ml are able to show inhibition.