The method of determining the presence or absence of activity in the inhibition of platelet aggregation (pa1), peeled and dedicated inhibitor activity of platelets (pa1) and/or the shortened form, the method of cleaning pa1 from snake venom, the pharmaceutical composition to prevent the formation of a blood clot, a method of inhibiting the formation of thrombus

 

(57) Abstract:

The invention relates to medicine and relates to a method of evaluation of snake venom on the presence or absence of platelet aggregation (PA1), based on the specific binding with receptor purified and isolated from snake venom PA1 and its shortened standards, allocation method PA1 from snake venom, and pharmaceutical compositions based on it. Essence: the invention includes a method of determining in snake venom ability to inhibit platelet aggregation, the method of purification of an inhibitor of platelet activity of different samples of snake venom, its characterization and a truncated form containing the modified lysine residue, which specifically inhibits the binding of fibrinogen or factor a background of Villebranda GPIIb-IIIA. The advantage of the invention is to obtain a therapeutic agent capable of blocking or inhibiting the formation of blood clots without danger of hemostasis. 13 C. and 32 C.p. f-crystals, 4 tab., 43 Il.

The invention relates to a group of peptides that represent or are related to platelet aggregation inhibitors, isolated and purified from various snake venoms. These peptides are useful as terapevticheskii relates to peptides which block specific receptors for adhesive proteins included in the adsorption and platelet aggregation. In addition, this invention describes methods for the detection and purification of the above-mentioned peptides from snake venoms to substantial homogeneity, as well as methods of using the primary amino acid sequences of these polypeptides to produce active peptides as synthetic, and using techniques of recombinant DNA.

The basis of the invention

Heart disease is the leading cause of death in most Western countries. Death from heart disease is often caused by thrombocytopaenia ischemic syndromes, which are triggered by atherosclerosis and arteriosclerosis and include, but are not limited to, acute myocardial infarction, chronic changeable angina, transient ischemic attacks and seizures, peripheral vascular disorders, arterial thrombosis, preeclampsia, embolism and/or thrombosis after angioplasty, carotid endarterectomy, anastomosis of vascular transplants and chronic cardiovascular devices, for example, "in dwelling" catheters or shunts "devices extrakorporale circulation"/. These sindrilaru platelet activation or on the walls of the vessel, or inside the lumen of using "blood-borne mediators, but are revealed by platelet aggregates, which form a blood clot, restricting blood flow.

There have been numerous studies to understand the mechanism of formation of aggregates of platelets and blood clots. Platelets respond to various damage to blood vessels, such as narrowing of the lumen, plaque formation and the presence of foreign bodies such as catheters/ and the like. The response of platelets to these injuries is the sequence of events, including adhesion and activation, and selection trombotsitnoy granular components, including the possible cell mitogenic factors. Activated units of platelets causes the formation of fibrin, which further stabilizes the clot.

Currently, much is known about the mechanisms regulating these reactions. Although estimulando platelets contain receptors for several adhesive proteins, including laminin /VLA2, VLA6/ and collagen /VLA2, GPIV and others), the initial adherence of platelets to the subendothelial layer, as expected, is an intermediate in the binding membrane glycoprotein of the platelet /GP/ Ib immobilizovannym facto is no known physiological substances, possessing affinity for the receptor, including: ADP, epinephrine, thrombin, collagen and thromboxane A2.

Platelet aggregation mediasuite GP IIb-IIIa complex on the membrane surface of the platelet. GP IIb-IIIa exists on the surface of unstimulated platelets in an inactive form. When platelets are activated by adhesion and physiological agonists, GP IIb-IIIa also becomes activated, such that it becomes a receptor for fibrinogen /Fg/, the factor a background of Villebranda /VWF/ and fibronectin /Fn/ /see Phillips et al., Blood /1988/ 71, 831-843/, but it is the binding of fibrinogen and/or factor von Willebrand's disease, which are believed to be mainly responsible for platelet aggregation and the formation of thrombus in vivo. Therefore, substances which specifically inhibit the binding of fibrinogen or factor von Willebrand with GP IIb-IIIa, inhibit platelet aggregation and could be candidates for inhibiting the formation of thrombus in vivo.

It is now known that GP IIb-IIIa platelet is a member of supercilii structurally related receptors, adhesion proteins, known collectively as "integrins". As hitherto known, all integrins, similar to GP IIb-IIIa represent the DDA IIIa/. There is a high degree of homology between known sequences of integrin subunits, indicating that integrins have evolved from conventional predecessors. Integrins function in a number of cell adsorbsia and were found in leukocytes, endothelial cells, smooth muscle cells and other cells in the vascular network. Because integrins are widely distributed, while the GP IIb-IIIa limited to platelets, the preferred antiregime agent would be inhibited selectively GP IIb-IIIa in contrast to other integrins.

It has been described several classes of peptides that block the binding of adhesive proteins to activated platelets and inhibit platelet aggregation /see Hawiger et al., U.S. patent 4661471 and Rouslahti et al., U.S. patents 4614517, 4578079, 4792525 and UK application GB 2207922 A/. In one class of peptide sequence RGD is crucial tetrapeptide sequence RGDS, RGDT, RGDC, in particular, were used. Amino acid sequence RGDK found in a number of adhesive proteins, including Fg, Vn, vWF and Fn. It was shown that this sequence plays an important role in the interaction of the adhesive protein receptors adgezivnosti, for example, Pierschbacher, M. D., et al., J. Biol. Chem. /1987/, 262, 17294-17298, Ruggeri et al., Proc. Natt. Acad. Sci (USA) /1986/ 83: 5708-5712, and Rouslanti et al., Cell /1986/ 44: 517-518. Tetrapeptide containing this sequence are described in the application for the European patent 319506, published on 7 June 1989. Short peptides containing homoarginine instead of arginine in RGD sequences disclosed in PCT application W089/07609, published on August 24, 1989.

Structural changes allowed in RGD-containing peptides have been studied Pierschbacher, M. D. et al., J. Biol. Chem. /supra/. In these studies it was found that the manipulation of RGD-containing sequence is not only influenced the activity associated with inhibition of binding of fibronectin or vitronectin with the substrate, but could make the differentiation between binding of the two ligands. GRGDSPC peptide sequence, which was taken from the cell domain of fibronectin was used as a model peptide. It turns out that some substitutions, such as replacing L-Arg D-Arg, with no effect on the binding of any ligand, but the substitution of D-Ala to Gly or D-Asp on Z-Asp destroys inhibitory activity. While the substitution of D-Ser in Z-Ser lowered inhibition interaction with vitronectin receptor VI is operating on Asn Ser resulted in a peptide which had increased the inhibition of binding of fibronectin and reduced effect on the binding of vitronectin. Other substitutions at Ser had other effects. Threonine is substituted for Ser, gave a peptide with increased inhibition of the receptor binding of vitronectin, the substitution of L-Pro was brought to an inactive peptide. The cyclic peptide was also obtained from the sequence Gly-Pen-Gly-Arg-Gly-Asp-Ser-Pro-Cys-Ala, where "Pen" represents penicillamine and disulfide bridge was formed between the Pen and Cys. According to the authors, penicillamine have a function of increasing conformational constraints on the cycle, whereas the N-terminal Gly and carboxy-terminal Ala were added to the distance of the free amino and carboxyl groups from the cycle. This cyclic peptide was able to inhibit the binding of vitronectin more strongly than the same peptide prior to cyclization, but was ineffective in the inhibition of binding of fibronectin.

Recently Samanen, J. , et al., J. Cell Biochem. /1990/ Suppl. 14:A229 been reported the antimicrobial peptide with a modification of RGD-sequence, with "R" alkilirovanny balance. A review of the correlation of structure and activity in RGD-containing peptides, was published Azi F. E. et al. , in Proc. 11-th Am. Peptide Symp., Marshall et al., ed ESCOM two groups of peptides, one linear and the other circular, which, as indicated above, connect the GP IIb-IIIa receptors on platelets and thus inhibit its ability to bind vWF, fibronectin and fibrinogen-fibrin. There are no data that relate to the binding specificity of these peptides. The group of cyclic peptides includes modification of RGD-sequence, in which R is replaced by D or Z homoarginine, dimethyl or diethylamine, lysine or alpha-alkilirovanie derived from these residues. Minimum cyclic structures include just "R" GD sequence contained between the two residues which form a disulfide bridge.

A separate class of inhibitory peptides uses peptide sequence, modeled on the carboxyl terminal sequence obtained from the gamma chain of fibrinogen, dodecapeptide HHL GGAQKAGDV (Kloczewiak et al., Biochemistry /1989/ 28: 2915-2919; Timmons et al. (ibid), 2919-2923; U.S. patent 4661471 /supra/; the application for the European patent 298820). Although this sequence inhibits the binding of Fg and vWF to GP IIb-IIIa and subsequent platelet aggregation, the usefulness of this peptide is limited because of its low epinasty interaction with receptors on the platelet /IC50= 10 - 100 microns/.

Included in this group of inhibitors peptides from snake venoms are "Alboran" isolated from Trimeresurus albolabris, elegantin isolated from T. elegans, flavoviridis isolated from T. flavoviridis, bitrocket statin isolated from Bothrops atrok, baystation isolated from Bitis arietans described Niewiarowski, S., et al., Fhroneb Haemostas /1989/ 62:319 /Abstr SY of Ukraine halys, which was described by Huang, T. F., et al., Fhrombhaemostas /1989/ 62:48 /Abstr 112/. All these peptides show a high degree of sequence homology. In addition, all peptides from snake venoms described to date, which inhibit the binding of adhesive proteins with integrin receptors contain PGD-sequence.

Although these described operating beginnings of snake venom are potent inhibitors of platelet aggregation in vivo, these peptides also bind with high affinity with other elements of receptors for adhesive proteins such as vitronectin receptor or fibronectin /Knudsen K. A., et al. , Exp. Cell. Res. /1988/ 179: 42-49, Rucinski, B., et al., Troneb Haemostas /1989/ 62: 50 /Abstr. 120//. This lack of specificity factors snake venom for GP IIb-IIIa is an undesirable feature of their therapeutic use as inhibitors of blood clots, as they have a possible influence on the adhesive properties of other cells in the vascular system, particularly the properties of adhesion through integrin.

Another approach, developed for the generation of inhibitors trombotsitnoy clots, was the use of "murine anti-GP IIb-IIIa monoclonal antibodies that block the binding of edgetrade coronary arterial reocclusion after reperfusion tissue plasminogen activator in dogs /Yaneda, T., et al., J. Clin Inwest /1988/ 81: 1284-1292/ and to prevent the cyclic reduction of flow in injured coronary arteries of dogs with a high degree of stenosis in dogs. Possible side effects of the use of such monoclonal antibodies in humans may be the result of their long-term impacts and their potential immunogenicity.

It is clear that in order to prevent or at least reduce unwanted blood clots necessary additional therapeutic treatment regimen. In particular, therapeutic agents capable of blocking or inhibiting the formation of blood clots at a particular location without danger of hemostasis and without affecting other cell adhesion, would provide major therapeutic results. Ideally, these agents should be potent, specific IIb-IIIa and non-immunogenic for most patients, they must also be easy to introduce, stable, and economical to manufacture. Further, these agents must act short-term and be able to act at the earliest stages of thrombus formation without taking the long-term hemostasis. The present invention satisfies these and other analogion is to identify factors with a low molecular weight /< 10 CD/ in snake venom or other biological sources that specifically inhibit thrombus formation by platelet aggregation. This technique uses the notion that platelet aggregation is mainly carried out through the binding of fibrinogen and/or vWF to GP IIb-IIIa on the surface of platelets if platelet counts are processed by the appropriate stimulator, such as ADP. Using these criteria, i.e. the inhibition of binding of fibrinogen and/or vWF with a separate receptor and similar criterion associated with inhibition of binding of fibronectin /Fn/ fibronectin receptor /Fn/ FnR-binding/ and vitronectin to vitronectin receptor /Vn/ VnR binding/ as well as the binding of other factors, such as Fn and Vn GP IIb-IIIa, can be quickly and easily obtained profile specificity for inhibitor of platelet aggregation /PAI/. This approach was used for screening and characteristics of a wide list of snake venoms on the presence or absence of PAI, for characterizing the specificity of PAI, identifikovano from this list, according to their specificity in the inhibition of binding to GP IIb-IIIa in contrast to the inhibition of other integrins, and for the identification of active peptides, cosob quick selection on the presence or absence of PAI in the biological fluid, which comprises contacting the fluid with a dedicated GP IIb-IIIa in the test reaction in the presence of fibrinogen and comparing the number of fibrinogen associated with GP IIb-IIIa in this test of the reaction, the amount of fibrinogen associated with GP IIb-IIIa in the control reaction. This method may further include reaction test and control reactions, which involve contacting Fn with Fn receptor, Vn with Vn receptor, Fn with GP IIb-IIIa or vWF to GP IIb-IIIa for characterizing the specificity PA1.

In another aspect the invention is directed to a new PAI in a dedicated form, which is identified in, and can be isolated from Echis colorata mollusks, Eristicophis macmahonii, A. hypnali, A. acutus, A. piscivorous leucostoma, A. piscivorous conanti; Bothrops asper, Bothrops cotiara, B. jararaca, B. jararacussu, B. lansbergi, B. medusa, B. nasuta, B. neuvudi, B. pradoi, B. schlegli, Crotalus atrox; C. basilicus, C. cerastes cerastes, C. durissus durissus, C. durissus totonatacus, C. horridus horridus, C. molossus molossus, C. ruber ruber, C. viridis cereberus, Crotalus V. helleri, Crotalus V. lutosus, Crotalus V. oreganus, Crotalus V. viridis; Zachesis mutas, Sistrures catenatus tergeminus, and Sistrurus nularus barbouri.

Preferred are the PAI in a dedicated form, derived from, or having the amino acid sequence of PAI obtained from Eristicophis macmahonii /eristicophis/, Bothrops cotiara /coterin/, B. jararacussu; Crotalus atrox /crotchrockets/, Crotalus basilicus /basilican/, C. cerasles cerastes /sarasti is n/ a, Crotalus viridis lutosus /litozin/, C. v. viridis /viridis/ Crotalus V. oreganus /oregonin/, Crotalus v. helleri, Lachesis muta /Lachesis/, Sistrurus catenatas tergemunus /trigeminy/ and S. milarus barbouri /barbury/.

Especially preferred are eristicophis, katarin, crotchrockets, cerstin, Bressan, harridan, Rubery, lagesen, basilican, litozin, Molossian, oregonin, viridin, trigeminy and Barbarin.

The invention also includes peptides from amino acid sequence, as described above, which are shortened and/or modified forms of the peptides of natural origin and/or have one or more peptide crosslinks alternative crosslinks, such as-CH2NH - or-CH2CH2-.

In a preferred aspect of the invention relates to PAI in the selected form, which can be obtained from the active snake venom identified using the method of the invention and showing the ability to specifically inhibit the binding of fibrinogen /Fg/ and/or von Willebrand factor /vWF/ GP IIb-IIIa and shortened and/or modified.

In yet another preferred aspect of the invention relates to PAI snake venom in the selected form in which the sequence responsible ZM aspect the invention is directed to a group of peptides or related peptides compounds, which are inhibitors of platelet aggregation, which are able to inhibit the binding of Fg or vWF to GP IIb-IIIa with significantly higher efficiency than the efficiency with which they inhibit the binding of vitronectin to vitronectin receptor or fibronectin to fibronectin receptor. These peptides are characterized by the presence of a linking sequence K*CDX instead RGDX binding sequence, which is, as you know, in PAI proteins, K*represents a substituted or unsubstituted protein diesel residue of the formula R'2N(CH2)4CHNHCO-, where R1represents independently H or Deputy, which is enough donor in order not to violate the basicity of the adjacent nitrogen, and in which one or two of metagenomic groups optionally may be replaced by O or S, as described below. Barbarisovyj PAI isolated from S. milarus barbouri, is one of the illustrations in this series of peptides. However, shorter forms of the peptide can also be used as similar sequences that also contain modifications of 1-10 amino acid residues at another location of the peptide chain, and/or replacement peptide native RGDX sequence, where this sequence is replaced with K*CDX. As in the case of Barbarina, these dedicated PAI can be different in native form, or may be reduced, and/or can contain substitution 1-10 amino acid residues, or deletions, and/or may have ones communication, replacement peptide crosslinking.

Another group of compounds which fall within the scope of the invention is the group where the previous connection as described, except that glilly residue in the RGD or K*GD sequence is replaced by sarcothalia balance. This class of compounds retains the activity and specificity related RGD or K*GD-containing peptides.

Another illustrative group of embodiments is a peptide or modified peptide having specific PAI activity, formulas

< / BR>
where K*represents a substituted or unsubstituted lazily the rest of the formula R'2N(CH2)4CHNHCO, as described above,

where each R1is independently H, alkyl /1-6C/ or at least one of R1is R2-C= NR3where R2represents H, alkyl /1-6C/ or substituted or unsubstituted phenyl or benzo is R3- H, alkyl/1-6C), phenyl or benzyl, or R2-C=NR3represents a radical selected from the group consisting of

< / BR>
< / BR>
where m is an integer of 2-3, and each R5is independently H or alkyl /1-6/,

and where one or two (CH2may(may) be replaced by(s) on or S, provided that these O (or S) is not adjacent to another heteroatom,

AA1is a small neutral /polar or nonpolar/ amino acid and n1 is an integer from 0-3,

AA2is a neutral, nonpolar big /aromatic or non-aromatic/ or polar aromatic amino acid and n2 is an integer from 0-3,

AA3represents polynomy residue or modified polynomy balance /as defined below/ and n3 is an integer from 0-1,

AA4is neutral, a small amino acid or N-alkilirovanny form and n4 is an integer from 0-3,

each of X1and X2is independently a residue capable of forming communication between the X1and X2to obtain a cyclic compound, as shown, and

each of Y1and Y2represents independently without interfering Deputy or moiety from the group consisting of: -CH2NH-, -CH2S-, -CH2CH2-, -CH= CH- /CIS-trance/, -COCH2-, -CH(OH)CH2- and-CH2SO-;

with the proviso that when n3 is 0, or

1) the sum of n2 and n4 must be at least 2,

or

2) K*must be other than Har or K, or

3) at sea one of X1and X2must be other than Cys /C/, penicillamine /Pen/, or 2-amino-3,3-cyclopentylmethyl-3-mercaptopropionate acid /APmP/ or

4) Y1or Y2must include at least one amino acid residue, or

5) one or more peptide linkages is replaced by the above-mentioned alternative communication.

Other aspects of the invention relate to recombinant methods and materials related to the synthesis of these and other related peptides, methods of their synthesis in vitro from the pharmaceutical compositions containing these compounds, and to methods of inhibiting platelet aggregation and clot formation when using these compounds and compositions.

Description of drawings

Fig. 1 shows the inhibition of binding of fibrinogen to GP IIb-IIIa using partially purified from snake venoms.

Fig. 2 shows the dependence of the inhibition of the adhesion of the dose Method-is Tina.

Fig. 3 shows the HPLC chromatogram of crude PAI of Eristicophis macmahoni venom. The shaded area contains the biologically active fraction.

Fig. 4 shows the HPLC chromatogram PAI fractions from Fig. 3. The shaded area contains the bioactive fraction.

Fig. 5 shows a view of analytical chromatography HPLC PAI-fractions from Fig. 4.

Fig. 6 shows the complete amino acid sequence of eristicophis, Barbarina, Cheremisina, ceratina, rubeena, Lactina, Katarina, crotchrockets, harridine, lutsina, virigina, molossia, basilicia, durisin, ferrozine, serebrin and Paganini and fragments of digestive enzymes by using the automated disintegration of Edman.

Fig. 7 depicts the HPLC chromatogram PAI obtained from G-50 fractions crude Sistrurus C. tergeminus poison.

Fig. 8 depicts the HPLC chromatogram PAI fractions from Fig. 7.

Fig. 9 shows the activity of the purified PAI Fig. 8 inhibition of binding analyses with multiple receptors.

Fig. 10 depicts an HPLC chromatogram of an inhibitor of platelet aggregation obtained from G-50 fractions crude Sistrurus c. tergeminus poison. The shaded area contains the bioactive fraction.

Fig. 11 depicts chromatography the amino acid sequences Barbarina.

Fig. 13 depicts the HPLC chromatogram of untreated PAI of Lachesia mutas poison. Shaded areas contain biologically active fraction.

Fig. 14 depicts the HPLC chromatogram of fractions of active PAI of Fig. 13. The shaded area contains the biologically active fraction.

Fig. 15 depicts an analytical HPLC chromatogram PAI fractions Fig. 14 of Lachesia mutas.

Fig. 16 depicts the HPLC chromatogram of crude PAI from Crotalus viridis viridis venom. The shaded area contains the biologically active fraction.

Fig. 17 depicts the HPLC chromatogram PAI fractions Fig. 16.

Fig. 18 shows the effect of dose of purified peptides snake venoms on the inhibition of binding of fibrinogen/GP IIb-IIIa compared to agitation.

Fig. 19 shows the effect of dose of purified snake venom peptides for inhibition of ADP /4um/ induced platelet aggregation in platelet-rich plasma /PRP/ compared to agitation.

Fig. 20 shows the area of activity of the chromatogram HPLC fractionation C. c. cerastes venom.

Fig. 21 shows the results of HPLC chromatographic analysis of the active fractions Fig. 20.

Fig. 22 shows the area of activity of HPLC fractionation PAI of C. ruber r the tion of C. atrox.

Fig. 24 shows an analytical HPLC chromatogram-homogeneous peptide isolated from Bothrops cotiara.

Fig. 25 shows the effect of dose cleaned Katarina on the inhibition of binding of fibrinogen to GP IIb-IIIa and the inhibition of the binding of vitronectin to vitronectin receptor.

Fig. 26 shows the effect of purified peptides snake venoms on the binding of fibrinogen to GP IIb-IIIa and vitronectin to the vitronectin receptor.

Fig. 27 shows the results of the activity of binding to analog #1, [E28L41C64] barbourin (28-73) with respect to GP IIb-IIIa and the vitronectin receptor.

Fig. 28 shows the ability of the synthetic analogue eristicophis to inhibit the binding of fibrinogen to GP IIIb-IIIa and the inability to inhibit the binding of vitronectin to vitronectin receptor.

Fig. 29 shows the ability of linear and cyclic KGDW compounds to inhibit the binding of fibrinogen to GP IIb-IIIa.

Fig. 30 shows the ability of various KGDW analogues to inhibit the binding of fibrinogen to GP IIb-IIIa and inhibit the binding of vitronectin to vitronectin receptor.

Fig. 31 shows the ability of various native and synthetic inhibi shows the ability of RGDS and cyclic RGD compounds to inhibit the adherence of cells M21 melanoma to vitronectin and the lack of ability cyclic KGDW analogue to inhibit the adherence of cells M21 melanoma to vitronectin.

Fig. 33 shows activity similar 60, Mpr-(Har)-G-D-W-P-C-NH2the inhibition of platelet aggregation and cell adhesion to vitronectin.

Fig. 34 shows the activity of Fig. 33 long analogue 19, Mpr-K-G-D-W-P-C-NH2.

Fig. 35 shows the occurrence of reduction of cyclic flow (CFRsin an open-chest model of thrombosis dogs /Fotts Model/.

Fig. 36 shows the effect of dose of 10 mg pill CFRsinitiated in an open-chest model of thrombosis dogs /Folds Model/.

Fig. 37 shows the effect of a dose of 40 mg pill CFRsinitiated in an open-chest model of thrombosis dogs /Folts Model/.

Fig. 38 shows the DNA sequence of the full length amino acid coding sequence Barbarina /1-73/.

Fig. 39 shows the DNA sequence that encodes a [M-1 L41] Barbarin /1-73/ sewn to PhoA leading sequence.

Fig. 40 shows the DNA sequence that encodes the analog #1 made with the leading sequence for expression in bacteria.

Fig. 41 shows the oligonucleotides used in PCR reactions to obtain DNA encoding the analog #1. Amino acids included in the analog themselves PC, coding the analog #1.

Fig. 43 shows a diagram of the truncated gene Barbarina as tandem replication.

Ways of carrying out the invention

The invention provides inhibitors of platelet aggregation /PAI/, which can be isolated from snake venom, which was identified as an active analytical methods of the invention and compounds that have similar structures and are synthesized using standard in vitro techniques, such as solid-phase peptide synthesis, or using recombinant methods, or a combination of these methods. Some of these inhibitors are highly specific for inhibition of platelet aggregation and does not otherwise inhibit the binding within the family of integrins. Other inhibitors have other areas of specificity. The sections below describe the allocation of PAI natural origin of snake venom, the design of inhibitors that have a significantly higher activity in the inhibition of platelet aggregation, such as the interaction of vitronectin/vitronectin receptor by introducing a sequence of K*GD is preferable RGD, methods of synthesis of these peptides, recombinant methods to pavati snake venoms, which contain PAI, introduction and application of PAI invention.

PAI is meant as a factor that can prevent aggregation stimulated platelets in standard assays, for example assays described Gan, Z., R. et al., and Huang., T-F. et al. (supra). In these analyses the washed platelets are combined with fibrinogen, Ca+2and the test material. Platelets stimulated by ADP /or other known stimulants, or combinations thereof and/ aggregation /or lack of/ observed when using, for example, commercially available aggregometry.

Some of the PAI of the invention are identified as specific for inhibiting the binding of fibrinogen and/or vWF to GP IIIb-IIIa. It is clear that the specificity is the power question, therefore, PAI-specific inhibition of binding of Fg or vWF to GP IIb-IIIa, inhibits this binding is significantly more than it inhibits the binding of Fn with FnR or Vn with VnR. Under the "much more" means that either the percentage of inhibition of at least twice for a given concentration of PAI, or the concentration of PAI, which causes 50% inhibition, at least two times less for inhibiting the binding of Fg or vWF/GP IIb-IIIa than for other svyazyvaem /PAI/ invention include peptides with low molecular weight, which can be obtained in a dedicated form, as described below, from snake venom, which was identified as "active", i.e. it is found that contains PAI, using the method of the invention, which is described below.

The method of the invention allows to quickly identify and characterize the presence of effective PAI in snake venom that selectively inhibits binding to GP IIb-IIIa in contrast to other integrins, such as vitronektinove receptor and the receptor for fibronectin. When such identification and, optionally, and optimally, the characterization of the PAI can be isolated and purified using various standard techniques illustrated here and known to experts. For example, there may be used a combination of separation based on the molecular weight of the /usually the extraction of substances < 10 KD/, ion-exchange chromatography and high performance liquid chromatography with a reverse phase. Other techniques can also be used, but the operating procedure is suitable for PAI from any active snake venom is as follows.

Approximately 10-1000 mg of venom dissolved in dilute acetic acid and passed through the bullet size is through the analysis of binding /Fg/ GP IIb-IIIa of the invention, the standard analysis of platelet aggregation /PAA/ or any similar analysis that relies on the activity of binding adhesive protein GP IIb-IIIa. Otherwise, fraction < 10 KD fraction of poison can be extracted using ultrafiltration and similarly analyzed.

Fractions with low molecular weight, selected by any procedure, then skipped through preparative C-18 HPLC column, such as column HPLC C-18 Delta Pak reverse phase, produced Waters, pre-equilibrated with 0.1% triperoxonane acid /TFA/ 8% acetonitrile. Absorbed PAI then suirable using gradient 8-60% acetonitrile in 0.1% TFA. Loop gradient and the flow rate is optimized using routine procedures. Active fractions are determined using PAA or method binding receptor of the invention. The active fractions are then collected, concentrated and tested for homogeneity using analytical high-performance liquid chromatography or SDS - PACE. Then apply a gradient purification obraniakowi high-performance liquid chromatography to until PAI will not be homogeneous.

PAI invention obtained by the above-described or other ivorous leucostoma, A. piscivorous conanti; Bothrops asper, Bothrops cotiara, B. jararaca, B. jararacussu, B. lansbergi, B. medusa, B. nasuta, B. neuwiedi, B. pradoi, B. schlegli, Crotalus atrox, C. basilicus, C. cerastes cerastes, C. durissus durissus, C. durissus totonatacus, C. horridus horridus, C. molossus molossus, C. ruber ruber, C. viridis cereberus, Crotalus v. helleri, Crotalus v. lutosus, Crotalus v. oreganus, Crotalus v. viridis, Lachesis mutas, Sistrurus catenatus terdeminus and Sistrurus milarus barbouri.

Preferred are PAI, in its pure form is obtained from, or having the amino acid sequence of PAI obtained from Eristicophis macmahonii /eristicophis/, Bothrops cotiara /coterin/, B. jararacussu, Crotalus atrox /crotchrockets/, Crotalus basilicus /basicily/, C. cerastes cerastes /cerstin/, C. durissus totonatacus /durisin/, C. durissus durissus /durisin/, C. h. horridus /norreen/, Crotalus m. molossus /molossid/, Crotalus ruber ruber /Rubery/, Crotalus viridis lutosus /litozin/, C. v. viridis /viridis/, Crotalus v. oreganus /oregonin/, Crotalus v. helleri, Lachesis mutas /lagesen/, Sistrurus catenatus tergeminus /trigeminy/ and S. milarus barbouri /barbury/. Especially preferred are PAI-specific inhibition of Fg or vWF/GP IIb-IIIa binding, such as PAI of Sistrurus m. barbouri.

Especially preferred are eristicophis, katarin, crotchrockets, cerstin, durisin, harridan, Rubery, lagesen, basicity, litozin, Molossian, oregonin, viridin, trigeminy and Barbarin.

In purified PAI can be the result of using standartizatcii species PAI/ or recombinant receipt. For example, Applied Biosystems Sequentor can be used after carboxamidotryptamine or pyridylmethylamine peptide, as described by Huang et al. , J. Biol. Chem. /1987/ 262: 16157-16163, followed by desalting of the sample on C-18 Delta Pak column using a 0.1% TFA and acetonitrile. It is clear that dedicated PAI sequence can synthesis in vitro to modify by changing the sequences that do not disrupt the activity. In General, these modified species will differ from the native forms of 1-10, preferably 1-4 amino acid substituents or will be shortened. In addition, one or more peptide linkages may be replaced by other bonds, as described herein below. Particularly preferred substitution is the replacement of RGD on the K*GD for giving GP IIb-IIIa specificity, as described below.

PAI from Sistrurus m. barbouri, was purified to homogeneity, defined sequence and is called "Barbarin". In contrast to adhesive proteins for GP IIb-IIIa identified so far, and peptides from snake venoms that block GP IIb-IIIa function, Barbarin not contains the standard Arg-Gly-Asp sequence of adhesive proteins, known to specialists. Obvious svyazyvavshej region of this peptide is especially surprising from the point of view of observation, that replacement of Arg to Lys in the small synthetic peptides based on RDGX sequences, greatly reduces the ability of these peptides to contact integrirovanie receptors /Pierschbacher et al., Proc. Natl. Acad. Sci /USA/ /1984/ 81: 5985-5988, Williams et al., Thromb. Res. /1987/, 46: 457-471, Huang et al., J. Biol. Chem. /1987/, 262: 16157-16163. I believe that this substitution may be partially responsible for the specificity barborikova peptide to inhibit Fg and vWF binding to GP IIb-IIIa in comparison with, for example, inhibition of the binding of vitronectin to vitronectin receptor.

K*GDX-containing peptides

It is shown that "barbarisovyj" peptide, selected using the method of the invention has a binding sequence KGDX, in contrast, found in PAI-known technical solutions. It turns out that the presence of KGDX in this PAI sequence associated with the preferred affinity for GP IIb-IIIa, as opposed to vitronectin receptor or fibronectin. It turns out that the substitution effect balance lisala for arginine in the sequence is associated with increased length of the side chain, along with remaining basicity of nitrogen as further described here. Surprisingly, it turns out that this is not lazily balance by itself, which is cnym extension replaced by arginine. Thus, the peptides of the invention that contain K*GDX into the binding sequences are significantly more active at inhibiting

the binding of Fg or vWF to GP IIb-IIIa compared to their ability to inhibit the binding of vitronectin with vitronectin receptor and the binding of fibronectin with fibronectin receptor. As stated above, under "significantly greater activity in the inhibition of preferred binding means that the percentage of inhibition is at least two times greater at a fixed concentration of inhibitor or the concentration of PAI, which causes 50% inhibition is at least two times smaller binding of Fg or vWF to GP IIb-IIIa, than to bind other ligands with other integrins. Used here, K*refers to lysiloma residue, which is unsubstituted or which contains the substituents of the hydrogens on the Epsilon amino group. Deputies should be sufficient electrondonor in order to maintain the basicity of the nitrogen to which they are attached. Thus, K*is defined as lazily the rest of the formula R21N(CH2)4CHNHCO, where each R1is independently researched the place a H, alkyl/1-6C/ or is substituted or unsubstituted phenyl or benzyl residue or represents NR24in which each R4is independently H or alkyl /1-6C/, and

represents H, alkyl /1-6C), phenyl or benzyl or R2-C=NR3is a radical selected from the group consisting of

< / BR>
< / BR>
where m is an integer of 2-3, and each R5is independently H or alkyl /1-6C/,

and where one or two /CH/ can be replaced by O or S

provided that the above O or S are not adjacent to another heteroatom.

"Alkyl" is usually defined as a linear or branched chain or cyclic hydrocarbon residue, characterized by the number of carbon atoms, such as methyl, ethyl, isopropyl, N-hexyl, 2-methylbutyl, cyclohexyl and the like.

Benzyl and phenyl residues represented by R2can be unsubstituted or can be substituted nemchausky substituents. The preferred models of substitutions are such models, in which only one Deputy is associated with the aromatic nucleus, preferably in position 4. Preferred substituents are electrondonor the I K include lysine residues, homoarginine, formirovaniya, ornithine, acetamidine, NGNG-antilegomena and familymedicine. Phenylimidazole residue, for example, has the formula

PL-C(=NH)-NH(CH2)4CH(NH-)CO-

Since the essential feature of the preferred inhibition of binding is replaced in the substitution of R RGDX to K*one class of peptides or related peptide compounds of the invention include inhibitors of platelet aggregation of natural origin, which usually contain RGDX in the binding sequence, whereby these species are modified by substitution of R for K*in this sequence. Included in the invention are native peptides, with this substitution, as well as fragments of sufficient length for the manifestation of the efficiency in the selective inhibition of the binding of adhesive proteins with GP IIb-IIIa and fragments or peptides of the full length, which are irrelevant to the substitution positions of the peptide, which does not violate this activity. For the most part, the fragments will contain residues corresponding to the length of the peptide chain of at least 7 amino acids, if the conformation is controlled, for example, cyclization, and will be bol and, may 4-10, preferably 1-4, and more preferably 1-3 amino acid substitutions in non-K*GDX part of the peptides.

In addition, G from RGDX or K*GDX can be replaced sarcosinates balance.

Moreover, one or more peptide bonds may be optionally substituted for the placeholder links, such as links, obtained by recovery or elimination. Thus, one or more peptide linkages can be replaced with other types of links, such as-CH2NH-, -CH2S-, -CH2CH2-, -CH= CH- /CIS-trance/, -COCH2, -CH(OH)CH2- and-CH2SO the methods known in the art. The following links describe the receipt of peptide analogs which include these alternative linking units: Spatola, A. F. , Vega Data (March 1983), V. 1, Issue 3, "Peptide Backbone Modifications" (general review); Spatola, A. F. in "Chemistry and Biochemistry of Amino Acids, Peptides and Proteins", B. weinstein, eds., Marcel Dekker, New York, p. 267 (1983) (general review); Morley, J. S. Trends. Pharm. Sci (1980) pp. 463-468 (general review); Hudson, D. et al. Int J. Pept. Prot. Res. (1979) 14: 177-185 (-CH2NH-, CH2CH2-); Spatola, A. F. et al., Life Sci (1986) 38: 1243-1249 (-CH2S); Hann, M. M., J. Chem. Soc. Perkin Trans. I (1982) 307-314 (-CH-CH-, cis and traus); Alnequist, R. G., et al., J. Med. Chem (1980) 23: 1393-1398 (-COCH2-); Jennings-white, C. et al. Tetrahedron Lett (1982) 23: 2533 (-COCH2); Szelke, M. , et al., European Appln. EP 45665 (1982) CA:97:3940"ptx2">

Especially preferred is-CH2NH-.

Examples of fragments and/or modified species PAI natural origin of snake venom include [E28L41C64] Barbarin (28-73) from the sequence

< / BR>
and [K29]eristicophis (4-51) from the sequence

< / BR>
In this post, the fragment size is noted in parentheses after the name using the number of amino acids that are included in the fragment, and enclosed in square brackets Preface letters and numbers indicate amino acid substitution at numbered positions in the native full length peptide. Thus, for the above barborikova fragment length fragment includes residues 28-73 inclusive of the native sequence and the amino acid originally in positions 28, 41 and 64 numbered native sequence were replaced with Glu /E/, Leu /L/ and Cys /C/, respectively.

As additional examples, the arginine of the RGD sequence, appearing in trigemina, elephantine, albolabris, crotalaria, flavoviridis, echistatin, Batistuta, viridine, malossini, luteine, basilicia, applewine, haleine, harridine, telemania, Lahaina, katiana K*the remainder to provide specific active PAI preferred affinity for GP IIb-IIIa. In addition, shortened forms of these peptides, containing at least 20, preferably at least 30 and more preferably at least 40 amino acids, can be obtained from the native peptide or in a modified form. In addition, or in alternative 1-10, preferably 1-4 amino acids are not suitable and RGD/K*GD sequence can be substituted or modified, preferably moderate conservative amino acid substitutions. Under conservative amino acid substitutions implied, for example, substitution of an acidic amino acid residue to an acidic amino acid residue, main on main, neutral to neutral, and so on, as further described below.

One additional group of examples includes a group in which glilly the residue of the RGD or K*GD can be replaced by sarcothalia balance with preservation activity. Thus, active PAI, which are selected and/or modified in other ways, as described above, may further be modified by means of this substitution.

While fragmenta compounds of the invention by substitution of the RGD to K*GD, in additional embodiments of the invention specifically active peptides based on a compatible extensions K*GD sequence itself. In this regard, a preferred group of peptides or related peptide compounds of the invention are cyclic peptides of General formula

< / BR>
where K*represents a substituted or unsubstituted lysyl, as defined above,

AA1small neutral / polar or non-polar amino acid and n1 is an integer from 0-3,

AA2neutral nonpolar big / aromatic or non-aromatic / or polar aromatic amino acid and n2 is an integer from 0-3,

AA3- polynomy residue or modified polynomy balance /as defined below/ and n3 is an integer from 0-1,

AA4neutral, a small amino acid or N-alkilirovanny their shape and n4 is an integer from 0-3,

each of X1and X2represents independently a residue capable of forming communication between the X1and X2to obtain a cyclic compound, as shown, and

each of Y1and Y2represents independently numerouse Deputy or may not be,

in which the th of CH2NH-, -CH2S-, -CH2CH2-, -CH=CH -, CIS and TRANS/ -COCH2-, -CH(OH)CH2- and-CH2SO-;

with the condition that if n3 = 0, or

1) the sum of n2 and n4 valley at least 2 or

2) K*must be other than Har or K, or

3) at least one of X1and X2must be other than Cys /C/: penicillamine /Pen/ or 2-amino-3,3-cyclopentylmethyl-3-mercaptopropionate acid /the APMP/ or

4) Y1or Y2must include at least one amino acid residue, or

5) one or more peptide linkages is replaced by the above-mentioned alternative communication.

Y1and Y2can be a peptide extensions of 0-25 amino acid residues and can be in the form of derivatives, Y1-N terminal elongation may, for example, be azetilirovanna or otherwise allyawan, Y2C-terminal may be liderovna NH2or primary or secondary amine of the formula R-NH2or R2NH, where R represents independently lower alkyl 1-4C, such as methyl, n-butyl, or tert-butyl, Y1can also be H or acyl, Y2can be /OH/, NH2or an amine, as described above. When the compound of the formula /I/ represents prosociality residues capable of cyclization, such as, for example, and most preferably, the cysteine residues capable of forming disulfide loop. However, other residues capable of forming disulfide bonds or other connections, can also be used, such as Pen /penicillamine/ balance described Pierschbacher (supra), or Mpr /mercaptopropionyl/ or Mvl /mercaptophenyl/ balance. Other types of covalent bonds include peptide bond, as for example an amide formed between the amino group of the side chain litelnogo residue from the carboxyl group of the side chain glutamine balance, and ester bonds, such as would be formed between the alcohol group of the side chain of threonine residue with a carboxyl group of the side chain bartiloro balance. Any similar residue capable of forming peptide bonds, with the remainder of the chain /or modified peptide bonds, as described above and are capable of forming covalent bonds to implement cyclization, can be used. This includes, for example, a simple cyclic peptide in which the peptide bond is formed directly between the NH2when the N-end and COOH at the C-end.

As described above, one or more specified peptide TRANS/, COCH2-, -CH(OH)CH2- and-CH2SO-.

When referring to amino acid residues AA1-AA4as described above, description was made on the basis of the classification method, in which amino acid residues can be generally divided into four main subclass. This classification is also presented here below in chart form.

Acid: the residue has a negative charge due to loss of H ion at physiological pH and the residue is attracted aqueous solution in order to try to occupy surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH.

Main: the residue has a positive charge as a result of Association with the H ion at physiological pH and the residue is attracted aqueous solution so as to try to occupy surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous solution at physiological pH.

Neutral / non-polar: the balance is not charged at physiological pH and the residue is repelled aqueous solution so as to try to occupy interior positions in the conformation of a peptide in which it is contained when / polar: the residues are not charged at physiological pH, but the balance is drawn aqueous solution so as to try to take external positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium.

Of course, it is clear that the statistical set of individual molecules remnants of some molecules will be charged, and some do not, and will be either attracted to the aquatic environment or repulsion to a greater or lesser extent. To meet the definition of "charged" a significant percentage of /for at least about 25%/ individual molecules are charged at physiological pH. The degree of attraction or repulsion for the classification of polar or nonpolar is arbitrary and therefore amino acids, in particular, is considered in the invention, have been classified, in particular, as one or the other. Most of the amino acids that are not specifically named, can be characterized on the basis of known behavior.

Amino acid residues can further be divided into cyclic and acyclic, aromatic and non-aromatic, self-explanatory classification with respect to lateral replacement residues and groups as small or large. The remainder is considered as small, elioda, of course, always non-aromatic residue.

For amino acids proteins of natural origin unit according to the previous scheme is as follows /see also the diagram below/:

Acidic: aspartic acid and glutamic acid.

Main / acyclic: arginine, lysine.

Main / circular: histidine.

Neutral / polar / small: glycine, serine and cysteine.

Neutral / polar / large / nonaromatic: threonine, asparagine, glutamine.

Neutral / polar / large / aromatic: tyrosine.

Neutral / nonpolar / small: alanine.

Neutral / nonpolar / large / nonaromatic: valine, isoleucine, leucine, methionine.

Neutral / nonpolar / large / aromatic: phenylalanine and tryptophan.

Enchondroma the amino acid Proline, although technically located within the group of neutral / nonpolar / large / cyclic and nonaromatic, is a special case due to its known effects on the secondary conformation and its peptide chains and therefore is not included in this defined group, but classified the finish is pleasant, represents a five-membered nitrogen-containing heterocycle with a carboxyl group in position 2. Modified prolinnova remains of all nitrogen-containing five - or shestichlennyi heterocycles with a carboxyl group in the position alpha to the nitrogen cycle may also include additional heterocyclic atoms. Thus, the modified prolinnova residues include residues pipecolinic acid /2-carboxytherapy, abbreviation PTP/ thiazolidin /Th2/. Thus, polynomy and modified polynomy balance formula

< / BR>
where one or two methylene groups may be replaced by NR, S or O and where any cyclic nitrogen may optionally be substituted nemesius Deputy, such as alkyl.

Some commonly encountered amino acids that are not encoded by the genetic code, include, for example, beta-alanine/beta-Ala/ or other amino acids, such as 3-aminopropanol, 4-aminobutanol and so forth, alpha-aminoisobutyric acid /Aib/, sarcosin /Sar/ ornithine /Orn/, citrulline /Cit/, homoarginine /Har/, tert-butylene /t-Bua/, tert-butylglycol /t-BuG/, N-methylisoleucine /N-Melle/ phenylglycine /Phg/, cyclohexylamin /Cha/ nonlatin /Nle/, cysteine acid /Cya/, ripec is given in a special category.

Based on the above definition, Sar and beta-Ala are neutral / nonpolar / small, tert-Bua, t-BuG, N-Melle, Nle and Cha - neutral / nonpolar / large / nonaromatic,

Har and Orn - main / acyclic,

Cys - acid,

Cit, Acetyl Lys and MSO - neutral / nonpolar / large / aromatic and

Pip and Thz modified prolinnova remains.

The foregoing can be shown in a diagram (see diagram 1 at the end of the description).

Various omega-amino acids are classified according to size as a neutral / nonpolar / small (beta-Ala, that is, 3-aminopropionic acid, 4-aminobutanoic acid) or large (all other amino acids).

Other amino acid substitutions to amino acids encoded in the gene, may also be included in peptide compounds within the scope of the invention, and can be classified in accordance with a common schema.

In the formula representing the selected specific embodiment of the present invention, the amino - and carboxyl end groups, although often not specifically shown, will be in the form that they acquire at physiological pH, unless otherwise noted. Thus understood is satalino are indicated and shown, or in specific examples or in the total formula. Of course, basic and acid additive salts, including salts, which are formed at non-physiological pH values, are also included in the compounds of the invention. If not mentioned specifically, the residues are in the L-form. In the General formula indicated residues can be either L or D. In General, the peptides of the invention have 0, 1 or 2 D-residues, inclusive, preferably 0 or 1, most preferably 0. In the presented peptides each encoded residue represented by symbols in a single letter corresponding to the trivial name of the amino acids, according to the following conventional list:

Amino acid - an Alpha character

Alanine - A

Arginine - R

Asparagine - N

Aspartic acid - D

Cysteine - C

Glutamine - O

Glutamic acid - E

Glycine - G

Histidine - H

Isoleucine - I

Leucine - L

Lysine - K

Methionine - M

Phenylalanine - F

Proline - P

Serine - S

Threonine - T

Tryptophan - W

Tyrosine - Y

Valine - V

Pyroglutamyl acid - Z

Abbreviation of the amino acids that are not genetically encoded, that is, as indicated above.

In the specific peptides presented in this per, what resicom top (t). While residues of the peptides of the invention generally are in the natural L optical isomer form, one or two, preferably one amino acid may be replaced with the optical isomer D form.

Free functional groups, including groups with carboxyl or amine ends, can also be modified by amidation, acylation or other substitution, which can be, for example, change the solubility of the compounds without affecting their activity.

When education emitirovannykh peptides of the present invention similar compounds can be synthesized, for example, using Boc-AA-pMBHA-resin or Boc-AA-BHA-resin, where AA is selected carboxyterminal amino acid of the desired peptide, as described later in detail. Otherwise, the peptides of the present invention can be chemically or enzymatically lidirovali after peptide synthesis using methods well known in the art, or prepared using standard techniques of peptide synthesis in solution.

Some embodiments of de novo peptides of the invention are preferred. In the sequence of K*(G)/Sar) DG/Sar is predpochiol for AA2are neutral / nonpolar / aromatic amino acids, particularly tryptophan and phenylalanine, especially tryptophan, n2 is preferably 1, X1and X2are preferably Cys, Mpr or Pen /penicillamine/ residues. Y1- preferably H, acetyl or Gly, Y2- preferably-NH2or A-NH2-. Generally preferred are the C-terminal amidarone form Y2.

Thus, the preferred embodiment analogues PAI invention include peptides of the following formula. Although all of these peptides are able to provide the formation of cyclic form through the formation of disulfide bonds, these bonds, in particular, not shown, other cyclic form are marked with "cyclo".

Preferred peptides

PAI 1: E-C-A-D-G-L-C-C-D-Q-C-R-F-L-K-K-G-T-V-C-R-V-A-K-G-D-W-N - D-D-T-C-T-G-Q-S-C-D-C-P-R-N-G-L-Y-G

PAI 2: E-E-P-C-A-T-G-P-C-C-R-R-C-K-F-K-R-A-G-K-V-C-R-V-A-K-G-D - W-N-N-D-Y-C-T-G-K-S-C-D-C-P-R-N-P-W-N-G

PAI 3: G-C-G-K-G-D-W-P-C-A-NH2;

PAI 4: G-C-K-G-D-W-P-C-A-NH2< / BR>
PAI 5: C-G-K-G-D-W-P-C-NH2< / BR>
PAI 7: C-K-G-D-W-C-A-NH2;

PAI 9: Mpr-K-G-D-Pen-NH2< / BR>
PAI 10: C-K-G-D-W-P-C-NH2< / BR>
PAI 12: C-K-G-D-Y-P-C-NH2< / BR>
PAI 13: C-K-G-D-F-P-C-NH2< / BR>
PAI 14: C-K-G-D-L-P-C-NH2< / BR>
PAI 15: C-K-G-D-V-P-C-NH2< / BR>
PAI 16: C-K-G-D-Y(AI 20: Mpr-K-G-D-Y-P-C-NH2< / BR>
PAI 21: Mpr-K-G-D-F-P-C-NH2< / BR>
PAI 22: Mpr-K-G-D-L-P-C-NH2< / BR>
PAI 23: Mpr-K-G-D-V-P-C-NH2< / BR>
PAI 24: Mpr-K-G-D-Y(OMe)-P-C-NH2< / BR>
PAI 25: Mpr-K-G-D-(2-Nal)-P-C-NH2< / BR>
PAI 26: Mpr-K-G-D-(Cha)-P-C-NH2< / BR>
PAI 27: cyclo(G-K-G-D-W-P)

PAI 28: cyclo(A-K-G-D-W-P)

PAI 29: cyclo(D-Ala-K-G-D-W-P)

PAI 30: cyclo(F-K-G-D-W-P)

PAI 31: cyclo(beta-Ala-K-G-D-W-P)

PAI 32: cyclo(gamma-Abu-K-G-D-W-P)

PAI 33: cyclo(R-K-G-D-W-P-)

PAI 34: C-K-G-D-W-G-C-NH2< / BR>
PAI 37: C-K-A-D-W-P-C-NH2< / BR>
PAI 39: C-K-G-D-W-(Sar)-C-NH2< / BR>
PAI 41: C-K-G-D-I-P-C-NH2< / BR>
PAI 42: C-K-G-D-(4-Cl-Phe)-P-NH2< / BR>
PAI 43: C-K-Sar-D-W-P-C-NH2< / BR>
PAI 44: C-K-G-D-(4-NO2-Phe)-P-C-NH2< / BR>
PAI 47: Acetyl-C-K-G-D-W-P-C-NH2< / BR>
PAI 48: Mpr-K-G-D-W(Formyl)-P-C-NH2< / BR>
PAI 49: Mvl-K-G-D-W-P-C-NH2< / BR>
PAI 51: Mpr-K-G-D-W-P-Pen-NH2< / BR>
PAI 52: Mpr-K-G-D-W-P-Pent-NH2< / BR>
PAI 54: Mpr-K-G-Dt-W-P-Pen-NH2< / BR>
PAI 55: Mpr-K-G-D-W-(Thz-C-NH2< / BR>
PAI 56: Mpr-K-G-D-H(2,4-DNF)-P-C-NH2< / BR>
PAI 57: Mpr-K-G-D-(2-Nal)-P-Pen-NH2< / BR>
PAI 58: Mvl-K-G-D-W-P-Pen-NH2< / BR>
PAI 59: Mpr-K-G-D-W-(Pip)-Pen-NH2< / BR>
PAI 60: Mpr-(Har)-G-D-W-P-C-NH2< / BR>
PAI 61: Mpr-K-G-D-W-P-Ct-NH2< / BR>
PAI 62: Mpr-Kt-G-D-W-P-Pen-NH2< / BR>
PAI 63: Mpr-(Har)-G-D-W-P-Pen-NH2< / BR>
PAI 64: Mpr-(Acetimidyl-Lys)-G-D-W-P-C-NH2< / BR>
PAI 65: Mpr-(Acetimidyl-Lys)-G-D-W-P-Pen-NH2< / BR>
PAI 66: Mpr-(NGNG-ethylene-Har)-G-D - W-P-C-NH2< / BR>
PAI 67: Mpr-(NGNPAI 70: Mpr-(Phenylimidyl-Lys)-G-D-W-P-C-NH2< / BR>
PAI 71: Mpr-Har-Sar-D-W-P-PenNH2< / BR>
PAI 72: Mpr-(Phenylimidyl-Lys)-G-D-W-P-PenNH2< / BR>
PAI 73: Mpr-Har-G-D-W-(3,4-dehydro-Pro-C-NH2< / BR>
PAI 74: Mpr-Har-C-D-Pen-NH2< / BR>
PAI 75: Mpr-(Phenylimidyl-Lys)-G-D - Pen-NH2< / BR>
Especially preferred are peptides of formula

PAI 3: G-C-G-K-G-D-W-P-C-A-NH2< / BR>
PAI 4: G-C-K-G-D-W-P-C-A-NH2< / BR>
PAI 5: C-G-K-G-D-W-P-C-NH2< / BR>
PAI 9: Mpr-K-G-D-Pen-NH2< / BR>
PAI 10: C-K-G-D-W-P-C-NH2< / BR>
PAI 12: C-K-G-D-Y-P-C-NH2< / BR>
PAI 13: C-K-G-D-F-P-C-NH2< / BR>
PAI 19: Mpr-K-G-D-W-P-C-NH2< / BR>
PAI 25: Mpr-K-G-D-(2-Nal)-P-C-NH2< / BR>
PAI 34: C-K-G-D-W-G-C-NH2< / BR>
PAI 39: C-K-G-D-W-(Sar)-C-NH2< / BR>
PAI 42: C-K-G-D-(4-Cl-Phe)-P-NH2< / BR>
PAI 43: C-K-Sar-D-W-P-C-NH2< / BR>
PAI 44: C-K-G-D-(4-NO2-Phe)-P-C-NH2< / BR>
PAI 47: Acetyl-C-K-G-D-W-P-C-NH2< / BR>
PAI 48: Mpr-K-D-D-W(Formyl)-P-C-NH2< / BR>
PAI 49: Mvl-K-G-D-W-P-C-NH2< / BR>
PAI 51: Mpr-K-G-D-W-P-Pen-NH2< / BR>
PAI 52: Mpr-K-G-D-W-P-(D-Pen)-NH2< / BR>
PAI 55: Mpr-K-G-D-W-(Thz-C-NH2< / BR>
PAI 56: Mpr-K-G-D-H(2,4-DNF)-P-C-NH2< / BR>
PAI 57: Mpr-K-G-D-(2-Nal)-P-Pen-NH2< / BR>
PAI 58: Mvl-K-G-D-W-P-Pen-NH2< / BR>
PAI 59: Mpr-K-G-D-W-(Pip)-Pen-NH2< / BR>
PAI 60: Mpr-(Har)-G-D-W-P-C-NH2< / BR>
PAI 61: Mpr-K-G-D-W-P-Ct-NH2< / BR>
PAI 62: Mpr-Kt-G-D-W-P-Pen-NH2< / BR>
PAI 63: Mpr-(Har)-G-D-W-P-Pen-NH2< / BR>
PAI 64: Mpr-(Acetimidyl-Lys)-G-D-W-P-C-NH2< / BR>
PAI 65: Mpr-(Acetimidyl-Lys)-G-D-W-P-Pen-NH2< / BR>
PAI 66: Mpr-(NGNG<-W-P-C-NH2< / BR>
PAI 69: Mpr-(Acetimidyl-Lys)-G-D-W-P-Pen-NH2< / BR>
PAI 70: Mpr-(Phenylimidyl-Lys)-G-D-W-P-C-NH2< / BR>
PAI 71: Mpr-Har-Sar-D-W-P-Pen-NH2< / BR>
PAI 72: Mpr-(Phenylimidyl-Lys)-G-D-W-P-PenNH2< / BR>
PAI 73: Mpr-Har-G-D-W-(3,4-dehydro-Pro-C-NH2< / BR>
Chemical synthesis of the peptides of the invention

Compounds within the scope of the invention may be chemically synthesized using methods well known in the art, such as, for example, solid-phase peptide synthesis. The synthesis begins with carboxyterminal end of the peptide using an alpha-amino protected amino acids. Tert-butyl oxycarbonyl // Boc protective group can be used for all amino groups, even though other protective groups, such as fluorenylmethoxycarbonyl (Fmoc) is appropriate. For example, Boc-Cly-OH, Boc-Ala-OH, Boc-His(Tos)-OH /that is selected carboxyterminal amino acids/ can be tarifitsirovana on substrates chlorotoluenes polystyrene resin, p-methylbenzhydrylamine /pMBHA/ or PAM resin. The substrate of the polystyrene resin is preferably a copolymer of styrene with about 0.5-2% divinylbenzene as a crosslinking agent, which contributes to the polystyrene polymer was completely soluble in certain organic the other methods of peptide synthesis are also provided in U.S. patent N 3862925, 3842067, 3972859 and 4105602.

During the synthesis may use manual methods of synthesis or automatic, for example, an Applied Biosystems 430A or 431A Peptide Synthesizer (Foster City, California), following the instructions supplied by the manufacturer. Cleavage of the peptides from the resin can be performed by using a "low-high" method of removing protection, as described Lu, G. S. et al., Int. T. Peptida of Protein Researsh /1987/ 29: 545-557. Obtaining analogues PAI snake venoms can be carried out using the procedure described in Lu, G. S. et al., Int T. Peptida Protein Pesearch /1989/ 86: 4022-4026, which describes the solid-phase synthesis of echistatin.

The cyclic peptides of this invention that do not contain disulfide bonds, can be easily obtained by a combination of solid-phase synthesis and formation of a cyclic ring structure in solution using the General methods as described in U.S. patent N 4612366 Natt. Thus, linear peptides obtained by standard Merrifield resin, can be derived from the resin with hydrazine, followed by cyclization of the corresponding azide with the formation of cyclic peptides.

Specialists in the field of peptide synthesis is easy to see that the intermediates that are formed in accordance with the present image is changed in the scope of the present invention.

Recombinant getting

Otherwise, the individual compounds of the present invention can be obtained by expression of recombinant DNA in accordance with well known techniques. This may be desirable for large quantities or alternative embodiments of such compounds. As the peptide sequence is relatively short, recombinant getting easier, but getting by recombinant methods is particularly preferred over standard solid-phase peptide synthesis of peptides of at least 8 amino acid residues.

DNA encoding PAI sequence, preferably obtained using the methods of synthesis are commercially available nucleic acid. Methods for constructing expression systems for the production of PAI in recombinant hosts also generally known to experts.

The expression can be carried out either in prokaryotic or kriticheskih hosts. Prokaryotes 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 Rseudomonas or other bacterial and control sequence, derived from species compatible with the host. For example, a work vector for E. coli is pBR 322 and its derivatives. Generally used prokaryotic control sequences, which contain the promoter to stimulate transcription, optionally with an operator, along with ribosome sidwashini sequences, include such well-known promoters as the beta-lactamase /penicillinase and/ Lac /lac/ promoter system, a tryptophan /trp/ promoter system and the lambda-derived PLthe promoter and N-gene ribosome binding site. However, it can be used any available promoter system compatible with prokaryotes.

Expression systems useful in eukaryotic hosts include the promoters obtained from suitable eukaryotic genes. Class of promoters useful in yeast include promoters for synthesis of glycolytic enzymes, such as promoters length 3-phosphoglyceraldehyde. Other yeast promoters include promoters from enolase gene or Leu 2 gene obtained from YEp13.

Suitable mammalian promoters include the early and late promoters from SV 40 or other viral promoters such as pirnie enhancers and enhancers mammals cited above. In the case of plant cells are used expression systems, the promoter synthesis nopaline, for example, is suitable.

Expression systems are constructed using well-known methods of restriction and stitching and are transformed into respective owners.

The transformation is done using standard techniques appropriate to such cells. Cells containing the expression system, culturious under conditions appropriate for production of PAI and PA2, then stand out and cleaned up.

Antibodies

The availability of purified PAI invention also enables production of antibodies specifically immunoreactive to these types of active peptide.

Compositions containing the purified PAI isolated from snake venom or synthesized by another method that can be used to stimulate the production of antibodies, which are immunoreactive to PAI peptide. Standard immunization protocols involving the introduction of a PAI of different vertebrate, such as rabbits, rats, mice, sheep and chickens, leads to the serum, which is immunoreactive to a purified peptide. PAI can be useful konjugierte with a suitable antigenic neutral nonet. In addition, the free peptide can be introduced with methylated BSA as an alternative to conjugation. Besides secreting antibodies cells immunized mammal can be immortalized, "immortalired" for groups of monoclonal antibodies, which can then be tested for reactivity with PAI.

The obtained polyclonal or monoclonal antibody preparations useful in the analysis of the levels of the corresponding PAI in biological samples using standard immunoassay techniques.

The analysis of the invention

Identification of the source material from snake venom, which contains the active PAI and PAI which has a known specificity, it becomes possible by means of analysis of the invention. Analytical samples show that compounds that block the binding of fibrinogen to GP IIb-IIIa complex in vitro, is also able to inhibit thrombin or ADP-induced aggregation of human platelets and the formation of platelet-thrombin in vivo. This observation provides the basis for obtaining active PAI by clarifying the ability of the test materials to disrupt the interaction of fibrinogen to GP IIb-IIIa.

In the analysis, GP IIb-IIIa obtained in purified form, as ity of the solid substrate, such as pellets, analytical tubes or plates microtitration. Coated substrate is then brought into contact with the fibrinogen and the test material and incubated for a time sufficient to allow maximal binding to immobilized fibrinogen GP IIb-IIIa. Fibrinogen is usually given at a concentration of about 5-50 nM and the tested material can be added, if desirable, a series of dilutions. Incubation is usually carried out for 2-4 hours at 35oC, and the time and temperature are interdependent.

After incubation solution containing fibrinogen and the tested material is removed and the binding of fibrinogen is measured by the quantitative determination of fibrinogen associated with GP IIb-IIIa. Can be used, any suitable means of detection, but it is convenient to use labeled fibrinogen, for example, using radioactive, fluorescent or biotinylated label. Such methods are well known and do not need a thorough description here.

Evaluation of results is carried out by using a control sample, is usually identical to the test sample, except when tested Fg associated to the control as being the basis, so

< / BR>
Other performance measurement of inhibition, such as IC50can also be used.

Analytical system of the invention further include characterization of the specificity of PAI using analysis of inhibition of binding, identical to above, but replacing Fg other adhesive proteins and other receptor GP IIb-IIIa. In particular, inhibition of the binding of vitronectin to vitronectin receptor, fibronectin with fibronectin receptor, fibronectin with GP IIb-IIIa and fibrinogen and/or GP IIb-IIIa can be determined. Adhesive protein and receptors for these analyses available to the specialists.

Other tests

In addition to the laminar analyses invention is also suitable are other tests activity of inhibiting platelet aggregation and related activities, as

described above. So, the list of the used tests is as follows:

1. Laminar analysis using specific receptors described in the previous paragraphs.

2. Standard tests directly applied to platelet aggregation, such as the assays described Gann Z., R. et al., J. Biol. Chem. /1988/, 263: 19827-19832, Huand T. F. et al., J. Bio 3. The model of thrombosis in vivo in dogs, as described below in example 1, and I. P. Folts et al., Circulations /1986/ 54: 365.

4. The effect on cell adhesion using S35 motionengine cells, as described below in example 19.

Introduction and application

PAI invention are therapeutically useful for preventing the formation of clots. Indications for such treatment include, without limitation, atherosclerosis and arteriosclerosis, acute myocardial infarction, chronic changeable angina, transient ischemic attacks and seizures, peripheral vascular disorders, arterial thrombosis, preeclampsia, embolism, restenosis and/or thrombosis following angioplasty, carotid endarterectomy, anastamose vascular transplants and chronic cardiovascular devices, for example, "in dwelling", catheters or shunts apparatus of extracorporeal circulation/. These syndromes represent a variety of stenotic and obtenerse vascular disorders, which are believed to have been caused by the activation of platelets on the vessel walls.

PAI can be used to prevent or stop the development of clotting in the case of volatile angina and arterial embolism or thrombosis, as well as to treat brain disorders include treatment and prevention of transient ischemic attacks and treatment of thrombotic stroke.

PAI can also be used to prevent platelet aggregation, embolization or total depletion in the extracorporeal circulation, including the improvement of renal dialysis, cardiopulmonary bypass, hemoperfusion and plasma exchange.

PAI prevent platelet aggregation, embolization or total depletion associated with intravascular disorders and introduction leads to improved application nutrientrich "balloon pumps", ventricular assistive devices and arterial catheters.

PAI will also be useful in the treatment or prevention of venous thrombosis, as deep vein thrombosis, IVC thrombosis or renal portal vein and thrombosis pulmonary veins.

Various violations, including the depletion of platelets, such as thrombocytopenic greengourmet also treatable.

In addition, the PAI of the present invention can be used in various non-therapeutic applications where it is desirable inhibition of platelet aggregation. For example, can be implemented improved storage of platelets and whole blood by adding sufficient quantities of peptides, the number of which will vary depending on LASS="ptx2">

Dosage PAI can widely vary depending on the desired effect.

Typically, the dosage will be between about 0.01 and 10 mg/kg, preferably between about 0.01 and 0.1 mg/kg of body weight. Introduction preferably parenterally, such as intravenously daily for up to one week, or as much as one or two months or more, all of which will vary depending on the size of the peptide. If the peptides are small enough /for example, less than about 8-10 amino acid residues/, can be used other routes of administration, such as intranasal /nose/, under the tongue, and the like.

Injectables can be prepared in convenient forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Suitable fillers are, for example, water, saline, dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate, cysteine hydrochloride, and the like. In addition, if desired, the injectable pharmaceutical composition can contain minor amounts of nontoxic auxiliary substances such as wetting liposomes/.

Example 1

Analysis of inhibitors of adhesion of platelets from snake venom

A. Description of methods of analysis - plastic methods of analysis

Purified trombotsitnoy GP IIb-IIIa receptor was prepared as described Fitrgerald L. A. et al., Anal. Biochem. /1985/ 151: 169-177. Vitronektinove receptor was obtained as described in Smith, I. W. J. Biol. Chem. /1988/ 263: 18726-18731. After cleaning, the receptors were placed in 0.1% Triton X-100 at 0.1-1.0 mg/ml

Receptors were applied to the cells in 96-porous flat plates ELISA /Linbro EIA-Plus microtiter plate, Flow Laboratories) after dilution 1: 200 solution of 20 mM Tris-HCl, 150 mM NaCl, 1 mM CaCl2, a pH of 7.4 to reduce the concentration of Triton-X to the lower micellar concentration and adding an aliquot of 100 μl to each cell. The cells were all placed in the incubator overnight at 4oC and then the liquid was removed until dry. Additional sites were blocked by adding serum albumin bull /BSA/ 35 mg/ml in the above buffer for 2 hours at 30oC to prevent nonspecific binding. Cells were then washed once binding buffer /50 mm Tris-HCl, 100 mm NaCl, 2 mm CaCl21 mg/ml BSA/.

Appropriate ligands /fibrinogen, factor a background of Villebranda or vitronectin/ were tagged with the major ligands were added to the coated receptor cells at the point of concentration of 10 nm/ /100 μl/ cell/ and were incubated for three hours at 30oC in the presence or absence of the tested samples. After incubation, the liquid is removed until dry and the associated ligand is determined quantitatively.

For125I-labeled ligand protein solubilizated 250 ál of SDS. For biotinylated ligands bound protein is detected by adding antibioticnevada antibodies to alkaline phosphatase, followed by the addition of the substrate /p-nitrophenylphosphate and determining the optical density of each cell at 405 nm. Reduced coloration or low content of 125I observed in the cells incubated with the test samples, which inhibit the binding of ligand to the receptor.

B. Determination of inhibition of adhesion in snake venom

Sixty-eight raw freeze-dried snake venoms were obtained from Sigma Chemical Company (St. Louis MO) or Miami Serpentarium Labs /salt lake city, Utah/ were dissolved at 1 mg/ml in buffer /50 mm Tris, 100 mm NaCl, 0.02% of azide, 2 mm CaCl2/. Onemillimeter aliquots of the solutions were subjected to ultrafiltration through Centrocon-10 /um membrane/ microconcentrators /Amicon, Danvers MA/. The filtrates were used as test samples in the analysis of receptor /ligand of paragraph A, using what you shown in the table. 1.

You can see that the activity is present in several, but not all types of Viperinae, but not in all species tested EI apidae.

Fig. 1 shows the results when different dilutions of the filtrate for four species. Even at the highest dilution of 25 ál/0.5 ml three active poison showed maximum inhibition.

C. determination of the activity of the peptides in the model of thrombosis

Purified peptides were tested for their ability to prevent the formation of blood clots in the coronary arteries of dogs in the model described Folts (Folts T. D., et al., Cerculation /1976/ 54: 365). In this model, it was shown that the reduction of blood flow in narrowed coronary arteries occurs because of the formation of platelet aggregates, and it was shown that agents that block the binding of fibrinogen to GP IIb-IIIa, prevent these reduce blood flow /Paste, R. S. et al., Blood /1986/, 68: 783/. Peptides were dissolved in normal saline and injected in a peripheral vein in a separate ball.

D. Effect of purified snake venom peptides on cell adherence to adhesive proteins

Melanoma cells M21, which Express high levels vitronektinove receptor, were the metabolic state of35the th accession provided an incubation period of 1 hour at 37oC and then followed by washing to remove leatheireannach cells. After washing adhered cells were solubilization and supernatant were placed in a liquid scintillation counter. The fraction of cells remaining adhered, was calculated by dividing the number of pulses per minute in solubilizing supernatant to the number of pulses per minute of the total number of cells added to each cell. The effect of purified snake venom peptides and synthetic cyclic peptides on cell adhesion was determined by placing them with cells M21 during the incubation period.

E. Specificity of inhibition of adhesion

The ultrafiltrates from three species of snake venom of Sistrurus m. barbouri, Crotalus ruber ruber, Crotalus Basilicus were tested in the analyses as fibrinogen /GP IIb-IIIa and vitronectin /vitronektinove receptor/ paragraph A. the Results were evaluated at various dilutions. As shown in Fig. 2, the venom of Sistrurus m. barbouri preferably inhibits the binding of fibrinogen to GP IIb-IIIa, the venom of Crotalus ruber ruber inhibits the binding of both systems is approximately equal way, and the venom of Crotalus basilicus preferably inhibits the binding of vitronectin/ vitronektinove receptor.

Table. 2 shows the results of the analysis of amino acid composition of the purified PAI examples 2-6, table. 3 shows the results for examples 8-11.

This analysis was carried out by hydrolysis of peptides using 6 N. HCl and analysis of the hydrolyzate, and used Weckman 121 HC analyzer, equipped with a system data Model 126. Cysteine acid was determined according to the method of Moore, J. Biol. Chem. /1969/ 230: 235-237. Tryptophan was not determined.

Example 2

Purification of an inhibitor of platelet aggregation /PAI/ from poison Eristocophis macmahoni

A solution of 45 mg Eristocophis macmahoni venom (Miami Serpentarium Labs, Lot # /EM23/ in 1.0 ml of 0.5% triperoxonane acid was cooled on ice for 20 minutes, tsentrifugirovanie at 14000 rpm for 3 minutes to remove insoluble material and loaded in the column, 3.9 mm x 30 cm, C-18, Delta-Pan reversal-phase column for high performance liquid chromatography /Waters, Milford MA/, equilibrated with 5% acetonitrile containing 1% triperoxonane acid. The gradient of passing from 5 to 15% acetonitrile in 5 minutes /2% in mi which was performed using a Waters 600 E liquid chromatograph. A flow rate of 1.5 ml/min was maintained throughout the gradient and effluent column was collected in 2 min fractions in polypropylene tubes.

Column effluent was tested at 220 nm/ 2.5 units of absorption FS /AUFS/.

Fractions were concentrated to one-half their original volume using a Speed-Vac concentrator /Savant/, followed by lyophilization. The samples were then dissolved in 1 ml of distilled water and aliquots /10-50 ál/ analyzed for their ability to inhibit the aggregation of human platelets in platelet-rich plasma induced by a 20 µm ADP, using aggregometer whole blood /Chiono-Log Corp., Havertown, PA/.

As shown in Fig. 3 activity was detected in fractions, which were suirable at 21-25% acetonitrile. These fractions were then dried and again passed through a column high-performance liquid chromatography /HPLC/ C-18 using a more shallow gradient of acetonitrile as described below, the initial conditions consisted of 8% acetonitrile, followed by a gradient up to 25% acetonitrile over 58 minutes /0,25%/min, then to 60% acetonitrile over 10 minutes. One-minute fractions were collected, dried and again about what is shown in Fig. 4, the active fractions were suirable at 24% acetonitrile. Active fractions were then analyzed by analytical high performance liquid chromatograph with detection at 220 nm and suirable as a single symmetric bioactive component, as shown in Fig. 5. Amino acid analysis of the material, purified by HPLC showed that the peptide contains 49 residues, including 7-8 cysteine, as shown in table. 2.

Attempts automated disintegration of Edman carboxymethylamino peptide gave no detectable sequence. Was therefore carried out the processing of the material with Lys-C and Asp-N endoproteinase, giving fragments, the sequence of amino acid residues which have been established and are shown in Fig. 6. This analysis revealed a sequence of 48 residues. However, because the two tryptophan residue are evident in this analysis the sequences that were not identified in the amino acid composition of intact peptide contains 51 amino acid residue. Thus, two Current and one Arg residues, loss of defined amino acid sequence, apparently, was present when the blocked amino end of the peptide. As it was quite likely that peptide thereof, we have removed this group from intact carboxymethylamino peptide with the enzyme pyroglutamyl aminopeptidase /L-pyroglutamyl peptide hydrolase, EC 3.4.11 . 8, Bochringer Mannheim, Biochemicals, Indianapolis /N/. The protocols described Podell and Abraham, Biochem., Biophys. Res. Commun. /1978/, 81:176-185 were used. Processing of 100 μg of peptide peptidases when the ratio of substrate-enzyme 100:1, followed by purification by high-performance liquid chromatograph with a reversible phase mixture on an analytical C-18 column (Waters, gave material, suitable for automated degradation of Edman. The results of this analysis and the distribution of the whole sequence of this peptide, which was named "ristocetin" shown in Fig. 6.

Complete amino acid sequence of this PAI shown in Fig. 6. This peptide has the RGD-binding region and shows significant homology to echistatin.

Example 3

Purification of PAI from the venom of Sistrurus catenatus tergeminus

Three hundred and sixty mg Sistrurus C tergeminus venom (Miami Serpentarium Labs, Lot # ST6SZ) was dissolved in 7.0 ml of 0.5 M acetic acid and filtered through a column of Sephadex C-50 (fine) Pharmacia 2.5 x 100 cm), equilibrated and washed with 0.5 M acetic acid. The column was turned away at a flow rate of approximately 25 ml/h and 5 ml fracc and so forth/ and lyophilized for analysis. Dried the collected fractions were again dissolved in water and aliquots were analyzed for inhibitory activity in ADP-induced aggregation of human platelets. Active fractions /31-40/ were dried and collected.

This material was dissolved in 2 ml of 0.5% triperoxonane acid and passed through a column 19 mm x 30 cm C-18 Delta Pak HPLC with reverse phase high-performance liquid chromatograph, balanced 8% acetonitrile containing 0.1% TFA. The gradient from 8% to 30% acetonitrile over 30 minutes and then up to 60% acetonitrile, twenty minutes was passed at a flow rate of 18 ml/min Effluent was collected in polypropylene tubes in 0.2 min fractions and tested at 220 nm /2.2 A UFA. Fractions were concentrated in a Speedvac concentrator /Savant/, lyophilized and analyzed by antiaggregatory activity with human platelets as previously described.

Fig. 7 shows that PAI-containing fractions elute at 24-25% acetonitrile. The analysis of these active fractions using HPLC with detection at 220 nm showed symmetric bioactive component, as shown in Fig. 8. Amino acid analysis of this material showed israsena and alkylated by iodization and purified on C-18 reversal-phase HPLC column. Analysis of the N-terminal sequence of this material showed the following amino acid sequence for 23 cycles of degradation of Edman Glu-Ala-Gly-Glu-Glu-Cys-Asp-Cys-Gly-Ser-Pro-Ala-Asn-Pro-Cys-Cys-Asp-Ala-Ala-Thr-Cys-Lys-Leu.

Complete amino acid sequence for this PAI, which was named "trigeminy" shown in Fig. 6.

The purified peptide was tested in the analyses based on the receptor described in example 1, paragraph A. the concentration of the pure peptide with less than 100 nm inhibited the binding of Fg and vWF to GP IIb-IIIa and VB and vWF with vitronectin receptor, as shown in Fig. 9.

Example 4

Purification of an inhibitor of platelet aggregation from the venom of Sistrurus milatas barbouri

Two hundred mg Sistrurus m. barbouri venom /Miami Serpentarium Labs, Lot # SM13SZ/ was dissolved in 7.0 ml of 0.5 M acetic acid and passed through the column Sephadex C-50 (fine) /Pharmacia 2.5 x 100 cm/, uravnoveshennoy and elyuirovaniya 0.5 M acetic acid. The column was at a flow rate of 26 ml/h and 5 ml fractions were collected and analyzed on antiaggregatory platelet activity, as previously described. Active fractions /41-50/ were combined and lyophilized. This material was again dissolved in 2.0 ml of 0.5% triperoxonane acid and passed through a preparative C-18 HPLC is tion were collected in polypropylene tubes, concentrated, lyophilized and analyzed for inhibitory activity of platelet aggregation.

Fig. 10 shows the activity profile of this column HPLC, which showed several factions /45-57/ that were more than 90% homogeneous. The peptide from the faction 46 /150 µg/ was purified to homogeneity by analytical C-18 column with manual collection symmetric peak, as shown in Fig. 11. Amino acid analysis of this material showed the peptide from 71-72 amino acids, including 12 cysteine residues, as shown in table. 2.

The purified peptide /150 µg/ was dissolved in 300 ál of reaction buffer /6 M guanidine HCl, 0.25 M Tris-HCl, 20 mm EDTA, 20 mm dithiothreitol /TT/, pH 7.5/ for 1.5 hours at room temperature to reduce the peptide. This was followed by the reaction of 3 μl of 4-vinylpyridine) - derivatives (Aldrich) at room temperature for an additional hour. The reaction was stopped by adding 200 μl of 1% triperoxonane acid and filtered through an analytical C-18 HPLC column and eleirovania gradient of acetonitrile in water containing 0.1% triperoxonane acid /TGA/ starting with 8% acetonitrile and passing to 25% acetonitrile over 20 min, then to 60% acetonitrile in 10 minutes.

Part of this clause which was carried out by exhaustive proteolytic cleavage restored and alkylated peptide using endoproteinase Lys-C and endoproteinase Asp-N peptide fragments, allocated either C-3 or C-18 reversal-phase HPLC columns using acetonitrile / water / TGA gradient elution. The amino acid sequence of the N-Terminus of intact peptide and isolated proteolytic fragments were determined as described Yarden, I. et al., Nature /1986/, 323: 226, when using an automated degradation of Edman on gas-phase sequencer.

Complete amino acid sequence of the selected peptide, named "Barbarin" shown in Fig. 6, along with sequences of proteolytic fragments. Comparison of this sequence with sequences of inhibitors of adhesion from another poison shown in Fig. 12.

Example 6

Purification of PAI from Lachesis mutas venom

99 mg Lachesis mutas venom (Miami Serp. Labs. Lot # LM15FZ) was dissolved in 2.0 ml of 0.5% triperoxonane acid, cooled on ice for 20 minutes, centrifuged at 14000 rpm for 3 minutes to remove insoluble material and was passed through the column, 3.9 mm x 30 cm, C-18 Delta Pak reversal-phase HPLC /Waters/, equilibrated with 5% acetonitrile containing 0.1% triperoxonane acid. A gradient from 5% to 15% acetonitrile in 5 minutes and then 30% after 35 minutes /2%/min and further to 60% acetonitrile over 20 minutes was the e fractions were collected, concentrated by Speedvac and dried. Fractions were analyzed for inhibitory activity of platelet aggregation.

Fig. 13 shows the active fractions that elute at 18% acetonitrile. These fractions were again banished through a column of C-18, using a smaller gradient, consisting of a 40 minute gradient from 5-28% acetonitrile. One-minute fractions were collected, concentrated, lyophilized and analyzed for the activity of inhibition of platelet aggregation, with the results shown in Fig. 14. These active fractions were run through an analytical C-18 column and a fraction lirovannomu Central peak was collected manually. Suirvey material, which is a single symmetric peak, as shown in Fig. 15, was subjected to amino acid analysis, which showed the peptide from 72-73 amino acids containing 12 cysteines, as shown in the table. 2.

Complete amino acid sequence of this PAI called "lagesen" shown in Fig. 6.

Example 6

Purification of PAI from the venom of Crotalus viridis viridis

47 mg of Crotalus viridis viridis venom Sigma Chemical Co. Lot # 24 F-0534/ was dissolved in 1 ml of 0.5% triperoxonane acid, cooled on ice for 20 minutes, centrifugals when the but-phase HPLC column /Waters/, balanced 5% acetonitrile containing 0.1% triperoxonane acid. A gradient from 5% to 15% acetonitrile in 5 minutes /2%/minute/, followed by a gradient from 15% to 30% acetonitrile in 35 minutes and then up to 80% acetonitrile over 60 minutes was banished. A flow rate of 1.5 ml/min was maintained throughout the gradient and column effluent was collected in a polypropylene test tube in a two-minute fractions. Column effluent was monitored at 220 nm/ 3.0 AUFS. The fractions were concentrated, lyophilized and analyzed for activity of inhibiting platelet aggregation.

Active fractions shown in Fig. 16, 18-19% acetonitrile, were run through a column of C-18 HPLC using a gradient of 8% to 20% acetonitrile in 48 minutes /0,25 /0,25% /min/. The fractions were concentrated, was liofilizirovanny and checked for activity and active fractions were run through a C-18 column using 8-16% acetonitrile over 10 minutes, 16-20% acetonitrile over 15 minutes and then 60% after 10 minutes. Effluent was monitored at 220 nm with individual peaks collected manually in polypropylene tubes. Re-analysis of the active peak on analytical HPLC gave the results shown in Fig. 17. Amino acid analysis providingquality the sequence PAI, named "viridis" shown in Fig. 5 and compared with other PAI in Fig. 12.

Example 7

Comparison of purified PAI with echistatin

The peptides were removed as described in examples 2 and 4, eristicophis and Barbarin were compared with the peptide of 49 residues by agitation in the inhibition of binding of fibrinogen to the GP IIb-IIIa, as described in example 1, paragraph A. Fig. 18 shows that these purified PAI are 2-3 times more active in this assay than the standard echistatin.

Peptides, purified to homogeneity from Echis carinatus Sistrurus m. barbouri and Eristicophis macmahoni poisons compared with agitation in the analysis of platelet aggregation stimulated by ADP. Increasing concentrations of purified peptides from snake venom was added /without pre-incubation at certain concentrations /Fig. 19/. The snake venom peptides from Ericticophis macmahoni and Sistrurus m. barbouri were at least two times more active than echistatin, which is consistent with their order of activity observed in the inhibition of binding of fibrinogen to GP IIb-IIIa, as provided above.

Example 8

Purification of PAI from Cratalus cerastes cerastes venom.

One gram Crotalus c. cerastis poison /Miami Serpentarium Labs, Lot # CE 4SZ/ was dissolved in 7.0 ml of 0.5 M acetic acid and skipped chere

The column was turned away at a flow rate of 25 ml/h with 5 ml fractions collected in polypropylene tubes. Aliquots of these fractions were analyzed for inhibitory activity aggregation activity, as described previously. Active fractions /71-80/ were collected and freeze-dried. The dried material was re-suspended in 2.0 ml of 0.5% triperoxonane acid, insoluble material removed by centrifugation and passed through a preparative C-18 Waters column high-performance liquid chromatography /HPLC/ as described in example 3 and blueraven, applying the terms of the gradient elution as described in example 3. Fractions from the column were collected in polypropylene tubes, concentrated and analyzed for activity in the inhibition of platelet aggregation. Fig. 20 shows the activity profile of this fractionation by high-performance liquid chromatography.

Active fractions with activity in the inhibition of platelet aggregation were collected and freeze-dried and re-passed through a preparative C-18 HPLC column, suirvey the same gradient. Fractions were collected manually in polypropylene tubes and were again tested for activity in the inhibition of the a W conditions described in example 4, and the homogeneous fractions were collected and lyophilized. Analysis of this material by analytical high performance liquid chromatograph shown in Fig. 21.

The purified peptide was subjected to amino acid analysis, showing that it was a peptide from 73-74 amino acids, contains 12 cysteine residues, as shown in table. 3.

The purified peptide /450 µg/ was dissolved in 750 μl of reaction buffer /6 M guanidine-HCl/, 0.25 M Tris-HCl 20 mm EDTA, 20 mm dithiothreitol /DDT/ pH 7,50 / for 1.5 hours at room temperature until complete "reduca" peptide, followed by reaction at room temperature for one hour with an excess of iodated /Fluka 16 mg/. The reaction was stopped by adding 500 μl of 1% triperoxonane acid and the reaction mixture is filtered through analytical column C-18 HPLC and eleirovania with a gradient of acetonitrile from 8 to 25% after 20 minutes, then up to 60% acetonitrile in 10 minutes. UV-absorbing peak was collected manually in a 1.5 ml tube Allendorf and dried.

Part of this carboxamido methylated peptide was analyzed by N-terminal sequence. Exhaustive proteolytic cleavage was carried out with iswidely on HPLC columns C-3 or C-13 reversible phase when using the conditions for gradient elution of acetonitrile / water / triperoxonane acid. Amino acid sequence was determined as described in example 4. Complete amino acid sequence defined for "kirstina" shown in Fig. 6 and compared with sequences from other PAI in Fig. 12.

Example 9

Purification of PAI from the venom of Crotalus ruber ruber

One gram of Crotalus ruber ruber venom /Miami Serpentarium Labs, Lot # CP17SZ) was dissolved in 8 ml of 0.5 M acetic acid and passed through a column of Sephadex C-50 (fine)(Pharmacia 2.5 x 100 cm), equilibrated at room temperature and elyuirovaniya acetic acid. The flow rate through the column was 25 ml/h with 5 ml fractions collected in polypropylene tubes. Aliquots of fractions were assayed for activity in the inhibition of platelet aggregation, as described. Active fractions /61-70/ were collected and freeze-dried. The dried material was re-suspended in 2.0 ml of 0.5% triperoxonane acid. Insoluble material was removed by centrifugation and passed through a preparative C-18 Waters HPLC column as described, and blueraven using the gradient conditions described in example 3. Fractions collected in polypropylene tubes were concentrated on a rotary evaporator and analyzed for activity in Engibarov the individual active fractions were lyophilized. Faction 43 and 50 were collected and passed through an analytical C-18 column with reverse phase and suirvey using the conditions described in example 4, which consisted of the combined acetonitrile gradient passing from 8% acetonitrile to 25% after twenty minutes, then ten minutes to 69% acetonitrile, giving a homogeneous peptide, which we call "suberin". Automated degradation of Edman carboxamidotryptamine peptide gives the sequence shown in Fig. 6.

Example 10

Purification of PAI from Crotalus atrox venom

One gram of Crotalus atrox venom (Miami Serpentarium Labs, Lot # CX16AZ) was dissolved in 10 ml of 0.5 M acetic acid and passed through a column of Sephadex C-50 (fine) (Pharmacia 2.5 x 110 cm), equilibrated and traversed at room temperature with 0.5 M acetic acid. The flow velocity during the passage of the column was 25 ml/h with 5 ml fractions collected in polypropylene tubes. Aliquots of fractions were assayed for activity in the inhibition of platelet aggregation, as described previously. Active fractions /81-100/ were collected and freeze-dried. The dried material was dissolved in 2.0 ml of 0.5% triperoxonane acid and passed through a preparative C-18 HPLC column as described in example 3. F. alisherovna on the activity in the inhibition of platelet aggregation as before. Active fractions were again passed through the analytical column C-18, and obtained a homogeneous peptide /Fig. 23/. Amino acid analysis of this material showed that the peptide contains 72 amino acids, including 12 cysteine residues, as shown in the table. 3. Amino acid sequence of the selected peptide, crotalaria shown in Fig. 6.

Example 11

Purification of PAI from Bothrops cotiara

Six hundred eighty milligrams of Bothrops cotiara poison (Miama Serpentarium Labs, Lot # BO5SZ) was dissolved in 10 ml of 0.5 M acetic acid and passed through the column Sephadex C-50 /thin/ /Pharmacia 2.5 x 110 cm/, balanced and elyuirovaniya 0.5 M acetic acid. The column was passed at a flow rate of 25 ml/h with 5 ml fractions collected in polypropylene tubes. Aliquots of fractions were assayed for activity in the inhibition of platelet aggregation, as described previously. Active fractions /71-80/ were collected and lyophilized, and the dried material was re-suspended in 2.0 ml of 0.5% triperoxonane acid and passed through a preparative column C-18 reverse phase. The column was eleirovania using conditions described in example 3. Fractions were collected in polypropylene tubes, concentrated on AI were separately dried. Fraction of several peaks were again passed through analytical C-18 column as described in example 4. Analytical HPLC profile of homogeneous peptide shown in Fig. 24. Amino acid analysis of this material shows that this peptide contains 72 amino acids,

including 12 cysteine residues, as shown in the table. 3. Full sincinaty the sequence of this peptide, which we call "katarin" shown in Fig. 6 and 12.

The purified peptide was tested in receptor assays described in example 1. Initial determination showed that low concentrations of Katerina /1-4 μm/ selectively inhibit the binding of vitronectin with vitronectin receptor, whereas the same concentration had a significantly lower inhibitory activity in binding of fibrinogen to GP IIb-IIIa, as shown in Fig. 25, however, subsequent experiments failed to confirm this result.

Example 12

Purification of PAI from Crotalus viridis lutosus

A. One gram Crotalus viridis lutosus venom (Miami Serpentarium Labs, Lot # CL18SZ) was dissolved in 8 ml of 0.5 M acetic acid and passed through a column of Sephadex C-50 (fine) (Pharmacia 2.5 x 110 cm) which was equilibrated and eleirovania 0.5 M acetic acid. Flow rate at yli analyzed for activity in the inhibition of platelet aggregation. Faction /71-100/ were collected and freeze-dried. The dried material was re-suspended in 2.0 ml of 0.5% triperoxonane acid. Insoluble material was removed by centrifugation and passed through a preparative C-18 Waters reversal-phase column and blueraven using conditions for gradient elution as described in example 3. Fractions from the column were collected in polypropylene tubes, concentrated on a rotary evaporator and analyzed for activity in the inhibition of platelet aggregation. Active fractions were lyophilized in individual test tubes. Fractions with activity were again passed through a Waters analytical C-18 column using the combined acetonitrile gradient as described in example 4. Fractions were collected manually in a 1.5 ml Eppendorf tube. Homogeneous fractions were collected and lyophilized. Analytical high performance liquid chromatography of this material showed a single symmetrical peak. Complete amino acid sequence of this peptide, which we call "litozin" shown in Fig. 6 and 12.

B. in a Manner analogous to the method described in paragraph A, PAI from B. jararacussu, C. basilicus, C. durissus dirissus, C. v. oreganus, C. h. horridus, C. v. Dov shown in the table. 3. Amino acid sequence of PAI C. h. horridus, C. basilicus, C. m. molossus, C. v. oreganus, and c. d. durissus marked as harriden, basicily, Molossian, oregonin and durisin respectively, as shown in Fig. 6. Data on receptor binding to purified peptides of examples 1-12 are shown in Fig. 26.

In examples 13-16 below, the peptides were synthesized using solid-phase methods on an Applied Biosystems 431A peptide synthesizer using t-Boc amino acid, activated as HOBt active esters in accordance with the manufacturer's instructions, which are presented briefly in the following way to obtain Boc-AA1.....AA/p-1/ AA - /p/-O-PAM-polystyrene resin.

Half millimole selected Vos-AA/p/-O-PAM-polystyrene resin is processed in accordance with the following scheme for the unification of Boc-AA/p-1/HE:

1) removing the protection triperoxonane acid: 30% TFA in D, 3 min, 50% TFA in DCM, 16 minutes

2) Rinsing and neutralization: DCM wash /5X/, 3 min, 5% DIEA in DCM, 2 min, 5% DIEA in NMP, 2 min NMP wash /6X/, 5 min.

3) Binding: 4 equivalent of Boc-AA-HOB ester in NMP /pre-activation 55 min, 38 min, DMCO for the preparation of 15% DMCO/ 85% NMP, 16 min, 3.8 equivalents of DIEA, 5 minutes.

4) Rinse and sample resin: NMP about the 4 minutes

Example 13

Receiving analog 1

[E28L41C64] Barbarin /28/73/:

E-C-A-D-G-L-C-C-D-Q-C-R-F-L-K-K-G-T-V-C-R-V-A-K-G-D-W - N-D-D-T-C-T-G-Q-S-C-D-C-P-R-N-G-L-Y-G

Half millimole PAM-Cly resin /0.6 mEq/g Applied Biosystems Foster City, CA) was subjected to procedure A with the required amino acids /put in order/. Boc-protected amino acids had the following protection in the side chain: Arg (Tos), Asp (OcHex), Cys (4-MeBzl), Glu (OcHex), Lys (Cl-Z), Thr (OBzl), Trp (CHO) and Tyr (Br-Z).

After ruffles complete the secure chain peptide-resin, aminoterminal Boc-group was removed triperoxonane acid and the resin dried as its TFA-salt form. Resin /1.3 g/ was subjected to a "low-high" HF procedures unprotect, followed by removal of HF under vacuum. The dried mixture of the peptide-resin was transferred to a glass filter /coarse/ with ethyl ether and was washed several times alternating washes of ether and chloroform to remove most of the organic protecting groups and substances used in the removal of protection.

The peptide mixture was transferred to 2 l of 0.4% acetic acid and the pH increased to 7.39 concentrated NH4OH. The resin was filtered from this solution and the solution is left to precipitate at 4oC without stirring in teeniees mixing. Precipitated material was removed by filtration and the filtrate was brought to pH = 3,0 acetic acid and lyophilized.

The raw material was dissolved in 8.0 ml of 0.5 M acetic acid and passed through Safadas C-50 /slim/ column /2.5 x 100 cm, equilibrated with 0.5 M acetic acid. The column was passed at 20 ml/h and fractions /4 ml were collected in polypropylene tubes. Aliquots of the fractions were dried, re-suspended in water and analyzed for activity in the inhibition of aggregation as described above. Active fractions /71-80/ were collected and lyophilized.

The dried material /66 mg/ was re-dissolved in 2.0 ml of 0.1 M acetic acid and passed through a preparative Waters C-18 column equilibrated with 8% acetonitrile, containing 0.1% triperoxonane acid. The gradient of passing from 8% acetonitrile, 20% in 10 minutes, followed by a slow gradient up to 30% acetonitrile in 40 min was carried out. The column was eleirovania at 18 ml/min and fractions /12/ were collected in polypropylene tubes. The fractions were concentrated on a rotary evaporator to 1.0 ml volume and 10 µl aliquots were tested in the analysis of platelet aggregation.

Active fractions /29-32/ were indie is m Fractions 29 and 30 were collected and passed through the analytical column at 1.0 ml of 0.5% triperoxonane acid. The main peak was collected manually and dried, yielding 1.6 mg of pure peptide.

Amino acid analysis of this material confirmed the identity of the peptide. Analysis of this material on its ability to inhibit the binding of fibrinogen to GP IIb-IIIa and vitronectin with VnR is shown in Fig. 26 and 27. These data demonstrate the high affinity of the analog for GP IIb-IIIa and the relative lack of epinasty for VnR at concentrations up to 1 μm.

Example 14

Receiving analog # 2, [K29]eristicophis /4-51/:

E-E-P-C-A-T-G-P-C-C-R-R-C-K-F-K-R-A-G-K-V-C-R - V-A-K-G-D-W-N-N-D-Y-C-T-G-K-S-D-C-P-R-N-W-N-G

Half millimole PAM-Cly resin /0.6 mEq/g Applied Biosystems, Foster City, CA) was subjected to procedure A with the required amino acids /put in order/. The BOC-protected amino acids had the following protection of the side chain: Arg (Tos), Asp (OcHex), Cys (4-MeBzl), Glu (O-cHex), Lys (ClZ), Ser (OBzl), Trp (CHO) and Tyr (Br-S).

Splitting, Assembly and purification of the peptide was identical to the above examples. Data binding receptor for this analogue is shown in Fig. 26 and 28.

Example 15

Receiving analog # 3: G-C-C-K-C-D-W-P-C-A-NH2< / BR>
Half myllyniemi in order/. Boc-protected amino acids had the following protection of the side chain: Asp (O-cHex), Cys (4-MeBzl); and Lys (Cl-Z) After Assembly is complete, substituted peptide-resin aminoterminal Boc group was removed using triperoxonane acid and the resin dried as its TFA-salt form. Resin /1.54 g/ was treated with anhydrous hydrogen fluoride /HF/ containing 10% anisole, 2% ethylmercury, for 30 min at -10oC and an additional 30 minutes at 0oC. the HF was removed in vacuo and the mixture of the peptide/resin was suspended in diethyl ether, followed alternately by washing with chloroform and ether 3X. After the last washing with ether, the peptide was extracted from the resin using 2.0 M acetic acid, diluted with distilled water and dried.

The crude peptide /370 mg was dissolved in obeskislorozhennuju 10 mm NH4OAc pH 8, 0.5 mg/ml and subjected to oxidation by adding dropwise a small excess of 0.01 M solution of ferrocyanide of potassium /K3Fe(CN)6/ mixed an additional 20 minutes, and brought to pH 5 with acetic acid. The peptide solution was treated with Dowex AC3 x 4 anion-exchange resin for 15 minutes with stirring and the resin filtered, diluted with H2O and dried, yielding crude cyklinowanie acid as eluent, with subsequent ion exchange chromatography on CM-Sepharose /Pharmacia/ using gradient elution produced by adding 100 mm NH4OAc to a solution of 10 mm NH4OAc pH 4.5. Fractions that had a minimum purity of 90% by HPLC analysis were collected and freeze-dried from H2O several times, giving 175 mg. Final cleaning consisted of preparative HPLC purification on a Waters C-18 reversal-phase column with acetonitrile/water, TFA gradient, giving the pure peptide. Data binding receptor for this analogue is shown in Fig. 26, 29 and 30.

Example 16

Additional analogs

The following analogs were synthesized: in most cases a manner similar to the shown in example 15. However, similar 60, shown below, was obtained in solution through grandravine the side chain of the lysine residue analog #19 using the procedure Bajuss S., FEBS Letts /1980/ 110: 85-87.

One mg of analog #19 was introduced in the interaction with 1 mg 1-amidino-3,5-dimethylpyrazole /Aldrich/ 1 ml of absolute ethanol in the presence of diisopropylethylamine /DIEA/ at room temperature for 4 days. Analogue 60 was purified from excess reagent and raw materials by reversal-phase HPLC on C-13 column primenyaite.

#48: Mpr-K-G-D-W(Formyl)-P-C-NH2< / BR>
#49: Mvl-K-G-D-W-P-C-NH2< / BR>
#50: Mpr-K-G-D-Wt-P-Pen-NH2< / BR>
#51: Mpr-K-G-D-W-P-Pen-NH2< / BR>
#52: Mpr-K-G-D-W-P-Pent-NH2< / BR>
#53: Mpr-K-G-D-W-Pt-Pen-NH2< / BR>
#54: Mpr-K-G-Dt-W-P-Pen-NH2< / BR>
#55: Mpr-K-G-D-W-(Thz-C-NH2< / BR>
#56: Mpr-K-G-D-H(2,4-DNP)-P-C-NH2< / BR>
#57: Mpr-K-G-D-(2-Nal)-P-Pen-NH2< / BR>
#58: Mvl-K-G-D-W-P-Pen-NH2< / BR>
#59: Mpr-K-G-D-W-(Pip)-Pen-NH2< / BR>
#60: Mpr-(Har)-G-D-W-P-C-NH2< / BR>
#61: Mpr-K-G-D-W-P-Ct-NH2< / BR>
#62: Mpr-(D-Lys)-G-D-W-P-Pen-NH2< / BR>
#63: Mpr-(Har)-G-D-W-P-Pen-NH2< / BR>
#64: Mpr-(Acetimidyl-Lys)-G-D-W-P-C-NH2< / BR>
#65: Mpr-(Acetimidyl-Lys)-G-D-W-P-Pen-NH2< / BR>
#66: Mpr-(NGNG-ethylene-Har)-G-D-W-P-C-NH2< / BR>
#67: Mpr-(NGNG-ethylene-Har)G-D-W-P-Pen-NH2< / BR>
#68: Mpr-Har-Sar-D-W-P-C-NH2< / BR>
#69: Mpr-(Acetimidyl-Lys)-G-D-W-P-Pen-NH2< / BR>
#70: Mpr-(Phenylimidyl-Lys)-G-D-W-P-C-NH2< / BR>
#71: Mpr-Har-Sar-D-W-P-PenNH2< / BR>
#72: Mpr-(Phenylimidyl-Lys)-G-D-W-P-PenNH2< / BR>
#73: Mpr-Har-G-D-W-(3,4-dehydro-P)-C-NH2< / BR>
#4: G-C-K-G-D-W-P-C-A-NH2< / BR>
#5: C-G-K-G-D-W-P-C-NH2< / BR>
#6: G-C-G-K-G-D-W-C-A-NH2< / BR>
#7: G-C-K-G-D-W-C-A-NH2< / BR>
#8: Acetyl-C-K-G-D-C-NH2< / BR>
#9: Mpr-K-G-D-Pen-NH2< / BR>
#10: C-K-G-D-W-P-C-NH2< / BR>
#11: Acetyl-C-R-G-D-Pen-NH2< / BR>
#12: C-K-G-D-Y-P-C-NH2< / BR>
#13: C-K-G-D-F-P-C-NH2< / BR>
#19: Mpr-K-G-D-W-P-C-NH2< / BR>
#34: C-K-G-D-W-G-C-NHSUB>2< / BR>
#39: C-K-G-D-W-(Sar)-C-NH2< / BR>
#40: C-K(Formyl)-G-D-W-P-C-NH2< / BR>
#41: C-K-G-D-I-P-C-NH2< / BR>
#42: C-K-G-D-(4-Cl-Phe)-P-NH2< / BR>
#43: C-K-Sar-D-W-P-C-NH2< / BR>
#44: C-K-G-D-(4-NO2-Phe)-P-C-NH2< / BR>
#45: C-K-G-D-(NMePhe)-P-C-NH2< / BR>
#46: C-H-G-D-W-P-C-NH2< / BR>
#47: Acetyl-C-K-G-D-W-P-C-NH2< / BR>
Example 17

PAI-active peptides

When tested in standard assays for inhibition of aggregation described above, analogs # 3-5 had the IC values of 5 ám for the ability to inhibit ADP-induced aggregation of human platelets. However, similar # 6 has the IC50more than 20 μm, and similar #7 - 100 microns.

The values of the IC50for analogues of the invention in this analysis are the values presented in table. 4.

Example 18

The activity of the linear peptides compared to the circular

When tested for inhibition of binding of fibrinogen to GP IIb-IIIa in laminar analysis, linear RGDW-NH2was quite similar to the activity of cyclic G-CGRGDWPCA-NH2/Fig. 29/. On the contrary, linear KGDW-NH2that was a lot less active than cyclic GCGKGDWPCA-NH2/Fig. 29/. For KGDW compounds, but not RGDW-compounds, cyclization led to a noticeable increase in the ability of the peptide ingibirovala synthetic peptides

The peptides synthesized in example 17, in addition to the assessment on the ability to inhibit platelet aggregation directly, were also tested in the analysis of the invention, as described above. Results for analogs 4-8 shown in Fig. 30. As can be seen from the drawing, these analogues are capable in different ways, changing the order to inhibit the binding of fibrinogen to GP IIb-IIIa, compared to the binding of vitronectin with vitronectin receptor. It turns out that the analogue of 4 among this group has the greatest difference. Analogues 7 and 5, on the other hand, it is also quite specific and have an excellent activity of inhibiting platelet aggregation.

Example 20

The effect of purified peptides on cell adhesion

Melanoma cells M21 was in the state of35S-methionine and then added to the coated with vitronectin plates in the presence of certain concentrations of purified peptides from snake venoms. Cell adherence was measured by solubilization of cells remaining after incubation and washing, as described in section C. As shown in Fig. 31, no Barbarin, neither peptide 1 /shortened Barbarin/ had no significant effect on cell adhesion to vitronectin, although both are potedia active inhibitor of the binding of vitronectin with vitronectin receptor, was very active in the inhibition of the binding of cells to vitronectin. In a similar experiment, peptide #3, peptide #3 with K replaced by R (GCGRGDWPCA-NH2), and RGDS were investigated on the accession of vitronectin to the cell M21. As shown in Fig. 32, RGDS GCGRGDWPCA-NH2are active inhibitors of the cellular connection, whereas GCGKCDWPCA-NH2were ineffective up to 60 microns.

Example 21

Comparison of analogs #60 and #19

Analogues 60 and 19, described above, are peptides of the invention containing a sequence of K*CDX and are identical, except for the realization of K*. Analogue 60 has the formula:

Mpr-(Har)-G-D-W-P-C-NH2< / BR>
analogue 18 - formula:

Mpr-K-G-D-W-P-C-NH2< / BR>
These analogues were tested using standard assays for inhibition of platelet aggregation and the analysis of cell adhesion example 20 described above. The results are presented in Fig. 33 and 34. As shown in Fig. 33, similar 60 is effective at vanishingly small concentrations in the inhibition of platelet aggregation and is relatively less effective in preventing cell adhesion to vitronectin. Fig. 34 shows that similar #19 has a good active on the inhibition of platelet aggregation than its counterpart # 60. Similar #60 has the IC50in platelet aggregation approximately 0.15 nM; similar #19 has the IC50from about 1 nM.

Example 22

"Filt" model of thrombosis in the coronary artery of dogs.

A. Initiation of the reduction of cyclic flow (CFRs) in open chest dogs. The obturator was placed in the left anterior descending (LAD) coronary artery 20 kg dog, as previously described. "Phasic" and "mean" blood flows measured by electromagnetically /EM/ sample flow and sample flow Doppler shown in Fig. 35.

B. the Influence of cyclic GCGKGDWPCA-NH2/analogue #3/ CFRsin open chest dogs

Dose of 100 mg of this peptide was introduced into a peripheral vein of the dog. It is shown in Fig. 36 is a diagram of blood flow in LAD, as described above. Note partial clipping of the CFR, as can be seen in the lower loop of flow reductions. Note also that the flow is not reduced to the same extent as in the control /A/.

C. Second injection 40 mg analogue # 3 was conducted in a peripheral vein. As shown in Fig. 37, full shutoff CFR shows that was restored full flow in the LAD.

Example 23

Construction of expression vectors for barbarisoviy oligonucleotides, as shown in Fig. 38, which were kinesiology, hardened and stitched into EcoRI-Hill I, recycled M13 mp 18 using standard techniques. A single sequence of bacterial alkaline phosphatase gene /pHoA/ Watson M. E. E., Nucleic Acids Research /1984/ 12:5145 was added to barborinou design by stitching of synthetic oligonucleotides into EcoR I/ NcoI sites [L14] Barbarin /1-73/ design, as shown in Fig. 39. Nucleotide sequences of all constructs were confirmed by Sandy method dideoxy chain breakage.

A shortened version of this peptide was also constructed from synthetic oligonucleotides that encode only amino acids 28-73 of the full length molecule. Two changes, O28E28and A64C64were entered using siteprovides mutagenesis as described by Kunkel et al. Meth. Enzymol. /1987/ 154: 367. pHoA single sequence was added to the shortened version, as described above /Fig. 40/. In addition, a single sequence for E. coli stable to heat enterotoxin II /Picken, R. W. , et al., Ynfect. Immun. /1983/, 42:269/ was added to a shortened version using synthetic oligonucleotides with EcoR I and NcoI compatible ends. All id:161, manufactured from CLONTECH Lab, Inc.) using EcoR I and Hind III restriction endonucleases.

The gene encoding the adjacent duplication of the desired peptide was obtained using polymerase chain reaction /PRC/ for multimerization unit barborikova peptide full length 1-73 containing L41 and C64.

Fig. 41 shows the oligonucleotides used for PCR synthesis. PCR reaction was carried out according to the method of Saiki R. K., et al., Science /1988/ 239: 487. The resulting polymer compound contains methionine at each end of the sequence, as shown in Fig. 42, and provides the desired restriction sites to construct.

Related duplications are formed from individual multiparametric components using, for example, stitching EcoR I/BamHI fragment with Bgl II /Nind III fragment into M13 mp18 vector cut with EcoR I/Hind III dimer formation. The resulting dimer cut EcoR I and BamHI and re-sewn with Bgl II/Nind III fragment to obtain a trimer, and so on up until you get the desired size. This design is presented in chart form in Fig. 43.

Multimer was then sewn into the E. coli vector pKK233-2 Amann E, bene /1985/ 40: 183 manufactured by Clontech processing vector Nco I / Hind III and stitched introduction protein is used above the revised vector along c NcoI-EcoRI-subfragments, contains slightly modified aminoterminal part /amino acids 1-72/ from chloramphenicole acetyltransferase gene Chang, C. N. et al., Gene /1987/ 55: 189 and EcoRI-Hind III subfragment multimeric structures.

Example 24

Expression of recombinant gases

The expression of the protein from all recombinantly plasmids described above, is carried out according Kanamari et al., Gene /1988/ 66: 295 after transfection in appropriate host strains of E. coli. The products are characterized by electrophoresis sodium dodecyl sulphate polyacrylamide gel and their ability to inhibit ADP-induced platelet aggregation in platelet-rich plasma. After cleaning multimeric proteins into Monomeric units Langenbrunner splitting and the products analyzed as described above.

1. The method of determining the presence or absence of activity in the inhibition of platelet aggregation (PA1) in a biological fluid, characterized in that carry out the contacting of the liquid sample with purified trombotsitnoy GP IIb-IIIa in the presence of a solution of fibrinogen (Fg) or factor von Willebrand's disease (vWF), under conditions in which Fg and vWF are associated with the above GP IIb-IIIa, and the definition of reduction iliary does not contain a biological fluid.

2. The method according to p. 1, characterized in that further perform at least one analysis selected from the group consisting of determining the inability or ability of the above-mentioned liquid to inhibit the binding of vitronectin with vitronectin receptor, determining the inability or ability of the above-mentioned liquid to inhibit the binding of fibronectin with fibronectin receptor, determining the inability or ability of the above-mentioned liquid to inhibit the binding of fibronectin with GP IIb-IIIa receptor and determining the ability or inability of the aforementioned fluid to inhibit the binding of factor a background of Villebranda vitronectin receptor.

3. The purified inhibitor of platelet aggregation (PA1), derived from snake venom, characterized by the fact that has a specific ability to inhibit the binding of fibrinogen and/or the factor a background of Villebranda with GP IIb-IIIa and the relative inability to inhibit the binding of vitronectin with vitronectin receptor or to inhibit the binding of fibronectin with fibronectin receptor.

4. Cleaned and selected PA1 or modified and/or truncated form, derived from snake venom, select the ra, B. jararaca, B. jararacussu, B. lansbergi, B. medusa, B. nasuta, B. neuwiedi, B. pradoi, B. schlegli; Crotalus atrox, C. basilicus, C. cerastes cerastes, C. durissus durissus, C. durissus totonatacus, C. horridus horridus, C. molossus molossus, C. ruber ruber, C. viridis cereberus, Crotalus v. helleri, Crotalus v. lutosus, Crotalus v. oreganus, Crotalus v. viridis; Lachesis mutas; Sistrurus catenatus tergeminus, and Sistrurus milarus barbouri, identifiable according to any one of paragraphs.1 and 2, with snake venom is not Echis carinatus, Trimeresurus gramineus, T. flavoviridis, T. Elegans, T. albolabris, Bitis arietans, Bothrops atrox, Ukraine halys, Ukraine p. Piscivorus or Ukraine rhodostoma.

5. PA1 under item 4, wherein the snake venom is selected from the group consisting of Eristicophis macmahonii (eristicophin); Bothrops cotiara (cotiarin); B. jararacussu; Crotalus atzox (cotratroxin); Crotalus basilicus ( basilicin); C. cerastes cerastes (cerastin); C. durissus totonatacus; C. h. horridus (horridin); Crotalus m. molossus (molossin); C. v.helleri; C. ruber ruber (ruberin); Crotalus viridis lutosus (lutosin); C. v.viridis(viridin); C. v. oreganus (oreganin); C. durissus durissus (durissin); Lachesis mutas (lachesin); Sistrurus catenatus tergeminus (tergemin); and S. milarus barbouri (barbourin).

6. PA1 on PP.4 and 5, characterized in that the isolation and purification of snake venom spend the transmission of the poison through the chromatographic column to obtain a solution containing a component with a molecular weight of less than 10 KD, with subsequent transmission of the above-mentioned fractions through highly permitting liquid chromatograph with a reverse phase with many factions and the allocation of those factions, kotlerman weighing less than 10 KD receive the transmission of the poison through the chromatographic column, using gel filtration size, elute fractions and allocate fractions of inhibiting the binding of fibrinogen to GP IIb-IIIa.

8. Cleaning method PA1 from snake venom, characterized in that carry out the transmission of the poison through the column, separating by size, to obtain a solution containing components with a molecular weight of less than 10 KD and subsequent transmission of the above-mentioned fractions through high-resolution liquid chromatograph with a reverse phase with many factions and the release of factions, inhibiting the binding of fibrinogen to GP IIb-IIIa.

9. The cleaning method on p. 8, characterized in that the fraction less than 10 KD is obtained by transmission of the poison through the chromatographic column, using the gel, separating by size, elution of the many factions and allocation of fractions, which inhibit the binding of fibrinogen to GP IIb-IIIa.

10. Pharmaceutical composition for preventing the formation of thrombus, characterized in that it contains PA1 under item 3 in an amount effective to prevent the formation of a blood clot in a mixture with a pharmaceutically acceptable filler.

11. Method of inhibiting the formation of thrombus at the animal, wherein the animal is administered the effect is icesto 0.01 to 10 µg/kg body weight.

12. Pharmaceutical composition for preventing the formation of thrombus, characterized in that it contains PA1 on p. 4 in a quantity effective to prevent the formation of thrombus, in a mixture with a pharmaceutically acceptable filler.

13. Method of inhibiting the formation of thrombus in animals, characterized in that it is administered an effective amount PA1 on PP.4 - 7, or pharmaceutical composition in an amount of 0.01 - 10 μg/kg body weight.

14. Inhibitor of platelet aggregation in a purified and selected as PA1, isolated from snake venom, and containing as an inhibitor sequence amino acid sequence Lys-Gly-Asp(KGD).

15. Inhibitor of platelet aggregation under item 14, characterized in that it contains the following amino acid sequence:

< / BR>
or modified and/or truncated form.

16. PA1, is able to inhibit the binding of Fg and vWF to GP IIb-IIIa on being more active than the inhibition of the binding of vitronectin with vitronectin receptor or fibronectin to fibronectin receptor, and PA1 contains the amino acid sequence K*(Sar|G)D, where K* is lazily OST the scrap (1-6C) or at most one R1is R2-C = NR3where R2- H, alkyl (1-6C) or is a substituted or unsubstituted phenyl or benzyl residue, or represents NR4in which each R4is independently H or alkyl (1-6C) and R3- H, alkyl (1-6C), phenyl or benzyl, or R2-C = NR3represents a radical selected from the group consisting of

< / BR>
< / BR>
where m is an integer of 2-3, and each R5is independently H or alkyl (1-6C), and where one or two CH2can be replaced by O or S provided that the above O or S are not adjacent to other heteroatoms,

and where the above K*(Sar/G)D is contained in the cyclic peptide of at least 8 amino acid residues.

17. PA1 under item 16, characterized in that it has a primary structure PA1 natural origin containing inhibitory sequence RGD or KGD or shortened and/or modified their form, where the residue in the RGD sequence found in the above PA1 or shortened and/or modified form, is replaced by K* or K KGD sequence found in the above PA1 or shortened and/or modified form, is replaced in the embodiment K*, which is not K

p. 18, characterized in that as PA1 natural origin used PA1 selected from the group consisting of trigemina, echistatin, elegantin, albolabris, Lahaina, flavoviridis, eristicophis, Cheremisina, rhodostoma, applewine, alisina, Batistuta, rubeena, ceratina, Katarina, crotalaria, harridine, basilicia, lutsina, molossia, durisin, carrazana, serebrin, oregonia, virigina and Barbarina.

20. PA1, is able to inhibit the binding of Fg or vWF to GP IIb-IIIa on being more active than the inhibition of the binding of vitronectin with vitronectin receptor or fibronectin to fibronectin receptor, and PA1 has the formula

< / BR>
where K* is lazily balance formula

R21N(CH2)4CHNHCO-

where each R1is independently H, alkyl (1-6C) or at most one R1is R2-C = NR3where R2- H, alkyl (1-6C) or is a substituted or unsubstituted phenyl or benzyl residue, or represents NR42in which each is independently H or alkyl (1-6C) and R3- H, alkyl (1-6C), phenyl or benzyl, or R2-C = NR3represents a radical selected from the th independently H or alkyl (1-6C) and one or two (CH2) can be replaced by O or S provided that the above O or S is not adjacent to another heteroatom;

AA1is a small, neutral (polar or nonpolar) amino acid;

n1 is an integer from 0-3;

AA2neutral, nonpolar large (aromatic or nonaromatic), or polar aromatic amino acid;

n2 is an integer from 0-3;

AA3- polynomy residue or modified polynomy the rest;

n3 is an integer from 0-1;

AA4neutral, a small amino acid or N-alkilirovanny its form;

n4 is an integer from 0-3;

each of X1and X2represents independently a residue capable of forming communication between the X1and X2to obtain cyclic compounds as shown;

each of Y1and Y2represents independently numerouse Deputy or may not exist where one or more peptide bonds may be optionally replaced by a linkage selected from the group consisting of-CH2NH-, -CH2S-, -CH2CH2-, -CH=CH-(CIS-TRANS), -COCH2-, -CH(OH)CH2- and-CH2SO-; with the proviso that n3 = 0 or 1) the sum of n2 and n4 must be at least 2, or (2) K* must be drugtown connection.

21. PA1 on p. 20, characterized in that Y1represents H, acyl, or a peptide residue or derivative, or absent, Y2- OH, H, or a peptide residue, or a derivative or missing.

22. PA1 on p. 21, characterized in that Y2represents NH2-A-NH2or not.

23. PA1 on p. 21, characterized in that Y1- H, acetyl, Q, or absent.

24. RAO p. 20, characterized in that X1and X2selected from the group consisting of cysteine (C), mercaptopropionyl (Mpr) and penitsillamin(Pen).

25. PA1 on p. 20, wherein AA1 is G and n1= 0 or 1.

26. PA1 on p. 20, wherein AA2 is selected from the group consisting of W, F, L, Y, and V

27. PA1 under item 26, wherein AA2 is a W.

28. PA1 on p. 20, characterized in that K*-K, Har acetimidoyl - Lys or phenylamides - Lys.

29. PA1 on p. 20, wherein is selected from the group consisting of:

PA1 1: E-C-A-D-G-L-C-C-D-Q-C-R-F-L-K-K-G-T-V-C-R-V-A-K-G-D-W-N-D-D-T-C-T-G-Q-S-C-D-C-P-R-N-G-L-Y-G

PA1 2: E-E-P-C-A-T-G-P-C-C-R-R-C-K-F-K-R-A-G-K-V-C-R-V-A-K-G-D-W-N-N-D-Y-C-T-G-K-S-C-D-C-P-R-N-P-W-N-G

PA1 3: G-C-G-K-G-D-W-P-C-A-NH2;

PA1 4: G-C-K-G-D-W-P-C-A-NH2;

PA1 5: C-G-K-G-D-W-P-C-NH2< / BR>
PA1 7: C-K-G-D-W-C-A-NH2< / BR>
PA1 10: C-K-G-D-W-P-C-NH2< / BR>
PA1 16: C-K-G-D-Y(Ome)-P-C-NH2< / BR>
PA1 17: C-K-G-D-(2-Nal)-P-C-NH2< / BR>
PA1 18: C-K-G-D-(Cha)-P-C-NH2< / BR>
PA1 19: Mpr-K-G-D-W-P-C-NH2< / BR>
PA1 20: Mpr-K-G-D-Y-P-C-NH2< / BR>
PA1 21: Mr-K-G-D-F-P-C-NH2< / BR>
PA1 22: Mpr-K-G-D-L-P-C-NH2< / BR>
PA1 23: Mpr-K-G-D-V-P-C-NH2< / BR>
PA1 24: Mpr-K-G-D-Y(Ome)-P-C-NH2< / BR>
PA1 25: Mpr-K-G-D-(2-Nal)-P-C-NH2< / BR>
PA1 26: Mpr-K-G-D-(Cha)-P-C-NH2< / BR>
PA1 27: cyclo (G-K-G-D-W-P)

PA1 28: cyclo (A-K-G-D-W-P)

PA1 29: cyclo (A-K-G-D-W-P)

PA1 30: cyclo (F-K-G-D-W-P)

PA1 31: cyclo (beta - Ala-K-G-D-W-P)

PA1 32: cyclo (gamma-Abu-K-G-D-W-P)

PA1 33:cyclo (R-K-G-D-W-P)

PA1 34: C-K-G-D-W-G-C-NH2< / BR>
PA1 39: C-K-G-D-W-(Sar)-C-NH2< / BR>
PA1 41: C-K-G-D-L-P-C-NH2< / BR>
PA1 42: C-K-G-D-(4-Cl-Phe)-P-NH2< / BR>
PA1 43: C-K-Sar-D-W-P-C-NH2< / BR>
PA1 44: C-K-G-D-(4-NO2-Phe)-P-C-NH2< / BR>
PA1 47: Acetyl-C-K-G-D-W-P-C-NH2< / BR>
PA1 48: Mpr-K-G-D-W(Formyl)-P-C-NH2< / BR>
PA1 49: Mv1-K-G-D-W-P-C-NH2< / BR>
PA1 51: Mpr-K-G-D-W-P-Pen-NH2< / BR>
PA1 52: Mpr-K-G-D-W-P-Pen-NH2< / BR>
PA1 54: Mpr-K-G-D-W-P-Pen-NH2< / BR>
PA1 55: Mpr-K-G-D-W-(Thz-C-NH2< / BR>
PA1 57: Mpr-K-G-D-(2-Nal)-P-Pen-NH2< / BR>
PA1 58: Mv1-K-G-D-W-P-Pen-NH2< / BR>
PA1 59: Mpr-K-G-D-W-(Pip)-Pen-NH2< / BR>
PA1 60: Mpr-(Har)-G-D-W-P-C-NH2< / BR>
PA1 61: Mpr-K-G-D-W-P-C-NH2< / BR>
PA1 62: Mpr-K-G-D-W-P-Pen-NH2< / BR>
PA1 63: Mpr-(Har)-G-D-W-P-Pen-NH2< / BR>
PA1 64: Mpr-(Acetimidyl-Lys)-G-D-W-P-C-NH2< / BR>
PA1 65: Mpr-(Acetimidyl-Lys)-G-D-W-P-Pen-NH2< / BR>
PA1 66: Mpr-(NGNG-W-P-C-NH2< / BR>
PA1 69: Mpr-(Acetimidyl-Lys)-G-D-W-P-Pen-NH2< / BR>
PA1 70: Mpr-(Phenylimidyl-Lys)-G-D-W-P-C-NH2< / BR>
PA1 71: Mpr-Har-Sar-D-W-P-PenNH2< / BR>
PA1 72: Mpr-((Phenylimidyl-Lys)-G-D-W-P-PenNH2< / BR>
PA1 73: Mpr-Har-G-D-W-(3,4-dehydro-Pro-C-NH2< / BR>
PA1 75: Mpr-(Phenylimidyl-Lys)-G-D-Pen-NH2< / BR>
30. PA1 on p. 29, characterized in that it is chosen from the group including:

PA1 3: G-C-G-K-G-D-W-P-C-A-NH2< / BR>
PA1 4: G-C-K-G-D-W-P-C-A-NH2< / BR>
PA1 5: C-G-K-G-D-W-P-C-NH2< / BR>
PA1 10: C-K-G-D-W-P-C-NH2< / BR>
PA1 12: C-K-G-D-Y-P-C-NH2< / BR>
PA1 13: C-K-G-D-F-D-C-NH2< / BR>
PA1 19: Mpr-K-G-D-W-P-C-NH2< / BR>
PA1 25: Mpr-K-G-D-(2-Nal)-P-C-NH2< / BR>
PA1 34: C-K-G-D-W-G-C-NH2< / BR>
PA1 39: C-K-G-D-W-(Sar)-C-NH2< / BR>
PA1 42: C-K-G-D-(4-Cl-Phe)-P-NH2< / BR>
PA1 43: C-K-Sar-D-W-P-C-NH2< / BR>
PA1 44: C-K-G-D-(4-NO2-Phe)-P-C-NH2< / BR>
PA1 47: Acetyl-C-K-G-D-W-P-C-NH2< / BR>
PA1 48: Mpr-K-G-D-W(Formyl)-P-C-NH2< / BR>
PA1 49: Mv1-K-G-D-W-P-C-NH2< / BR>
PA1 51: Mpr-K-G-D-W-P-Pen-NH2< / BR>
PA1 52: Mpr-K-G-D-W-P-Pen-NH2< / BR>
PA1 55: Mpr-K-G-D-W-(Thz-C-NH2< / BR>
PA1 57: Mpr-K-G-D-(2-Nal)-P-Pen-NH2< / BR>
PA1 58: Mv1-K-G-D-W-P-Pen-NH2< / BR>
PA1 59: Mpr-K-G-D-W-(Pip)-Pen-NH2< / BR>
PA1 60: Mpr-(Har)-G-D-W-P-C-NH2< / BR>
PA1 61: Mpr-K-G-D-W-P-C-NH2< / BR>
PA1 62: Mpr-K-G-D-W-P-Pen-NH2< / BR>
PA1 63: Mpr-(Har)-G-D-W-P-Pen-NH2< / BR>
PA1 64: Mpr-(Acetimidyl-Lys)-G-D-W-P-C-NH2< / BR>
PA1 65: Mpr-(Acetimidyl-Lys)-G-D-W-P-Pen-NH2< / BR>
PA1 66: Mpr-(NGNG-ethylene-Har)-G-D>PA1 70: Mpr-(Phenylimidyl-Lys)-G-D-W-P-C-NH2< / BR>
PA1 71: Mpr-Har-Sar-D-W-P-PenNH2< / BR>
PA1 72: Mpr-((Phenylimidyl-Lys)-G-D-W-P-PenNH2< / BR>
PA1 73: Mpr-Har-G-D-W-(3,4-dehydro-Pro-C-NH2< / BR>
31. PA1 under item 30, wherein the inhibitor of platelet aggregation is a

Mpr-(Har)-G-D-W-P-C-NH2.

32. PA1 under item 30, wherein the inhibitor of platelet aggregation is a

Mpr-(Har)-G-D-W-P-Pen-NH2.

33. PA1 under item 30, wherein the inhibitor of platelet aggregation is a

Mpr-(Phenylimidyl-Lys)-G-D-W-P-C-NH2.

34. PA1 under item 30, wherein the inhibitor of platelet aggregation is a

Mpr-(Phenylimidyl-Lys)-G-D-W-P-Pen-NH2.

35. PA1 on p. 29, wherein selected from the group consisting of:

PA1 19: Mpr-K-G-D-W-P-C-NH2< / BR>
PA1 24: Mpr-K-G-D-Y(Ome)-P-C-NH2< / BR>
PA1 47: Acetyl-C-K-G-D-W-P-C-NH2< / BR>
PA1 48: Mpr-K-G-D-W(Formyl)-P-C-NH2< / BR>
PA1 49: Mv1-K-G-D-W-P-C-NH2< / BR>
PA1 51: Mpr-K-G-D-W-P-Pen-NH2< / BR>
PA1 54: Mpr-K-G-D-W-P-Pen-NH2< / BR>
PA1 59: Mpr-K-G-D-W-(Pip)-Pen-NH2< / BR>
PA1 60: Mpr-(Har)-G-D-W-P-C-NH2< / BR>
PA1 63: Mpr-(Har)-G-D-W-P-Pen-NH2< / BR>
PA1 64: Mpr-(Acetimidyl-Lys)-G-D-W-P-C-NH2< / BR>
PA1 65: Mpr-(Acetimidyl-Lys)-G-D-W-P-Pen-NH2< / BR>
PA1 66: Mpr-(NGNG-ethylene-Har)-G-D-W-P-C-NH2< / BR>
PA1 68: Mpr-Har-Sar-D-W-P-C-NH2< / BR>
PA1
36. PA1 on p. 35, wherein the inhibitor of platelet aggregation is a

Mpr-K-G-D-W-P-C-NH2.

37. PA1 on p. 35, wherein the inhibitor of platelet aggregation is a

Mpr-K-G-D-W(Formyl)-P-C-NH2.

38. PA1 on p. 35, wherein the inhibitor of platelet aggregation is a

Mv1-K-G-D-W-P-C-NH2.

39. PA1 on p. 35, wherein the inhibitor of platelet aggregation is a

Mpr-K-G-D-W-P-Pen-NH2.

40. PA1 on p. 35, wherein the inhibitor of platelet aggregation is a

Mpr-K-G-D-W-(Pip)-Pen-NH2.

41. PA1 on p. 35, characterized in that it is a

Mpr-(Acetimidyl-Lys)-G-D-W-P-C-NH2.

42. PA1 on p. 35, characterized in that it is a

Mpr-(Acetimidyl-Lys)-G-D-W-P-Pen-NH2.

43. PA1 on p. 35, characterized in that it is a

Mpr-Har-G-D-W-(3,4-dehydro-Pro-C-NH2.

44. Pharmaceutical composition for preventing the formation of thrombus, characterized in that it contains PA1 on PP.14, 16 or 20 in an effective amount in a mixture with a pharmaceutically acceptable filler.

45. Method of inhibiting the formation of thrombus in animals, characterized in that kind of composition in an amount of 0.01 to 10 µg/kg body weight.

Priority points and features:

16.06.89 on PP.1 - 9;

06.10.89 on PP.14 - 41 and PA1 1 - 34;

20.02.90 - PA1 35 - 70;

15.06.90 - PA1 37 - 75;

PP.11 - 13, 44 and 45, depending on PA1.

 

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