WO1992008476A1

WO1992008476A1 - Peptides that inhibit platelet binding of adhesion molecules

Info

Publication number: WO1992008476A1
Application number: PCT/US1991/008328
Authority: WO
Inventors: Zaverio M. Ruggeri; Richard A. Houghten
Original assignee: The Scripps Research Institute
Priority date: 1990-11-07
Filing date: 1991-11-07
Publication date: 1992-05-29
Also published as: AU9131091A

Abstract

Peptides comprising interchain-linked multimers containing the Arg-Gly-Asp sequence held together by an interchain stable bonds, which inhibit the binding of fibrinogen or other adhesion molecules to platelets or integrin expressing cells, prevents platelet-to-platelet or cell-to-cell aggregation and are useful in preventing, retarding, or detecting thrombus formation.

Description

o PEPTIDES THAT INHIBIT PLATELET

BINDING OF ADHESION MOLECULES

The invention described herein was made in the course of work under a grant or award (Grant No. HL 31950) from The Department of Health, Education and U.S. Welfare and the Government has certain rights therein.

FIELD OF INVENTION

The present invention relates to thrombotic disorders, and, more particularly to peptides that

10 inhibit cell-to-cell binding.

BACKGROUND ART

A group of proteins collectively referred to as

,c adhesion or adhesive molecules are involved in binding of cells to each other and binding of cells to the extracellular matrix. The extracellular matrix is the extracellular space in tissue that is filled by a network of macromolecules. The macromolecules comprise a variety

2 of polysaccharides and proteins that are secreted locally and assembled into an organized meshwork. Such interactions of cell-to-cell binding and cell binding to extracellular matrix involving adhesion molecules include platelet aggregation and platelet adhesion to blood 5 vessel walls. The members of the adhesion molecule group, such as fibronectin, vitronectin, thrombospondin, fibrinogen, and von Willebrand factor (vWF) , all contain the tripeptide seguence Arg-Gly-Asp (RGD) . This seguence is present in other naturally occurring peptides 0 including but not limited to collagen α 1(1) and α2 (I), thrombin, α lytic protease, and lambda receptor protein. The RGD sequence is recognized by one or more integrins on the cell surface and thus constitutes the integrin recognition sequence. Integrins are a superfamily of 5 adhesion molecule receptors [Hynes, Cell 48:549-554 (1987) ]. These receptors play a major role in binding of cells to other cells and binding of cells to the extracellular matrix. Integrins include platelet membrane glycoprotein lib/Ilia, fibronectin receptor, vitronectin receptor, MAC-1, and LFA-1. All of the integrins form α/β heterodimers on the cell surface and some share a common β subunit. Many of the adhesion molecules either bind to or constitute part of the extracellular matrix in addition to other structural molecules such as collagen, glycosaminoglycans, and proteoglycans. The extracellular matrix includes the subendothelial matrix.

This invention relates generally to regulation of cell-to-cell aggregation or regulation of cells binding to the extracellular matrix, and relates specifically to the inhibition of binding of fibrinogen and other adhesion molecules such as, von Willebrand Factor (vWF) , fibronectin, and vitronectin, to platelets and other cells expressing the membrane glycoprotein Ilb/IIIa (GPIIb/IIIa) integrin or i munologically or structurally related integrins, which results inter alia in inhibition or prevention of cell-to-cell or platelet-to-platelet aggregation and thrombosis.

Platelets are specialized circulating cell fragments that perform the biological functions of adhering to areas of blood vessel wall injury and aggregating with one another to form a hemostatic plug. Both adhesion and aggregation are mediated by the interaction of specific molecules with membrane glycoprotein (GP) receptors on the platelet surface. The adhesive properties of platelets are of central importance in normal hemostasis as well as in the development of pathological vascular occlusion. There is significant evidence that rupture of an atherosclerotic plaque with superimposed thrombosis occurs in the majority of cases of unstable angina, myocardial infarction and sudden death of ischemic origin [Davies et al.. Br. Heart J. 63:363 (1985); Fuster et al. , Circulation 77:1213 (1988); Badimon et al.. Arteriosclerosis 6:312 (1986); Ver ylen et al.. Science 238:491 (1987)].

Platelet adhesion to surfaces may be mediated by several different binding sites on the platelet membrane which may interact with distinct adhesion molecules. In contrast, platelet aggregation (the interaction of platelets with one another which results in the mass of the thrombus) appears to be essentially dependent on the .- function of the GP lib/Ilia integrin, an activation- modulated binding site for four adhesive molecules: fibrinogen, vWF, fibronectin, and vitronectin. Although fibrinogen is considered the physiological GP lib/Ilia ligand, evidence is accumulating from ex vivo experiments ^5 that vWF is involved in mediating platelet aggregation. Small molecules and synthetic peptides containing the integrin recognition sequence, RGD, have been shown to inhibit GP Ilb-IIIa mediated platelet functions by competing with the binding of physiologic ligands and, 0 thus, have been considered as potential anti-thrombotic agents [Hawiger et al.. Proc. Na l. Acad. Sci USA 79:2068-2071 (1982); Pierschbacher et. al.. J. Biol. Chem. 257:9593 (1982); Pierschbacher et al.. Proc. Natl. Acad. Sci USA 80:1224 (1983); Pierschbacher and 5 Ruoslahti, Proc. Natl. Acad. Sci USA 81:5985 (1984); Kloczewiak et al.. Biochemistry 23:1767 (1984); Pierschbacher and Ruoslahti, Nature 309:30 (1984);. Gartner and Bennett, J. Biol. Chem. 260:11891 (1985); Plow et al.. Proc. Natl. Acad. Sci USA 82: 8057 (1985); 0 Ruggeri et al. _f Proc. Natl. Acad. Sci. USA 83:5708 (1986); Plow et al.. Blood 70:110 (1987); Pierschbacher and Ruoslahti, J. Biol. Chem. 262:17294 (1987); Huang et al.. J. Biol. Chem. 262:16157 (1987); Garsky, et al.. Proc. Natl Acad. Sci. USA 86:4022 (1989); Chao et al.. 5 Proc. Natl. Acad. Sci USA 86:8050 (1989); Parker and Gralnick , Blood 74:1226 (1989); Hautanen et al.. J. Biol. Chem 264:1437 (1989)]. This concept has found support in a limited number of preliminary animal studies using different experimental models of thrombosis [Cadroy et.al.. J. Clin. Invest. 84:939 (1989); Shebuski et al. Thromb. Haemost. 61:183 (1989) Cook et al. Am J. Physiol. 256:H1038 (1989); Haskel et al. Circulation 80:1775 (1989)].

Another peptide sequence occuring at the carboxyl terminal end of the gamma-chain of fibrinogen (His-His- Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val) has also been

10 shown to have similar inhibitory effects, although with lesser efficacy resulting from lower affinity for platelets rKloczewiak et al. Biochemistry 23:1767 (1984); Ruggeri et al. Proc. Natl. Acad. Sci USA 83:5708 (1986)].

, ~ Hawiger et al. report the use of synthetic peptides as a method of inhibiting the binding of vWF to platelets (U.S. Patent No. 4,666,884, issued May 19, 1987) and Ruoslahti et al. report a tetrapeptide containing Arg- Gly-Asp that has cell attachment-promoting-activity when

2o bound to a substrate (U.S. Patent 4,578,079 issued Mar. 25, 1986).

There are some significant problems in using monomeric peptide molecules as anti-thrombotic agents. One is that the small synthetic compounds of the prior

25 art usually exhibit lower affinity for a given binding site compared to the affinity of the parent macromolecule. Second, these small synthetic peptides have a short survival time in the circulation.

The peptide subunits linked by interchain stable

30 bonds, preferably covalent bonds such as disulfide bonds or amide bonds, of the present invention are superior to the prior art small synthetic peptides in their ability to inhibit fibrinogen-platelet binding, vWF-platelet binding, and platelet aggregation due to greater

35 affinities of binding and/or better stability in vivo. u and thus, are better candidates as anti-thrombotic agents. Moreover, the peptides of the present invention can be effective at lower concentrations in the blood, resulting in less likelihood of undesired side effects and lower cost of administration. The background information for these studies is predicated upon the knowledge obtained previously from Ruggeri et al. Proc. Nat'l Acad. Sci. USA 83:5708-5712 (1986) and from Zimmerman et al. U.S. Patent No. 4,683,291 issued July 28, 1987, Reexam certificate issued July 3, 1990, the contents of which are hereby incorporated by reference.

10 These references show that peptides containing positively charged residues on the amino terminal side of the RGD sequence display increased inhibitory activity.

The peptides linked by covalent bonds of the present

,- invention would also be expected to inhibit the binding of any of the adhesion molecules to any cell that has an analogous integrin, so that the utility of the invention is not limited to the inhibition of platelet aggregation and the treatment of thrombosis. For example, two cell o lines derived from human solid tumors, human cervical carcinoma and human colon carcinoma, express molecules immunologically related to GpIIb/IIIa [Grossi et al.. The FASEB Journal 2:2385-2395, (1988)]; as do normal endothelial cells. Thus, these cells would be expected 5 to be inhibited by RGD containing peptides.

SUMMARY OF INVENTION

The peptides of the present invention include 0 peptides that inhibit binding of adhesion molecules to cells expressing integrins comprising multimers having at least a first and second subunit, the sequence of each subunit containing at least the Arg-Gly-Asp sequence, and the subunits are joined by a interchain stable bond 5 preferably a covalent bond, more preferably a disulfide and amide bond. The bond may be located on the amino terminal, carboxyl terminal side or alternating sides of the Arg-Gly-Asp sequence of each subunit. The sequence of each subunit may be identical or different.

Peptides of the present invention are formed from subunits to make di ers, trimers, tetramers, pentamers, or the like.

The present invention has the utility of preventing or retarding the formation of a clot or thrombus, both venous and arterial, in the blood. The peptides of the present invention have higher inhibitory activity than 0 previously known synthetic peptides and therefore may be superior anti-thrombotic agents.

The another aspect of this invention relates to pharmaceutical compositions using peptides of the present - invention as the active ingredient in a pharmaceutically acceptable medium for intravenous, subcutaneous, intraperitoneal, oral or nasal administration into mammals.

Another aspect of this invention relates to methods o f°^r inhibiting the aggregation of cells to each other, or inhibiting the binding of adhesion molecules to cells or inhibiting the binding of cells to extracellular matrices, by contacting the cells with a peptide of the present invention in an amount of said peptide effective 5 to inhibit such aggregation or binding. The cells of the present methods are preferably cells expressing integrins, most perferably platelets or tumor cells and the adhesion molecules of the present methods are preferably vWF or fibrinogen. 0 Another aspect of this invention relates to methods for preventing and /or treating thrombosis in mammals using peptides of the present invention, alone or in combination with thrombolytic agents.

Another aspect of this invention relates to methods 5 for detecting thrombi or platelet aggregates using the peptides of the present invention.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1 shows the correlation between number of arginine residues in a synthetic peptide and inhibitory activity.

Figure 2 shows the effect of the residue preceding the Arg-Gly-Asp sequence on the inhibitory activity of synthetic peptides. ._ Figure 3 shows the effect of the number of residues intervening between the polyarginine chain and the Arg- Gly-Asp sequence on the inhibitory activity of synthetic peptides.

Figure 4 shows the dose-response curves of various nc synthetic peptides. Dimeric peptides have the lowest IC₅₀ values.

DISCLOSURE OF INVENTION

0 ^For purposes of this disclosure, accepted short-hand designations of the amino acids have been used. A complete listing is provided herein below:

AMINO ACID ABBREVIATIONS

5

0

5

The nomeclature used to define the peptides is in accordance with conventional representation of the N- terminus is to the left and the C-terminus appears to the right. In the peptide structures described herein, each amino acid residue can be in the (L) or (D) configuration, preferably the (L) configuration. The peptides of the present invention have the utility of inhibiting the binding of adhesion molecules to cells, and specifically inhibiting fibrinogen-platelet or vWF-platelet binding, which results inter alia in inhibition or prevention of cell-to-cell or platelet-to- platelet binding. Specifically, the present invention has the utility of preventing or retarding the formation of a clot or thrombus, both venous and arterial, in the blood. The peptides of the present invention have higher inhibitory activity than previously known synthetic peptides and therefore may be superior anti-thrombotic agents.

The peptides of the present invention comprise peptides that inhibit binding of adhesion molecules to cells expressing integrins comprising multimers having at least a first and second subunit, the sequence of each subunit contains at least the Arg-Gly-Asp sequence, and the subunits are joined by an interchain stable bond preferably a covalent bond, more preferably a disulfide and amide bond. The bond may be located on the amino terminal or carboxyl terminal side of the Arg-Gly-Asp sequence. The sequence of each subunit may be identical or different.

The present invention also includes peptides that inhibit binding of adhesion molecules to cells expressing integrins comprising multimers of at least a first and second subunit held together by interchain stable bonds having the following general formula:

(X_z-Arg-Gly-Asp-X₂)_y wherein X = any amino acid or combination of amino acids, z is an integer from 1 to 18, and y is an integer from 2 to 5, and the sequence of each subunit is either identical or different. The stable bonds that link subunits include covalent bonds, preferably amide bonds formed between Glu and Lys, Asp and Lys, Glu and Orn, or Asp and Orn and disulfide bonds formed between Cys residues on each subunit. The bonds may be formed on the

10 amino terminal side, the carboxyl terminal side , or alternating sides of the Arg-Gly-Asp sequence of each subunit.

Peptides of the present invention are formed from _je subunits to make dimers, trimers, tetramers, pentamers, and the like.

The peptides of the present invention also includes multimers of at least a first and second subunit held together by interchain stable bonds further defined by o the formula:

[X_z-Arg-Gly-Asp-(Cx)-Cys_w]_y wherein X is any amino acid or combination of amino acids; z is an integer from 1 to 18;

(Cx) is a chain of 1 to 18 amino acid residues 5 selected from the group consisting of Ala, Arg, Asn, Asp,

Glu, Gin, Gly, His, He, Leu, Met, Phe, Pro, Trp, Tyr,

Val, or Orn; w is an integer from 0 to 1; y is an integer from 2 to 5; and the sequence of each subunit is either identical or different. The stable bonds that link 0 subunits include covalent bonds, preferably amide bonds formed between Glu and Lys, Asp and Lys, Glu and Orn, or

Asp and Orn and disulfide bonds formed between Cys residues on each subunit. The bonds may be formed on the amino terminal side, the carboxyl terminal side , or 5 alternating sides of the Arg-Gly-Asp sequence of each subunit. These peptides are formed from subunits to make dimers, trimers, tetramers, pentamers, and the like.

The peptides of the present invention further comprises peptides that inhibit binding of adhesion molecules to cells expressing integrins comprising multimers of at least a first and second subunit held together by interchain stable bonds having the following formula:

(Arg_m-Cys-Arg_n-X_z-Arg-Gly-Asp-(Cx)-Cys_w)_y wherein m is an integer from 0 to 8, n is an integer from 1 to 5, z is an integer from 1 to 4, X is any amino acid or combination of amino acids, (Cx) is a chain of 1 to 18 amino acid residues selected from the group consisting of Ala, Arg, Asn, Asp, Glu, Gin, Gly, His, He, Leu, Met, Phe, Pro, Trp, Tyr, Val, or Orn; w is an integer from 0 to 1; and y is an integer from 2 to 5; and the sequence of each subunit is identical or different. The stable bond that link subunits include covalent bonds, preferably amide bonds formed between Glu and Lys, Asp and Lys, Glu and Orn, or Asp and Orn and disulfide bonds formed between Cys residues on each subunit. The bonds may be formed on the amino terminal side, the carboxyl terminal side , or alternating sides of the Arg-Gly-Asp sequence of each subunit. The peptides are formed from subunits to make dimers, trimers, tetramers, pentamers, and the like.

Preferably, the peptides of the present invention comprises peptides that inhibit binding of fibrinogen and other adhesion molecules to cells expressing integrins such as platelets comprising a multimer of at least a first and second subunit held together by an interchain stable bond having the following formula:

(Arg_m-cys-Arg_n-x_z-Arg-Giy-Asp- (^Cχ) -^Cvs _w) _y wherein m is an integer from 0 to 3, n is an integer from l to 5, z is an integer from 1 to 4, and x is selected from the amino acids Gin, Phe, Pro, Thr, Leu, Ala, Gly, or Ser, Cx is Val, w is an integer from 0 to 1, and y is an integer from 2 to 5.

More preferably, the peptides of the present invention includes peptides that inhibit binding of adhesion molecules to cells expressing integrins comprising multimers of at least a first and second subunit held together by an interchain stable bond having the following formula:

(Arg₃-Cys-Arg-X_z-Arg-Gly-Asp-Val-Cys_w)_y (SEQ ID NO. : l) wherein X is any amino acid; z is 1; w is an integer from 0 to 1; and y is an integer from 2 to 5, and the sequence

10 of each subunit is identical or nonidentical. The stable bonds that link subunits include covalent bonds, preferably amide bonds formed between Glu and Lys, Asp and Lys, Glu and Orn, or Asp and Orn and disulfide bonds ,c formed between Cys residues on each subunit. The bonds may be formed on the amino terminal side, the carboxyl terminal side, or alternating sides of the Arg-Gly-Asp sequence of each subunit. The peptides are formed from subunits to make dimers, trimers, tetramers, pentamers, o and the like.

Most preferably, the peptides of the present invention comprises a peptide that inhibits binding of adhesion molecules to cells expressing integrins comprising multimers of at least a first and second 5 subunit held together by interchain stable bond having the following formula:

(Arg₃-Cys-Arg-Ser-Arg-Gly-Asp-Val-Cys_w)_y (SEQ ID No. : 2) and w is an integer from 0 to 1; and y is an integer from 2 to 5, and the subunits are linked by covalent bonds, 0 preferably disulfide bonds between Cys residues formed on the amino terminal side, the carboxyl terminal side, or alternating sides of the Arg-Gly-Asp sequence of each subunit.

The invention further comprises a method for 5 inhibiting the binding of adhesion molecules to cells with integrins comprising contacting the cells with a interchain covalently-linked peptide of the present invention in an amount of said peptide effective to inhibit said binding.

The invention further comprises a method for inhibiting the binding of adhesion molecules to platelets comprising contacting the platelets with a interchain covalently-linked peptide of the present invention in an amount of said peptide effective to inhibit said binding. The invention further comprises a method for inhibiting the binding of fibrinogen to platelets comprising contacting platelets with an interchain covalently-linked peptide of the present invention in an amount of said peptide effective to inhibit said binding.

The invention further comprises a method for inhibiting the binding of von Willebrand factor to platelets comprising contacting platelets with an interchain covalently-linked peptide of the present invention in an amount of said peptide effective to inhibit said binding. ^Tne invention further comprises a method for inhibiting the binding of cells with integrins to extracellular matrices comprising contacting cells with a interchain covalently-linked peptide of the present invention in an amount of said peptide effective to inhibit said binding.

The invention further comprises a method for inhibiting the binding of platelets to extracellular matrices comprising contacting platelets with an interchain covalently-linked peptide of the present invention in an amount of said peptide effective to inhibit said binding.

The invention further comprises a method for inhibiting aggregation of cells to each other comprising contacting the cells with an interchain covalently-linked peptide of the present invention in an amount of said peptide effective to inhibit said binding.

The invention further comprises a method for inhibiting aggregation of platelets to each other comprising contacting the platelets with an interchain covalently-linked peptide of the present invention in an amount of said peptide effective to inhibit said binding. Peptides of formula Arg_m-Arg-Gly-Asp-Val were found to give progressively lower 1C₅₀ values (the concentration of the peptide which would inhibit binding by 50%, as determined by the procedure described below) ._ when the value of m increased between 1 and 8 (Fig. 1). This was in agreement with results published previously by Ruggeri et al.. Proc Natl. Acad. Sci USA 83:5708 (1986) . The effect of various substitutions in the position following the polyarginine chain and preceding ^5 the sequence Arg-Gly-Asp-Val (the integrin recognition sequence) was determined by synthesizing and testing peptides of formula Arg₈-X-Arg-Gly-Asp-Val (SEQ ID NO. : 3) , where X corresponded to any amino acid. Hydrophobic non-polar residues at position X gave the lower IC₅₀ 0 values, with serine giving the lowest value of all (Fig. 2) . In contrast, the two acidic residues, aspartic acid and glutamic acid, gave the highest IC₅₀ values (Fig. 2) . The effect of the number of residues inserted between the polyarginine sequence and the integrin recognition 5 sequence was evaluated in peptides of formula Arg₈-Ala_z- Arg-Gly-Asp-Val where z was varied from 1 to 5. As shown in Fig. 3, the lowest IC₅₀ was obtained with z = 1.

On the basis of the results mentioned above, peptides of the formula Arg_m-Cys-Arg-Ser-Arg-Gly-Asp-Val, 0 were m = 0 to 3, were synthesized and tested to evaluate the hypothesis that interchain covalently-linked subunits might exhibit lower IC₅₀ values, and thus better inhibitory activity, than single chain molecules. This hypothesis was based on the consideration that 5 covalently-linked dimeric molecules, possessing two integrin recognition sequences could interact with two adjacent GP Ilb-IIIa binding sites and exhibit, because of the divalent interaction, higher affinity of binding. The short distance between the two Arg-Gly-Asp-Val sequences was likely to permit binding of the synthetic molecules to adjacent GP Ilb-IIIa sites on the same platelet, and not to act as linkers between different platelets promoting aggregation the way fibrinogen does. The results shown in Table 1 demonstrate that, indeed, the disulfide-linked dipeptide with formula (Arg₃-Cys-

-₀ Arg-Ser-Arg-Gly-Asp-Val)₂ (SEQ ID NO.: 4) showed IC₅₀ values approximately 100-fold lower than those of the reference peptide Tyr-Arg-Gly-Asp-Val and of the native, nonlabelled fibrinogen (assay procedures pp.21 & 22). When the disulfide bond in the divalent peptide was

^e reduced and the cysteine residue carboxyamidomethylated with iodoacetamide, the IC₅₀ value became closer to that of the reference peptide (Table 1) . Figure 4 shows the dose response curve for the synthetic peptides from which the IC₅₀ values were calculated for Table 1.

20

25

30

35 Table 1 EFFECT OF DISULFIDE BOND LINKED PEPTIDES ON THE INHIBITORY ACTIVITY OF SYNTHETIC PEPTIDES SYNTHETIC PEPTIDE IC₅₀ fumoles/L,

Tyr-Arg-Gly-Asp-Val 2.100 Arg -Cys-Arg-Ser-Arg-Gly-Asp-Val 0.028

Arg₃-Cy Is-Arg-Ser-Arg-Gly-Asp-Val (SEQ ID NO.:4) Cys-Arg-Ser-Arg-Gly-Asp-Val 0.660

I

Cys-Arg-Ser-Arg-Gly-Asp-Val (SEQ ID NO. : 5) -^Q Arg₃-Cys-Arg-Ser-Arg-Gly-Asp-Val 10.000

Non-labelled Fibrinogen 2.000 **

, c *The cysteine residue was carboxyamidomethylated

^**Measured using 5 μg/ml of ¹²⁵I labelled fibrinogen

The importance of the location of the bond between monomeric peptides on inhibitory activity was o investigated. Cysteinyl-linked dimeric peptides with a penta-arginine sequence preceding the Ser-Arg-Gly-Asp-Val sequence of the peptide were synthesized, with the only variable being the position of the cysteine residue. The lowest IC₅₀ using these dimeric peptides was obtained 5 using the peptide with the formula:

Arg₂-Cys-Arg₃-Ser-Arg-Gly-Asp-Val

Arg₂-Cys-Arg₃-Ser-Arg-Gly-Asp-Val (SEQ ID NO. :9)

as shown in Table 2. This demonstrates that the position 0 of the bond affects the inhibitory activity of the dimeric peptides.

5 t Table 2

EFFECT OF LOCATION OF THE DISULFIDE BOND IN A DIMERIC PEPTIDE ON INHIBITORY ACTIVITY PEPTIDE SEQUENCE IC_Ξ0 -Umoles/L-

Arg₅-Cys-Ser-Arg-Gly-Asp-Val 0.820

Arg₅-Cys-Ser-Arg-Gly-Asp-Val (SEQ ID NO.:6)

Arg₄-Cys-Arg₁-Ser-Arg-Gly-Asp-Val 0.230 Arg₄-Cys-Arg₁-Ser-Arg-Gly-Asp-Val (SEQ ID NO.:7)

Arg₃-Cys-Arg₂-Ser-Arg-Gly-Asp-Val 0.110 Arg₃-Cys-Arg₂-Ser-Arg-Gly-Asp-Val (SEQ ID NO.:8)

- Arg₂-Cys-Arg₃-Ser-Arg-Gly-Asp-Val 0.082 Arg₂-Cys-Arg₃-Ser-Arg-Gly-Asp-Val (SEQ ID NO.:9)

Arg₁-Cys-Arg₄-Ser-Arg-Gly-Asp-Val 0.180

Arg₁-Cys-Arg₄-Ser-Arg-Gly-Asp-Val (SEQ ID NO.:10)

Cys-Arg₅-Ser-Arg-Gly-Asp-Val 0.250

Cys-Arg₅-Ser-Arg-Gly-Asp-Val (SEQ ID NO.:11)

Tyr-Arg-Gly-Asp-Val 5.400

Peptides within the scope of the present invention are peptides covalently linked by interchain amide bonds. Peptides are synthesized in a manner analogous to that used to prepare the peptides in Table 2. In place of 0 Cys. , a Glu, Lys, Asp, or Orn is substituted and the amide bond is made using an amide condensation agent. Examples of such interchain amide bond linked peptides are as follows (Table 3) . 5 It Table 3

DIMERIC PEPTIDES LINKED BY INTERCHAIN AMIDE BONDS

Arg_R-Glu-Ser-Arg-Gly-Asp-Val (SEQ ID NO.:12) Arg I₅-Lys-Ser-Arg-Gly-Asp-Val (SEQ ID NO.:13) _ Arg_ή-Ly>.s-A_r,g_i„-Ser-Arg-Gly_-A-s,p--V_al „(SEQ.ID.NO..:.„15,)

Arg₃-Glu-Arg -Ser-Arg-Gly-Asp-Val (SEQ ID NO.:16) 0 Arg₃-Lys-Arg₂-Ser-Arg-Gly-Asp-Val (SEQ ID NO.:17)

Arg₂-Glu-Arg₃-Ser-Arg-Gly-Asp-Val (SEQ ID NO.:18) Arg₂-Lys-Arg₃-Ser-Arg-Gly-Asp-Val (SEQ ID NO.:19)

Arg₁-Glu-Arg₄-Ser-Arg-Gly-Asp-Val (SEQ ID NO.:20) Arg₁-Lys-Arg₄-Ser-Arg-Gly-Asp-Val (SEQ ID NO.:21)

Glu-Arg₅-Ser-Arg-Gly-Asp-Val (SEQ ID NO.:22) Lys-Arg₅-Ser-Arg-Gly-Asp-Val (SEQ ID NO.:23)

Arg₃-Glu-Arg-Ser-Arg-Gly-Asp-Val (SEQ ID NO.:24) Arg₃-Lys-Arg-Ser-Arg-Gly-Asp-Val (SEQ ID NO.:25)

The sequences identified above are specific examples of peptides falling within the defined formulas and should not be interpreted as limiting the scope of the present invention.

Interchain covalent bonds may be located on the amino terminal or carboxyl terminal side of the Arg-Gly- Asp peptide sequence to form dimers, trimers, tetramers, pentamers and the like. Some examples of dimers that are formed by placement of the bond on the carboxyl side of the Arg-Gly-Asp sequence are as follows (Table 4) . Table 4

PEPTIDES LINKED BY INTERCHAIN COVALENT

BONDS ON THE CARBOXYL TERMINAL SIDE OF

THE ARG-GLY-ASP SEQUENCE

Arg_ς-Ser-Arg-Gly-Asp-Val-Cys I Arg₅-Ser-Arg-Gly-Asp-Val-Cys (SEQ ID NO.:26)

Arg₈-Arg-Gly-Asp-Val-Cys

Arg₈-Arg-Gly-Asp-Val-Cys (SEQ ID NO. :27)

Arg₇-Arg-Gly-Asp-Val-Cys

Arg₇-Arg-Gly-Asp-Val-Cy Is (SEQ ID NO.:28)

Arg₆-Arg-Gly-Asp-Val-Cys Arg₆-Arg-Gly-Asp-Val-Cy Is (SEQ ID NO.:29)

Arg₅-Arg-Gly-Asp-Val-Cys

Arg₅-Arg-Gly-Asp-Val-Cy Is (SEQ ID NO.:30)

Arg₄-Arg-Gly-Asp-Val-Cys

Arg₄-Arg-Gly-Asp-Val-Cy Is (SEQ ID NO.:31)

Arg₃-Arg-Gly-Asp-Val-Cys

Arg₃-Arg-Gly-Asp-Val-Cy Is (SEQ ID NO.:32)

Arg₂-Arg-Gly-Asp-Va1-Cys

Arg₂-Arg-Gly-Asp-Val-Cy Is (SEQ ID NO.:33)

Arg-_L-Arg-Gly-Asp-Val-Cys Arg-_L-Arg-Gly-Asp-Val-Cys (SEQ ID NO.:34)

Arg₈-Ser-Arg-Gly-Asp-Val-Cys Arg₈-Ser-Arg-Gly-Asp-Val-Cys (SEQ ID NO. :35) Arg₈-Gly-Arg-Gly-Asp-Val-Cys Arg₈-Gly-Arg-Gly-Asp-Val-Cys (SEQ ID NO.:36)

Arg₈-Ala-Arg-Gly-Asp-Val-Cys Arg₈-Ala-Arg-Gly-Asp-Val-Cys (SEQ ID NO.:37)

Arg₈-Leu-Arg-Gly-Asp-Va1-Cys Arg₈-Leu-Arg-Gly-Asp-Val-Cys (SEQ ID NO.:38)

Ser-Arg-Gly-Asp-Arg₅-Cys Ser-Arg-Gly-Asp-Arg₅-Cys (SEQ ID NO.:39)

Arg-Arg-Gly-Asp-Arg₈-Cys Arg-Arg-Gly-Asp-Arg₈-Cys (SEQ ID NO.:40)

Gly-Arg-Gly-Asp-Arg_ft-Cys

Gly-Arg-Gly-Asp-Arg₈-Cy Is (SEQ ID NO.:41)

Arg₅-Cys-Ser-Arg-Gly-Asp-Glu (SEQ ID NO.:42) Arg₅-Cys-Ser-Arg-Gly-Asp-Orn (SEQ ID NO.:43)

Arg₄-Cys-Arg-,-Ser-Arg-Gly-Asp-Glu (SEQ ID NO.:44)

Arg₄-Cys-Arg₁-Ser-Arg-Gly-Asp-Or In (SEQ ID NO.:45)

The location of the interchain covalent bond may be on the amino terminal side of of the first subunit and on the carboxyl terminal side of the second subunit, or vise versa, as generally illustrated in the following formula:

X-Arg-Gly-Asp-X

X-Arg-Gly-Asp-X or alternatively,

wherein X is an amino acid capable of forming a stable bond, preferably a covalent bond. Interchain covalent bonds for trimers can be similarly arranged as generally illustrated in the following formula: -Arg-Gly-Asp-X

or alternatively,

wherein X is an amino acid capable of forming a stable bond, preferably a covalent bond. In this instance, the second subunit is bonded at two locations, one bond connects the first subunit with the second and the other bond connects the third subunit with the second subunit. Interchain covalent bonds between subunits forming tetramers, pentamers and the like can be similarly arranged. The sequences identified above are specific examples of peptides falling within the sequences defined in formulas and should not be interpreted as limiting the scope of the present invention.

Interchain covalently-linked peptides of the present invention also have utility in inhibiting binding of other proteins including other adhesion molecules to platelets or other integrin containing cells. For example, the peptides of the present invention were quite effective in inhibiting binding of von Willebrand factor to platelets (Table 5) . o Table 5

INHIBITION OF VWF BINDING TO PLATELETS BY THE PEPTIDES

SYNTHETIC PEPTIDE I C _{5 0}

.umoles/L)

Arg-Gly-Asp-Val 1.56

Arg^-Cys-Arg-Ser-Arg-Gly-Asp-Val 0.01

Arg₃-Cy Is-Arg-Ser-Arg-Gly-Asp-Val (SEQ ID NO.:4)

Arg₃-Cys-Arg-Ser-Arg-Gly-Asp-Val 1.20

*The cysteine residue was carboxyamidomethylated

Peptides are synthesized by the method of simultaneous multiple peptide synthesis (SMPS) as described in detail by Houghten, Proc. Natl. Acad. Sci. USA 82:5131 (1985) and as generally set forth in US Patent No. 4,683,291, issued July 28, 1987, Reexam. issued July 3, 1990, the disclosure of which is incorporated herein by reference, or by other suitable methods. All peptides were purified by reversed phase high performance liquid chromatography (HPLC) using methodology reported by Ruggeri et al.. Proc. Natl. Acad. Sci. USA 83:5708 (1986) although other equivalent purification techniques may also be used. A purity of at least 95 to 99% (based on all peptides present) is reasonably obtainable and preferable. Formation of intermolecular disulfide bonds to obtain homodimeric peptides was achieved by air oxidation of peptide solutions prepared directly into 25 mM ammonium acetate, pH 8.5, at a concentration of 1 x 10^~3 moles/liter, although other methods of forming disulfide bonds may be used including oxidation using potassium ferricyanide or iodine. After overnight exposure to air, isolation of dimers from monomeric species was obtained by reversed- phase HPLC chromatography. A purity of 95 to 99% dimers is expected and preferred. Trimers, tetramers, pentamers and the like may be similarly obtained. Other purification techniques may also be employed. Methods for making amide bonds are well known in the art and include the use of amide condensation agents such as diisopropylcarbodiimide and the like. Peptide solutions for testing were prepared by carefully weighing a lyophilized dry peptide and dissolving it into a buffer composed of 20 mM HEPES, 150 mM NaCl, pH 7.35, to give the desired final concentration. The composition and concentration of single amino acids was verified with an automated amino acid analyzer (LKB) following complete hydrolysis after 24 hours in 6 M HCl at 110°C , as described by Ruggeri et al.. Proc. Natl. Acad. Sci. USA 83:5708 (1986). A purity of 95 to 99% is expected and preferred. Most of the peptides of the present invention may also be obtained using recombinant DNA techniques in prokaryotic or eucaryotic expression systems.

Assessment of fibrinogen binding to platelets and inhibition of binding was performed as previously described by Ruggeri et al.. Proc Natl. Acd. Sci. USA 83:5708 (1986). Fibrinogen was purified from freshly collected plasma using the glycine precipitation method of Kazal et al.. Proc. Soc. Exp. Biol. Med. 113: 989 (1963) . Traces of contaminating vWF were removed by gel filtration through an 80 x 5 cm Sepharose 4B-C1 column (Pharmacia) . Purified fibrinogen was labeled with ¹²⁵I using Iodogen (Pierce) following the procedure described by Fraker and Speck, Biochem. Biophys. Res. Commun. .80:849-857 (1978). Platelet suspensions devoid of plasma proteins were prepared by the albumin density gradient centrifugation technique of Walsh et al.. Br. J. Haematol. 36:281 (1977) with modifications described by Trapani-Lombardo et al.. J. Clin. Invest.76:1950 (1985). Binding of radiolabeled fibrinogen to platelets was measured after platelet stimulation with alpha-thrombin (a generous gift of Dr. John W. Fenton, II) at 22-25°C for 10 minutes (3.125 x 10 -¹ platelets per liter and thrombin at 0.5 NIH units/ml). Hirudin (Sigma) was then added at a 25-fold excess (unit/unit) for 5 minutes before addition of the radiolabeled ligand and any competing ligand. After these additions, the final platelet count in the mixture was 1 x 10¹¹/liter. After incubation for an additional 30 minutes at 22-25°C, bound and free ligand were separated by centrifuging 50 μl of the mixture through 300 μl of 20% sucrose at 12,000 x g for 4 minutes as described by Ruggeri et al.. J. Clin. Invest. 72:1-12 (1983). The platelet pellet was separated from the rest of the mixture to determine platelet-bound radioactivity. Inhibition of fibrinogen binding to platelets was evaluated at a constant ¹²⁵ι- fibrinogen concentration of 25 μg/ml, by testing the effect of a series of peptide concentrations and calculating from the dose-response curve the peptide concentration necessary to inhibit specific fibrinogen binding by 50% (IC₅₀) • Nonspecific binding was arbitrarily defined as that measured in the presence of a saturating concentration of the anti-GP Ilb-IIIa monoclonal antibody LJ-CP3 (U.S. Patent application, Serial No. 294,471 filed Jan. 6, 1989, ATCC Accession No. HB 10046) . The nonspecific binding corresponded typically to less than 10% of the total binding, or 0.5% of added counts.

Von Willebrand factor was purified from human cryoprecipitate, iodinated and the von Willebrand factor binding studies performed as previously described by Ruggeri et al. J. Clin. Invest. 72:1-12 (1983).

The peptides of the present invention can be formulated into pharmaceutical preparations for therapeutic, diagnostic or other uses in mammals. The peptides of the present invention are especially useful u in prevention and/or treatment of vascular disorders including venous or arterial thromboembolisms, thrombotic occlusion or stenosis in coronary, cerebral or perepheral arteries, and vascular grafts and in disease states predisposed to thrombosis such as disseminated intravascular coagulation, major trauma, major surgery, cancer, acute myocardial infarction, and thrombotic stroke, and hereditary diseases such as anti-thrombin III deficiencies, congenital protein C deficiencies or other hereditary diseases associated with platelet aggregation and thrombus formation. Other candidates for treatment 0 with peptides of the present invention include mammals with coronary heart disease or those in high risk categories for heart attack or stroke such as those with high blood pressure, diabetes, high cholesterol, or 5 smokers.

The peptides may be used alone or in combination with a thrombolytic agent or more than one thrombolytic agent for prevention or treatment of thrombosis. Such thrombolytic agents include but are not limited to o tissue-type plasminogen activator or derivatives thereof, streptokinase or derivatives thereof, urokinase or derivatives thereof, prourokinase or derivatives thereof, an acylated form of plasminogen or plasmin and derivatives thereof, and activated protein C and 5 derivatives thereof. Derivatives include natural products, fragments, synthetic peptides or recombinant products that have the desired anti-thrombotic activity of the natural product.

The biologically active peptides produced by peptide 0 synthesis or by the prokaryotic or eukaryotic expression of cloned peptide genes purified in accordance with the present invention can be used for the in vivo treatment of mammalian species by physicians and/or veterinarians. The amount of active peptide will, of course, depend upon 5 the severity of the condition being treated, the route of administration chosen, and the specific activity of the active peptides, and ultimately will be decided by the attending physician or veterinarian. As a guide, a concentration of peptide which is 3 to 4 times the concentration of the IC₁₀₀ value, using the platelet inhibition assay, would be an approximate minimum dose. The active peptide may be administered by any route appropriate to the condition being treated including intravenous, intraperitoneal, intramuscular, subcutaneous, oral, nasal and the like. Preferably, the . peptide is injected into the blood stream of the mammal being treated. It will be readily appreciated by those skilled in the art that the preferred route will vary with the condition being treated.

While it is possible for the active peptide to be i c administered as the pure or substantially pure compound, it is preferable to present it as a pharmaceutical formulation or preparation.

The formulations of the present invention, both for veterinary and for human use, comprise an active peptide, o as above described, together with one or more pharmaceutically acceptable carriers and optionally other therapeutic ingredients. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious 5 to the recipient thereof. The formulations may conveniently be presented in unit dosage form and may be prepared by any method well known in the pharmaceutical art.

All methods include the step of bringing into 0 association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid 5 carriers or both, and then, if necessary, shaping the product into the desired formulation.

Formulations suitable for intravenous, subcutaneous, or intraperitoneal administration conveniently comprise sterile aqueous solutions of the active ingredient with solutions which are preferably isotonic with the blood of the recipient. Such formulations may be conveniently prepared by dissolving solid active ingredient in water containing physiologically compatible substances such as sodium chloride (e.g. 0.1-2.0 M) , glycine, and the like, and having a buffered pH compatible with physiological conditions to produce an aqueous solution, and rendering

10 said solution sterile. These may be presented in unit or multi-dose containers, for example, sealed ampoules or vials.

The formulations of the present invention may

, - incorporate a stabilizer. Illustrative stabilizers are polyethylene glyσol, proteins, saccharides, amino acids, inorganic acids, and organic acids which may be used either on their own or as admixtures. These stabilizers are preferably incorporated in an amount of 0.11-10,000

20 parts by weight per part by weight of peptide. If two or more stabilizers are to be used their total amount is preferably within the range specified above. These stabilizers are used in aqueous solutions at the appropriate concentration and pH. The specific osmotic

25 pressure of such aqueous solutions is generally in the range of 0.1-3.0 osmoles, preferably in the range of 0.8- 1.2. The pH of the aqueous solution is adjusted to be within the range of 5.0-9.0, preferably within the range of 6-8. In formulating the therapeutic agent of the

30 present invention, anti-adsorption agent may be used.

When oral preparations are desired, the compositions may be combined with typical carriers, such as lactose, sucrose, starch, talc magnesium stearate, crystalline cellulose, methyl cellulose, carboxymethyl cellulose,

35 glycerin, sodium alginate or gum arabic among others. When nasal administration is desired, surface active agents used in accordance with the present invention may be those which promote the absorption of peptides through the nasal mucous membrane, that is to say, those which work as absorption promotors. Such surface active agents include bile salts, cationic, anionic, nonionic, and amphoteric agents and phospholipids and the like that are well known in the art. The amount of such surface active agent may usually be in the range from 0.001 to 10% w/v and preferably 0.01 to 1% w/v, the amount depending on ._ the specific surfactant used. Aqueous or nonaqueous media may be used either alone or in admixture in the form of solution, suspension, emulsion or ointment, if applicable to nasal mucosa. The preparations of the present invention may also contain other additives, such _je as stabilizers, adsorption preventing agents and preservatives. If desired, the preparations of the present invention may be formulated in the form of a gel by adding gel bases such as cellulose derivatives, natural gums, vinyl polymers, acrylic acid polymers, and 0 ^tne like.

The peptides of the present invention also have utility for the in vivo or in vitro detection of platelet aggregates or thrombi, and other aggregates of cells containing integrins, and for measuring cell surface 5 expression of integrins, and the like. The peptides of the invention may be used in diagnostic tests, such as immunoassays, for detecting platelet aggregates. Such diagnostic techniques include for example, enzyme-linked immunosorbent assays, radioimmunoassays, fluorescence 0 immunoassays and other techniques in which peptides are labelled with a detectable tag. Such tags include radioactive, enzymes, biotin, fluorochromes, electron dense reagents and the like. The method for detecting platelet aggregates or thrombi includes exposing the 5 suspected platelet aggregate or thrombus to an effective amount of the tagged peptide of the present invention and then measuring the presence of bound, tagged peptides. Detection means for the labelled peptides include radioimagery, spectroscopic, photochemical, immunochemical, biochemical or chemical means. Methods for detecting the presence of platelet aggregates or thrombi bound by tagged peptides of the present invention, in a mammal include, contrast angiography, arteriography, computerized axial tomography scanning and the like. Such methods of detecting platelet aggregates or thrombi are useful in diagnosing thrombotic diseases such as stroke, myocardial infarction or the like. Such in vitro and in vivo detection methods may be quantitative or qualitative for the presence of platelet aggregates, thrombi, or other aggregates of cells containing integrins.

SEQUENCE LISTING (1) GENERAL INFORMATION:

(i) APPLICANT: Ruggeri, Zaverio M. Houghten, Richard A. (ii) TITLE OF INVENTION: PEPTIDES THAT INHIBIT PLATELET BINDING OF ADHESION MOLECULES (iii) NUMBER OF SEQUENCES: 47 (iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Kathryn M. Brown, MORGAN & FINNEGAN

(B) STREET: 345 Park Avenue

(C) CITY: New York

(D) STATE: New York

(E) COUNTRY: USA

(F) ZIP: 10154 (V) COMPUTER READABLE FORM:

^•(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

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

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US 07/610,363

(B) FILING DATE: 07-NOV-1990

(C) CLASSIFICATION: (viϋ) ATTORNEY/AGENT INFORMATION:

(A) NAME: Brown, Kathryn M.

(B) REGISTRATION NUMBER: 34,556

(C) REFERENCE/DOCKET NUMBER: 1198-4007 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (212)415-8646

(B) TELEFAX: (212)751-6849

(C) TELEX: 421792

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 11 amino acids (B) TYPE: amino acid

(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE:

(A) NAME/KEY: Cross-links

(B) LOCATION: 4..>11

(D) OTHER INFORMATION: /note= "Multimers of sequence linked by interchain bonds at

C or X;X = any amino acid"

(x) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US 4,683,291

(I) FILING DATE: 28-OCT-1985

(J) PUBLICATION DATE: 28-JUL-1987 (X) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US Bl 4,683,291

(I) FILING DATE: 28-OCT-1985

(J) PUBLICATION DATE: 03-JUL-1990

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: Arg Arg Arg Cys Arg Xaa Arg Gly Asp Val Cys 1 5 10

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE:

(A) NAME/KEY: Cross-links

(B) LOCATION: 4..XL1 (D) OTHER INFORMATION: /note= "Multimers of sequence linked by interchain bonds at Cys on amino terminal or carboxyl terminal side of RGD sequence (X) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US 4,683,291

(I) FILING DATE: 28-OCT-1985

(J) PUBLICATION DATE: 28-JUL-1987

(X) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US Bl 4,683,291

(I) FILING DATE: 28-OCT-1985

(J) PUBLICATION DATE: 03-JUL-1990

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

Arg Arg Arg Cys Arg Ser Arg Gly Asp Val Cys 1 5 10 (2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(A) NAME/KEY: Cross-links

(B) LOCATION: 9..>13 (D) OTHER INFORMATION: /note= "Multimers of sequence linked by interchain bonds at X;X = any amino acid" (X) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US 4,683,291 (I) FILING DATE: 28-OCT-1985 (J) PUBLICATION DATE: 28-JUL-1987 (x) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US Bl 4,683,291 (I) FILING DATE: 28-OCT-1985 (J) PUBLICATION DATE: 03-JUL-1990 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

Arg Arg Arg Arg Arg Arg Arg Arg Xaa Arg Gly Asp Val 1 5 10

(2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(ix) FEATURE:

(A) NAME/KEY: Cross-links

(B) LOCATION: 4..>10 (D) OTHER INFORMATION: /note= "Multimers of sequence linked by interchain disulfide bonds at C"

(X) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US 4,683,291 (I) FILING DATE: 28-OCT-1985

(J) PUBLICATION DATE: 28-JUL-1987

(X) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US Bl 4,683,291

(I) FILING DATE: 28-OCT-1985 (J) PUBLICATION DATE: 03-JUL-1990

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

Arg Arg Arg Cys Arg Ser Arg Gly Asp Val 1 5 10

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(A) NAME/KEY: Cross-links

(B) LOCATION: l..>7 (D) OTHER INFORMATION: /note= "Multimers of sequence linked by interchain disulfide bonds at Cys residues"

(X) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US 4,683,291

(I) FILING DATE: 28-OCT-1985

(J) PUBLICATION DATE: 28-JUL-1987

(x) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US Bl 4,683,291

(I) FILING DATE: 28-OCT-1985

(J) PUBLICATION DATE: 03-JUL-1990

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

Cys Arg Ser Arg Gly Asp Val 1 5

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(A) NAME/KEY: Cross-links

(B) LOCATION: 6..>11 (D) OTHER INFORMATION: /note= "Multimers of sequence linked by interchain disulfide bonds at Cys residues"

(X) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US 4,683,291

(I) FILING DATE: 28-OCT-1985

(J) PUBLICATION DATE: 28-JUL-1987 (X) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US Bl 4,683,291

(I) FILING DATE: 28-OCT-1985

(J) PUBLICATION DATE: 03-JUL-1990

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Arg Arg Arg Arg Arg Cys Ser Arg Gly Asp Val 1 5 10 (2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(A) NAME/KEY: Cross-links

(B) LOCATION: 5..>11

(D) OTHER INFORMATION: /note= "Multimers of sequence linked by interchain disulfide bonds at Cys residues'*

(X) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: US 4,683,291

(I) FILING DATE: 28-OCT-1985

(J) PUBLICATION DATE: 28-JUL-1987

(x) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US Bl 4,683,291 (I) FILING DATE: 28-OCT-1985

(J) PUBLICATION DATE: 03-JUL-1990

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

Arg Arg Arg Arg Cys Arg Ser Arg Gly Asp Val 1 5 10

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(A) NAME/KEY: Disulfide-bond (B) LOCATION: 4..>11

(D) OTHER INFORMATION: /note= "Sequence linked by interchain disulfide bond at Cys residue with Cys residue on

Arg3-Cys-Arg2-Ser-Arg-Gly-Asp-Val

(X) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US 4,683,291

(I) FILING DATE: 28-OCT-1985

(J) PUBLICATION DATE: 28-JUL-1987

(x) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US Bl 4,683,291

(I) FILING DATE: 28-OCT-1985

(J) PUBLICATION DATE: 03-JUL-1990

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

Arg Arg Arg Cys Arg Arg Ser Arg Gly Asp Val 1 5 10

(2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(A) NAME/KEY: Cross-links

(B) LOCATION: 3..>11 (D) OTHER INFORMATION: /note= "Multimers of sequence linked by interchain disulfide bonds at Cys residues" (x) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US Bl 4,683,291 (I) FILING DATE: 28-OCT-1985 (J) PUBLICATION DATE: 03-JUL-1990

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Arg Arg Cys Arg Arg Arg Ser Arg Gly Asp Val 1 5 10

(2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: (A) NAME/KEY: Cross-links

(B) LOCATION: 2..>11 (D) OTHER INFORMATION: /note= "Multimers of sequence linked by interchain disulfide bonds at Cys residues" (X) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US Bl 4,683,291 (I) FILING DATE: 28-OCT-1985 (J) PUBLICATION DATE: 03-JUL-1990 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

Arg Cys Arg Arg Arg Arg Ser Arg Gly Asp Val 1 5 10

(2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(A) NAME/KEY: Cross-links (B) LOCATION: 1..>11

(D) OTHER INFORMATION: /note= "Multimers of sequence linked by interchain disulfide bonds at Cys residues"

(X) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US 4,683,291

(I) FILING DATE: 28-OCT-1985

(J) PUBLICATION DATE: 28-JUL-1987

(x) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US Bl 4,683,291

(I) FILING DATE: 28-OCT-1985

(J) PUBLICATION DATE: 03-JUL-1990

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

Cys Arg Arg Arg Arg Arg Ser Arg Gly Asp Val 1 5 10

(2) INFORMATION FOR SEQ ID NO:12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(A) NAME/KEY: Cross-links

(B) LOCATION: 6..>11 (D) OTHER INFORMATION: /note= "Sequence linked by interchain amide bonds at Glu position to Lys residue on Arg5-Lys-Ser- Arg-Gly-Asp-Val sequence" (X) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US 4,683,291 (I) FILING DATE: 28-OCT-1985 (J) PUBLICATION DATE: 28-JUL-1987 (X) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: US Bl 4,683,291

(I) FILING DATE: 28-OCT-1985 (J) PUBLICATION DATE: 03-JUL-1990

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

Arg Arg Arg Arg Arg Glu Ser Arg Gly Asp Val 1 5 10

(2) INFORMATION FOR SEQ ID NO:13:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 11 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(ix) FEATURE:

(A) NAME/KEY: Cross-links

(B) LOCATION: 6..>11 (D) OTHER INFORMATION: /note= "Sequence linked by interchain amide bond at Lys residue with Glu residue on Arg5-Glu-Ser-Arg-

Gly-Asp-Val sequence"

(X) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US 4,683,291

(I) FILING DATE: 28-OCT-1985

(J) PUBLICATION DATE: 28-JUL-1987

(x) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US Bl 4,683,291

(I) FILING DATE: 28-OCT-1985

(J) PUBLICATION DATE: 03-JUL-1990

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

Arg Arg Arg Arg Arg Lys Ser Arg Gly Asp Val 1 5 10

(2) INFORMATION FOR SEQ ID NO:14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(A) NAME/KEY: Cross-links

(B) LOCATION: 5..>11

(D) OTHER INFORMATION: /note= "Sequence linked by interchain amide bond at Glu residue with Lys residue on Arg4-Lys-Arg-Ser-

Arg-Gly-Asp-Val

(X) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US 4,683,291 (I) FILING DATE: 28-OCT-1985

(J) PUBLICATION DATE: 28-JUL-1987

(X) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US Bl 4,683,291

(I) FILING DATE: 28-OCT-1985 (J) PUBLICATION DATE: 03-JUL-1990

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

Arg Arg Arg Arg Glu Arg Ser Arg Gly Asp Val 1 5 10

(2) INFORMATION FOR SEQ ID NO:15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(A) NAME/KEY: Cross-links

(B) LOCATION: 5..>11 (D) OTHER INFORMATION: /note= "Sequence linked by interchain amide bond at Lys residue with Glu residue on Arg4-Glu-Arg-Ser- Arg-Gly-Asp-Va1 (x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: US 4,683,291

(I) FILING DATE: 28-OCT-1985 (J) PUBLICATION DATE: 28-JUL-1987

It

(x) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US Bl 4,683,291

(I) FILING DATE: 28-OCT-1985

(J) PUBLICATION DATE: 03-JUL-1990

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

Arg Arg Arg Arg Lys Arg Ser Arg Gly Asp Val 1 5 10

(2) INFORMATION FOR SEQ ID NO:16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO 5 (ix) FEATURE:

(A) NAME/KEY: Cross-links

(B) LOCATION: 4..>11 (D) OTHER INFORMATION: /note= "Sequence linked by interchain amide bond at Glu residue 0 with Lys residue on Arg3-Lys-Arg2-Ser-

Arg-Gly-Asp-Val

(X) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US 4,683,291

(I) FILING DATE: 28-OCT-1985 5

(J) PUBLICATION DATE: 28-JUL-1987

(x) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US Bl 4,683,291

(I) FILING DATE: 28-OCT-1985

(J) PUBLICATION DATE: 03-JUL-1990 0

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

Arg Arg Arg Glu Arg Arg Ser Arg Gly Asp Val 1 5 10

(2) INFORMATION FOR SEQ ID NO:17:

(i) SEQUENCE CHARACTERISTICS: 5 (A) LENGTH: 11 amino acids It (B) TYPE: amino acid

(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(ix) FEATURE:

5

(A) NAME/KEY: Cross-links

(B) LOCATION: 4..>11

(D) OTHER INFORMATION: /note= "Sequence linked by interchain amide bond at Lys residue with Glu residue on Arg3-Glu-Arg2-Ser- 0

Arg-Gly-Asp-Val"

(x) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US 4,683,291

(I) FILING DATE: 28-OCT-1985 ₅ (J) PUBLICATION DATE: 28-JUL-1987

(X) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US Bl 4,683,291

(I) FILING DATE: 28-OCT-1985

(J) PUBLICATION DATE: 03-JUL-1990 o (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:

Arg Arg Arg Lys Arg Arg Ser Arg Gly Asp Val 1 5 10

(2) INFORMATION FOR SEQ ID NO:18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(A) NAME/KEY: Cross-links

(B) LOCATION: 3..>11

(D) OTHER INFORMATION: /note= "Sequence linked by interchain amide bond at residue Glu with Lys residue on Arg2-Lys-Arg3-Ser- Arg-Gly-Asp-Val

(x) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US 4,683,291

(I) FILING DATE: 28-OCT-1985

(J) PUBLICATION DATE: 28-JUL-1987

(X) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US Bl 4,683,291 (I) FILING DATE: 28-OCT-1985 (J) PUBLICATION DATE: 03-JUL-1990 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:

Arg Arg Glu Arg Arg Arg Ser Arg Gly Asp Val 1 5 10

(2) INFORMATION FOR SEQ ID NO:19: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(A) NAME/KEY: Cross-links

(B) LOCATION: 3..>11 (D) OTHER INFORMATION: /note= "Sequence linked by interchain amide bond at Lys residue with Glu residue on Arg2-Glu-Arg3-Ser- Arg-Gly-Asp-Val" (X) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US 4,683,291 (I) FILING DATE: 28-OCT-1985 (J) PUBLICATION DATE: 28-JUL-1987

(X) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US Bl 4,683,291 (I) FILING DATE: 28-OCT-1985 (J) PUBLICATION DATE: 03-JUL-1990 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: Arg Arg Lys Arg Arg Arg Ser Arg Gly Asp Val 1 5 10

(2) INFORMATION FOR SEQ ID NO:20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(B) LOCATION: 2..>11

(D) OTHER INFORMATION: /note= "Sequence linked by interchain amide bond at residue Glu with Lys residue on Arg-Lys-Arg4-Ser- Arg-Gly-Asp-Val" (x) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US 4,683,291 (I) FILING DATE: 28-OCT-1985 (J) PUBLICATION DATE: 28-JUL-1987 (X)^' PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US Bl 4,683,291 (I) FILING DATE: 28-OCT-1985 (J) PUBLICATION DATE: 03-JUL-1990 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:

Arg Glu Arg Arg Arg Arg Ser Arg Gly Asp Val 1 5 10

(2) INFORMATION FOR SEQ ID NO:21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(B) LOCATION: 2..>11

(D) OTHER INFORMATION: /note= "Sequence linked by interchain amide bond at Lys residue with Glu residue on Arg-Glu-Arg4-Ser-

Arg-Gly-Asp-Val"

(x) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US 4,683,291

(I) FILING DATE: 28-OCT-1985

(J) PUBLICATION DATE: 28-JUL-1987 (x) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US Bl 4,683,291

(I) FILING DATE: 28-OCT-1985

(J) PUBLICATION DATE: 03-JUL-1990

( i) SEQUENCE DESCRIPTION: SEQ ID NO:21: Arg Lys Arg Arg Arg Arg Ser Arg Gly Asp Val 1 5 10

(2) INFORMATION FOR SEQ ID NO:22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(A) NAME/KEY: Cross-links

(B) LOCATION: 1..>11 (D) OTHER INFORMATION: /note= "Sequence linked by interchain amide bond at Glu residue with Lys residue on Lys-Arg5-Ser-Arg- Gly-Asp-Val" (x) PUBLICATION INFORMATION:

(x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: US Bl 4,683,291

(I) FILING DATE: 28-OCT-1985

(J) PUBLICATION DATE: 03-JUL-1990

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:

Glu Arg Arg Arg Arg Arg Ser Arg Gly Asp Val 1 5 10 (2) INFORMATION FOR SEQ ID NO:23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(A) NAME/KEY: Cross-links

(B) LOCATION: 1..>11 (D) OTHER INFORMATION: /note= "Sequence linked by interchain amide bond at Lys residue with Glu residue on Glu-Arg5-Ser-Arg- Gly-Asp-Val" (x) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US Bl 4,683,291 (I) FILING DATE: 28-OCT-1985 (J) PUBLICATION DATE: 03-JUL-1990 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:

Lys Arg Arg Arg Arg Arg Ser Arg Gly Asp Val 1 5 10

(2) INFORMATION FOR SEQ ID NO:24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE:

(A) NAME/KEY: Cross-links

(B) LOCATION: 4..>10

(D) OTHER INFORMATION: /note= "Sequence linked by interchain amide bond at Glu residue with Lys residue on Arg3-Lys-Arg-Ser-

Arg-Gly-Asp-Val"

(X) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US 4,683,291

(I) FILING DATE: 28-OCT-1985

(J) PUBLICATION DATE: 28-JUL-1987

(X) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: US Bl 4,683,291

(I) FILING DATE: 28-OCT-1985

(J) PUBLICATION DATE: 03-JUL-1990

( i) SEQUENCE DESCRIPTION: SEQ ID NO:24:

Arg Arg Arg Glu Arg Ser Arg Gly Asp Val 1 5 10

(2) INFORMATION FOR SEQ ID NO:25: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(A) NAME/KEY: Cross-links

(B) LOCATION: 4..>10

(D) OTHER INFORMATION: /note= "Sequence linked by interchain amide bond at Lys residue with Glu residue on Arg3-Glu-Arg-Ser- Arg-Gly-Asp-Val"

(x) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: US 4,683,291 o

(I) FILING DATE: 28-OCT-1985 (J) PUBLICATION DATE: 28-JUL-1987 (x) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US Bl 4,683,291

(I) FILING DATE: 28-OCT-1985

5

(J) PUBLICATION DATE: 03-JUL-1990

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:

Arg Arg Arg Lys Arg Ser Arg Gly Asp Val 1 5 10

(2) INFORMATION FOR SEQ ID NO:26: ° (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide 5 (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE:

(A) NAME/KEY: Disulfide-bond

(B) LOCATION: 1..>11 (D) OTHER INFORMATION: /note= "Sequence linked by interchain disulfide bond at Cys residue with Cys residue on Arg5-Ser- Arg-Gly-Asp-Val-Cys" (x) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US Bl 4,683,291 (I) FILING DATE: 28-OCT-1985 (J) PUBLICATION DATE: 03-JUL-1990 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:

Arg Arg Arg Arg Arg Ser Arg Gly Asp Val Cys 1 5 10

(2) INFORMATION FOR SEQ ID NO:27: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(A) NAME/KEY: Disulfide-bond

(B) LOCATION: l..>13 (D) OTHER INFORMATION: /note= "Sequence linked by interchain disulfide bond at Cys residue to Cys residue on Arg8-Arg-Gly- Asp-Val-Cys" (X) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: US 4,683,291

(H) DOCUMENT NUMBER: US Bl 4,683,291 (I) FILING DATE: 28-OCT-1985

(J) PUBLICATION DATE: 03-JUL-1990 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:

Arg Arg Arg Arg Arg Arg Arg Arg Arg Gly Asp Val Cys 1 5 10

(2) INFORMATION FOR SEQ ID NO:28:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(A) NAME/KEY: Disulfide-bond (B) LOCATION: l..>12

(D) OTHER INFORMATION: /note= "Sequence linked by interchain amide bond at Cys residue with Cys residue on Arg7-Arg-Gly-Asp-

Val-Cys"

(x) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US 4,683,291

(I) FILING DATE: 28-OCT-1985

(J) PUBLICATION DATE: 28-JUL-1987

(X) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US Bl 4,683,291

(I) FILING DATE: 28-OCT-1985

(J) PUBLICATION DATE: 03-JUL-1990

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:

Arg Arg Arg Arg Arg Arg Arg Arg Gly Asp Val Cys 1 5 10

(2) INFORMATION FOR SEQ ID NO:29:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(A) NAME/KEY: Disulfide-bond

(B) LOCATION: 1..>11 (D) OTHER INFORMATION: /note= "Sequence linked by interchain disulfide bond at Cys residue with Cys residue on Arg6-Arg- Gly-Asp-Va1-Cys" (x) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US Bl 4,683,291 (I) FILING DATE: 28-OCT-1985

(J) PUBLICATION DATE: 03-JUL-1990 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:

Arg Arg Arg Arg Arg Arg Arg Gly Asp Val Cys 1 5 10

(2) INFORMATION FOR SEQ ID NO:30:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids (B) TYPE: amino acid

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO (ix) FEATURE:

(A) NAME/KEY: Disulfide-bond

(B) LOCATION: l..>10

(D) OTHER INFORMATION: /note= "Sequence linked by interchain disulfide bond at Cys residue with Cys residue on Arg5-Arg-

Gly-Asp-Val-Cys"

(X) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US 4,683,291

(I) FILING DATE: 28-OCT-1985

(J) PUBLICATION DATE: 28-JUL-1987

(x) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US Bl 4,683,291

(I) FILING DATE: 28-OCT-1985

(J) PUBLICATION DATE: 03-JUL-1990

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:

Arg Arg Arg Arg Arg Arg Gly Asp Val Cys 1 5 10

(2) INFORMATION FOR SEQ ID NO:31:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

It

(A) NAME/KEY: Disulfide-bond

(B) LOCATION: l..>9

(D) OTHER INFORMATION: /note= "Sequence linked by interchain disulfide bond at Cys residue with Cys residue on Arg4-Arg-

Gly-Asp-Val-Cys"

(X) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US 4,683,291

(I) FILING DATE: 28-OCT-1985

(J) PUBLICATION DATE: 28-JUL-1987

(x) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US Bl 4,683,291

(I) FILING DATE: 28-OCT-1985

(J) PUBLICATION DATE: 03-JUL-1990 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:

Arg Arg Arg Arg Arg Gly Asp Val Cys 1 5

(2) INFORMATION FOR SEQ ID NO:32:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(A) NAME/KEY: Disulfide-bond

(B) LOCATION: l..>8 (D) OTHER INFORMATION: /note= "Sequence linked by interchain disulfide bond at Cys residue with Cys residue on Arg3-Arg- Gly-Asp-Val-Cys" (X) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US 4,683,291 (I) FILING DATE: 28-OCT-1985

(J) PUBLICATION DATE: 28-JUL-1987 (X) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US Bl 4,683,291

(I) FILING DATE: 28-OCT-1985

(J) PUBLICATION DATE: 03-JUL-1990

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:

Arg Arg Arg Arg Gly Asp Val Cys l 5

(2) INFORMATION FOR SEQ ID NO:33:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(A) NAME/KEY: Disulfide-bond

(B) LOCATION: l..>7 (D) OTHER INFORMATION: /note= "Sequence linked by interchain disulfide bond at Cys residue with Cys residue on Arg2-Arg- Gly-Asp-Val-Cys ^Ώ (X) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US Bl 4,683,291 (I) FILING DATE: 28-OCT-1985 (J) PUBLICATION DATE: 03-JUL-1990 (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:

Arg Arg Arg Gly Asp Val Cys 1 5

(2) INFORMATION FOR SEQ ID NO:34:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid u (D) TOPOLOGY: unknown

(A) NAME/KEY: Disulfide-bond

(B) LOCATION: l..>6

(D) OTHER INFORMATION: /note= "Sequence linked by interchain disulfide bond at Cys residue with Cys residue on Arg-Arg-

Gly-Asp-Val-Cys sequence"

(x) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US 4,683,291

(I) FILING DATE: 28-OCT-1985

(J) PUBLICATION DATE: 28-JUL-1987 (x) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US Bl 4,683,291

(I) FILING DATE: 28-OCT-1985

(J) PUBLICATION DATE: 03-JUL-1990

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: ^Ar9 ^Al*g Gly ^AsP ^Val Cys 1 ^' 5

(2) INFORMATION FOR SEQ ID NO:35:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(A) NAME/KEY: Disulfide-bond

(B) LOCATION: l..>14

(D) OTHER INFORMATION: /note= "Sequence linked by interchain disulfide bond at Cys residue with Cys residue on Arg8-Ser-

Arg-Gly-Asp-Val-Cys sequence" (X) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US 4,683,291

(I) FILING DATE: 28-OCT-1985

(J) PUBLICATION DATE: 28-JUL-1987

(X) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US Bl 4,683,291

(I) FILING DATE: 28-OCT-1985

(J) PUBLICATION DATE: 03-JUL-1990

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:

Arg Arg Arg Arg Arg Arg Arg Arg Ser Arg Gly Asp Val Cys 1 5 10 ⁰ (2) INFORMATION FOR SEQ ID NO:36:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown 5 (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE:

(A) NAME/KEY: Disulfide-bond 0 (B) LOCATION: l..>14 (D) OTHER INFORMATION: /note= "Sequence linked by interchain disulfide bond at Cys residue with Cys residue on Arg8-Gly-

Arg-Gly-Asp-Val-Cys" 5

(X) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US 4,683,291

(I) FILING DATE: 28-OCT-1985

(J) PUBLICATION DATE: 28-JUL-1987

(X) PUBLICATION INFORMATION: 0

(H) DOCUMENT NUMBER: US Bl 4,683,291

(I) FILING DATE: 28-OCT-1985

(J) PUBLICATION DATE: 03-JUL-1990

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:

-e Arg Arg Arg Arg Arg Arg Arg Arg Gly Arg Gly Asp Val Cys -³⁰ 1 5 10 υ (2) INFORMATION FOR SEQ ID NO:37:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(A) NAME/KEY: Disulfide-bond

(B) LOCATION: l..>14

(D) OTHER INFORMATION: /note= "Sequence linked by interchain disulfide bond at Cys residue with Cys residue on Arg8-Ala- Arg-Gly-Asp-Val-Cys sequence" (X) PUBLICATION INFORMATION:

(I) FILING DATE: 28-OCT-1985 (J) PUBLICATION DATE: 03-JUL-1990 (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:

Arg Arg Arg Arg Arg Arg Arg Arg Ala Arg Gly Asp Val Cys 1 5 10

(2) INFORMATION FOR SEQ ID NO:38: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: (A) NAME/KEY: Disulfide-bond

(B) LOCATION: l..>14 (D) OTHER INFORMATION: /note= "Sequence linked by interchain bond at Cys residue with

Cys residue on Arg8-Leu-Arg-Gly-Asp-

Val-Cys sequence"

(X) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US 4,683,291

(I) FILING DATE: 28-OCT-1985

(J) PUBLICATION DATE: 28-JUL-1987

(x) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US Bl 4,683,291

(I) FILING DATE: 28-OCT-1985

(J) PUBLICATION DATE: 03-JUL-1990

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:

Arg Arg Arg Arg Arg Arg Arg Arg Leu Arg Gly Asp Val Cys 1 5 10

(2) INFORMATION FOR SEQ ID NO:39:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(A) NAME/KEY: Disulfide-bond

(B) LOCATION: l..>10

(D) OTHER INFORMATION: /note= "Sequence linked by interchain disulfide bond at Cys residue with Cys residue on Ser-Arg- Gly-Asp-Arg5-Cys sequence" (x) PUBLICATION INFORMATION:

(I) FILING DATE: 28-OCT-1985 (J) PUBLICATION DATE: 03-JUL-1990

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:

Ser Arg Gly Asp Arg Arg Arg Arg Arg Cys 1 5 10

(2) INFORMATION FOR SEQ ID NO:40:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 13 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(ix) FEATURE:

(A) NAME/KEY: Disulfide-bond

(B) LOCATION: l..>13 (D) OTHER INFORMATION: /note= "Sequence linked by interchain disulfide bond at Cys residue with Cys residue on Arg-Arg-Gly-

Asp-Arg8-Cys"

(X) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US 4,683,291

(I) FILING DATE: 28-OCT-1985

(J) PUBLICATION DATE: 28-JUL-1987

(X) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US Bl 4,683,291

(I) FILING DATE: 28-OCT-1985

(J) PUBLICATION DATE: 03-JUL-1990

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:

Arg Arg Gly Asp Arg Arg Arg Arg Arg Arg Arg Arg Cys 1 5 10

(2) INFORMATION FOR SEQ ID NO:41:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 13 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(A) NAME/KEY: Disulfide-bond

(B) LOCATION: l..>13

(D) OTHER INFORMATION: /note= "Sequence linked by interchain disulfide bond at Cys residue with Cys residue on Gly-Arg-Gly-

Asp-Arg8-Cys"

(x) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US 4,683,291 (I) FILING DATE: 28-OCT-1985

(J) PUBLICATION DATE: 28-JUL-1987

(X) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US Bl 4,683,291

(I) FILING DATE: 28-OCT-1985 (J) PUBLICATION DATE: 03-JUL-1990

( i) SEQUENCE DESCRIPTION: SEQ ID NO:41:

Gly Arg Gly Asp Arg Arg Arg Arg Arg Arg Arg Arg Cys 1 5 10

(2) INFORMATION FOR SEQ ID NO:42: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(A) NAME/KEY: Cross-links

(B) LOCATION: 1..>11 (D) OTHER INFORMATION: /note= "Sequence linked by interchain amide bond at Glu residue with Orn residue on Arg5-Cys-Ser-Arg- Gly-Asp-Orn" (X) PUBLICATION INFORMATION: (H) DOCUMENT NUMBER: US 4,683,291

(I) FILING DATE: 28-OCT-1985 (J) PUBLICATION DATE: 28-JUL-1987

(X) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US Bl 4,683,291

(I) FILING DATE: 28-OCT-1985

(J) PUBLICATION DATE: 03-JUL-1990

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:

Arg Arg Arg Arg Arg Cys Ser Arg Gly Asp Glu 1 5 10

(2) INFORMATION FOR SEQ ID NO:43:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(A) NAME/KEY: Cross-links

(B) LOCATION: 1..>11 (D) OTHER INFORMATION: /note= "Sequence linked by interchain amide bond at X=Orn residue with Glu residue on Arg5-Cys-

Ser-Arg-Gly-Asp-Glu"

(X) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US 4,683,291

(I) FILING DATE: 28-OCT-1985

(J) PUBLICATION DATE: 28-JUL-1987

(X) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US Bl 4,683,291

(I) FILING DATE: 28-OCT-1985

(J) PUBLICATION DATE: 03-JUL-1990

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:

Arg Arg Arg Arg Arg Cys Ser Arg Gly Asp Xaa 1 5 10

(2) INFORMATION FOR SEQ ID NO:44:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids (B) TYPE: amino acid

(A) NAME/KEY: Cross-links

(B) LOCATION: 1..>11

(D) OTHER INFORMATION: /note= "Sequence linked by interchain amide bond at Glu residue with Orn residue on Arg4-Cys-Arg-Ser-

Arg-Gly-Asp-Orn"

(x) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US 4,683,291

(I) FILING DATE: 28-OCT-1985 (J) PUBLICATION DATE: 28-JUL-1987

(X) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US Bl 4,683,291

(I) FILING DATE: 28-OCT-1985

(J) PUBLICATION DATE: 03-JUL-1990 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:

Arg Arg Arg Arg Cys Arg Ser Arg Gly Asp Glu 1 5 10

(2) INFORMATION FOR SEQ ID NO:45:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(A) NAME/KEY: Cross-links

(B) LOCATION: 1..>11

(D) OTHER INFORMATION: /note= "Sequence linked by interchain amide bond at X = Orn residue with Glu residue on Arg4-Cys- Arg-Ser-Arg-Gly-Asp-Glu" (x) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US Bl 4,683,291 (I) FILING DATE: 28-OCT-1985 (J) PUBLICATION DATE: 03-JUL-1990 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:

Arg Arg Arg Arg Cys Arg Ser Arg Gly Asp Xaa 1 5 10

(2) INFORMATION FOR SEQ ID NO:46: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(A) NAME/KEY: Cross-links

(B) LOCATION: 1..>11 (D) OTHER INFORMATION: /note= "Sequence linked by interchain amide bond at Glu residue with Orn residue on Arg3-Cys-Arg2-Ser- Arg-Gly-Asp-Orn (X) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US Bl 4,683,291 (I) FILING DATE: 28-OCT-1985 (J) PUBLICATION DATE: 03-JUL-1990 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: Arg Arg Arg Cys Arg Arg Ser Arg Gly Asp Glu 1 5 10

(2) INFORMATION FOR SEQ ID NO:47:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: unknown

(B) LOCATION: 1..>11

(D) OTHER INFORMATION: /note= "Sequence linked by interchain amide bond at X=Orn residue with Glu residue on Arg3-Cys- Arg2-Ser-Arg-Gly-Asp-Glu (X) PUBLICATION INFORMATION:

(H) DOCUMENT NUMBER: US Bl 4,683,291 (I) FILING DATE: 28-OCT-1985 (J) PUBLICATION DATE: 03-JUL-1990 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:

Arg Arg Arg Cys Arg Arg Ser Arg Gly Asp Xaa 1 5 10

Claims

u WHAT IS CLAIMED IS:

1. A peptide that inhibits binding of adhesion molecules to cells expressing integrins comprising:

A) multimers comprising at least a first and second subunit;

B) each sequence of every subunit containing at least the sequence Arg-Gly-Asp;

C) the subunits being joined by an interchain stable bond.

2. A peptide of claim 1 wherein the stable bond is a covalent bond.

3. A peptide of claim 2 wherein the covalent bond is selected from the group consisting of disulfide or amide.

4. A peptide of claim 1 wherein the sequence of each of the subunits is identical.

5. A peptide of claim 1 wherein the sequence of each of the subunits is different.

6. A peptide that inhibits binding of adhesion molecules to cells expressing integrins comprising: a multimer of at least a first and second subunit held together by an interchain stable bond, the peptide having the following formula:

(X_z-Arg-Gly-Asp-X_z)_y wherein X = any amino acid or combination of amino acids; z is an integer from 1 to 18; and y is an integer from 2 to 5.

7. A peptide of claim 6 wherein the interchain stable bond is a covalent bond.

8. A peptide of claim 7 wherein the covalent bond is selected from the group consisting of disulfide or amide.

9. A peptide of claim 7 wherein the interchain covalent bond is an amide bond and at least one X on each subunit is selected from Glu, Lys, Asp, or Orn.

10. A peptide of claim 7 wherein the interchain υ covalent bond is a disulfide bond and at least one X on each subunit is Cys.

11. A peptide of claim 6 wherein the subunits have sequences which are identical.

12. A peptide of claim 6 wherein the subunits have sequences which are different.

13. A peptide of claim 6 wherein the interchain stable bond is located on the amino terminal side of the Arg-Gly-Asp sequence.

14. A peptide of claim 6 wherein the interchain . .. stable bond is located on the carboxyl terminal side of the Arg-Gly-Asp sequence.

15. A peptide that inhibits binding of adhesion molecules to cells expressing integrins comprising: a multimer of at least a first and second subunit held

^ together by an interchain stable bond, the peptide having the following formula:

[X_z-Arg-Gly-Asp-(Cx)-Cys_w]_y wherein X is any amino acid or combination of amino acids; z is an integer from 1 to 18; 20 (Cx) is a chain of 1 to 18 amino acid residues selected from the group consisting of Ala, Arg, Asn, Asp, Glu, Gin, Gly, His, He, Leu, Met, Phe, Pro, Trp, Tyr, Val, or Orn; w is an integer from 0 to l; 25 wherein at least one of X_z or Cx is capable of forming an interchain stable bond when w = 0; y is an integer from 2 to 5.

16. A peptide of claim 15 wherein the interchain stable bond is a covalent bond.

30 17. A peptide of claim 16 wherein the covalent bond is selected from the group consisting of disulfide or amide.

18. A peptide of claim 15 wherein the subunits have sequences which are identical.

35 19. A peptide of claim 15 wherein the subunits have sequences which are different.

20. A peptide of claim 15 wherein the interchain stable bond is located on the amino terminal side of the Arg-Gly-Asp sequence.

21. A peptide of claim 15 wherein the interchain stable bond is located on the carboxyl terminal side of the Arg-Gly-Asp sequence.

22. A peptide of claim 17 wherein, one x on each subunit is Cys and w is 0, the bond between subunits is a disulfide bond.

23. A peptide of claim 17 wherein w is 1, and the 0 bond between subunits is a disulfide bond.

24. A peptide of claim 17 wherein one X on the first subunit is Cys and w is 0; and w is 1 on the second subunit, and the bond between subunits is a disulfide 5 bond and the bond is formed on the amino terminal side the Arg-Gly-Asp sequence of the first subunit and on the carboxyl terminal side of the Arg-Gly-Asp of the second subunit, alternating with each subunit.

25. A peptide of claim 15 wherein X is selected o from the group consisting of Arg, Cys, Gin, Phe, Pro,

Thr, Leu, Ala, Gly or Ser.

26. A peptide according to claim 17 wherein one X on the first subunit is selected from Glu or Asp and one X on the second subunit is selected from Lys or Orn, w is 0, and the bond between subunits are amide bonds between the amino acids.

27. A peptide according to claim 17 wherein one Cx on the first subunit is selected from Glu or Asp and one Cx on the second subunit is Orn and the bond between the subunits are amide bonds between the amino acids.

28. A peptide according to claim 17 wherein one X on the first subunit is selected from Glu or Asp and one Cx is Orn on the second subunit and the bonds between subunits are amide bonds between the amino acids.

29. A peptide that inhibits binding of adhesion molecules to cells expressing integrins comprising: a multimer of at least a first and second subunit held together by an interchain stable bond, the peptide having the following formula:

(Arg_m-Cys-Arg_n-X_z-Arg-Gly-Asp-(Cx)-Cys_w)_y wherein m is an integer from 0 to 8, n is an integer from 1 to 5, z is an integer from 1 to 4; X is any amino acid or combination of amino acids;

(Cx) is a chain of 1 to 18 amino acid residues selected from the group consisting of Ala, Arg, Asn, Asp, Glu, Gin, Gly, His, He, Leu, Met, Phe, Pro, Trp, Tyr, Val, or Orn; w is an integer from 0 to 1; and y is an integer from 2 to 5.

30. A peptide of claim 29 wherein the interchain stable bond is a covalent bond.

31. A peptide of claim 30 wherein the covalent bond is selected from the group consisting of disulfide or amide.

32. A peptide of claim 29 wherein the subunits have sequences which are identical.

33. A peptide of claim 29 wherein the subunits have sequences which are different.

34. A peptide of claim 29 wherein the interchain stable bond is located on the amino terminal side of the Arg-Gly-Asp sequence.

35. A peptide of claim 29 wherein the interchain stable bond is located on the carboxyl terminal side of the Arg-Gly-Asp sequence.

36. A peptide according to claim 31 wherein one X on the first subunit is selected from Glu or Asp and one

X on the second subunit is selected from Lys or Orn and the bond between the subunits are amide bonds between the amino acids.

37. A peptide according to claim 31 wherein one Cx on the first subunit is selected from Glu or Asp and Cx u on the second subunit is Orn and the bond between subunits are amide bonds between the amino acids.

38. A peptide accoding to claim 31 wherein one X on the first subunit is selected from Glu or Asp and Cx is Orn on the second subunit and the bonds between the subunits are amide bonds between the amino acids.

5 39. A pepti.de of clai.m 29 wherei.n Cx i.s Val.

40. A peptide of claim 29 wherein, y equals 2.

41. A peptide of claim 31 wherein, the covalent bond is a disulfide bond which is formed on the amino terminal side of each Arg-Gly-Asp sequence and w is 0. 0

42. A peptide of claim 31 wherein, the covalent bond is a disulfide bond which is formed on the carboxyl terminal side of each Arg-Gly-Asp sequence and w is 1.

43. A peptide of claim 31 wherein, the covalent 5 bond is a disulfide bond which is formed on the amino terminal side the Arg-Gly-Asp sequence of the first subunit and w is 0, and on the carboxyl terminal side of the Arg-Gly-Asp of the second subunit and w is 1, alternating with each subunit. o

44. A peptide according to claim 29 wherein, m is an integer from 0 to 3 and z is i.

45. A peptide that inhibits binding of adhesion molecules to cells expressing integrins comprising: a multimer of at least a first and second subunit held together by an interchain stable bond, the peptide having the following formula: (Arg₃-Cys-Arg-X_z-Arg-Gly-Asp-Val-Cys_w)_y (SEQ ID NO.:l) wherein X is any amino acid; z is 1; w is an integer from

0 to 1; and y is an integer from 2 to 5; 46. A peptide of claim 45 wherein the interchain stable bond is a covalent bond.

47. A peptide of claim 46 wherein the covalent bond is selected from the group consisting of disulfide or amide.

48. A peptide of claim 45 wherein the subunits have sequences which are identical.

49. A peptide of claim 45 wherein the subunits have sequences which are different.

50. A peptide of claim 45 wherein the interchain stable bond is located on the amino terminal side of the Arg-Gly-Asp sequence.

51. A peptide of claim 45 wherein the interchain stable bond is located on the carboxyl terminal side of the Arg-Gly-Asp sequence.

52. A peptide according to claim 47 wherein X on the first subunit is selected from Glu or Asp and X on

10 the second subunit is selected from Lys or Orn and the bonds between subunits are amide bonds between the amino acids.

53. A peptide according to claim 45 wherein, X is ,c selected from the group consisting of Gin, Phe, Pro, Thr,

Leu, Ala, Gly, or Ser.

54. A peptide of claim 45 wherein, y equals 2.

55. A peptide of claim 47 wherein, the covalent bond is a disulfide bond which is formed on the amino

2o terminal side of each Arg-Gly-Asp sequence and w is 0.

56. A peptide of claim 47 wherein, the covalent bond is a disulfide bond which is formed on the carboxyl terminal side of each Arg-Gly-Asp sequence and w is 1.

57. A peptide of claim 47 wherein, the covalent 25 bond is a disulfide bond which is formed on the amino terminal side the Arg-Gly-Asp sequence of the first subunit and w is 0, and on the carboxyl terminal side of the Arg-Gly-Asp of the second subunit and w is 1, alternating with each subunit.

30 58. A peptide that inhibits binding of adhesion molecules to cells expressing integrins comprising: a multimer of at least a first and second subunit held together by an interchain stable bond, the peptide having the following formula:

35 (Arg₃-Cys-Arg-Ser-Arg-Gly-Asp-Val-Cys_w) (SEQ ID NO. :2) ϋ and w is an integer from 0 to l; and y is an integer from

2 to 5.

59. A peptide of claim 58 wherein the interchain stable bond is a covalent bond.

60. A peptide of claim 59 wherein the covalent bond is a disulfide bond.

61. A peptide of claim 58 wherein the subunits have sequences which are identical.

62. A peptide of claim 58 wherein the subunits have sequences which are different.

63. A peptide of claim 58 wherein y equals 2.

10

64. A peptide of claim 60 wherein the disulfide bond is formed on the amino terminal side of each Arg- Gly-Asp sequence and w is 0.

65. A peptide of claim 60 wherein the disulfide . - bond is formed on the carboxyl terminal side of each Arg- Gly-Asp sequence and w is 1.

66. A peptide of claim 58 wherein the bond is formed on the amino terminal side the Arg-Gly-Asp sequence of the first subunit and on the carboxyl

2o terminal side of the Arg-Gly-Asp of the second subunit, alternating with each subunit.

67. A peptide that inhibits binding of adhesion molecules to cells expressing integrins selected from the group of peptides consisting essentially of:

25 Arg -Cys-Arg₁-Ser-Arg-Gly-Asp-Val

Arg₄-Cys-Arg₁-Ser-Arg-Gly-Asp-Val, (SEQ ID NO.:7)

Arg₃-Cys-Arg -Ser-Arg-Gly-Asp-Val

Arg₃-Cys-Arg₂-Ser-Arg-Gly-Asp-Val, (SEQ ID NO.:8)

- - Arg₂-Cys-Arg₃-Ser-Arg-Gly-Asp-Val

Arg₂-Cys-Arg₃-Ser-Arg-Gly-Asp-Val, (SEQ ID NO.:9)

Arg₁-Cys-Arg₄-Ser-Arg-Gly-Asp-Val

Arg₁-Cys-Arg₄-Ser-Arg-Gly-Asp-Val, (SEQ ID NO.:10) 35 Cys-Arg₅-Ser-Arg-Gly-Asp-Val

I

Cys-Arg₅-Ser-Arg-Gly-Asp-Val , (SEQ ID NO. : 11) or Arg,-Cys-Arg-. -Ser-Arg-Gly-Asp-Val

I

Arg₃-Cys-Arg₁-Ser-Arg-Gly-Asp-Val (SEQ ID NO . : 4 ) .

68. A method for inhibiting the binding of adhesion molecules to cells with integrins comprising contacting the cells with an effective amount of the peptide of claim 1, 6, 15, 29, 45, 58, or 67.

69. A method for inhibiting the binding of fibrinogen to platelets comprising contacting the platelets with an effective amount of the peptide of claim 1, 6, 15, 29, 45, 58, or 67.

70. A method for inhibiting the binding of von Willebrand factor to platelets comprising contacting the cells with an effective amount of the peptide of claim 1, 6, 15, 29, 45, 58, or 67.

71. A method for inhibiting the binding of cells with integrins to extracellular matrices comprising contacting the cells with an effective amount of the peptide of claim 1, 6, 15, 29, 45, 58, or 67.

72. A method for inhibiting the binding of platelets to extracellular matrices comprising contacting the platelets with an effective amount of the peptide of claim 1, 6, 15, 29, 45, 58, or 67.

73. A method for inhibiting the aggregation of cells with integrins to each other comprising contacting the cells with an effective amount of the peptide according to claim 1, 6, 15, 29, 45, 58, or 67.

74. A method for inhibiting the aggregation of platelets to each other comprising contacting the platelets with an effective amount of the peptide of claim 1, 6, 15, 29, 45, 58, or 67.

75. A pharmaceutical composition for prevention or treatment of thrombosis comprising one or more peptides of claim 1, 6, 15, 29, 45, 58, or 67 in a pharmaceutically acceptable medium.

76. A pharmaceutical composition of claim 75 administered by a route selected from the group consisting of: intravenous, subcutaneous, intramuscular, intranasal, oral or intraperitoneal.

77. A pharmaceutical composition according to claim 75 wherein the pharmaceutically acceptable medium is aqueous and is selected from the group consisting of: water, physiological saline solutions and buffers.

78. A method of preventing or treating thrombosis _lft in a mammal comprising: administering an effective amount of a pharmaceutical composition of claim 75 to the mammal.

79. A method of preventing or treating thrombosis comprising: administering an effective amount of the _jc composition of claim 75 and a thrombolytic agent.

80. A method of claim 79, wherein said thrombolytic agent is selected from the group consisting of: tissue-type plasminogen activator, urokinase, prourokinase, activated protein C, plasmin, plasminogen, o or streptokinase, or derivatives and combinations thereof.

81. A method of detecting platelet-platelet aggregates or thrombi comprising: exposing suspected platelet-platelet aggregates or thrombi to an effective 5 amount of the peptide of claim 1, 6, 15, 29, 45, 58, or 67, the peptide being attached to a detectable tag, and detecting the tagged peptides.

82. A method of claim 81, wherein the exposing step is performed in vivo. 0 83. A method of claim 81, wherein the tag is selected from the group consisting of: radioactive tags, fluorescent tags, biotin tags, or electron dense tags.

5