# US 5,344,783 A · Platelet aggregation inhibitors

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- Number: US 5,344,783 A
- Document ID: US5344783A
- Status: Granted
- Kind: A
- Application number: 08/091,392
- Filed: 1993-07-12
- Granted: 1994-09-06
- Earliest claimed: 1989-06-16
- Assignees: COR Therapeutics, Inc.
- Inventors: Robert M. Scarborough; Israel F. Charo
- Web page: https://patentgrep.com/patent/US5344783A

## CPC

- C07K 14/755: Factors VIII {, e.g. factor VIII C (AHF), factor VIII Ag (VWF)}
- A61K 38/12: Cyclic peptides {, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C (A61K38/043 - A61K38/046 take precedence)}
- A61K 38/1703: {from vertebrates}
- A61K 38/57: from animals; from humans {(A61K38/553, A61K38/556 take precedence)}
- C07K 14/46: from vertebrates
- C07K 14/75: Fibrinogen
- C07K 14/78: Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)

## Abstract

An assay for screening snake venom for the presence or absence of platelet aggregation inhibitors (PAIs) based on specific receptor binding is described. Using this assay, the identification and characterization of the PAI in a wide range of snake venom samples were accomplished. The purified PAI from several of these active snake venoms is described. In addition, PAIs lacking the Arg-Gly-Asp adhesion sequence but containing Lys-Gly-Asp are prepared and shown to specifically inhibit the binding of fibrinogen or von Willebrand Factor to GP IIb-IIIa.

## Claims

### Claim 1

A method for selecting a compound that selectively inhibits platelet aggregation over other cellular activities mediated by integrins, which method comprises:
(a) contacting a sample of said compound with purified GP IIb-IIIa receptor coated onto a solid support in the presence of a solution of labeled fibrinogen or von Willebrand Factor under conditions wherein fibrinogen or von Willebrand factor binds to said GP IIb-IIIa;
(b) measuring the binding of fibrinogen or von Willebrand factor to GP IIb-IIIa in comparison to the binding of fibrinogen or von Willebrand factor to GP IIb-IIIa in a control which does not contain said compound by measuring the amount of bound labeled fibrinogen;
(c) determining the inhibition in (b);
(d) contacting a sample of said compound with purified vitronectin receptor coated onto a solid support in the presence of a solution of labeled vitronectin under conditions wherein vitronectin binds to said vitronectin receptor;
(e) measuring the binding of vitronectin to vitronectin receptor in comparison to the binding of vitronectin to vitronectin receptor in a control which does not contain said compound by measuring the amount of bound labeled vitronectin;
(f) determining the inhibition in (e); and
(g) selecting a sample in which the relative inhibition determined in (c) is at east twice that determined in (f).

### Claim 2

A method for selecting a compound that selectively inhibits platelet aggregation over other cellular activities mediated by integrins, which method comprises:
(a) contacting a sample of said compound with purified GP IIb-IIIa receptor coated onto a solid support in the presence of a solution of labeled fibrinogen or von Willebrand Factor under conditions wherein fibrinogen or von Willebrand factor binds to said GP IIb-IIIa;
(b) measuring the binding of fibrinogen or von Willebrand factor to GP IIb-IIIa in comparison to the binding of fibrinogen or von Willebrand factor to GP IIb-IIIa in a control which does not contain said compound by measuring the amount of bound labeled fibrinogen;
(c) determining the inhibition in (b);
(d) contacting a sample of said compound with purified fibronectin receptor coated onto a solid support in the presence of a solution of labeled fibronectin under conditions wherein fibronectin binds to said fibronectin receptor;
(e) measuring the binding of fibronectin to fibronectin receptor in comparison to the binding of fibronectin to fibronectin receptor in a control which does not contain said compound by measuring the amount of bound labeled fibronectin;
(f) determining the inhibition in (e); and
(g) selecting a sample in which the relative inhibition determined in (c) is at least twice that determined in (f).

### Claim 3 (depends on 1)

The method of claim 1, comprising the further steps of:
(h) contacting a sample selected in (g) with purified fibronectin receptor coated onto a solid support in the presence of a solution of labeled fibronectin under conditions wherein fibronectin binds to said fibronectin receptor;
(i) measuring the binding of fibronectin to fibronectin receptor in comparison to the binding of fibronectin to fibronectin receptor in a control which does not contain said compound by measuring the amount of bound labeled fibronectin;
(j) determining the inhibition in (i); and
(k) selecting a sample in which the relative inhibition determined in (c) is at least twice that determined in (j).