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Details for Patent: 5,344,783

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Details for Patent: 5,344,783

Title: Platelet aggregation inhibitors
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.
Inventor(s): Scarborough; Robert M. (Hayward, CA), Charo; Israel F. (Lafayette, CA)
Assignee: COR Therapeutics, Inc. (South San Francisco, CA)
Filing Date:Jul 12, 1993
Application Number:08/091,392
Claims: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).

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).

3. 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).
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