Guinea pig proteinase-activated receptor 4 and its activating peptide

Abstract

This invention relates to nucleic acid encoding guinea pig PAR 4 and to the protein encoded thereby. The guinea pig DNA and protein are useful for the development of models of human platelet aggregation. The invention further relates to an animal model to assess the role of PAR antagonists in thrombosis.

1 . A Protease Activated Receptor (PAR) activating peptide comprising the sequence SFPGQ(X) n , where X is any amino acid or amino acid derivative and wherein n is 0-30. 
     
     
         2 . A method comprising the steps of:
 (a) admixing in an aqueous environment a guinea pig PAR-4 activating peptide (AP) with a cell or tissue;   (b) incubating the PAR-4 activating peptide and the cell or tissue for a predetermined amount of time; and   (c) measuring the interaction of the peptide with the cells.   
     
     
         3 . The method of  claim 2  wherein the cell or tissue is in vitro. 
     
     
         4 . The method of  claim 2  wherein the cell is in vivo. 
     
     
         5 . The method of  claim 2  wherein the interaction of the peptide with the cell is detected by measuring the activation of a Protease Activated Receptor (PAR). 
     
     
         6 . A method for promoting human platelet aggregation comprising the step of combining an aggregating amount of an activating peptide with human platelets wherein the activating peptide comprises the sequence SFPGQ(X) n  where x is any amino acid and n is a number between 0 and 30. 
     
     
         7 . A method for determining whether a substance is capable of inhibiting guinea pig PAR-4 activity in a guinea pig comprising the steps of:
 giving a test substance and an activating peptide to a guinea pig, wherein the activating peptide comprises the sequence SFPGQ(X) n  where x is any amino acid and n is a number between 0 and 30; and   measuring the ability of the test substance to inhibit PAR-4 activation in the guinea pig as compared to a control guinea pig that did not receive the test substance.   
     
     
         8 . A method for determining whether a substance capable of interfering with the interaction between an activating peptide and a PAR-4 protein comprising the steps of:
 adding a test substance and an activating peptide to a cell membrane composition comprising a PAR-4 protein wherein the activating peptide comprises the sequence SFPGQ(X) n  where x is any amino acid and n is a number between 0 and 30;   measuring the amount of binding of the activating peptide to the cell membrane composition; and   comparing the amount of binding of the activating peptide to the cell membrane composition with the amount of binding of the activating peptide in control cells receiving no test substance.   
     
     
         9 . The method of  claim 8  wherein the cell membrane composition is a cell membrane fraction. 
     
     
         10 . The method of  claim 8  wherein the cell membrane composition is part of an intact cell, an isolated tissue or organ sample. 
     
     
         11 . The method of  claim 10  wherein the cells are platelets and the measuring the amount of binding step comprises measuring the amount of platelet aggregation, calcium mobilization, ADP degranulation or cell shape change. 
     
     
         12 . The method of  claim 10  wherein the method is performed in a guinea pig. 
     
     
         13 . The method of  claim 8  wherein the activating peptide is labeled and the measuring the amount of binding step comprises measuring the amount of label bound to the cell membrane composition.