PAR-1 Activation by Metalloproteinase-1 (MMP-1)

Abstract

Matrix metalloproteases (MMPs) play many important roles in normal and pathological remodeling processes including atherothrombotic disease, inflammation, angiogenesis and cancer. This invention relates to the activation of protease-activated receptor-1 (PAR-1) by endogenous platelet MMP-1 collagenase on the surface of platelets. Exposure of platelets to fibrillar collagen converts the surface-bound pro-MMP-1 zymogen to active MMP-1, which promotes aggregation through PAR-1, MMP-1 is shown to cleave the PAR-1 extracellular domain at a novel site, which then strongly activates Rho-GTP signaling pathways, cell shape change and motility, and MAPK signaling. Blockade of MMP-PAR 1 suppresses thrombogenesis under arterial flow conditions and inhibited thrombosis in animals. These studies provide a link between matrix-dependent activation of metalloproteases and platelet-G protein signaling and identify MMP-1/PAR-1 as a new target for the treatment and prevention of arterial thrombosis and other thrombotic diseases.

1 - 71 . (canceled) 
     
     
         72 . An isolated polypeptide, its sequence comprising no less than 5 contiguous amino acid residues of one of the two fragments that result from a proteolytic cleavage between aspartic acid at position 39 (D39) and proline at position 40 (P40) of human protease-activated receptor-1 (PAR-1), said polypeptide further terminates at one end with a cleavage site that would have resulted from said proteolytic cleavage. 
     
     
         73 . The isolated polypeptide of  claim 72 , wherein said polypeptide has a proline at its N terminus. 
     
     
         74 . The isolated polypeptide of  claim 72 , comprising the polypeptide sequence of PRSFLLRN (SEQ ID NO:1). 
     
     
         75 . A method of diagnosing a thrombotic disease state in a patient, comprising measuring an amount of the polypeptide of  claim 72  in platelets taken from a patient. 
     
     
         76 . A method of identifying a PAR-1 antagonist comprising the steps of:
 (a) providing the isolated polypeptide of  claim 72 ;   (b) providing a candidate agent;   (c) contacting platelets with said isolated polypeptide in the presence of said candidate agent;   (d) measuring PAR-1 signaling activity, and   (e) comparing said PAR-1 signaling activity in the presence of said candidate agent to said PAR-1 signaling activity in the absence of said candidate agent,   wherein a decrease of at least 10% in PAR-1 signaling activity in the presence of said candidate agent as compared to PAR-1 signaling activity in the absence of said candidate agent identifies said candidate agent as a PAR-1 antagonist.   
     
     
         77 . The method of  claim 76 , wherein said PAR-1 signaling activity comprises Rho-GTP or MAPK pathway signaling. 
     
     
         78 - 79 . (canceled) 
     
     
         80 . A medical device coated with a matrix layer comprising an agent that substantially inhibits proteolytic cleavage between aspartic acid at position 39 (D39) and proline at position 40 (P40) of said patient's protease-activated receptor-1 (PAR-1). 
     
     
         81 . A medical device coated with a matrix layer comprising an agent that substantially inhibits protease-activated receptor-1 (PAR-1) signaling activity that results from proteolytic cleavage of PAR-1 between aspartic acid at position 39 (D39) and proline at position 40 (P40). 
     
     
         82 . The medical device of  claim 81 , wherein said agent comprises SCH 530348. 
     
     
         83 . The medical device of  claim 81 , wherein said matrix layer is a biocompatible peptide matrix. 
     
     
         84 . The medical device of  claim 81 , wherein said device is implantable. 
     
     
         85 . The medical device of  claim 81 , wherein said agent comprises a pepducin lipopeptide of a PAR family member. 
     
     
         86 . The medical device of  claim 85 , wherein said pepducin lipopeptide of a PAR family member comprises a PAR-1 pepducin lipopeptide. 
     
     
         87 . The medical device of  claim 86 , wherein said PAR-1 pepducin lipopeptide is selected from the group consisting of P1i3pal-7, P1i3pal-12, P1i3pal-12S, P1i3pal-10S, P1i1pal-11, P1i2pal-7, P1i2pal-11, P1i2pal-16, P1i2pal-21, P1i4pal13 and P1i4pal13R.