Methods of screening for inhibitors of antiplasmin cleaving enzyme

Abstract

Human α 2 -antiplasmin (α 2 AP) is the major inhibitor of the proteolytic enzyme plasmin that digests fibrin. Two forms of α 2 AP circulate in human plasma: a 464-residue protein, which we have termed “pro”-form, or α 2 AP pro and an N-terminally-shortened 452-residue “activated”-form, or α 2 AP act . The latter becomes crosslinked to fibrin by activated factor XIII about 5-fold more rapidly than α 2 AP pro and makes fibrin resistant to digestion by plasmin. A new human plasma proteinase has been identified herein that cleaves the Pro12-Asn13 bond of α 2 AP pro to yield α 2 AP act . This enzyme is identified herein as Antiplasmin Cleaving Enzyme (APCE). Novel inhibitors of circulating APCE can diminish α 2 AP inhibitory capacity within forming fibrin or blood clots thereby making fibrin deposits or blood clots more susceptible to removal by plasmin. Patients who are susceptible to atherosclerotic plaque formation or are susceptible to developing thrombi that compromise organ function will benefit by therapies providing such inhibitors on a long term basis.

1 . A method of screening for inhibitors of antiplasmin cleaving enzyme, comprising: 
 providing a fluorescent resonance energy transfer peptide comprising a P 1 —P 1 ′ bond and comprising a fluorophore and a quenching group separated by the P 1 —P 1 ′ bond wherein the P 1  comprises a proline or proline analog and the P 1 ′ comprises an asn, ser, tyr, or other amino acid such that the peptide can be cleaved by α 2 -antiplasmin cleaving enzyme;    providing a quantity of α 2 -antiplasmin cleaving enzyme;    exposing the α 2 -antiplasmin cleaving enzyme to an α 2 -antiplasmin cleaving enzyme inhibitor candidate to form a test mixture;    combining the test mixture with the fluorescent resonance energy transfer peptide; and    measuring the fluorescence emission from the test mixture to identify whether or not the α 2 -antiplasmin cleaving enzyme inhibitor candidate inhibits the activity of α 2 -antiplasmin cleaving enzyme.    
     
     
         2 . The method of  claim 1  wherein the fluorescent resonance energy transfer peptide comprises the quenching group upstream of the proline-asparagine bond and the fluorophore downstream of the proline-asparagine bond.  
     
     
         3 . The method of  claim 1  wherein the fluorescent resonance energy transfer peptide comprises the quenching group downstream and the fluorophore upstream of the proline-asparagine bond.  
     
     
         4 . The method of  claim 1  wherein the P 1 —P 1 ′ bond is a proline-asparagine bond.  
     
     
         5 . The method of  claim 1  wherein the fluorescent resonance energy transfer peptide comprises SEQ ID NO:9.  
     
     
         6 . The method of  claim 1  wherein the α 2 -antiplasmin cleaving enzyme comprises: 
 a purified protein having a molecular weight of about 180 kDa as determined by SDS-PAGE in a dimeric form, wherein each subunit of the dimeric form has a molecular weight of about 97 kDa (SDS-PAGE), and has an N-terminal amino acid consisting of iisoleucine as set forth in SEQ ID No.: 1, said protein further comprising internal sequences as set forth in SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, and wherein the enzyme cleaves precursor α 2 -antiplasmin at the pro12-asn13 bond    
     
     
         7 . An inhibitor of O 2 -antiplasmin cleaving enzyme identified by the screening method of  claim 1 .  
     
     
         8 . An inhibitor of antiplasmin cleaving enzyme which is effective in binding to or blocking the α 2 -antiplasmin binding site, or α 2 -antiplasmin pro-asn or cleaving site of antiplasmin cleaving enzyme.  
     
     
         9 . A method for identifying an enzyme inhibitor, comprising: 
 combining a dimeric molecule having α 2 -antiplasmin cleaving enzymatic activity, said dimeric molecule having a molecular weight of about 180 kilodaltons as determined by SDS-PAGE, a substrate for said molecule, and a test substance to be tested as an enzyme inhibitor; and    determining activity of the dimeric molecule on the substrate, wherein a decrease in activity of the dimeric molecule when the test substance is present indicates that said test substance is an inhibitor.