REGIONS REMOTE FROM THE SITE OF CLEAVAGE DETERMINE MACROMOLECULAR SUBSTRATE RECOGNITION BY THE PROTHROMBINASE COMPLEX

The proteolytic formation of thrombin is catalyzed by the prothrombina se complex of blood coagulation. The kinetics of prethrombin 2 cleavag e was studied to delineate macro-molecular substrate structures necess ary for recognition at the exosite(s) of prothrombinase. The product, cu-thrombin, was a linear competitive inhibitor of prethrombin 2 activ ation without significantly inhibiting peptidyl substrate cleavage by prothrombinase. Prethrombin 2 and alpha-thrombin compete for binding t o the exosite without restricting access to the active site of factor Xa within prothrombinase, Inhibition by alpha-thrombin was not altered by saturating concentrations of low molecular weight heparin, Further more, proteolytic removal of the fibrinogen recognition site in alpha- thrombin only had a modest effect on its inhibitory properties. Both a lpha-thrombin and prethrombin 2 were cleaved with chymotrypsin at Trp( 148) and separated into component domains. The C-terminal-derived xi 2 fragment retained the ability to selectively inhibit macromolecular s ubstrate cleavage by prothrombinase, while the xi 1 fragment was witho ut effect. As the xi 2 fragment lacks the fibrinogen recognition site, the P1-P3 residues or the intact cleavage site, specific recognition of the macromolecular substrate by the exosite in prothrombinase is ac hieved through substrate regions, distinct from the fibrinogen recogni tion or heparin-binding sites, and spatially removed from structures s urrounding the scissile bond.