FINDING OF FIBRIN MONOMER AND HEPARIN TO THROMBIN IN A TERNARY COMPLEX ALTERS THE ENVIRONMENT OF THE THROMBIN CATALYTIC SITE, REDUCES AFFINITY FOR HIRUDIN, AND INHIBITS CLEAVAGE OF FIBRINOGENS

Interaction of the blood clotting proteinase, thrombin, with fibrin mo nomer and heparin to form a thrombin fibrin monomer heparin ternary co mplex is accompanied by a change in thrombin catalytic specificity, Eq uilibrium binding interactions in the assembly of the ternary complex were characterized quantitatively using thrombin labeled at the active site with a fluorescent probe and related to changes in thrombin spec ificity toward exosite I-dependent binding of hirudin and cleavage of fibrinogen, Changes in the active site environment accompanying bindin g of heparin or fibrin to thrombin in binary complexes were reported b y fluorescence enhancements which contributed additively to the pertur bation accompanying formation of the ternary complex, Quantitative ana lysis of the interactions supports a preferentially ordered path of te rnary complex assembly, in which initial binding of heparin to thrombi n facilitates binding of fibrin monomer with an similar to 40-fold inc reased affinity, Binding of fibrin monomer in the ternary complex decr eased the affinity of native thrombin for hirudin by > 100-fold and in hibited cleavage of fibrinogen, but this inhibition was overcome when fibrin(ogen)-fibrin interactions occurred, These results support a ter nary complex model in which heparin binding through exosite II of thro mbin facilitates fibrin monomer binding via exosite I, with accompanyi ng changes in thrombin catalytic specificity resulting from perturbati ons in the active site and reduced accessibility of exosite I to hirud in and fibrinogen.