Late-fibrin(ogen) fragment E modulates human alpha-thrombin specificity

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Abstract
Fibrinogen contains at least two independent sites having demonstrable affinity for α‐thrombin. One of these two sites, located in the fibrin E domain, binds to structures within the anion‐binding exosite of α‐thrombin. Taking advantage of its solubility, we have used late‐fibrin(ogen) fragment E in competition experiments to examine its effect on α‐thrombin specificity. We show that fragment E modulates α‐thrombin enzymic activity towards small synthetic substrates, suggesting that fibrin‐thrombin interaction might induce subtle changes in the conformation near the catalytic center of the enzyme. In addition, fragment E behaved as a competitive inhibitor of α‐thrombin‐catalyzed fibrinopeptide‐A cleavage (Ki= 5.2±1.3 μM), indicating that α‐thrombin interaction with the fibrin moiety of fibrinogen makes a major contribution to the efficacy of fibrinogen hydrolysis. Fragment E inhibited α‐thrombin‐induced serotonin release by platelets (concentration required to obtain 50% inhibition IC50= 10 μM) and α‐thrombin binding to GPIb. Fragment E competitively inhibited α‐thrombin binding to thrombomodulin (Ki= 18.3±0.8 μM) but did not inhibit protein‐C activation in the absence of thrombomodulin. The data are consistent with the proposal that fibrin, platelet GPIb and thrombomodulin bind to overlapping, but probably non‐identical sites, while protein C binds to an independent site on α‐thrombin.