Evidence that both exosites on thrombin participate in its high affinity interaction with fibrin

Exosite 1 on thrombin mediates low affinity binding to sites on the NH2 termini of the alpha- and beta-chains of fibrin. A subpopulation of fibrin molecules (gamma(A)/gamma'-fibrin) has an alternate COOH terminus of the normal gamma-chain (gamma(A)/gamma(A)-fibrin) that binds thrombin with high affinity. To determine the roles of exosites 1 and 2 in the high affinity interaction of thrombin with gamma(A)/gamma'-fibrin, binding studies were done with thrombin variants and exosite 1- or 2-directed ligands. alpha-Thrombin bound gamma(A)/gamma'-fibrin via high and low affinity binding sites. A peptide analog of the COOH terminus of the gamma'-chain that binds alpha-thrombin via exosite 2 blocked the high affinity binding of alpha-thrombin to gamma(A)/gamma'-fibrin, suggesting that the interaction of alpha-thrombin with the gamma'-chain is exosite 2-mediated. In support of this concept, (a) gamma-thrombin, which lacks a functional exosite 1, bound to gamma(A)/gamma'-fibrin, but not to gamma(A)/gamma(A)-fibrin; ( b) thrombin R93A/R97A/R101A, an exosite 2-defective variant, bound only to gamma(A)/gamma'-fibrin via low affinity sites; and ( c) exosite 2-directed ligands reduced alpha-thrombin binding to gamma(A)/gamma'-fibrin. However, several lines of evidence indicate that exosite 1 contributes to the high affinity interaction of thrombin with gamma(A)/gamma-fibrin. First, the affinity of gamma-thrombin for gamma(A)/gamma'-fibrin was lower than that of alpha-thrombin. Second, removal of a low affinity binding site on the beta-chain of gamma(A)/gamma'-fibrin reduced its affinity for alpha-thrombin. Third, exosite 1- directed ligands reduced alpha-thrombin binding to gamma(A)/gamma'-fibrin. Taken together, these data suggest that, although exosite 2 mediates the interaction of thrombin with the gamma'-chain of gamma(A)/gamma'-fibrin, simultaneous ligation of exosite 1 by low affinity binding sites is essential for the high affinity interaction of thrombin with gamma(A)/gamma'-fibrin.