THROMBIN-FIBRINOGEN INTERACTION - PH-DEPENDENCE AND EFFECTS OF THE SLOW-]FAST TRANSITION

A recently developed strategy capable of measuring the equilibrium dissociation constant for thrombin-fibrinogen interaction has been used to explore the pH dependence of the interaction and the effects of thrombin conformational transitions. The dependence of fibrinogen binding to thrombin in the pH range 6-10 is bell-shaped and remarkably similar to that obtained in the case of the small synthetic amide substrate tosyl-Gly-Pro-Arg-p-nitroanilide-AcOH. Since the synthetic substrate contains no groups that can ionize in the pH range 6-10, the bell-shaped curve must reflect ionization reactions of two groups of the enzyme with pK1 = 7.53 +/- 0.09 and pK2 = 8.80 +/- 0.09. These groups can be identified as the catalytic histidine, His57, and the amino terminus of the B chain, Ilel6, respectively. Deprotonation of His57 in the acidic region is important for optimal binding, while protonation of Ile16 in the alkaline region is critical for the formation of a salt bridge with Asp194, which guarantees the conformational stability of the enzyme. The loss of binding free energy at low (<7.0) and high (>9.0) pH values is linked to protonation of His57 and deprotonation of Ile16, respectively. The first 51 residues of the Aalpha chain of fibrinogen are known to be necessary and sufficient for optimal recognition by thrombin, but none of them contributes to the pH dependence of fibrinogen binding in the pH range examined. Hence, the two possible ionizable groups of the Aalpha chain, i.e., the amino terminus Alal and His24, make no contacts with the thrombin surface. This result is consistent with crystallographic analysis of thrombin bound to fibrinopeptide A and supports a recently proposed structural model for the interaction of the first 51 residues of the Aalpha chain with thrombin. The slow (Na+-free) and fast (Na+-bound) forms of thrombin, that differ in their catalytic competence toward small synthetic amide substrates, have also widely different properties in the recognition of fibrinogen. The slow form binds fibrinogen with an affinity nearly 20 times smaller than that of the fast form. It follows from linkage principles that fibrinogen binding to thrombin must stabilize the fast form and that the slow-->fast transition plays a key role in molecular recognition by thrombin. Analysis of the kinetic mechanism of hydrolysis of fibrinogen to yield fibrin I monomer shows that deacylation becomes rate-limiting in the slow form, as seen for small synthetic amide substrates. Clotting of fibrinogen is also drastically affected, with the slow form showing reduced clotting activity. Under conditions of physiological relevance (pH 7.4 and 37-degrees-C), no clotting activity is observed in the presence of the slow form over a time scale during which clotting in the presence of the fast form is complete.