The Binding Of Thrombin By Clots Formed From Fragment X

Previous studies showed that: 1. thrombin was specifically and reversibly bound by fibrin, 2. Scatchard analysis of the data suggested high and low affinity binding sites, and 3. the bound thrombin was quantitatively released following proteolysis of the fibrin by plasmin. In the present study thrombin binding to clots formed from fragment X was studied. The binding of thrombin to fibrin decreased progressively in relation to the original fibrinogen concentration as the fibrin was formed from fibrinogen progressively degraded with plasmin and thus progressively less clottable. Fragment X was then isolated by Sephadex G-200 filtration of partially proteolysed fibrinogen. The fragment X preparation exhibited 76% clottability with thrombin, was heterogeneous with an average molecular weight of 292,000 ±36,000, and contained 2 moles fibrinopeptide A and 0.25 moles fibrinopeptide B per mole. Fibrin formed from clottable fragment X bound thrombin with a molar binding ratio of 0.32 compared to 0.35 for fibrin formed from intact fibrinogen and a binding constant of 7.5 × 10⁵ M^-1 compared to 6.6 × 10⁵ M^-1 for the high affinity site on fibrin from intact fibrinogen. The data indicate that the NH₂-terminal end of the Bβ chain and the COOH-terminal portion of the Aα chain arenot required for high affinity thrombin binding. Because the demonstrated binding is to clottable plasmin degradation products and the molar binding ratio is less than one, it is suggested that the higher affinity thrombin binding site is not present in the fibrinogen molecule but is formed by two or more fibrin molecules present in a polymer.