Prothrombin Is a Cofactor for the Binding of Factor XI to the Platelet Surface and for Platelet-Mediated Factor XI Activation by Thrombin

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Abstract
To study the physiological significance of thrombin as an initiator of intrinsic blood coagulation, activated human platelets were compared with dextran sulfate as a surface for thrombin-catalyzed factor XI activation. Activated gel-filtered platelets promoted factor XI activation by thrombin at initial rates 2−5-fold greater than dextran sulfate in the presence of high molecular weight kininogen (HK, 45 nM), ZnCl2 (25 μM), and CaCl2 (2 mM), conditions optimal for factor XI binding to platelets. Physiological concentrations of HK (636 nM) inhibited factor XI activation by thrombin in a concentration-dependent manner, and this inhibition was reversed by prothrombin (1−3 μM) and by prothrombin fragment 1.2 (PF1.2), but not by prothrombin fragment 1 (PF1). Since prothrombin and PF1.2 (but not PF1) also displaced HK from its binding site on the Apple 1 domain of factor XI, we conclude that the Kringle II domain of prothrombin competes with HK for binding to the Apple 1 domain of factor XI. Prothrombin (1−3 μM) and PF1.2 (but not PF1) in the presence of CaCl2 (2 mM) were able to replace HK (45 nM) in the presence of ZnCl2 (25 μM) as a cofactor for the specific, reversible, high-affinity (Kd ∼25 nM) binding of factor XI to 947 ± 150 sites per platelet. This binding is mediated by residues Asn 235−Arg 266 in the Apple 3 domain since a conformationally constrained, synthetic peptide analogue of this sequence inhibits both factor XI binding to activated platelets and platelet-mediated, thrombin-catalyzed factor XI activation in the presence of prothrombin and CaCl2. Finally, prothrombin (1.2 μM) and CaCl2 (2 mM) could substitute for HK (45 nM) and ZnCl2 (25 μM) in promoting optimal rates of thrombin-catalyzed factor XI activation on the platelet surface, thereby initiating the intrinsic coagulation pathway by mechanisms completely independent of the contact phase proteins, factor XII, HK, and prekallikrein.