ENHANCED THROMBIN SENSITIVITY OF A FACTOR-VIII HEPARIN-COFACTOR-II HYBRID

Generation of thrombin at a site of vascular injury is a key event in the arrest of bleeding, In addition to the conversion of fibrinogen in to the insoluble fibrin, thrombin can initiate a number of positive an d negative feedback mechanisms that either sustain or down-regulate cl ot formation. We have modulated the thrombin sensitivity of human bloo d coagulation factor VIII, an essential cofactor in the intrinsic path way of blood coagulation, We have substituted an acidic region of fact or VIII corresponding to amino acid sequence Asp(712)-Ala(736) by amin o acid sequence Ile(51)-Leu(80) of the thrombin inhibitor heparin cofa ctor II. Functional analysis of the resulting factor VIII-heparin cofa ctor II hybrid, termed des-(868-1562)-factor VIII-HCII, revealed an in crease in procoagulant activity as measured in a one-stage clotting as say. Incubation of purified des-(868-1562)-factor VIII-HCII with diffe rent amounts of thrombin showed that this protein was more readily act ivated by thrombin when compared with des-(868-1562)-factor VIII, a co ntrol protein lacking amino acid sequence Ile(51)-Leu(80) of heparin c ofactor II. This was manifested by an increase in the second order rat e constant of activation by thrombin for des-(868-1562) factor VIII-HC II (12.0 +/- 0.48 x 10(6) M(-1) s(-1)) compared with des-(868-1562)-fa ctor VIII (1.77 +/- 0.21 x 10(6) M(-1) s(-1)). Our data suggest that a mino acid sequence Ile(51)-Leu(80) of heparin cofactor II endows facto r VIII with increased sensitivity towards thrombin which results in ac celerated clot formation.