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 into the insoluble fibrin, thrombin can initiate a number of positive and negative feedback mechanisms that either sustain or down-regulate clot formation. We have modulated the thrombin sensitivity of human blood coagulation factor VIII, an essential cofactor in the intrinsic pathway of blood coagulation. We have substituted an acidic region of factor VIII corresponding to amino acid sequence Asp 712 -Ala 736 by amino acid sequence Ile 51 -Leu 80 of the thrombin inhibitor heparin cofactor II. Functional analysis of the resulting factor VIII-heparin cofactor II hybrid, termed des-(868-1562)-factor VIII-HCII, revealed an increase in procoagulant activity as measured in a one-stage clotting assay. Incubation of purified des-(868-1562)-factor VIII-HCII with different amounts of thrombin showed that this protein was more readily activated by thrombin when compared with des-(868-1562)-factor VIII, a control protein lacking amino acid sequence Ile 51 -Leu 80 of heparin cofactor II. This was manifested by an increase in the second order rate constant of activation by thrombin for des-(868-1562)-factor VIII-HCII (12.0 ± 0.48 × 10 6 −1 s −1 ) compared with des-(868-1562)-factor VIII (1.77 ± 0.21 × 10 6 −1 s −1 ). Our data suggest that amino acid sequence Ile 51 -Leu 80 of heparin cofactor II endows factor VIII with increased sensitivity towards thrombin which results in accelerated clot formation.