Proteolysis of blood coagulation factor VIII by the factor VIIa-tissue factor complex: Generation of an inactive factor VIII cofactor

Activation of factor VIII. by thrombin occurs via limited proteolysis at R- 372, R-740, and R-1689 The resultant active factor VIIIa molecule consists of three noncovalently associated subunits: A1-a1, A2-a2, and A3-C1-C2 (50, 45, and 73 kDa respectively). Further proteolysis of factor VIIIa at R-336 and R-562 by activated protein C subsequently inactivates this cofactor. W e now find that the factor VIIa-tissue factor complex (VIIa-TF/PL), the tri gger of blood coagulation with restricted substrate specificity, can also c atalyze limited proteolysis of factor VIII. Proteolysis of factor VIII was observed at 10 sites, producing 2 major fragments (47 and 45 kDa) recognize d by an anti-factor VIII A2 domain antibody. Time courses indicated the slo w conversion of the large fragment to 45 kDa, followed by further degradati on into at least two smaller fragments. N-Terminal sequencing along with ti me courses of proteolysis indicated that VIIa-TF/PL cleaved factor VIII fir st at R-740, followed by concomitant cleavage at R-336 and R-372 Although c leavage of the light chain at R-1689 was observed, the majority remained un cleaved after 17 h. Consistent with this, only a transient 2-fold increase in factor VIII clotting activity was observed. Thus, heavy chain cleavage o f factor VIII by VIIa-TF/PL produces an inactive factor VIII, cofactor no l onger capable of activation by thrombin. In addition, VIIa-TF/PL was found to inactivate thrombin-activated factor Vm. We hypothesize that these prote olyses may constitute an alternative pathway to regulate coagulation under certain conditions. In addition, the ability of VIIa-TF/PL to cleave factor VIII at 10 sites greatly expands the known protein substrate sequences rec ognized by this enzyme-cofactor complex.