Activated protein C, protein S, and tissue factor pathway inhibitor cooperate to inhibit thrombin activation

Thrombin, the enzyme which converts fibrinogen into a fibrin clot, is produced by the prothrombinase complex, composed of factor Xa (FXa) and factor Va (FVa). Down-regulation of this process is critical, as excess thrombin can lead to life-threatening thrombotic events. FXa and FVa are inhibited by the anticoagulants tissue factor pathway inhibitor alpha (TFPIα) and activated protein C (APC), respectively, and their common cofactor protein S (PS). However, prothrombinase is resistant to either of these inhibitory systems in isolation. We hypothesized that these anticoagulants function best together, and tested this hypothesis using purified proteins and plasma-based systems. In plasma, TFPIα had greater anticoagulant activity in the presence of APC and PS, maximum PS activity required both TFPIα and APC, and antibodies against TFPI and APC had an additive procoagulant effect, which was mimicked by an antibody against PS alone. In purified protein systems, TFPIα dose-dependently inhibited thrombin activation by prothrombinase, but only in the presence of APC, and this activity was enhanced by PS. Conversely, FXa protected FVa from cleavage by APC, even in the presence of PS, and TFPIα reversed this protection. However, prothrombinase assembled on platelets was still protected from inhibition, even in the presence of TFPIα, APC, and PS. We propose a model of prothrombinase inhibition through combined targeting of both FXa and FVa, and that this mechanism enables down-regulation of thrombin activation outside of a platelet clot. Platelets protect prothrombinase from inhibition, however, supporting a procoagulant environment within the clot. •Neither TFPIα nor APC individually inhibits thrombin activation by prothrombinase.•TFPIα, APC, and their common cofactor PS combine to inhibit prothrombinase.•TFPIα renders factor Va more susceptible to cleavage by APC.•Factor Va degradation enhances the apparent inhibitory activity of TFPIα.•Platelets protect FVa from APC, even in the presence of TFPIα and PS.