Abstract
Current thought holds that factor Xa (FXa) bound in the prothrombinase complex is resistant to regulation by protein protease inhibitors during prothrombin activation. Here we provide evidence that, contrary to this view, the FXa-specific serpin inhibitor, protein Z-dependent protease inhibitor (ZPI), complexed with its cofactor, protein Z (PZ), functions as a physiologically significant inhibitor of prothrombinase-bound FXa during prothrombin activation. Kinetics studies showed that the rapid rate of inhibition of FXa by the ZPI-PZ complex on procoagulant membrane vesicles (ka(app) ∼107 m-1 s-1) was decreased ∼10-fold when FXa was bound to FVa in prothrombinase and a further ∼3-4-fold when plasma levels of S195A prothrombin were present (ka(app) 2 × 105 m-1 s-1). Nevertheless, the ZPI-PZ complex produced a major inhibition of thrombin generation during prothrombinase-catalyzed activation of prothrombin under physiologically relevant conditions. The importance of ZPI-PZ complex anticoagulant regulation of FXa both before and after incorporation into prothrombinase was supported by thrombin generation assays in plasma. These showed enhanced thrombin generation when the inhibitor was neutralized with a PZ-specific antibody and decreased thrombin generation when exogenous ZPI-PZ complex was added whether prothrombin was activated directly by FXa or through extrinsic or intrinsic pathway activators. Moreover, the PZ antibody enhanced thrombin generation both in the absence and presence of activated protein C (APC) anticoagulant activity. Taken together, these results suggest an important anticoagulant role for the ZPI-PZ complex in regulating both free FXa generated in the initiation phase of coagulation as well as prothrombinase-bound FXa in the propagation phase that complement prothrombinase regulation by APC.
Highlights
Current thought holds that factor Xa (FXa) bound in the prothrombinase complex is resistant to regulation by protein protease inhibitors during prothrombin activation
We demonstrate that the Z-dependent protease inhibitor (ZPI)-protein Z (PZ) complex suppresses thrombin generation in plasma independent of the suppression produced by activated protein C (APC), consistent with complementary roles for these two anticoagulants in regulating prothrombinase function
Progress curves were in all cases best fit by a single exponential process, consistent with factor Xa binding to and equilibrating with factor Va to form prothrombinase significantly faster than it was inactivated by the ZPI-PZ complex
Summary
Current thought holds that factor Xa (FXa) bound in the prothrombinase complex is resistant to regulation by protein protease inhibitors during prothrombin activation. Paulina St., Chicago, Previous studies have shown that membrane-associated FXa is rapidly inhibited by the ZPI-PZ complex in the presence of plasma levels of FV and prothrombin, but have suggested that the protease is fully protected from the serpin after FV is activated and binds FXa to form prothrombinase [3]. Such findings have implied that the ZPI-PZ complex functions primarily to dampen the coagulation response by inhibiting FXa generated from the extrinsic or intrinsic FXase complexes prior to its incorporation into prothrombinase and to minimally regulate FXa during prothrombinase activation of prothrombin. We demonstrate that the ZPI-PZ complex suppresses thrombin generation in plasma independent of the suppression produced by activated protein C (APC), consistent with complementary roles for these two anticoagulants in regulating prothrombinase function
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