The Anticoagulant Systems Cooperate Synergistically on Phospholipid Vesicle and Endothelial Cell Membranes, but Not on Platelets

Introduction: 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). TFPIα, APC, and PS function synergistically on the surface of phospholipid vesicles (PLs). We have also recently reported a shear-dependent interaction between PS and von Willebrand factor (VWF), which partially blocks its anticoagulant function. Here, we investigated the function of the TFPIα/APC/PS anticoagulant system on physiologic membrane surfaces (platelets and endothelial cells, or ECs), and the effect of VWF on prothrombinase inhibition in these different membrane environments.

Methods: Thrombin generation was measured in plasma and platelet-rich plasma (PRP) by calibrated automated thrombography, purified prothrombinase activity was assayed using a chromogenic substrate, and FVa cleavage was monitored by immunoblotting. Experiments were performed with synthetic PLs, washed platelets, or cultured EA.hy926 cells, an EC line. Experiments involving VWF were performed in the presence or absence of shear.

Results: In plasma TG, as we have previously published, PS anticoagulant activity was greatly enhanced by addition of thrombomodulin (TM), to promote protein C (PC) activation. TM decreased thrombin generation in a PS-dependent manner. The effect of TM and PS was enhanced by the addition of TFPIα, particularly at low PS concentrations. Conversely, inhibitory antibodies against TFPI and PC had an additive effect on plasma TG which was mimicked by inhibiting PS alone. Similar results were obtained in plasma supplemented with ECs (which provide TM) or in PRP. However, in PRP, the antibodies reversed TG beyond what was seen with tissue factor-initiation alone.

In purified protein systems, performed using either PLs or ECs, physiologic TFPIα concentrations had no detectable effect on thrombin activation by prothrombinase, unless APC was present in the system. The greatest inhibitory activity was observed when TFPIα, APC, and PS were all present, and was similar on ECs and PLs. Similarly, inhibition of FXa by TFPIα was found to render FVa susceptible to proteolysis by APC on either PLs or ECs. However, prothrombinase assembled on activated platelets was protected from inhibition, even in the presence of TFPIα, APC, and PS, as measured either by thrombin activation or FVa cleavage.

Finally, we assessed the functional consequences of the PS/VWF interaction. In purified systems with PLs, sheared VWF dose-dependently reversed FXa inhibition by PS/TFPIα, measured using a chromogenic FXa substrate. Conversely, APC cofactor function, as measured by FVa cleavage, was unaffected on either PLs or activated platelets. Consistent with this, thrombin activation by prothrombinase was unaffected by sheared VWF on these surfaces. Interestingly, sheared VWF promoted prothrombinase function on ECs, increasing the rate of thrombin activation beyond that observed in the absence of the anticoagulants.

Conclusions: 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. On PL or EC membranes in the plasma environment, APC, TFPIα, and PS cooperate synergistically to inhibit prothrombinase activity, preventing excessive clot propagation. Conversely, platelets protect prothrombinase from inhibition, supporting a procoagulant environment within the clot. The anticoagulant system appears to function similarly on PLs and on ECs, except that VWF has greater effect on ECs, suggesting that the protection mediated by platelets is due to a platelet component not present in ECs. We hypothesize that these regulatory mechanisms serve to localize procoagulant activity within the platelet plug and suppress it on ECs downstream of a clot.