Background: Protein S (PS) is a cofactor for the anticoagulants activated protein C (APC) and tissue factor pathway inhibitor alpha (TFPIα), which inhibit factors Va (FVa) and Xa (FXa), respectively. FVa and FXa form prothrombinase, which activates prothrombin. Neither the PS/APC nor PS/TFPIα system is independently effective at inhibiting thrombin generation by prothrombinase. We hypothesize that they instead function cooperatively.

Aims: To elucidate the regulation of prothrombinase by PS/APC/TFPIα.

Methods: The anticoagulant activities of PS, APC, and TFPIα were measured in purified protein and plasma-based assays, utilizing recombinant proteins to assess the individual cofactor functions of PS.

Results: FXa protected FVa from degradation by APC/PS, as described by other groups, but TFPIα dose-dependently reversed this protection. Similarly, exogenous TFPIα reduced plasma thrombin generation (ETP) 2-fold more when thrombomodulin was added to promote APC activation. Conversely, antibodies against TFPIα and APC had a synergistic effect and, when combined, promoted plasma thrombin generation and fibrin formation to the same extent as an antibody against PS alone.
We expressed a protein consisting of the APC-binding domains of PS (EGF1-2) to assess the effect of APC on PS/TFPIα function. In a purified protein assay, EGF1-2 dose-dependently blocked the APC cofactor activity of 50nM PS, but had no effect on the TFPIα activity. In the presence of EGF1-2, APC still promoted prothrombinase inhibition by PS/TFPIα (ETP decreasing 58.2% vs control).
We similarly assessed the impact of PS/APC on TFPIα, using a protein from the saliva of black flies, “black fly protease inhibitor” (BFPI), which mimics TFPIα but does not bind PS. In the absence of APC, with or without PS, BFPI had no effect on thrombin generation. However, in the presence of APC and PS, BFPI decreased the maximum rate of thrombin generation by 17.3+/-3.3%.

Conclusions: The PS/APC and PS/TFPIα systems work synergistically to downregulate prothrombinase and prevent thrombosis.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/protein-s-coordinates-the-synergistic-inhibition-of-prothrombinase-by-tfpi%ce%b1-and-activated-protein-c/