Saha Cardiovascular Research Center, University of Kentucky, Lexington, KY, United States of America.
Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, KY, United States of America.
Thromb Res. 2023 Oct;230:84-93. doi: 10.1016/j.thromres.2023.08.012. Epub 2023 Aug 23.
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.
凝血酶是将纤维蛋白原转化为纤维蛋白凝块的酶，由凝血酶原酶复合物产生，该复合物由因子 Xa（FXa）和因子 Va（FVa）组成。该过程的下调至关重要，因为过量的凝血酶会导致危及生命的血栓事件。FXa 和 FVa 分别被抗凝剂组织因子途径抑制剂 alpha（TFPIα）和活化蛋白 C（APC）抑制，它们的共同辅助因子蛋白 S（PS）。然而，凝血酶原酶在单独存在的情况下对这两种抑制系统都具有抗性。
我们假设这些抗凝剂一起作用效果最佳，并使用纯化蛋白和基于血浆的系统来测试这一假设。
在血浆中，TFPIα 在 APC 和 PS 的存在下具有更强的抗凝活性，最大的 PS 活性需要 TFPIα 和 APC，针对 TFPI 和 APC 的抗体具有相加的促凝作用，这可被单独针对 PS 的抗体模拟。在纯化蛋白系统中，TFPIα 剂量依赖性地抑制凝血酶原酶激活凝血酶，但仅在 APC 存在的情况下，并且这种活性被 PS 增强。相反，FXa 保护 FVa 免受 APC 的裂解，即使在 PS 存在的情况下也是如此，而 TFPIα 逆转了这种保护。然而，即使在存在 TFPIα、APC 和 PS 的情况下，血小板上组装的凝血酶原酶仍不受抑制。
我们提出了一种通过同时靶向 FXa 和 FVa 来抑制凝血酶原酶的模型，并且该机制能够下调血小板凝块外的凝血酶激活。然而，血小板保护凝血酶原酶免受抑制，从而在凝块内支持促凝环境。
