Department of Translational Medicine, Lund University, University Hospital, Malmö, Sweden.
J Thromb Haemost. 2022 Jan;20(1):58-68. doi: 10.1111/jth.15547. Epub 2021 Oct 19.
Factor V-Short (FV756-1458) is a natural splice variant in which 702 residues are deleted from the B domain. It exposes an acid region (AR2; 1493-1537) that binds tissue factor pathway inhibitor alpha (TFPIα). Protein S also interacts with TFPIα and serves as TFPIα-cofactor in factor Xa (FXa) inhibition. FV-Short and protein S function as synergistic TFPIα-cofactors in inhibition of FXa. FV810-1492 is an artificial FV-Short variant that cannot synergize with protein S as TFPIα cofactor even though it contains AR2 and binds TFPIα.
To elucidate the mechanisms for the synergism between FV756-1458 and protein S as TFPIα cofactors.
Four FV-Short variants were created, FV756-1458 and FV712-1458 contained the preAR2 region (1458-1492), whereas FV810-1492 and FV713-1492 lacked this region. The synergistic TFPIα cofactor activity between FV-Short variants and protein S was analyzed by FXa-inhibition. A microtiter-based assay tested binding between FV-Short variants, protein S, and TFPIα.
The two preAR2-containing FV-Short variants were active as synergistic TFPIα cofactors, whereas the other two were inactive. All variants bound to TFPIα. None of the FV-Short variants bound directly to protein S. The combination of TFPIα and preAR2-containing FV-Short variants bound protein S, whereas TFPIα together with the preAR2-minus variants did not. Protein S potentiated TFPIα-binding to the preAR2-containing variants and binding between TFPIα and protein S was stimulated only by the preAR2-containing variants.
The preAR2 region is demonstrated to be crucial for the synergistic TFPIα-cofactor activity between FV-Short and protein S and for the assembly of a trimolecular FXa-inhibitory complex comprising FV-Short, protein S, and TFPIα.
因子 V-Short（FV756-1458）是一种天然剪接变异体，其中 B 结构域缺失了 702 个残基。它暴露了一个酸性区域（AR2；1493-1537），该区域可与组织因子途径抑制剂 alpha（TFPIα）结合。蛋白 S 也与 TFPIα相互作用，并作为 FXa 抑制因子 Xa（FXa）的 TFPIα 辅助因子。FV-Short 和蛋白 S 作为协同 TFPIα 辅助因子，共同抑制 FXa。FV810-1492 是一种人工 FV-Short 变体，尽管它含有 AR2 并与 TFPIα 结合，但不能与蛋白 S 协同作为 TFPIα 辅助因子。
阐明 FV756-1458 和蛋白 S 作为 TFPIα 辅助因子协同作用的机制。
构建了四种 FV-Short 变体，FV756-1458 和 FV712-1458 包含前 AR2 区（1458-1492），而 FV810-1492 和 FV713-1492 缺失该区域。通过 FXa 抑制分析，研究了 FV-Short 变体与蛋白 S 之间协同 TFPIα 辅助因子的活性。基于微孔板的测定法检测了 FV-Short 变体、蛋白 S 和 TFPIα 之间的结合。
两种含有前 AR2 的 FV-Short 变体作为协同 TFPIα 辅助因子具有活性，而另外两种变体则没有活性。所有变体均与 TFPIα 结合。FV-Short 变体均不直接与蛋白 S 结合。TFPIα 与含有前 AR2 的 FV-Short 变体结合，而蛋白 S 则与不含前 AR2 的变体结合。蛋白 S 增强了 TFPIα 与含有前 AR2 的变体的结合，并且仅含有前 AR2 的变体刺激了 TFPIα 与蛋白 S 之间的结合。
证明前 AR2 区域对于 FV-Short 和蛋白 S 之间的协同 TFPIα 辅助因子活性以及包含 FV-Short、蛋白 S 和 TFPIα 的三分子 FXa 抑制性复合物的组装至关重要。
