Division of Hematology and the Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.
Center of Experimental and Molecular Medicine, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands.
J Biol Chem. 2021 Jan-Jun;296:100234. doi: 10.1074/jbc.RA120.016341. Epub 2021 Jan 7.
Coagulation factor V (FV) plays an anticoagulant role but serves as a procoagulant cofactor in the prothrombinase complex once activated to FVa. At the heart of these opposing effects is the proteolytic removal of its central B-domain, including conserved functional landmarks (basic region, BR; 963-1008 and acidic region 2, AR2; 1493-1537) that enforce the inactive FV procofactor state. Tissue factor pathway inhibitor α (TFPIα) has been associated with FV as well as FV-short, a physiologically relevant isoform with a shortened B-domain missing the BR. However, it is unclear which forms of FV are physiologic ligands for TFPIα. Here, we characterize the binding and regulation of FV and FV-short by TFPIα via its positively charged C-terminus (TFPIα-BR) and examine how bond cleavage in the B-domain influences these interactions. We show that FV-short is constitutively active and functions in prothrombinase like FVa. Unlike FVa, FV-short binds with high affinity (K ∼1 nM) to TFPIα-BR, which blocks procoagulant function unless FV-short is cleaved at Arg, removing AR2. Importantly, we do not observe FV binding (μM detection limit) to TFPIα. However, cleavage at Arg and Arg displaces the FV BR, exposing AR2 and allowing TFPIα to bind via its BR. We conclude that for full-length FV, the detachment of FV BR from AR2 is necessary and sufficient for TFPIα binding and regulation. Our findings pinpoint key forms of FV, including FV-short, that act as physiologic ligands for TFPIα and establish a mechanistic framework for assessing the functional connection between these proteins.
凝血因子 V（FV）在凝血酶原酶复合物中发挥抗凝作用，但在被激活为 FVa 后充当促凝辅因子。这些相反作用的核心是其中心 B 结构域的蛋白水解去除，包括保守的功能地标（碱性区域，BR；963-1008 和酸性区域 2，AR2；1493-1537），这些地标强制处于无活性 FV 辅因子状态的 FV。组织因子途径抑制剂 α（TFPIα）与 FV 以及 FV-短型相关，后者是一种具有缩短的 B 结构域而缺失 BR 的生理相关同工型。然而，哪种形式的 FV 是 TFPIα 的生理配体尚不清楚。在这里，我们通过其带正电荷的 C 末端（TFPIα-BR）来描述 TFPIα 对 FV 和 FV-短型的结合和调节，并研究 B 结构域中的键裂解如何影响这些相互作用。我们表明 FV-短型是组成型激活的，并且在凝血酶原酶中像 FVa 一样起作用。与 FVa 不同，FV-短型以高亲和力（K∼1 nM）结合至 TFPIα-BR，除非 FV-短型在 Arg 处被切割，从而去除 AR2，否则这种结合会阻断促凝功能。重要的是，我们没有观察到 FV 与 TFPIα 的结合（检测限为μM）。然而，Arg 和 Arg 的切割会使 FV BR 脱离 AR2，从而使 AR2 暴露出来，并使 TFPIα 通过其 BR 结合。我们得出结论，对于全长 FV，FV BR 与 AR2 的分离是 TFPIα 结合和调节所必需且充分的。我们的发现确定了关键的 FV 形式，包括 FV-短型，它们是 TFPIα 的生理配体，并为评估这些蛋白质之间的功能联系建立了一个机制框架。
