Regulation of factor V and factor V-short by TFPIα: Relationship between B-domain proteolysis and binding.
Regulation of factor V and factor V-short by TFPIα: Relationship between B-domain proteolysis and binding.
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DOI：
10.1074/jbc.ra120.016341
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发表时间：
2021-01
期刊：
影响因子：
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通讯作者：
Camire RM
中科院分区：
文献类型：
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作者：
Petrillo T;Ayombil F;Van't Veer C;Camire RM
凝血因子V(Fv)具有抗凝作用，但一旦被FVA激活，则在凝血酶原酶复合体中起促凝血因子的作用。这些相反作用的核心是其中心B结构域的蛋白水解性移除，包括保守的功能标志物(碱性区域BR；963-1008和酸性区域2 AR2；1493-1537)，它们加强了FV原因子的失活状态。组织因子途径抑制物α(TFP Iα)与FV和FV-Short有关，Fv-Short是一种生理上相关的亚型，B结构域缩短，缺失BR。然而，目前还不清楚哪些形式的Fv是TFP Iα的生理配体。在这里，我们表征了TFP Iα通过其带正电的C末端(TFP Iα-BR)与Fv和Fv-Short的结合和调节，并研究了B-结构域的键断裂如何影响这些相互作用。我们发现Fv-Short在结构上是活性的，并且像FVA一样发挥凝血酶原酶的功能。与FVA不同，Fv-Short与TfpI∼-BR具有高亲和力(KD AR1)结合，TfpIα-BR阻断促凝血功能，除非Fv-Short在Arg1545上被切割，去除AR2。重要的是，我们没有观察到Fv与TfpIα的结合(TfpI M的检测限)。然而，在Arg709和Arg1018处的切割取代了Fv BR，暴露了AR2，并允许TFP Iα通过其BR结合。我们的结论是，对于全长Fv，Fv BR从AR2上分离是TFP Iα结合和调节所必需的，也是充分的。我们的发现明确了Fv的关键形式，包括Fv-Short，它们作为TFP Iα的生理配体，并建立了一个机制框架来评估这些蛋白质之间的功能联系。
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 (Kd ∼1 nM) to TFPIα-BR, which blocks procoagulant function unless FV-short is cleaved at Arg1545, removing AR2. Importantly, we do not observe FV binding (μM detection limit) to TFPIα. However, cleavage at Arg709 and Arg1018 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.