Ayombil Francis, Toso Raffaella, Camire Rodney M
Division of Hematology and the Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA.
Division of Hematology and the Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA; Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
J Thromb Haemost. 2025 Aug;23(8):2449-2460. doi: 10.1016/j.jtha.2025.04.028. Epub 2025 May 12.
Factor (F)V plays a pivotal role in coagulation, having both pro- and anticoagulant functions. Conserved acidic and basic regions (AR and BR) within FVs central B-domain play a key role in regulating procoagulant function. Removing the BR (residues 963-1008) through proteolysis or splicing, as in FV-short, produces an active cofactor, which is partly regulated by tissue factor pathway inhibitor-α (TFPIα) through its C-terminal BR. Although AR2 (residues 1493-1537) is known to be involved, other FV regions that may interact with the FV-BR or TFPIα-BR have not been defined.
To uncover binding interactions between the BRs and other regions on FV-short.
We used a unique chemical cleavage agent, FeBABE (Fe-p-bromoacetamidobenzyl-ethylenediaminetetraacetic acid) tethered to the FV-BR or TFPIα-BR (BR-FeBABE) to footprint areas of engagement with FV-short using biochemical methods and western blotting.
BR-FeBABE bound FV-short and FV-810, another B-domainless form of FV, with high affinity and blocked procoagulant activity, similar to untethered BR. When BR-FeBABE was stimulated, it cut FV-short and FV-810 at 3 sites including the beginning of AR2, near residues 678 to 692 comprising part of AR1 (residues 659-697), and at the beginning of the A2 domain. Cleavage by BR-FeBABE was specific as untethered FV-BR, TFPIα-BR, or TFPIα blocked proteolysis, and forms of FV that do not bind BR-FeBABE, such as FV, FVa, and any form of FV lacking AR2, were not cut.
These results define areas of interaction between the BR and FV-short and advance our knowledge about how FV is regulated by the B-domain and TFPIα.
凝血因子（F）V在凝血过程中起关键作用，兼具促凝和抗凝功能。FV中央B结构域内保守的酸性和碱性区域（AR和BR）在调节促凝功能中起关键作用。通过蛋白水解或剪接去除BR（963 - 1008位氨基酸残基），如在FV - short中，会产生一种活性辅因子，其部分受组织因子途径抑制剂 - α（TFPIα）通过其C末端BR的调节。尽管已知AR2（1493 - 1537位氨基酸残基）参与其中，但其他可能与FV - BR或TFPIα - BR相互作用的FV区域尚未明确。
揭示BR与FV - short上其他区域之间的结合相互作用。
我们使用一种独特的化学切割剂FeBABE（铁 - 对溴乙酰氨基苄基 - 乙二胺四乙酸）连接到FV - BR或TFPIα - BR（BR - FeBABE）上，通过生化方法和蛋白质印迹法来确定与FV - short相互作用的区域。
BR - FeBABE以高亲和力结合FV - short和FV - 810（FV的另一种无B结构域形式），并阻断促凝活性，类似于未连接的BR。当BR - FeBABE受到刺激时，它在3个位点切割FV - short和FV - 810，包括AR2起始处、包含AR1一部分的678至692位氨基酸残基附近（659 - 697位氨基酸残基）以及A2结构域起始处。BR - FeBABE的切割具有特异性，因为未连接的FV - BR、TFPIα - BR或TFPIα会阻断蛋白水解，而不与BR - FeBABE结合的FV形式，如FV、FVa以及任何缺乏AR2的FV形式，均未被切割。
这些结果确定了BR与FV - short之间的相互作用区域，并推进了我们对FV如何受B结构域和TFPIα调节的认识。
