Centre for Haematology, Imperial College London, London, UK.
Blood Adv. 2022 Jan 25;6(2):704-715. doi: 10.1182/bloodadvances.2021005382.
Protein S is a cofactor in the tissue factor pathway inhibitor (TFPI) anticoagulant pathway. It enhances TFPIα-mediated inhibition of factor (F)Xa activity and generation. The enhancement is dependent on a TFPIα-protein S interaction involving TFPIα Kunitz 3 and protein S laminin G-type (LG)-1. C4b binding protein (C4BP), which binds to protein S LG1, almost completely abolishes its TFPI cofactor function. However, neither the amino acids involved in TFPIα enhancement nor the mechanisms underlying the reduced TFPI cofactor function of C4BP-bound protein S are known. To screen for functionally important regions within protein S LG1, we generated 7 variants with inserted N-linked glycosylation attachment sites. Protein S D253T and Q427N/K429T displayed severely reduced TFPI cofactor function while showing normal activated protein C (APC) cofactor function and C4BP binding. Based on these results, we designed 4 protein S variants in which 4 to 6 surface-exposed charged residues were substituted for alanine. One variant, protein S K255A/E257A/D287A/R410A/K423A/E424A, exhibited either abolished or severely reduced TFPI cofactor function in plasma and FXa inhibition assays, both in the presence or absence of FV-short, but retained normal APC cofactor function and high-affinity C4BP binding. The C4BP β-chain was expressed to determine the mechanisms behind the reduced TFPI cofactor function of C4BP-bound protein S. Like C4BP-bound protein S, C4BP β-chain-bound protein S had severely reduced TFPI cofactor function. These results show that protein S Lys255, Glu257, Asp287, Arg410, Lys423, and Glu424 are critical for protein S-mediated enhancement of TFPIα and that binding of the C4BP β-chain blocks this function.
蛋白质 S 是组织因子途径抑制剂 (TFPI) 抗凝途径中的辅助因子。它增强了 TFPIα 介导的因子 (F)Xa 活性和生成的抑制作用。这种增强作用依赖于 TFPIα-蛋白质 S 相互作用，涉及 TFPIα Kunitz 3 和蛋白质 S 层粘连蛋白 G 型 (LG)-1。C4b 结合蛋白 (C4BP) 结合蛋白质 S LG1，几乎完全消除其 TFPI 辅助因子功能。然而，与 TFPIα 增强相关的氨基酸以及 C4BP 结合的蛋白质 S 的 TFPI 辅助因子功能降低的机制尚不清楚。为了筛选蛋白质 S LG1 内具有功能重要性的区域，我们生成了 7 种带有插入的 N-连接糖基化附着位点的变体。蛋白质 S D253T 和 Q427N/K429T 显示出严重降低的 TFPI 辅助因子功能，同时表现出正常的激活蛋白 C (APC) 辅助因子功能和 C4BP 结合。基于这些结果，我们设计了 4 种蛋白质 S 变体，其中 4 到 6 个表面暴露的带电残基被替换为丙氨酸。一种变体，蛋白质 S K255A/E257A/D287A/R410A/K423A/E424A，在存在或不存在 FV-短的情况下，在血浆和 FXa 抑制测定中表现出要么完全消除要么严重降低的 TFPI 辅助因子功能，但保留正常的 APC 辅助因子功能和高亲和力 C4BP 结合。表达 C4BPβ 链以确定 C4BP 结合的蛋白质 S 的 TFPI 辅助因子功能降低的机制。与 C4BP 结合的蛋白质 S 一样，C4BPβ 链结合的蛋白质 S 具有严重降低的 TFPI 辅助因子功能。这些结果表明，蛋白质 S 的赖氨酸 255、谷氨酸 257、天冬氨酸 287、精氨酸 410、赖氨酸 423 和谷氨酸 424 对于蛋白质 S 介导的 TFPIα 增强是至关重要的，并且 C4BPβ 链的结合阻断了这种功能。
