Department of Haematology, Faculty of Medicine, Imperial College London, London, UK.
Blood. 2010 Jun 10;115(23):4878-85. doi: 10.1182/blood-2009-11-256610. Epub 2010 Mar 22.
Protein S has an established role in the protein C anticoagulant pathway, where it enhances the factor Va (FVa) and factor VIIIa (FVIIIa) inactivating property of activated protein C (APC). Despite its physiological role and clinical importance, the molecular basis of its action is not fully understood. To clarify the mechanism of the protein S interaction with APC, we have constructed and expressed a library of composite or point variants of human protein S, with residue substitutions introduced into the Gla, thrombin-sensitive region (TSR), epidermal growth factor 1 (EGF1), and EGF2 domains. Cofactor activity for APC was evaluated by calibrated automated thrombography (CAT) using protein S-deficient plasma. Of 27 variants tested initially, only one, protein S D95A (within the EGF1 domain), was largely devoid of functional APC cofactor activity. Protein S D95A was, however, gamma-carboxylated and bound phospholipids with an apparent dissociation constant (Kd(app)) similar to that of wild-type (WT) protein S. In a purified assay using FVa R506Q/R679Q, purified protein S D95A was shown to have greatly reduced ability to enhance APC-induced cleavage of FVa Arg306. It is concluded that residue Asp95 within EGF1 is critical for APC cofactor function of protein S and could define a principal functional interaction site for APC.
蛋白质 S 在蛋白 C 抗凝途径中具有明确的作用，它增强了激活蛋白 C（APC）对因子 Va（FVa）和因子 VIIIa（FVIIIa）的失活作用。尽管其具有生理作用和临床重要性，但对其作用的分子基础尚未完全了解。为了阐明蛋白质 S 与 APC 相互作用的机制，我们构建并表达了人类蛋白质 S 的复合或点变异文库，在 Gla、凝血酶敏感区（TSR）、表皮生长因子 1（EGF1）和 EGF2 结构域中引入了残基取代。使用缺乏蛋白质 S 的血浆通过校准自动血栓形成（CAT）评估 APC 的辅助因子活性。在最初测试的 27 种变体中，只有一种，即蛋白质 S D95A（在 EGF1 结构域内），几乎没有 APC 辅助因子活性。然而，蛋白质 S D95A 被 γ-羧化，与磷脂结合的表观解离常数（Kd(app)）与野生型（WT）蛋白质 S 相似。在使用 FVa R506Q/R679Q 的纯化测定中，表明纯化的蛋白质 S D95A 大大降低了增强 APC 诱导的 FVa Arg306 裂解的能力。结论是，EGF1 内的残基 Asp95 对于蛋白质 S 的 APC 辅助因子功能至关重要，并且可以定义 APC 的主要功能相互作用位点。
