Giri T K, Villoutreix B O, Wallqvist A, Dahlbäck B, de Frutos P G
Department of Clinical Chemistry, Lund University, University Hospital, Malmö, Sweden.
Thromb Haemost. 1998 Nov;80(5):798-804.
Protein S is an important anticoagulant protein acting as cofactor to activated protein C (APC) in the degradation of membrane-bound factors Va and VIIIa. Binding of protein S to the membrane depends on the Gla-domain, whereas sites for APC-interaction are located in the thrombin-sensitive region (TSR) and the first EGF domain. The aims of the present investigation were to localize the sites on protein S which are involved in APC-cofactor function and to elucidate possible orientations of the TSR in relation to the membrane. For these purposes, we determined the epitope for a calcium-dependent monoclonal antibody (HPS67) against the TSR, which inhibits APC cofactor activity even though it does not impede protein S binding to the membrane. HPS67 did not recognize wild-type mouse protein S but gained reactivity against a recombinant mouse protein in which G49 and R52 were mutated to R and Q (found in human protein S), respectively, suggesting these two residues to be part of a surface exposed epitope for HPS67. This information helped in the validation and refinement of the structural model for the Gla-TSR-EGF1-modules of protein S. The X-ray structure of a Fab-fragment mimicking HPS67 was docked onto the protein S model. The observation that HPS67 did not inhibit phospholipid binding of protein S has implications for the possible orientation of protein S on the membrane surface. In the proposed model for membrane-bound protein S, there is no contact between the TSR and the membrane. Rather, the TSR is free to interact with membrane-bound APC.
蛋白S是一种重要的抗凝蛋白，在膜结合因子Va和VIIIa的降解过程中作为活化蛋白C（APC）的辅因子发挥作用。蛋白S与膜的结合取决于Gla结构域，而与APC相互作用的位点位于凝血酶敏感区（TSR）和第一个表皮生长因子（EGF）结构域。本研究的目的是确定蛋白S上参与APC辅因子功能的位点，并阐明TSR相对于膜的可能取向。为了实现这些目的，我们确定了一种针对TSR的钙依赖性单克隆抗体（HPS67）的表位，该抗体即使不阻碍蛋白S与膜的结合，也能抑制APC辅因子活性。HPS67不识别野生型小鼠蛋白S，但对一种重组小鼠蛋白具有反应性，其中G49和R52分别突变为R和Q（在人蛋白S中发现），这表明这两个残基是HPS67表面暴露表位的一部分。这些信息有助于验证和完善蛋白S的Gla-TSR-EGF1模块的结构模型。将模拟HPS67的Fab片段的X射线结构对接至蛋白S模型上。HPS67不抑制蛋白S与磷脂结合这一观察结果对蛋白S在膜表面的可能取向具有启示意义。在所提出的膜结合蛋白S模型中，TSR与膜之间没有接触。相反，TSR可自由地与膜结合的APC相互作用。
