Giri T K, García de Frutos P, Dahlbäck B
Department of Laboratory Medicine, Lund University, University Hospital Malmö, Sweden.
Thromb Haemost. 2000 Sep;84(3):413-9.
Protein S functions as a cofactor to activated protein C (APC) in the degradation of FVa and FVIIIa. In protein S, the thrombin sensitive region (TSR) and the first EGF-like domain are important for expression of the APC cofactor activity. A naturally occurring Thr103Asn (T103N) mutation in the first EGF-like domain of protein S has been associated with functional (type II) protein S deficiency. To elucidate the functional consequences of the T103N mutation, recombinant protein S mutant was expressed in mammalian cells and functionally characterised. The expression level of protein S T103N from transiently transfected COS 1 cells was equal to that of wild type protein S. The mutant protein S and wild type protein S were also expressed in 293 cells after stable transfection, and the recombinant proteins purified. In APTT- and PT-based coagulation assays, the mutant protein demonstrated approximately 50% lower anticoagulant activity as compared to wild type protein S. The functional defect was further investigated in FVa- and FVIIIa-degradation assays. The functional defect of mutant protein S was attenuated at increasing concentrations of APC. The results demonstrate the region around residue 103 of protein S to be of functional importance, possibly through a direct interaction with APC.
蛋白S在凝血因子Va（FVa）和凝血因子VIIIa（FVIIIa）的降解过程中作为活化蛋白C（APC）的辅因子发挥作用。在蛋白S中，凝血酶敏感区域（TSR）和首个表皮生长因子样结构域对于APC辅因子活性的表达至关重要。蛋白S首个表皮生长因子样结构域中天然存在的苏氨酸103天冬酰胺（T103N）突变与功能性（II型）蛋白S缺乏有关。为了阐明T103N突变的功能后果，在哺乳动物细胞中表达了重组蛋白S突变体并对其进行了功能表征。瞬时转染的COS 1细胞中蛋白S T103N的表达水平与野生型蛋白S的表达水平相当。稳定转染后，突变蛋白S和野生型蛋白S也在293细胞中表达，并对重组蛋白进行了纯化。在基于活化部分凝血活酶时间（APTT）和凝血酶原时间（PT）的凝血试验中，与野生型蛋白S相比，突变蛋白的抗凝活性降低了约50%。在FVa和FVIIIa降解试验中进一步研究了功能缺陷。随着APC浓度的增加，突变蛋白S的功能缺陷有所减轻。结果表明，蛋白S第103位残基周围区域具有重要功能，可能是通过与APC的直接相互作用实现的。
