Evenäs P, García de Frutos P, Nicolaes G A, Dahlbäck B
Department of Clinical Chemistry, Lund University, University Hospital, Malmö, Sweden.
Thromb Haemost. 2000 Aug;84(2):271-7.
Vitamin K-dependent protein S is a cofactor to the anticoagulant serine protease activated protein C (APC) in the proteolytic inactivation of the procoagulant, activated factor V (FVa) and factor VIII (FVIIIa). In the FVa degradation, protein S selectively accelerates the cleavage at Arg306, having no effect on the Arg506 cleavage. In the FVIIIa inactivation, the APC-cofactor activity of protein S is synergistically potentiated by FV, which thus has the capacity to function both as a pro- and an anticoagulant protein. The SHBG-like region of protein S, containing two laminin G-type domains, is required for the combined action of protein S and FV. To elucidate whether both G domains in protein S are needed for expression of APC-cofactor activities, chimeras of human protein S were created in which the individual G domains were replaced by the corresponding domain of the homologous Gas6, which in itself has no anticoagulant activity. In a plasma-based assay, chimera I (G1 from Gas6) was as efficient as wild-type recombinant protein S, whereas chimera II (G2 from Gas6) was less effective. The synergistic cofactor activity with FV in the inactivation of FVIIIa was lost by the replacement of the G2 domain in protein S (chimera II). However, chimera I did not exert full APC-cofactor activity in the FVIIa degradation, indicating involvement of both G domains or the entire SHBG-like region in this reaction. Chimera I was fully active in the degradation of FVa in contrast to chimera II, which exhibited reduced cofactor activity compared to protein S. In conclusion, by using protein S-Gas6 chimeric proteins, we have identified the G2 domain of protein S to be indispensable for an efficient inactivation of both FVIIa and FVa, whereas the G1 domain was found not to be of direct importance in the FVa-inactivation experiments.
维生素K依赖蛋白S是抗凝丝氨酸蛋白酶活化蛋白C（APC）的辅因子，在促凝活化因子V（FVa）和因子VIII（FVIIIa）的蛋白水解失活过程中发挥作用。在FVa降解过程中，蛋白S选择性加速在精氨酸306处的裂解，对精氨酸506处的裂解无影响。在FVIIIa失活过程中，FV协同增强蛋白S的APC辅因子活性，因此FV既能作为促凝蛋白又能作为抗凝蛋白发挥作用。蛋白S的SHBG样区域包含两个层粘连蛋白G型结构域，是蛋白S和FV联合作用所必需的。为了阐明蛋白S中的两个G结构域是否都是APC辅因子活性表达所必需的，构建了人蛋白S的嵌合体，其中单个G结构域被同源Gas6的相应结构域取代，Gas6本身没有抗凝活性。在基于血浆的检测中，嵌合体I（来自Gas6的G1）与野生型重组蛋白S一样有效，而嵌合体II（来自Gas6的G2）效果较差。在FVIIIa失活过程中，蛋白S中G2结构域的替换（嵌合体II）导致与FV协同的辅因子活性丧失。然而，嵌合体I在FVIIa降解过程中并未发挥完全的APC辅因子活性，表明两个G结构域或整个SHBG样区域参与了该反应。与嵌合体II相比，嵌合体I在FVa降解中完全有活性，嵌合体II与蛋白S相比辅因子活性降低。总之，通过使用蛋白S-Gas6嵌合蛋白，我们确定蛋白S的G2结构域对于FVIIa和FVa的有效失活不可或缺，而在FVa失活实验中发现G1结构域并非直接重要。
