Thorelli E, Kaufman R J, Dahlbäck B
Department of Clinical Chemistry, Lund University, University Hospital Malmö, S-205 02, Malmö, Sweden.
J Biol Chem. 1998 Jun 26;273(26):16140-5. doi: 10.1074/jbc.273.26.16140.
Factor V (FV) is recently shown to express anticoagulant activity. It functions as a synergistic cofactor with protein S to activated protein C (APC) in the degradation of factor VIIIa (FVIIIa). FV is composed of multiple domains, A1-A2-B-A3-C1-C2. Thrombin cleaves FV at Arg-709, Arg-1018, and Arg-1545 that leads to the generation of a procoagulant FV species which functions as a cofactor to factor Xa (FXa) in the activation of prothrombin to thrombin. During the activation process, the B-domain is released from the heavy (A1-A2) and light chains (A3-C1-C2) which constitute the active FV (FVa). To elucidate which effect the different thrombin cleavages in FV have on the ability of FV to express APC-cofactor activity, seven recombinant FV mutants containing all possible combinations of mutated and native thrombin cleavage sites were tested in a FVIIIa degradation assay. Thrombin cleavage at Arg-709 and/or Arg-1018 yielded FV molecules that were still able to function as APC cofactors, whereas cleavage at Arg-1545 led to a complete loss in APC-cofactor function. This suggests that the APC-cofactor function of FV depends on the B-domain remaining attached to the A3 domain. The importance of the FV B-domain for expression of APC-cofactor activity was further investigated using two B-domain deleted FV molecules, FV des-709-1545 (with the whole B-domain deleted) and FV des-709-1476 (with amino acids 710-1476 of the B-domain being removed). FV des-709-1476 expressed APC-cofactor activity, whereas the FV des-709-1545 was completely devoid of such activity. Thus, the C-terminal part of the B-domain (residues 1477-1545) was crucial for the APC-cofactor function. FV and factor VIII (FVIII) are homologous proteins having similar domain organization. A FV/FVIII chimera, harboring the B-domain from FVIII (FVBVIII) instead of the FV B-domain did not work as an APC cofactor, further illustrating the importance of the FV B-domain for the APC-cofactor function.
最近研究表明，凝血因子V（FV）具有抗凝活性。在因子VIIIa（FVIIIa）的降解过程中，它作为蛋白S与活化蛋白C（APC）的协同辅因子发挥作用。FV由多个结构域组成，即A1 - A2 - B - A3 - C1 - C2。凝血酶在Arg - 709、Arg - 1018和Arg - 1545处切割FV，导致产生一种促凝FV物种，该物种在凝血酶原激活为凝血酶的过程中作为因子Xa（FXa）的辅因子发挥作用。在激活过程中，B结构域从构成活性FV（FVa）的重链（A1 - A2）和轻链（A3 - C1 - C2）上释放出来。为了阐明FV中不同的凝血酶切割对FV表达APC辅因子活性能力的影响，在FVIIIa降解试验中测试了七个包含突变和天然凝血酶切割位点所有可能组合的重组FV突变体。在Arg - 709和/或Arg - 1018处的凝血酶切割产生的FV分子仍能够作为APC辅因子发挥作用，而在Arg - 1545处的切割导致APC辅因子功能完全丧失。这表明FV的APC辅因子功能取决于B结构域与A3结构域保持连接。使用两个缺失B结构域的FV分子，即FV des - 709 - 1545（整个B结构域缺失）和FV des - 709 - 1476（B结构域的氨基酸710 - 1476被去除），进一步研究了FV B结构域对APC辅因子活性表达的重要性。FV des - 709 - 1476表达了APC辅因子活性，而FV des - 709 - 1545完全没有这种活性。因此，B结构域的C末端部分（残基1477 - 1545）对APC辅因子功能至关重要。FV和因子VIII（FVIII）是具有相似结构域组织的同源蛋白。一个携带FVIII的B结构域（FVBVIII）而非FV B结构域的FV/FVIII嵌合体不能作为APC辅因子发挥作用，这进一步说明了FV B结构域对APC辅因子功能的重要性。
