Safa O, Morrissey J H, Esmon C T, Esmon N L
Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, University of Oklahoma Health Sciences Center, Oklahoma City 73104, USA.
Biochemistry. 1999 Feb 9;38(6):1829-37. doi: 10.1021/bi981730a.
Factor VIIa, in complex with tissue factor (TF), is the serine protease responsible for initiating the clotting cascade. This enzyme complex (TF/VIIa) has extremely restricted substrate specificity, recognizing only three previously known macromolecular substrates (serine protease zymogens, factors VII, IX, and X). In this study, we found that TF/VIIa was able to cleave multiple peptide bonds in the coagulation cofactor, factor V. SDS-PAGE analysis and sequencing indicated the factor V was cleaved at Arg679, Arg709, Arg1018, and Arg1192, resulting in a molecule with a truncated heavy chain and an extended light chain. This product (FVTF/VIIa) had essentially unchanged activity in clotting assays when compared to the starting material. TF reconstituted into phosphatidylcholine vesicles was ineffective as a cofactor for the factor VIIa cleavage of factor V. However, incorporation of phosphatidylethanolamine in the vesicles had little effect over the presence of 20% phosphatidylserine. FVTF/VIIa was as sensitive to inactivation by activated protein C (APC) as thrombin activated factor V as measured in clotting assays or by the appearance of the expected heavy chain cleavage products. The FVTF/VIIa could be further cleaved by thrombin to release the normal light chain, albeit at a significantly slower rate than native factor V, to yield a fully functional product. These studies thus reveal an additional substrate for the TF/VIIa complex. They also indicate a new potential regulatory pathway of the coagulation cascade, i.e., the production of a form of factor V that can be destroyed by APC without the requirement for full activation of the cofactor precursor.
与组织因子（TF）结合的凝血因子VIIa是启动凝血级联反应的丝氨酸蛋白酶。这种酶复合物（TF/VIIa）具有极其有限的底物特异性，仅识别三种先前已知的大分子底物（丝氨酸蛋白酶原、凝血因子VII、IX和X）。在本研究中，我们发现TF/VIIa能够切割凝血辅因子凝血因子V中的多个肽键。SDS-PAGE分析和测序表明，凝血因子V在Arg679、Arg709、Arg1018和Arg1192处被切割，产生了一个重链截短和轻链延长的分子。与起始材料相比，该产物（FVTF/VIIa）在凝血试验中的活性基本未变。重构到磷脂酰胆碱囊泡中的TF作为凝血因子VIIa切割凝血因子V的辅因子无效。然而，在囊泡中加入磷脂酰乙醇胺对20%磷脂酰丝氨酸的存在影响很小。在凝血试验中或通过预期重链切割产物的出现来测量，FVTF/VIIa对活化蛋白C（APC）失活的敏感性与凝血酶活化的凝血因子V相同。FVTF/VIIa可被凝血酶进一步切割以释放正常轻链，尽管其速度明显慢于天然凝血因子V，从而产生一种功能完全正常的产物。因此，这些研究揭示了TF/VIIa复合物的另一种底物。它们还表明了凝血级联反应的一种新的潜在调节途径，即产生一种形式的凝血因子V，其可被APC破坏而无需辅因子前体的完全激活。
