A NOVEL EXOSITE IN THE LIGHT-CHAIN OF HUMAN ACTIVATED PROTEIN-C ESSENTIAL FOR INTERACTION WITH BLOOD-COAGULATION FACTOR VA
A NOVEL EXOSITE IN THE LIGHT-CHAIN OF HUMAN ACTIVATED PROTEIN-C ESSENTIAL FOR INTERACTION WITH BLOOD-COAGULATION FACTOR VA
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DOI：
10.1021/bi00210a014
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发表时间：
1993-11-30
期刊：
影响因子：
2.9
通讯作者：
GRIFFIN, JH
中科院分区：
文献类型：
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作者：
MESTERS, RM;HEEB, MJ;GRIFFIN, JH
活化蛋白C(APC)通过蛋白水解性失活凝血辅助因子Va和VIIIa发挥生理性抗凝作用。为了确定APC轻链表面介导抗凝血活性的区域，制备了10个合成肽，并测试了它们抑制APC抗凝活性的能力。合成肽-(142-155)在正常血浆和去蛋白S血浆的Xa-1期凝血试验中抑制APC的抗凝血活性，在5-25um时抑制50%。在使用纯化凝血因子的系统中，在磷脂存在或不存在的情况下，多肽-(142-155)可抑制APC催化的Va因子的失活，在50um多肽处抑制50%。但是，肽-(142-155)不影响APC的氨解活性，也不影响APC与重组丝氨酸[Arg3581-α1-抗胰蛋白酶]的反应。此外，在正常血浆和凝血酶原酶测定中，多肽-(142-155)对凝血因子Xa的凝血活性也有抑制作用，在本实验条件下，抑制作用分别为5um和50um，抑制率为50%。该多肽对凝血因子Xa或凝血酶的氨解活性无明显影响，对凝血酶对纯化的纤维蛋白原的凝血也无明显影响，提示该多肽不直接抑制这些酶的活性部位。多肽-(142-155)可直接与固定化因子Va结合。这些数据与以下假设一致，即APC轻链上的142-155残基序列提供了一个凝血因子Va结合部位，并且(142-155)肽与Va因子结合，从而干扰了APC对Va因子的失活和凝血酶原酶复合体中Xa因子活性的表达。
Activated protein C (APC) exerts its physiologic anticoagulant role by proteolytic inactivation of the blood coagulation cofactors Va and VIIIa. To identify regions on the surface of the light chain of APC that mediate anticoagulant activity, 10 synthetic peptides were prepared and tested for their ability to inhibit APC anticoagulant activity. The synthetic peptide-(142-155) inhibited APC anticoagulant activity in Xa-1-stage coagulation assays in normal and protein S-depleted plasma with 50% inhibition at 5-25 muM peptide. In a system using purified clotting factors, peptide-(142-155) inhibited APC catalyzed inactivation of factor Va in the presence or absence of phospholipids with 50% inhibition at 50 muM peptide. However, peptide-(142-155) had no effect on APC amidolytic activity or on the reaction of APC with the serpin, recombinant [Arg3581-alpha1-antitrypsin. Moreover, peptide-(142-155) inhibited factor Xa clotting activity in normal plasma as well as in a prothrombinase assay in the presence of factor Va with 50% inhibition at 5 muM and 50 muM peptide, respectively, under the assay conditions. The peptide had no significant effect on factor Xa or thrombin amidolytic activity and no effect on the clotting of purified fibrinogen by thrombin, suggesting that it does not directly inhibit these enzymes' active sites. Peptide-(142-155) was shown to bind directly to immobilized factor Va. These data are consistent with the hypothesis that the sequence of residues 142-155 in the light chain of APC provides a factor Va binding site and that peptide-(142-155) binds factor Va, thereby interfering with both APC inactivation of factor Va and expression of factor Xa activity in the prothrombinase complex.