Heeb M J, Rosing J, Bakker H M, Fernandez J A, Tans G, Griffin J H
Scripps Research Institute, La Jolla, CA 92037.
Proc Natl Acad Sci U S A. 1994 Mar 29;91(7):2728-32. doi: 10.1073/pnas.91.7.2728.
Although human protein S binds to human factor Va and inhibits prothrombinase activity, this inhibition is not totally dependent on factor Va. Hence, we investigated possible interaction of protein S with human factor Xa. Factor Xa, diisopropylphospho-factor Xa and their biotin derivatives ligand blotted specifically to protein S and protein S ligand blotted specifically to factor X and factor Xa. Biotinylated factors X and Xa bound to immobilized protein S and, reciprocally, protein S bound to immobilized factor Xa with a Kd of approximately 19 nM. In fluid phase, protein S bound to factor Xa with a Kd of approximately 18 nM. Protein S at 33 nM reversibly inhibited 50% of factor Xa amidolytic activity. Protein S inhibition of prothrombin conversion to thrombin by factor Xa was phospholipid-independent and was 1.6 times stimulated by Ca2+ ions. Inhibition of prothrombinase activity by protein S was 2.3-fold more potent in the presence of factor Va, with 50% inhibition at approximately 8 nM protein S. Protein S prolonged the factor Xa one-stage clotting time of protein S-depleted plasma in a dose-dependent manner. These data demonstrate mechanisms of anticoagulant action for protein S that are independent of activated protein C and that involve direct binding to factors Xa and Va and direct inhibition of factor Xa.
虽然人蛋白S与人因子Va结合并抑制凝血酶原酶活性，但这种抑制并不完全依赖于因子Va。因此，我们研究了蛋白S与人因子Xa之间可能的相互作用。因子Xa、二异丙基磷酸化因子Xa及其生物素衍生物在配体印迹中能特异性地与蛋白S结合，而蛋白S在配体印迹中能特异性地与因子X和因子Xa结合。生物素化的因子X和Xa与固定化的蛋白S结合，反之，蛋白S与固定化的因子Xa结合，解离常数（Kd）约为19 nM。在液相中，蛋白S与因子Xa结合，Kd约为18 nM。33 nM的蛋白S可逆性地抑制50%的因子Xa酰胺水解活性。蛋白S对因子Xa介导的凝血酶原向凝血酶转化的抑制作用不依赖磷脂，且受Ca2+离子刺激增强1.6倍。在因子Va存在的情况下，蛋白S对凝血酶原酶活性的抑制作用增强2.3倍，约8 nM的蛋白S可产生50%的抑制作用。蛋白S以剂量依赖的方式延长了蛋白S缺乏血浆的因子Xa一步凝血时间。这些数据证明了蛋白S的抗凝作用机制，该机制独立于活化蛋白C，涉及与因子Xa和Va的直接结合以及对因子Xa的直接抑制。
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