Han J H, Côté H C, Tollefsen D M
Department of Internal Medicine, Washington University, St. Louis, Missouri 63110, USA.
J Biol Chem. 1997 Nov 7;272(45):28660-5. doi: 10.1074/jbc.272.45.28660.
Meizothrombin and meizothrombin(desF1) are intermediates formed during the conversion of prothrombin to thrombin by factor Xa, factor Va, phospholipids, and Ca2+ (prothrombinase). These intermediates are active toward synthetic peptide substrates but have limited ability to interact with platelets or macromolecular substrates such as fibrinogen. Meizothrombin and meizothrombin(desF1) activate protein C, however, and may exert primarily an anticoagulant effect. In this study, we investigated the inhibition of meizothrombin and meizothrombin(desF1) by two glycosaminoglycan-dependent protease inhibitors, heparin cofactor II (HCII) and antithrombin (AT). Purified recombinant meizothrombin and meizothrombin(desF1) were inhibited by HCII in the presence of dermatan sulfate with maximal second-order rate constants of 8 x 10(6) M-1.min-1 and 1.8 x 10(7) M-1.min-1, respectively, but were inhibited less than one-tenth as fast by AT in the presence of heparin. Similarly, the products of the prothrombinase reaction were inhibited in situ more effectively by HCII than by AT. When HCII and dermatan sulfate were present continuously during the prothrombinase reaction, meizothrombin was trapped as a sodium dodecyl sulfate-stable complex with HCII and no amidolytic activity could be detected with a thrombin substrate. Our findings indicate that HCII is an effective inhibitor of meizothrombin and meizothrombin(desF1) and, therefore, might regulate the anticoagulant activity of these proteases.
中凝血酶和中凝血酶（去F1）是在凝血酶原被因子Xa、因子Va、磷脂和Ca2+（凝血酶原酶）转化为凝血酶的过程中形成的中间体。这些中间体对合成肽底物具有活性，但与血小板或纤维蛋白原等大分子底物相互作用的能力有限。然而，中凝血酶和中凝血酶（去F1）可激活蛋白C，可能主要发挥抗凝作用。在本研究中，我们研究了两种糖胺聚糖依赖性蛋白酶抑制剂——肝素辅因子II（HCII）和抗凝血酶（AT）对中凝血酶和中凝血酶（去F1）的抑制作用。在硫酸皮肤素存在的情况下，纯化的重组中凝血酶和中凝血酶（去F1）被HCII抑制，最大二级速率常数分别为8×10⁶ M⁻¹·min⁻¹和1.8×10⁷ M⁻¹·min⁻¹，但在肝素存在的情况下，被AT抑制的速度不到前者的十分之一。同样，凝血酶原酶反应的产物在原位被HCII抑制的效果比被AT抑制的效果更有效。当在凝血酶原酶反应过程中持续存在HCII和硫酸皮肤素时，中凝血酶会与HCII形成十二烷基硫酸钠稳定的复合物而被捕获，并且用凝血酶底物检测不到酰胺水解活性。我们的研究结果表明，HCII是中凝血酶和中凝血酶（去F1）的有效抑制剂，因此可能调节这些蛋白酶的抗凝活性。
