Jandrot-Perrus M, Clemetson K J, Huisse M G, Guillin M C
Laboratoire de Recherche sur l'Hémostase et la Thrombose, Faculté Xavier Bichat, Paris, France.
Blood. 1992 Dec 1;80(11):2781-6.
We describe here the alteration of thrombin specificity induced by its interaction with glycocalicin. Glycocalicin is the external part of platelet glycoprotein Ib alpha (GPIb alpha) and contains binding sites for von Willebrand factor and thrombin. Taking advantage of its solubility, we have used glycocalicin in competition assays on various thrombin activities. Glycocalicin did not inhibit chromogenic substrate hydrolysis nor diisopropylfluorophosphate iPr2 (PF) incorporation, indicating that thrombin binding to GPIb does not alter access to or the conformation of the thrombin catalytic site. Glycocalicin competitively inhibited thrombin binding to fibrin (Ki = 0.1 mumol/L) and blocked fibrinogen clotting activity of thrombin. Glycocalicin also inhibited thrombin binding to thrombomodulin in a competitive manner (Ki = 3 to 5 mumol/L), but failed to prevent thrombin interaction with protein C in the absence of thrombomodulin. Previous results have indicated that GPIb binds to thrombin within the anion binding exosite masked by the carboxy-terminal hirudin peptide 54-65. The present results confirm the implication of the anion binding exosite in GPIb recognition, and further indicate that the thrombin binding site for GPIb overlaps with the thrombin binding sites for fibrin and thrombomodulin, whereas it is distinct from the thrombin binding site for protein C. Some of the structural requirements for thrombin binding to GPIb appear to be very similar to those reported for binding to its platelet receptor. However, thrombin-GPIb interaction does not appear to compete with receptor hydrolysis but rather increases the sensitivity and the rate of platelet responses elicited by the receptor.
我们在此描述凝血酶与糖萼素相互作用所诱导的凝血酶特异性改变。糖萼素是血小板糖蛋白Ibα（GPIbα）的外部部分，含有与血管性血友病因子和凝血酶的结合位点。利用其溶解性，我们在各种凝血酶活性的竞争试验中使用了糖萼素。糖萼素不抑制生色底物水解，也不抑制二异丙基氟磷酸（iPr2PF）掺入，这表明凝血酶与GPIb的结合不会改变凝血酶催化位点的可及性或构象。糖萼素竞争性抑制凝血酶与纤维蛋白的结合（Ki = 0.1 μmol/L），并阻断凝血酶的纤维蛋白原凝血活性。糖萼素还以竞争性方式抑制凝血酶与血栓调节蛋白的结合（Ki = 3至5 μmol/L），但在不存在血栓调节蛋白的情况下未能阻止凝血酶与蛋白C的相互作用。先前的结果表明，GPIb在被羧基末端水蛭素肽54 - 65掩盖的阴离子结合外位点内与凝血酶结合。目前的结果证实了阴离子结合外位点在GPIb识别中的作用，并进一步表明凝血酶与GPIb的结合位点与凝血酶与纤维蛋白和血栓调节蛋白的结合位点重叠，而与凝血酶与蛋白C 的结合位点不同。凝血酶与GPIb结合的一些结构要求似乎与报道的与其血小板受体结合的结构要求非常相似。然而，凝血酶 - GPIb相互作用似乎并不与受体水解竞争，而是增加了受体引发的血小板反应的敏感性和速率。
