Hayes K L, Tracy P B
Department of Biochemistry, University of Vermont College of Medicine, Burlington, Vermont 05405, USA.
J Biol Chem. 1999 Jan 8;274(2):972-80. doi: 10.1074/jbc.274.2.972.
The platelet high affinity binding site for thrombin appears to be described by a classical receptor-ligand interaction that is distinct from the platelet thrombin receptor/substrate, PAR-1. However, the identification and function of the high affinity binding site with respect to its physiological importance have continued to elude investigators. Prior studies using two mutant thrombins suggested that thrombin interaction with the platelet high affinity binding site is mediated through an extensive portion of the thrombin molecule involving residues within the substrate binding pocket and the anion binding exosite (Leong, L., Henriksen, R. A., Kermode, J. C., Rittenhouse, S. E., and Tracy, P. B. (1992) Biochemistry 31, 2567-2575) and may mimic a thrombin-hirudin interaction. To test this hypothesis, an anti-hirudin peptide antibody (anti-hirpeptide Ab) was raised against a peptide mimicking the COOH terminus of hirudin. The Ab recognized adherent platelets and those in suspension as determined by enzyme-linked immunosorbent assay and immunofluorescence microscopy, respectively. 125I-Thrombin binding to platelets was inhibited in the presence of the anti-hirpeptide Ab in a dose-dependent manner with maximal inhibition >90%. Analyses of data from binding studies of 125I-thrombin to platelets at a fixed Ab concentration indicated that the anti-hirpeptide Ab inhibited the high affinity binding interaction exclusively. In addition, thrombin-induced increases in platelet [Ca2+]i were enhanced by blocking the high affinity binding site with the Ab due to redistribution of the agonist to PAR-1. Thrombin Quick I-induced platelet calcium mobilization was unaffected by the presence of the Ab, consistent with the inability of thrombin Quick I to bind to the high affinity site. Even though glycoprotein (GP) Ib contains a hirudin-like region within the alpha subunit, the postulated high affinity binding site, direct binding of 125I-thrombin could not be demonstrated to transfected Chinese hamster ovary and L cells expressing the GP Ib-IX-V complex. Furthermore, an anti-GP Ib Ab, raised to the peptide region proposed as the thrombin high affinity site, did not enhance thrombin-induced platelet calcium mobilization. The anti-hirpeptide Ab recognized a population of platelet membrane proteins distinct from PAR-1 and GP Ib by three-color immunofluorescence using confocal microscopy. These combined studies demonstrate that the high affinity binding site for thrombin is a unique platelet protein distinct from GP Ib which modulates the effective thrombin concentration localized at the human platelet surface.
凝血酶的血小板高亲和力结合位点似乎由一种经典的受体 - 配体相互作用所描述，该相互作用不同于血小板凝血酶受体/底物PAR - 1。然而，关于高亲和力结合位点的鉴定及其在生理重要性方面的功能，一直让研究人员难以捉摸。先前使用两种突变凝血酶的研究表明，凝血酶与血小板高亲和力结合位点的相互作用是通过凝血酶分子的一个广泛区域介导的，该区域涉及底物结合口袋和阴离子结合外位点内的残基（梁，L.，亨里克森，R. A.，凯莫德，J. C.，里滕豪斯，S. E.，和特雷西，P. B.（1992年）《生物化学》31卷，2567 - 2575页），并且可能模拟凝血酶 - 水蛭素的相互作用。为了验证这一假设，针对模拟水蛭素COOH末端的肽段制备了一种抗水蛭素肽抗体（抗水蛭肽抗体）。通过酶联免疫吸附测定和免疫荧光显微镜分别确定，该抗体识别贴壁血小板和悬浮血小板。在抗水蛭肽抗体存在的情况下，125I - 凝血酶与血小板的结合以剂量依赖性方式受到抑制，最大抑制率>90%。在固定抗体浓度下对125I - 凝血酶与血小板结合研究的数据进行分析表明，抗水蛭肽抗体仅抑制高亲和力结合相互作用。此外，由于激动剂重新分布到PAR - 1，通过用该抗体阻断高亲和力结合位点，凝血酶诱导的血小板[Ca2 +]i增加得到增强。凝血酶快速I诱导的血小板钙动员不受该抗体存在的影响，这与凝血酶快速I无法结合高亲和力位点一致。尽管糖蛋白（GP）Ib的α亚基内含有一个类似水蛭素的区域，即假定的高亲和力结合位点，但在转染表达GP Ib - IX - V复合物的中国仓鼠卵巢细胞和L细胞中，未能证明125I - 凝血酶的直接结合。此外，针对被提议作为凝血酶高亲和力位点的肽段区域制备的抗GP Ib抗体，并未增强凝血酶诱导的血小板钙动员。使用共聚焦显微镜通过三色免疫荧光法，抗水蛭肽抗体识别了一群不同于PAR - 1和GP Ib的血小板膜蛋白。这些综合研究表明，凝血酶的高亲和力结合位点是一种独特的血小板蛋白，不同于GP Ib，它调节定位于人血小板表面的有效凝血酶浓度。
