Kamiguti A S, Hay C R, Zuzel M
Department of Haematology, Royal Liverpool University Hospital, University of Liverpool, Liverpool L69 3BX, U.K.
Biochem J. 1996 Dec 1;320 ( Pt 2)(Pt 2):635-41. doi: 10.1042/bj3200635.
Jararhagin is a high-molecular-mass (52 kDa) haemorrhagic metalloproteinase from Bothrops jararaca venom and a member of the metalloproteinase/disintegrin/cysteine-rich protein family. The disintegrin domain of jararhagin has been implicated in the inhibition of platelet responses to collagen by a mechanism that is not entirely known. The present investigation demonstrates that both active and 1,10-phenanthroline-inactivated jararhagin inhibit platelet aggregation by collagen with an IC50 of 40 and 140 nM respectively. The apparently higher inhibitory effect of the active enzyme clearly indicates that, in addition to the disintegrin region, the metalloproteinase domain of jararhagin also participates in this inhibition. As collagen interacts with platelets via alpha 2 beta 1-integrin, we investigated the effects of jararhagin on this integrin using selected function-blocking monoclonal antibodies against both of its subunits. Flow cytometry of platelets treated with native jararhagin and immunoprecipitation of platelet surface glycoproteins from lysates after jararhagin treatment showed an apparently selective reduction of alpha 2 beta 1-integrin immunoreactivity with both anti-alpha 2 and anti-beta 1 monoclonal antibodies. The loss of immunoreactivity was not due to integrin internalization, since it also took place in cytochalasin D-treated platelets. Here we show that jararhagin cleaved isolated alpha 2 beta 1-integrin resulting in the generation of a 115 kDa beta 1 fragment. We therefore propose that the inhibition by jararhagin of platelet response to collagen is mediated through the binding of jararhagin to platelet alpha 2-subunit via the disintegrin domain, followed by proteolysis of the beta 1-subunit with loss of the integrin structure (conformation) necessary for the binding of macromolecular ligands.
矛头蝮蛇毒金属蛋白酶是一种来自巴西矛头蝮蛇毒液的高分子量（52 kDa）出血性金属蛋白酶，属于金属蛋白酶/去整合素/富含半胱氨酸蛋白家族。矛头蝮蛇毒金属蛋白酶的去整合素结构域通过一种尚不完全清楚的机制参与抑制血小板对胶原蛋白的反应。本研究表明，活性矛头蝮蛇毒金属蛋白酶和经1,10 - 菲咯啉失活的矛头蝮蛇毒金属蛋白酶均可抑制胶原蛋白诱导的血小板聚集，其IC50分别为40 nM和140 nM。活性酶明显更高的抑制作用清楚地表明，除了去整合素区域外，矛头蝮蛇毒金属蛋白酶的金属蛋白酶结构域也参与了这种抑制作用。由于胶原蛋白通过α2β1整合素与血小板相互作用，我们使用针对其两个亚基的选定功能阻断单克隆抗体研究了矛头蝮蛇毒金属蛋白酶对该整合素的影响。用天然矛头蝮蛇毒金属蛋白酶处理血小板后的流式细胞术以及在矛头蝮蛇毒金属蛋白酶处理后从裂解物中免疫沉淀血小板表面糖蛋白显示，用抗α2和抗β1单克隆抗体检测时，α2β1整合素的免疫反应性明显选择性降低。免疫反应性的丧失不是由于整合素内化，因为在细胞松弛素D处理的血小板中也会发生这种情况。在这里我们表明，矛头蝮蛇毒金属蛋白酶可切割分离的α2β1整合素，产生一个115 kDa的β1片段。因此，我们提出，矛头蝮蛇毒金属蛋白酶对血小板对胶原蛋白反应的抑制作用是通过其去整合素结构域与血小板α2亚基结合，随后β1亚基被蛋白水解，导致失去大分子配体结合所需的整合素结构（构象）来介导的。
