Calcium-dependent proteolysis occurs during platelet aggregation.
Calcium-dependent proteolysis occurs during platelet aggregation.
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DOI：
10.1016/s0021-9258(17)44593-5
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发表时间：
1983-08
期刊：
影响因子：
--
通讯作者：
J. Fox;C. Reynolds;D. Phillips
中科院分区：
文献类型：
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作者：
J. Fox;C. Reynolds;D. Phillips
通过二维聚丙烯酰胺凝胶电泳分析对照和刺激血小板，以确定蛋白质在血小板活化过程中是否发生改变。用凝血酶、胶原蛋白或钙离子载体 A23187 刺激血小板，并在 Ca2+ 存在下通过搅拌引起聚集。这些活化的血小板含有至少三种在对照血小板中未发现的多肽：1)Mr=200,000，pI在6.2和6.4之间； 2）Mr=100,000，pI=6.3； 3) Mr = 91,000，pI = 6.1。另一种多肽，即多肽 4，Mr = 97,000，pI = 5.9，仅存在于被凝血酶激活的血小板中。当通过向血小板悬浮液中添加5mM乙二醇双(β-氨基乙基醚)-N,N,N',N'-四乙酸(EGTA)或通过在不搅拌的情况下孵育血小板悬浮液来防止聚集时，不形成多肽1-3。用金黄色葡萄球菌 V8 蛋白酶部分水解多肽 2 和 4，产生不同组的肽水解片段。这些不同于α-肌动蛋白（一种主要的血小板蛋白）水解产生的那些，其分子量与多肽2和4相似。多肽1-3也在Ca2+存在下孵育血小板裂解物的过程中产生。通过使用 EGTA 螯合 Ca2+ 或添加 N-乙基马来酰亚胺、亮肽素或 mersalyl（钙依赖性蛋白酶抑制剂），可以防止裂解物中这些多肽的产生。这些数据表明，钙依赖性蛋白酶在血小板聚集过程中被生理制剂激活，并表明该蛋白酶可能在血小板对刺激的反应中发挥作用。
Control and stimulated platelets were analyzed by two-dimensional polyacrylamide gel electrophoresis to determine whether proteins are altered during platelet activation. Platelets were stimulated with thrombin, collagen, or the calcium ionophore A23187, and aggregation was brought about by stirring in the presence of Ca2+. These activated platelets contained at least three polypeptides not found in control platelets: 1) Mr = 200,000, pI between 6.2 and 6.4; 2) Mr = 100,000, pI = 6.3; and 3) Mr = 91,000, pI = 6.1. An additional polypeptide, polypeptide 4, with Mr = 97,000 and pI = 5.9, was present only in platelets activated by thrombin. When aggregation was prevented, either by adding 5 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) to the platelet suspension or by incubating the platelet suspension without stirring, polypeptides 1-3 were not formed. Partial hydrolysis of polypeptides 2 and 4 with Staphylococcus aureus V8 protease yielded distinct sets of peptide hydrolytic fragments. These differed from those produced by the hydrolysis of alpha-actinin, a major platelet protein, which has a molecular weight similar to polypeptides 2 and 4. Polypeptides 1-3 were also produced during incubation of platelet lysates in the presence of Ca2+. Generation of these polypeptides in lysates was prevented either by chelation of Ca2+ with EGTA or by the addition of N-ethylmaleimide, leupeptin, or mersalyl, inhibitors of the calcium-dependent protease. These data show that the calcium-dependent protease is activated during aggregation of platelets by physiological agents and suggest that this protease could have a role in platelet response to stimulation.