Activation of calpain I and hydrolysis of calpain substrates (actin-binding protein, glycoprotein Ib, and talin) are not a function of thrombin-induced platelet aggregation.
Activation of calpain I and hydrolysis of calpain substrates (actin-binding protein, glycoprotein Ib, and talin) are not a function of thrombin-induced platelet aggregation.
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钙蛋白酶 I 的激活和钙蛋白酶底物（肌动蛋白结合蛋白、糖蛋白 Ib 和 talin）的水解不是凝血酶诱导的血小板聚集的功能。
DOI：
10.1161/01.atv.11.4.882
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发表时间：
1991
期刊：
影响因子：
--
通讯作者：
Kunicki,TJ
中科院分区：
文献类型：
--
作者：
Wencel-Drake,JD;Okita,JR;Annis,DS;Kunicki,TJ
钙激活的中性蛋白酶（钙蛋白酶）已被证明切割蛋白质参与维持细胞结构。在人血小板中，钙蛋白酶的底物包括糖蛋白Ib（GPIb）、肌动蛋白结合蛋白（ABP）和塔林蛋白。GPIb-ABP复合物可以在去污剂提取物中分离，并被认为代表膜-细胞骨架附着位点。据推测，钙蛋白酶对GPIb-ABP的水解受该蛋白酶与血小板活化的质膜-细胞骨架界面结合程度的调节。最近，另一种钙蛋白酶底物（塔林）已被证明重新分配从细胞质的质膜细胞骨架界面作为凝血酶刺激的结果。为了研究钙蛋白酶I的细胞内分布，我们使用了对钙蛋白酶I的重链（催化亚基）具有特异性的单克隆抗体B27 D8。静息人血小板的间接免疫荧光染色显示检测不到表面抗原。然而，与Triton X-100透化，揭示了一个弥漫性的细胞内抗原与胞质分布一致。为了确定这种抗原分布是否反映了钙蛋白酶I的酶原或活化形式，并评估ABP、GPIb和talin的水解程度，我们采用了B27 D8和抗ABP（1B 3和3D 1）、GPIb（LJIb 10）的鼠单克隆抗体以及抗talin（A2和B11）的兔多克隆抗体进行定量免疫印迹分析。静息血小板的检查显示，钙蛋白酶I存在的85 kd酶原的形式和ABP，GPIb，和塔林存在于其原生的完整形式。当血小板与凝血酶聚集时，钙蛋白酶I的自蛋白水解在使血小板聚集体完全溶解于含十二烷基硫酸钠的缓冲液中所需的30秒内发生，而不是凝血酶诱导的活化的直接结果。（250字处删节）
Calcium-activated neutral proteinase (calpain) has been shown to cleave proteins involved in the maintenance of cell structure. In human platelets, substrates of calpain include glycoprotein Ib (GPIb), actin-binding protein (ABP), and talin. GPIb-ABP complexes can be isolated in detergent extracts and are thought to represent membrane-cytoskeleton attachment sites. It has been hypothesized that the hydrolysis of GPIb-ABP by calpain is regulated by the extent of binding of this proteinase to the plasma membrane-cytoskeleton interface with platelet activation. Recently, another calpain substrate (talin) has been shown to redistribute from the cytoplasm to the plasma membrane-cytoskeleton interface as the result of thrombin stimulation. To investigate the intracellular distribution of calpain I, we employed the monoclonal antibody B27D8, specific for the heavy chain (catalytic subunit) of calpain I. Indirect immunofluorescent staining of resting human platelets revealed undetectable surface antigen. Permeabilization with Triton X-100, however, revealed a diffuse intracellular antigen consistent with a cytosolic distribution. To determine whether this antigen distribution reflected the proenzyme or the activated form of calpain I and to assess the degree of hydrolysis of ABP, GPIb, and talin, we employed B27D8 and murine monoclonal antibodies against ABP (1B3 and 3D1), GPIb (LJIb10), and rabbit polyclonal antibodies against talin (A2 and B11) in a quantitative immunotransblot assay. Examination of resting platelets revealed that calpain I existed as the 85-kd proenzyme form and that ABP, GPIb, and talin existed in their native intact forms. When platelets were aggregated with thrombin, autoproteolysis of calpain I occurred within the 30 seconds required to completely solubilize platelet aggregates in sodium dodecyl sulfate-containing buffer and not as a direct result of thrombin-induced activation.(ABSTRACT TRUNCATED AT 250 WORDS)