Disruption of the mouse μ-calpain gene reveals an essential role in platelet function
Disruption of the mouse μ-calpain gene reveals an essential role in platelet function
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DOI：
10.1128/mcb.21.6.2213-2220.2001
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发表时间：
2001-03-01
影响因子：
5.3
通讯作者：
Chishti, AH
中科院分区：
文献类型：
--
作者：
Azam, M;Andrabi, SS;Chishti, AH
传统的钙蛋白酶是普遍存在的钙调节的半胱氨酸蛋白酶，其涉及细胞骨架组织、细胞增殖、凋亡、细胞运动和止血。有两种形式的常规钙蛋白酶：μ-钙蛋白酶或钙蛋白酶I，其需要微摩尔的钙来进行半最大激活，以及m钙蛋白酶或钙蛋白酶II，其在毫摩尔的钙浓度下起作用。我们评估的功能作用的80 kDa的催化亚基的mu -钙蛋白酶的基因失活，在胚胎干细胞中使用同源重组。mu -钙蛋白酶缺陷小鼠是可存活和可生育的。μ-钙蛋白酶的完全缺乏导致血小板聚集和凝块收缩的显著减少，但令人惊讶的是，突变小鼠显示出正常的出血时间。在钙蛋白酶底物（如α IIb β 3整联蛋白的β 3亚基、talin和ABP-280（细丝蛋白））的裂解模式和动力学中未观察到可检测的差异。然而，无μ-钙蛋白酶的血小板表现出包括α IIb β 3整联蛋白的β 3亚基在内的几种蛋白质的酪氨酸磷酸化受损，这与激动剂诱导的血小板聚集减少相关。这些结果提供了第一个直接证据，即μ-钙蛋白酶对正常血小板功能是必需的，不是通过影响细胞骨架蛋白的切割，而是通过潜在地调节血小板蛋白的酪氨酸磷酸化状态。
Conventional calpains are ubiquitous calcium-regulated cysteine proteases that have been implicated in cytoskeletal organization, cell proliferation, apoptosis, cell motility, and hemostasis. There are two forms of conventional calpains: the mu -calpain, or calpain I, which requires micromolar calcium for half-maximal activation, and the m calpain, or calpain II, which functions at millimolar calcium concentrations. We evaluated the functional role of the 80-kDa catalytic subunit of mu -calpain by genetic inactivation using homologous recombination in embryonic stem cells. The mu -calpain-deficient mice are viable and fertile. The complete deficiency of mu -calpain causes significant reduction in platelet aggregation and clot retraction but surprisingly the mutant mice display normal bleeding times. No detectable differences were observed in the cleavage pattern and kinetics of calpain substrates such as the beta3 subunit of alpha IIb beta3 integrin, talin, and ABP-280 (filamin). However, mu -calpain null platelets exhibit impaired tyrosine phosphorylation of several proteins including the beta3 subunit of alpha IIb beta3 integrin, correlating with the agonist-induced reduction in platelet aggregation. These results provide the first direct evidence that mu -calpain is essential for normal platelet function, not by affecting the cleavage of cytoskeletal proteins but by potentially regulating the state of tyrosine phosphorylation of the platelet proteins.