Effects of plasma membrane Ca2+-ATPase tyrosine phosphorylation on human platelet function
Effects of plasma membrane Ca2+-ATPase tyrosine phosphorylation on human platelet function
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DOI：
10.1111/j.1538-7836.2007.02508.x
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发表时间：
2007-05-01
影响因子：
10.4
通讯作者：
Dean, W. L.
中科院分区：
文献类型：
--
作者：
Bozulic, L. D.;Malik, M. T.;Dean, W. L.
背景：质膜Ca ~（2+）-ATP酶（PMCA）在维持静息血小板低胞内Ca ~（2+）（[Ca ~（2 +]（i））中起重要作用。早期的研究表明，凝血酶激活血小板导致PMCA的酪氨酸磷酸化，从而抑制泵活性。目的：目的是确定PMCA酪氨酸磷酸化的功能后果。研究方法：合成了一个含有PMCA酪氨酸磷酸化位点的十肽和一个乱序肽，并利用皂苷将其导入人血小板。Fura-2钙监测和聚集测定法用于表征抑制酪氨酸磷酸化的效果。结果如下：免疫沉淀的Western印迹分析表明，与乱序对照肽相比，在皂苷透化的凝血酶处理的血小板中，引入抑制肽使PMCA的酪氨酸磷酸化降低了近60%。在凝血酶介导的血小板活化过程中，PMCA酪氨酸磷酸化的抑制伴随着[Ca 2 +]（i）的显著降低。PMCA酪氨酸磷酸化减少和[Ca 2 +]（i）减少的功能结果是凝血酶介导的血小板聚集的发生显著延迟。结论：结果表明，PMCA酪氨酸磷酸化调节血小板活化过程中的[Ca 2 +]（i），从而影响活化过程中的下游事件。此外，PMCA酪氨酸磷酸化和由此产生的PMCA活性抑制通过增强伴随血小板活化的[Ca 2 +]（i）的增加而产生正反馈环机制。
Background: The plasma membrane Ca2+-ATPase (PMCA) plays an essential role in maintaining low intracellular Ca2+ ([Ca2+](i)) in resting platelets. Earlier studies demonstrated that platelet activation by thrombin results in tyrosine phosphorylation of PMCA, which inhibits pump activity. Objectives: The objective was to determine the functional consequences of PMCA tyrosine phosphorylation. Methods: A decapeptide including the tyrosine phosphorylation site of PMCA and a scrambled version were synthesized and introduced into human platelets using saponin. Fura-2 calcium monitoring and aggregometry were used to characterize the effects of inhibition of tyrosine phosphorylation. Results: Western blot analysis of immunoprecipitates showed that introduction of the inhibitory peptide decreased tyrosine phosphorylation of PMCA by nearly 60% in saponin-permeabilized, thrombin-treated platelets as compared with the scrambled control peptide. Concomitant with inhibition of PMCA tyrosine phosphorylation was a significant decrease in [Ca2+](i) during thrombin-mediated platelet activation. The functional consequence of reduced PMCA tyrosine phosphorylation and decreased [Ca2+](i) was a significant delay in the onset of thrombin-mediated platelet aggregation. Conclusions: The results demonstrate that PMCA tyrosine phosphorylation regulates [Ca2+](i) during platelet activation, which affects downstream events in the activation process. Moreover, PMCA tyrosine phosphorylation and resultant inhibition of PMCA activity produces a positive feedback loop mechanism by enhancing the increase in [Ca2+](i) accompanying platelet activation.