Protein kinase C isoform ε negatively regulates ADP-induced calcium mobilization and thromboxane generation in platelets.
Protein kinase C isoform ε negatively regulates ADP-induced calcium mobilization and thromboxane generation in platelets.
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DOI：
10.1161/atvbaha.111.242388
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发表时间：
2012-05
期刊：
影响因子：
--
通讯作者：
Kunapuli SP
中科院分区：
文献类型：
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作者：
Bynagari-Settipalli YS;Lakhani P;Jin J;Bhavaraju K;Rico MC;Kim S;Woulfe D;Kunapuli SP
蛋白激酶C(PKC)家族成员对血小板活化有正向和负向调节作用。尽管对PKC的正向调控作用进行了广泛的研究，但对PKC的负向调控作用却知之甚少。在这项研究中，我们研究了PKC介导的对ADP诱导的功能反应的负性调节的机制和特定的亚型。PAN-PKC抑制剂GF109203X增强了ADP诱导的cPLA2的磷酸化和血栓素A的生成，以及cPLA2激活的上游的ERK激活和细胞内钙(Ca~(2+))动员。因此，PKCs通过抑制ERK和Ca~(2+)i动员来抑制cPLA2的激活。由于经典PKC异构体的抑制剂GO-6976不影响ADP介导的血栓素生成，因此我们研究了新型PKC异构体的作用。与野生型(WT)产仔小鼠血小板相比，PKCε缺失小鼠血小板中腺苷二磷酸诱导的血栓烷生成、钙动员和ERK磷酸化增强。有趣的是，当血栓素释放被阻断时，腺苷二磷酸诱导的PKCεKO和WT的聚集是相似的，这表明PKCε不直接影响腺苷二磷酸诱导的聚集。在FeCl3诱导的动脉损伤模型中，PKCε基因敲除小鼠表现出较短的阻断时间，在尾部出血实验中表现出较短的出血时间。我们的结论是，PKCε负性调节二磷酸腺苷诱导的血小板内血栓素的产生，并提供抗血栓形成的保护。
Members of Protein Kinase C (PKC) family are shown to positively and negatively regulate platelet activation. Although positive regulatory roles are extensively studied, negative regulatory roles of PKCs are poorly understood. In this study we investigated the mechanism and specific isoforms involved in PKC-mediated negative regulation of ADP-induced functional responses. A pan-PKC inhibitor GF109203X potentiated ADP-induced cPLA2 phosphorylation and thromboxane generation, as well as ERK activation and intracellular calcium (Ca2+i) mobilization, two signaling molecules, upstream of cPLA2 activation. Thus, PKCs inhibit cPLA2 activation by inhibiting ERK and Ca2+i mobilization. Since, the inhibitor of Classical PKC isoforms, GO-6976 did not affect ADP-mediated thromboxane generation, we investigated the role of novel class of PKC isoforms. ADP- induced thromboxane generation, calcium mobilization and ERK phosphorylation were potentiated in PKCε null murine platelets compared to platelets from wild type (WT) littermates. Interestingly, when thromboxane release is blocked, ADP-induced aggregation in PKCε KO and WT was similar, suggesting that PKCε does not affect ADP-induced aggregation directly. PKCε knockout mice exhibited shorter times to occlusion in FeCl3-induced arterial injury model and shorter bleeding times in tail bleeding experiments. We conclude that PKCε negatively regulates ADP-induced thromboxane generation in platelets and offers protection against thrombosis.