GRK6 regulates the hemostatic response to injury through its rate-limiting effects on GPCR signaling in platelets
GRK6 regulates the hemostatic response to injury through its rate-limiting effects on GPCR signaling in platelets
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DOI：
10.1182/bloodadvances.2019000467
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发表时间：
2020-01-14
期刊：
影响因子：
7.5
通讯作者：
Ma, Peisong
中科院分区：
文献类型：
--
作者：
Chen, Xi;Gupta, Shuchi;Ma, Peisong
G蛋白偶联受体（GPCR）介导大多数血小板活化反应激动剂。然而，关于向活化的GPCR提供负反馈以限制血小板活化和血栓形成的机制仍存在问题。在这里，我们提供了GPCR激酶6（GRK 6）在血小板中发挥这一作用的第一个证据，使用CRISPR-Cas9基因组编辑产生的GRK 6（-/-）小鼠来检查GRK 6敲除对GPCR依赖性信号传导的影响。在血栓形成的早期阶段，GRK 6（-/-）小鼠中形成的止血血栓大于野生型（WT）对照，损伤部位血小板聚集迅速增加。GRK 6（-/-）血小板具有增加的血小板活化，但以激动剂选择性方式。与WT同窝仔相比，GRK 6（-/-）血小板对PAR 4激动剂或腺苷5 '-二磷酸刺激的反应增加，而对血栓素A（2）（TxA（2））的反应正常。GRK 6（-/-）血小板中这些变化的基础是Ca 2+动员、Akt活化和颗粒分泌的增加。此外，在人MEG-01细胞中GRK 6的缺失导致响应于凝血酶的Ca 2+应答和PAR 1表面表达的增加。最后，我们表明，人血小板活化凝血酶引起GRK 6与PAR 1结合增加，以及PAR 1的磷酸化增加。在MEG-01细胞中GRK 6的缺失导致PAR 1磷酸化的减少。总而言之，这些数据表明GRK 6通过PAR和P2 Y（12）介导的作用调节对损伤的止血反应，有助于限制血栓生长期间血小板活化的速率并防止不当的血小板活化。
G protein-coupled receptors (GPCRs) mediate the majority of platelet activation in response to agonists. However, questions remain regarding the mechanisms that provide negative feedback toward activated GPCRs to limit platelet activation and thrombus formation. Here we provide the first evidence that GPCR kinase 6 (GRK6) serves this role in platelets, using GRK6(-/-) mice generated by CRISPR-Cas9 genome editing to examine the consequences of GRK6 knockout on GPCR-dependent signaling. Hemostatic thrombi formed in GRK6(-/-) mice are larger than in wild-type (WT) controls during the early stages of thrombus formation, with a rapid increase in platelet accumulation at the site of injury. GRK6(-/-) platelets have increased platelet activation, but in an agonist-selective manner. Responses to PAR4 agonist or adenosine 5'-diphosphate stimulation in GRK6(-/-) platelets are increased compared with WT littermates, whereas the response to thromboxane A(2) (TxA(2)) is normal. Underlying these changes in GRK6(-/-) platelets is an increase in Ca2+ mobilization, Akt activation, and granule secretion. Furthermore, deletion of GRK6 in human MEG-01 cells causes an increase in Ca2+ response and PAR1 surface expression in response to thrombin. Finally, we show that human platelet activation in response to thrombin causes an increase in binding of GRK6 to PAR1, as well as an increase in the phosphorylation of PAR1. Deletion of GRK6 in MEG-01 cells causes a decrease in PAR1 phosphorylation. Taken together, these data show that GRK6 regulates the hemostatic response to injury through PAR- and P2Y(12)-mediated effects, helping to limit the rate of platelet activation during thrombus growth and prevent inappropriate platelet activation.