Department of Clinical and Experimental Medicine, Division of Clinical Chemistry, Medical Faculty, University of Linköping, University Hospital, Linköping 581 85, Sweden.
Biochem J. 2011 Jun 1;436(2):469-80. doi: 10.1042/BJ20101360.
PARs (protease-activated receptors) 1 and 4 belong to the family of G-protein-coupled receptors which induce both G(α12/13) and G(αq) signalling. By applying the specific PAR1- and PAR4-activating hexapeptides, SFLLRN and AYPGKF respectively, we found that aggregation of isolated human platelets mediated via PAR1, but not via PAR4, is abolished upon homologous receptor activation in a concentration- and time-dependent fashion. This effect was not due to receptor internalization, but to a decrease in Ca²⁺ mobilization, PKC (protein kinase C) signalling and α-granule secretion, as well as to a complete lack of dense granule secretion. Interestingly, subthreshold PAR4 activation rapidly abrogated PAR1 signalling desensitization by differentially reconstituting these affected signalling events and functional responses, which was sufficient to re-establish aggregation. The lack of ADP release and P2Y₁₂ receptor-induced G(αi) signalling accounted for the loss of the aggregation response, as mimicking G(αi/z) signalling with 2-MeS-ADP (2-methylthioadenosine-5'-O-diphosphate) or epinephrine (adrenaline) could substitute for intermediate PAR4 activation. Finally, we found that the re-sensitization of PAR1 signalling-induced aggregation via PAR4 relied on PKC-mediated release of both ADP from dense granules and fibrinogen from α-granules. The present study elucidates further differences in human platelet PAR signalling regulation and provides evidence for a cross-talk in which PAR4 signalling counteracts mechanisms involved in PAR1 signalling down-regulation.
蛋白酶激活受体 (PARs) 1 和 4 属于 G 蛋白偶联受体家族，可诱导 G(α12/13)和 G(αq)信号转导。通过应用特异性 PAR1 和 PAR4 激活六肽 SFLLRN 和 AYPGKF，我们发现，通过 PAR1 介导的人血小板分离物的聚集，而不是通过 PAR4 介导的聚集，在同源受体激活时，以浓度和时间依赖的方式被废除。这种效应不是由于受体内化，而是由于 Ca²⁺动员、PKC（蛋白激酶 C）信号转导和α-颗粒分泌减少，以及致密颗粒分泌完全缺失。有趣的是，亚阈值 PAR4 激活通过差异重建这些受影响的信号事件和功能反应，迅速消除 PAR1 信号脱敏，足以重新建立聚集。缺乏 ADP 释放和 P2Y₁₂受体诱导的 G(αi)信号转导是聚集反应丧失的原因，因为用 2-MeS-ADP（2-甲基硫腺苷-5'-O-二磷酸）或肾上腺素（肾上腺素）模拟 G(αi/z)信号转导可以替代中间 PAR4 激活。最后，我们发现，通过 PAR4 重新敏化 PAR1 信号诱导的聚集依赖于 PKC 介导的致密颗粒中 ADP 和α-颗粒中纤维蛋白原的释放。本研究进一步阐明了人血小板 PAR 信号转导调节的差异，并提供了证据表明 PAR4 信号转导相互作用可以抵消 PAR1 信号转导下调所涉及的机制。
