Calcium mobilization and protein kinase C activation downstream of protease activated receptor 4 (PAR4) is negatively regulated by PAR3 in mouse platelets.
Calcium mobilization and protein kinase C activation downstream of protease activated receptor 4 (PAR4) is negatively regulated by PAR3 in mouse platelets.
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DOI：
10.1371/journal.pone.0055740
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Nieman MT
中科院分区：
文献类型：
--
作者：
Arachiche A;de la Fuente M;Nieman MT
凝血酶通过蛋白酶激活受体（PARs）激活血小板。小鼠血小板表达PAR3和PAR4。PAR3在血小板中没有信号。然而，PAR4是一个相对较差的凝血酶底物，在低凝血酶浓度下需要PAR3作为辅助因子。在这项研究中，我们发现PAR3也调节PAR4信号。在凝血酶（30-100 nM）或PAR4激活肽（AYPGKF）的作用下，来自PAR3 - / -小鼠的血小板与野生型小鼠相比，Gq信号传导增加，细胞内钙（Ca2+）动员最大增加1.6倍，蛋白激酶C （PKC）底物磷酸化水平增加，细胞内Ca2+释放增加2倍。此外，杂合小鼠的血小板（PAR3+/−）在最大Ca2+动员方面有中等程度的增加。用P2Y12拮抗剂（2MeSAMP）治疗PAR3−/−小鼠血小板不影响凝血酶或AYPGKF对PAR4 Ca2+动员的反应。RhoA-GTP下游G12/13信号在凝血酶作用下的激活在野生型和PAR3−/−小鼠之间没有显著差异。由于PAR3对PAR4信号的影响不依赖于激动剂，我们研究了PAR3和PAR4与生物发光共振能量转移（BRET）的直接相互作用。PAR3和PAR4形成同质二聚体和异源二聚体。总之，我们的研究结果表明，除了在低凝血酶浓度下增强PAR4激活外，PAR3还通过物理相互作用负调控PAR4介导的小鼠血小板中最大Ca2+动员和PKC激活。
Thrombin activates platelets through protease activated receptors (PARs). Mouse platelets express PAR3 and PAR4. PAR3 does not signal in platelets. However, PAR4 is a relatively poor thrombin substrate and requires PAR3 as a cofactor at low thrombin concentrations. In this study we show that PAR3 also regulates PAR4 signaling. In response to thrombin (30–100 nM) or PAR4 activating peptide (AYPGKF), platelets from PAR3−/− mice had increased Gq signaling compared to wild type mice as demonstrated by a 1.6-fold increase in the maximum intracellular calcium (Ca2+) mobilization, an increase in phosphorylation level of protein kinase C (PKC) substrates, and a 2-fold increase of Ca2+ release from intracellular stores. Moreover, platelets from heterozygous mice (PAR3+/−) had an intermediate increase in maximum Ca2+ mobilization. Treatment of PAR3−/− mice platelets with P2Y12 antagonist (2MeSAMP) did not affect Ca2+ mobilization from PAR4 in response to thrombin or AYPGKF. The activation of RhoA-GTP downstream G12/13 signaling in response to thrombin was not significantly different between wild type and PAR3−/− mice. Since PAR3 influenced PAR4 signaling independent of agonist, we examined the direct interaction between PAR3 and PAR4 with bioluminescence resonance energy transfer (BRET). PAR3 and PAR4 form constitutive homodimers and heterodimers. In summary, our results demonstrate that in addition to enhancing PAR4 activation at low thrombin concentrations, PAR3 negatively regulates PAR4-mediated maximum Ca2+ mobilization and PKC activation in mouse platelets by physical interaction.
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