Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands; Leibniz-Institut für Analytische Wissenschaften-ISAS-e.V, Dortmund, Germany; Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom.
Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands; Synapse Research Institute Maastricht, 6217 KD Maastricht, The Netherlands.
Cell Calcium. 2023 Jun;112:102738. doi: 10.1016/j.ceca.2023.102738. Epub 2023 Apr 8.
In platelets, elevated cytosolic Ca is a crucial second messenger, involved in most functional responses, including shape change, secretion, aggregation and procoagulant activity. The platelet Ca response consists of Ca mobilization from endoplasmic reticulum stores, complemented with store-operated or receptor-operated Ca entry pathways. Several channels can contribute to the Ca entry, but their relative contribution is unclear upon stimulation of ITAM-linked receptors such as glycoprotein VI (GPVI) and G-protein coupled receptors such as the protease-activated receptors (PAR) for thrombin. We employed a 96-well plate high-throughput assay with Fura-2-loaded human platelets to perform parallel [Ca] measurements in the presence of EGTA or CaCl. Per agonist condition, this resulted in sets of EGTA, CaCl and Ca entry ratio curves, defined by six parameters, reflecting different Ca ion fluxes. We report that threshold stimulation of GPVI or PAR, with a variable contribution of secondary mediators, induces a maximal Ca entry ratio of 3-7. Strikingly, in combination with Ca-ATPase inhibition by thapsigargin, the maximal Ca entry ratio increased to 400 (GPVI) or 40 (PAR), pointing to a strong receptor-dependent enhancement of store-operated Ca entry. By pharmacological blockage of specific Ca channels in platelets, we found that, regardless of GPVI or PAR stimulation, the Ca entry ratio was strongest affected by inhibition of ORAI1 (2-APB, Synta66) > Na/Ca exchange (NCE) > P2× (only initial). In contrast, inhibition of TRPC6, Piezo1/2 or STIM1 was without effect. Together, these data reveal ORAI1 and NCE as dominating Ca carriers regulating GPVI- and PAR-induced Ca entry in human platelets.
在血小板中，升高的细胞质 Ca2+是一种关键的第二信使，参与大多数功能反应，包括形状变化、分泌、聚集和促凝活性。血小板 Ca2+反应包括内质网储存的 Ca2+动员，辅以储存操作或受体操作的 Ca2+进入途径。几种通道可以有助于 Ca2+进入，但在刺激 ITAM 连接受体（如糖蛋白 VI [GPVI]）和 G 蛋白偶联受体（如凝血酶的蛋白酶激活受体 [PAR]）时，它们的相对贡献尚不清楚。我们使用 Fura-2 加载的人血小板在 96 孔板高通量测定中，在 EGTA 或 CaCl2 存在下平行进行[Ca2+]测量。对于每种激动剂条件，这会产生一组 EGTA、CaCl2 和 Ca2+进入比曲线，由六个参数定义，反映不同的 Ca2+离子通量。我们报告说，GPVI 或 PAR 的阈值刺激，伴随着次要介质的可变贡献，诱导 3-7 的最大 Ca2+进入比。引人注目的是，与 thapsigargin 抑制 Ca-ATPase 相结合，最大 Ca2+进入比增加到 400（GPVI）或 40（PAR），表明强烈的受体依赖性增强了储存操作的 Ca2+进入。通过在血小板中用药理学方法阻断特定的 Ca2+通道，我们发现，无论刺激 GPVI 还是 PAR，Ca2+进入比都受到 ORAI1 抑制（2-APB、Synta66）> Na/Ca 交换（NCE）> P2×（仅初始）的强烈影响。相比之下，抑制 TRPC6、Piezo1/2 或 STIM1 则没有影响。总的来说，这些数据揭示了 ORAI1 和 NCE 作为调节人血小板中 GPVI 和 PAR 诱导的 Ca2+进入的主要 Ca2+载体。
