Ajanel Abigail, Andrianova Izabella, Kowalczyk Mia, Menéndez Pérez Javier, Bhatt Sradha R, Portier Irina, Boone Taylor, Ballard-Kordeliski Abigail, Kosaka Yasuhiro, Chaudhuri Dipayan, Paul David S, Bergmeier Wolfgang, Denorme Frederik, Campbell Robert A
Department of Emergency Medicine, Washington University School of Medicine, Saint Louis, MO (A.A., I.A., J.M.P., I.P., F.D., R.A.C.).
Program in Molecular Medicine, University of Utah, Salt Lake City. (M.K., Y.K.).
Circ Res. 2025 Jul 1. doi: 10.1161/CIRCRESAHA.125.326443.
Platelet activation relies on changes in cytoplasmic calcium flux. However, little is known about the role mitochondrial calcium flux plays in platelet activation. Activation induces release of calcium from intracellular stores, which enters the mitochondrial matrix through the MCU (mitochondrial calcium uniporter) to regulate bioenergetics and reactive oxygen species (ROS) formation, as demonstrated in other cells. However, whether MCU contributes to platelet function is unclear.
We generated platelet-specific Mcu-deficient mice () and compared them to littermate wild-type controls (). In vitro approaches assessed mitochondrial calcium flux and platelet activation responses to stimulation of immunoreceptor tyrosine-based activation motif (ITAM) receptors and GPCRs (G protein-coupled receptors). In addition, we examined in vivo hemostasis and thrombosis. We also treated human platelets with MCU inhibitors, and platelet function was assessed.
platelets had significantly reduced mitochondrial calcium flux in response to activation of ITAM receptors, whereas mitochondrial calcium flux in response to GPCR activation was unchanged. Platelet aggregation was significantly reduced by ITAM activation in platelets, but GPCR-induced aggregation was unchanged. Similar findings were observed when MCU was inhibited in human platelets. In vivo, mice had reduced arterial thrombosis and less ischemic stroke brain injury. Hemostasis was mildly altered in mice. Mechanistically, mitochondrial ROS generation was significantly reduced in platelets compared with platelets after ITAM-dependent activation, but not GPCR activation. Reduced mitochondrial ROS was associated with decreased ITAM signaling based on p-Syk (phospho-spleen tyrosine kinase) and p-PLCγ2 (phospho-phospholipase C-gamma 2) in platelets. Inhibiting mitochondrial ROS decreased aggregation as well as downstream ITAM signaling in platelets. Conversely, treating platelets with MitoParaquat to induce mitochondrial ROS increased platelet ITAM-dependent aggregation and signaling.
Our data support a role for mitochondrial calcium flux in regulating ITAM-dependent platelet activation through the generation of mitochondrial ROS.
血小板活化依赖于细胞质钙通量的变化。然而，关于线粒体钙通量在血小板活化中所起的作用知之甚少。如在其他细胞中所证实的那样，活化诱导细胞内钙库释放钙，钙通过线粒体钙单向转运体（MCU）进入线粒体基质以调节生物能量学和活性氧（ROS）的形成。然而，MCU是否对血小板功能有贡献尚不清楚。
我们构建了血小板特异性Mcu缺陷小鼠（），并将其与同窝野生型对照（）进行比较。体外方法评估了线粒体钙通量以及血小板对免疫受体酪氨酸激活基序（ITAM）受体和G蛋白偶联受体（GPCR）刺激的活化反应。此外，我们检查了体内止血和血栓形成情况。我们还用MCU抑制剂处理人血小板，并评估了血小板功能。
血小板在ITAM受体激活时线粒体钙通量显著降低，而对GPCR激活的线粒体钙通量未改变。ITAM激活使血小板的聚集显著减少，但GPCR诱导的聚集未改变。当人血小板中的MCU被抑制时也观察到类似结果。在体内，小鼠的动脉血栓形成减少，缺血性脑卒中脑损伤减轻。小鼠的止血有轻度改变。机制上，与血小板在ITAM依赖性激活后相比，血小板中依赖ITAM激活后的线粒体ROS生成显著减少，但GPCR激活后未减少。基于血小板中磷酸化脾酪氨酸激酶（p-Syk）和磷酸化磷脂酶Cγ2（p-PLCγ2），线粒体ROS减少与ITAM信号传导降低有关。抑制线粒体ROS可降低血小板的聚集以及下游ITAM信号传导。相反，用百草枯处理血小板以诱导线粒体ROS增加了血小板ITAM依赖性聚集和信号传导。
我们的数据支持线粒体钙通量通过线粒体ROS生成在调节ITAM依赖性血小板活化中发挥作用。
