Mitochondrial Calcium Uniporter Regulates ITAM-Dependent Platelet Activation.

Abstract

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 (<i>Mcu</i>^plt^-/-) and compared them to littermate wild-type controls (<i>Mcu</i>^plt+/+). 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. <i>Mcu</i>^plt^-/^- 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 <i>Mcu</i>^plt^-/^- platelets, but GPCR-induced aggregation was unchanged. Similar findings were observed when MCU was inhibited in human platelets. In vivo, <i>Mcu</i>^plt^-/^- mice had reduced arterial thrombosis and less ischemic stroke brain injury. Hemostasis was mildly altered in <i>Mcu</i>^plt^-/^- mice. Mechanistically, mitochondrial ROS generation was significantly reduced in <i>Mcu</i>^plt^-/^- platelets compared with <i>Mcu</i>^plt+/+ 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 <i>Mcu</i>^plt^-/^- platelets. Inhibiting mitochondrial ROS decreased aggregation as well as downstream ITAM signaling in <i>Mcu</i>^plt+/+ platelets. Conversely, treating <i>Mcu</i>^plt^-/^- 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.