Platelet Mitochondria Calcium Uniporter Regulates ITAM-Dependent Platelet Activation and Signaling
Authors: Ajanel Gomez Abigail , Denorme Frederik , Portier Irina , Kowalczyk Mia , Rameshbhatt Sradha , Kosaka Yasuhiro , Campbell Robert A. 
Published: December 2023

Introduction: The ability of platelets to multitask is critical to arrest bleeding after injury and for the development of arterial thrombosis, which underlies myocardial infarction and stroke. Platelet activation relies heavily on changes in cytoplasmic calcium flux. However, little is known on the role mitochondrial calcium flux directly plays in platelet activation. In other cells, release of calcium from intracellular stores results in calcium entry into channels located in the outer mitochondrial membrane. Once calcium enters the intermembrane space, entry into the mitochondrial matrix is regulated by a multimeric complex, composed of channel-forming subunits and regulatory elements called the mitochondrial calcium uniporter (MCU). MCU has been extensively studied in cardiac tissue where calcium flux through MCU is critical for regulating bioenergetics, ROS formation, and cytoplasmic calcium levels. As these processes are important for platelet activation, these studies would indicate mitochondrial calcium flux may regulate platelet activation. However, the role of MCU in platelet function and activation is poorly understood.
Aim: The goals of the current study are 1) to examine the role of MCU and mitochondrial calcium flux in platelet function and 2) to establish if mitochondrial calcium regulates thrombosis.
Methods: We generated a platelet-specific MCU-deficient mouse (MCU fl/fl-PF4-cre, KO) and compared them to littermate wild-type controls (MCU fl/fl, WT). Platelet function including activation, aggregation, and mitochondrial calcium flux in response to PAR4 activating peptide (PAR) and ADP (P2Y12), both GPCRs or to collagen (GPVI) and rhodocytin (CLEC-2), both ITAMs, were examined. In addition, we examined ex vivo platelet adhesion under arterial and venous shear to collagen and in vivo thrombosis using a ferric chloride carotid artery model.
Results: Mice deficient inplatelet MCU were viable and fertile. In addition, platelet-specific MCU deletion did not alter platelet counts, mean platelet volume, or platelet half-life. MCU KO platelets had significantly reduced (p&lt;0.05) GPVI and CLEC-2-dependent aIIbb3 activation and P-selectin expression while platelet activation in response to P2Y12 and PAR4 stimulation was unchanged. Consistent with our activation results, platelet aggregation was significantly reduced in response to collagen and rhodocytin (p&lt;0.05) in MCU KO platelets, but not thrombin or ADP. MCU KO platelets adhered significantly less (p=0.0012) to collagen compared to MCU WT platelets under arterial and venous shear conditions. In vivo, MCU KO mice had longer occlusion time compared to the WT (p=0.0044).
Mechanistically, mitochondrial calcium flux was significantly reduced (p&lt;0.05) in MCU KO platelets compared to WT platelets after GPVI stimulation, but not PAR4 activation. Furthermore, mitochondrial reactive oxygen species (ROS) generation was significantly reduced in MCU KO platelets compared to WT platelets (p=0.0097) after GPVI-dependent activation while PAR4 activation induced no change in mitochondrial ROS. Mitochondrial ROS is known to regulate signaling in other cells. Consistent with this hypothesis, we observed a significant reduction in the ITAM signaling molecules pSyK (p=0.0084) and pPLCγ2 (p=0.012) in MCU KO platelets after GPVI stimulation. Furthermore, inhibiting mitochondrial ROS using MitoTempo, a specific mitochondrial ROS inhibitor, decreased aggregation (p=0.0004) as well as downstream signaling, including pSyk (p=0.0046) and pPLCγ2 (p=0.0493) in WT platelets when treated with a GPVI agonist. In parallel, treating MCU KO platelets with H 2O 2 to induce ROS production increased platelet aggregation (p=0.0036) after GPVI activation.
Conclusion(s): Platelet MCU mediates platelet activation and thrombosis in an ITAM-dependent manner by regulating mitochondrial calcium flux and ROS generation as well as downstream ITAM signaling through GPVI and CLEC-2. Our data support a novel role for mitochondria and mitochondria calcium flux in regulating ITAM-dependent platelet activation and demonstrate platelet MCU as a novel anti-platelet target to reduce thrombosis