OC 18.1 - The role of Mitochondrial Calcium Uniporter in platelet function

Background: Platelet cytoplasmic calcium is important for platelet activation and downstream signaling events. However, the role of mitochondrial calcium in platelets is unclear. Mitochondrial calcium is mediated through the mitochondrial calcium uniporter (MCU), but the role of MCU in platelet function is poorly understood.

Aims: To examine the role of MCU in platelet function.

Methods: We generated a platelet-specific MCU-deficient mouse (MCUfl/fl-PF4-cre, KO). Platelet function and in vivo thrombosis was examined in both littermate wild-type (MCUfl/fl, WT) and KO mice.

Results: GPVI-dependent IIb3 activation and P-selectin expression were significantly (p < 0.05) reduced in KO platelets. Activation was similar between WT and KO platelets upon P2Y12 and PAR4 stimulation. Platelet aggregation was significantly reduced in response to collagen (p=0.001) in KO platelets, but not thrombin. Mechanistically, calcium flux was reduced in KO platelets compared to WT platelets after GPVI stimulation. Furthermore, mitochondrial reactive oxygen species (ROS) generation was significantly reduced in KO platelets compared to WT platelets (p=0.0097) in response to GPVI stimulation. In contrast, PAR4 activation induced little change in mitochondrial ROS in both WT and KO platelets. Mitochondrial ROS can regulate signaling. Consistent with this hypothesis, we observed a significant reduction in pSyK (p=0.0084) and p-PLC2 (p=0.012) in KO platelets after GPVI activation. To examine if MCU-deficiency altered thrombosis, we employed an ex vivo flow chamber model and measured vessel occlusion in vivo using a ferric chloride carotid artery thrombosis model. MCU KO platelets adhered significantly less to collagen compared to MCU WT platelets under arterial (p=0.0012) and venous (p=0.03) shear conditions while no difference was observed with fibrinogen. In vivo, MCU KO mice took longer to form an occlusion thrombus compared to WT mice (p=0.0044).

Conclusion(s): Platelet MCU mediates platelet activation and thrombosis in a GPVI-dependent manner by regulating mitochondrial calcium flux and ROS generation as well as downstream GPVI signaling.