Spencer Fox Eccles School of Medicine
43 Platelet Mitochondria Calcium Uniporter: Role in ITAM-Dependent Platelet Activation and Signaling
Mia Kowalczyk; Robert Campbell; Abigail Anjanel; and Frederik Denorme
Faculty Mentor: Robert Campbell (Internal Medicine, University of Utah)
This study aims to investigate the role of the mitochondrial calcium uniporter (MCU) in platelets during platelet activation. Platelet activation is crucial for arresting a bleeding event, however hyper-activation can pose the risk of thrombotic complications. When a thrombotic occlusion originating from arterial clots dislodges from the vessel wall, it navigates the circulatory system and bears the possibility of organ obstruction and subsequent functional impairment. Platelet activation to participate in thrombosis depends on many things including cytoplasmic calcium flux. Previous studies have shown calcium flux into the cytoplasm is vital in platelet activation and downstream signaling events. Rapid calcium influx into the intermembrane space of platelets causes a cellular calcium imbalance, triggering the multimeric complex responsible for facilitating calcium influx into mitochondria. This complex is referred to as the mitochondrial calcium uniporter (MCU). Under physiological conditions, calcium flux through the MCU generates ATP, however excess calcium flux may result in cellular necrosis. Though other studies have examined mitochondrial calcium flux mediation through MCU in other cell types, the role of mitochondrial calcium in platelet activation remains largely understudied.
Murine experiments were performed with platelet-specific MCU-depleted mice (MCUfl/fl-PF4- cre, KO) alongside wild-type littermate mice (MCUfl/fl, WT). MCU deletion did not alter mice viability or fertility, platelet counts, platelet volume, or platelet half-life. Three main protocols were used to assess platelet function: mitochondrial calcium flux, aggregation, and activation. Mitochondrial calcium flux was analyzed by using agonists PAR4 activating peptide, ADP (P2Y12 activator), rhodocytin (CLEC-2 activator), and collagen (GPVI activator). Aggregation assays were performed using collagen. Activation assays utilized thrombin (PAR4 activator), convulxin (GPVI activator), and rhodocytin to measure the level of platelet activation.
MCU deficiency demonstrated a prominent impact on platelet activation pathways. Specifically, MCU KO platelets exhibited significantly (p<0.05) reduced activation dependent on the GPVI and CLEC-2 receptors, while responses to PAR4 or P2Y12 activation remained unchanged. MCU KO platelets showed a significant (p<0.05) reduction in aggregation under collagen and rhodocytin conditions, with no change under thrombin or ADP stimulation. Under shear conditions, MCU KO platelets demonstrated significantly (p=0.0012) lower adhesion to collagen. KO platelet mitochondrial calcium flux was significantly (p<0.05) decreased under GPVI excitation, however not under PAR4 activation. Mitochondrial ROS generation in KO platelets was significantly (p=0.0097) reduced under GPVI activation but remained unchanged under PAR4 activation. Consequently, reduced ITAM signaling in pSyK (p=0.0084) and pPCLγ2 (p=0.012) was observed. Treatment with MitoTempo, inhibiting mitochondrial ROS, in WT platelets upon GPVI agonist exposure presented decreased aggregation (p=0.0004) and downstream signaling in pPCLγ2 (p=0.0493) and pSyK (p=0.0046). MCU KO platelets exhibited increased aggregation (p=0.0036) after GPVI activation when exposed to H2O2, which induces ROS production.
These findings suggest the MCU plays a crucial role in mediating platelet activation and thrombosis in a GPVI ITAM-dependent manner, primarily through the regulation of mitochondrial calcium flux, ROS generation, and downstream ITAM signaling pathways. Our data supports a novel role for mitochondria and mitochondria calcium flux in regulating GPVI-dependent platelet activation; this demonstrates platelet MCU as a novel anti-platelet target to reduce thrombosis. Thus, targeting the platelet MCU may offer a potential strategy for reducing thrombotic events.
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