Mitochondrial Calcium Entry Is Essential in Agonist-Induced Procoagulant Platelet Formation

Procoagulant platelets are a subpopulation of activated platelets with high-level phosphatidylserine (PSer) externalization. When initiated by co-stimulation with thrombin (THR) and a GPVI agonist, such as convulxin (CVX) or collagen, both extracellular calcium entry and mitochondrial permeability transition pore (mPTP) formation are required to mediate the transition to the procoagulant platelet phenotype. The intracellular mechanisms coordinating these processes remain unclear. Here we investigated the role of the mitochondrial calcium uniporter in the regulation of procoagulant platelet formation.

The flux of calcium from the cytosol to the mitochondrial matrix is limited by the ion-impermeable inner mitochondrial membrane. Initially, a pharmacologic approach was utilized to investigate the importance of mitochondrial calcium entry and elevation. Washed human or murine platelets were stimulated with THR and/or CVX in physiologic buffer with 2 mM CaCl₂. Mitochondrial transmembrane potential was evaluated using 0.5 μM tetramethylrhodamine methylester (TMRM). Limitation of mitochondrial calcium entry either through alteration of pre-stimulatory mitochondrial membrane potential (i.e. inhibition of F₀F₁ ATP-ase, mitochondrial uncoupling etc.) or using the ruthenium red analog (Ru360) abrogated both mPTP and procoagulant platelet formation consistent with the hypothesis that elevated calcium levels within the mitochondrial matrix drives these processes.

The molecular pathway mediating this rapid mechanism of calcium entry was investigated using mitochondrial calcium uniporter (MCU) null platelets. MCU is a transmembrane ion channel that allows the passage of Ca²⁺ from the cytosol into the mitochondrial matrix. Neither platelet aggregatory ability nor granule release was altered in response to single or dual-agonist stimulation. In contrast, procoagulant platelet formation in MCU null platelets was significantly decreased coincident with decreased mPTP formation as measured by loss of mitochondrial transmembrane potential. Whereas the number of annexin V positive platelets decreased from 60% in WT to 32% in MCU null (p<0.05, n=4). Thus, these results identify a key role of mitochondrial calcium uptake channel in the regulation of strong agonist-initiated procoagulant platelet formation and suggest a novel pharmacologic target that could be used the treatment of procoagulant-platelet related pathologies.