Regulation of Platelet Procoagulant Activity by Mitochondrial Calcium Influx

Abstract 2193

Phosphatidylserine (PS) exposure is critical for blood coagulation. On platelets PS exposure provides a surface for the assembly of coagulation enzyme complexes. Two pathways of platelet PS exposure have been identified, an apoptotic Bax/Bak-mediated pathway and an agonist-initiated and cyclophilin D (CypD)-regulated pathway. In the agonist-initiated pathway sustained high levels of intracellular calcium that occur in strongly-stimulated platelets cause mitochondrial permeability transition pore (mPTP) formation and platelet PS exposure. Even in strongly-stimulating conditions PS exposure is limited to a subpopulation of the activated platelets. In this study we investigated how mitochondrial calcium (Ca²⁺_mit) elevations link the intracellular processes of cytoplasmic calcium (Ca²⁺_cyt) elevation, mPTP formation and platelet PS exposure, and identify Ca²⁺_mit influx as a novel potential therapeutic target to manipulate platelet procoagulant activity independent of other platelet activation events.

We have previously characterized the sequence of calcium and mitochondrial events that occur in procoagulant platelets and demonstrated that Ca²⁺_cyt elevations occur prior to mPTP formation in platelets. Experiments were performed to investigate whether PS exposure could be modulated independently of elevations in Ca²⁺_cyt through alterations in mPTP sensitivity. First we tested whether PS exposure initiated by calcium ionophore treatment was affected by the absence of CypD, a critical regulatory component of the mPTP that potentiates its calcium responsiveness. In the absence of CypD a two-threefold greater concentration of ionophore was required to initiate platelet PS exposure. Phenylarsine oxide (PAO) potentiates the calcium sensitivity of the mPTP. PAO-treated platelets required two-three fold less calcium ionophore to initiate PS exposure, and this increase was independent of Ca²⁺_cyt elevation. Together, these experiments establish the concept that modulation of the mPTP response can affect platelet PS exposure independent of Ca²⁺_cyt elevations.

Ca²⁺_mit influx regulates mPTP formation. Using the mitochondrial-specific and calcium-sensitive dye rhod-2, Ca²⁺_mit was found to be rapidly elevated in strongly-stimulated platelets, and the degree of Ca²⁺_mit elevation was closely associated with platelet PS exposure. Calcium influx in the mitochondria occurs as the result of passive flow of Ca²⁺_cyt down its electrochemical gradient through the mitochondrial calcium uniporter (MCU), a recently identified inner-mitochondrial transmembrane channel. Treatment with the MCU-specific inhibitor Ru360 effectively prevented agonist-initiated Ca²⁺_mit influx and platelet PS exposure. To further test the importance of Ca²⁺_mit in the regulation of platelet PS exposure, the effects of altering prestimulatory mitochondrial transmembrane potential (ΔΨ_m) were examined using inhibitors of mitochondrial respiration and oxidative phosphorylation. Decreased prestimulatory ΔΨ_m should decrease the passive flow of calcium into the mitochondria down its electrical gradient. In these preconditioned platelets prestimulatory ΔΨ_m was strongly positively correlated with both agonist-initiated Ca²⁺_mit elevation and PS exposure (R²=0.9153). Minimal correlation was observed between the degree of Ca²⁺_cyt elevation and PS exposure (R²=0.4254). The therapeutic effects of the anti-diabetic agent metformin have been proposed to occur through inhibition of respiratory complex I, an effect that should decrease prestimulatory ΔΨ_m. Metformin treatment of platelets decreased prestimulatory ΔΨ_m, Ca²⁺_mit elevation, and platelet PS exposure in strongly-stimulated platelets. Together, these data emphasize the importance of Ca²⁺_mit elevation in the regulation of agonist-initiated PS exposure, and identify inhibition of Ca²⁺_mit uptake either by MCU antagonists or by decreasing prestimulatory ΔΨ_m as a novel potential target in the prevention of platelet procoagulant activity.