Dynamics and Regulation of Cyclophilin D-Dependent Necrosis in Platelets

Strong physiological activation of blood platelets leads to the emergence of a non-aggregatable procoagulant subpopulation characterized by the appearance of phosphatidylserine on the outer leaflet of the plasma membrane, providing a surface for coagulation reactions. The formation of procoagulant platelets is attributed to cyclophilin D-dependent opening of the mitochondrial permeability transition pore in response to mitochondrial calcium overload; however, the mechanisms of calcium entry and subsequent necrosis have not been definitively established. We used continuous flow cytometry to analyze the main stages of this process following platelet stimulation by monitoring cytosolic calcium dynamics (Fura Red), reactive oxygen species (ROS) levels (CM-H₂DCFDA), plasma membrane integrity (calcein), mitochondrial potential [DiOC₆(3)], fluorescent fibrinogen binding, and procoagulant activity (annexin V). Receptor stimulation induced procoagulant platelet formation through an intermediate platelet subpopulation characterized by depolarized mitochondria, but polarized plasma membrane and elevated phosphatidylserine. During the early activation (less than 5 min), ROS increased sharply in the subpopulation with elevated phosphatidylserine content and steadily increased in the non-procoagulant subpopulation; by 15 min, ROS levels were comparable between the two populations. Addition of the protonophore CCCP at any point during activation did not affect the formation of procoagulant platelets. However, CCCP addition during activation of platelets preincubated with the cyclophilin D inhibitor cyclosporin A resulted in explosive necrosis. CCCP also increased calcium concentrations in resting platelets. These data help explain the contradictory effects of CCCP on platelet necrosis and highlight the importance of the voltage-dependent calcium accumulation in mitochondria. They further suggest a significance of ROS in platelet necrosis, while the loss of membrane integrity appears to be a secondary process.