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Background: Platelet activation leads to the formation of two distinct subpopulations: one of them consists of amoeboid well-aggregating platelets with operating mitochondria and no phosphatidylserine (PS) on their surface, while another (called “procoagulant”) includes balloon-like cells with externalized PS, depolarized mitochondria and a cap of alpha-granule proteins, but without ability for aggregate formation. It is established that the subpopulations are not pre-existing: the fraction of procoagulant platelets increases with the degree of activation, and can be changed between 0 and 90%. Signal transduction mechanisms that define formation of these subpopulations are presently unclear. Aims: Revealing relationship between calcium dynamics and mitochondrial collapse in procoagulant platelet formation. Methods: Signal transduction in platelets during activation was measured with real-time confocal microscopy of single fibrinogen-bound platelets loaded with fluorescent dyes sensitive for calcium in cytosol, mitochondria and mitochondrial membrane potential in presence of the PS marker Annexin V. Results: Stimulation of platelets using thrombin or PAR1 agonist SFLRRN initially produces a series of stochastic cytosolic calcium spikes. Some platelets remain in this state. In others, there is a transition from spikes to the sustained high calcium, that could be the result of the uptake of cytosolic calcium spikes by mitochondria leading to its overloading, mPTP opening, and PS externalization. Conclusions: These results support the model of procoagulant subpopulation development following a series of stochastic cytosolic calcium spikes that are accumulated by mitochondria leading to the collapse, and suggest important roles of individual platelet reactivity and signal exchange between different mitochondria of a single platelet. Higher cytosolic calcium in a resting platelet increases its chances of death after activation.