Key Points• All blood coagulation factors predominantly bind to a small "cap"-like region on procoagulant-activated platelets.• Their concentration in this small region promotes acceleration of the membrane-dependent reactions of coagulation.Binding of coagulation factors to phosphatidylserine (PS)-exposing procoagulant-activated platelets followed by formation of the membrane-dependent enzyme complexes is critical for blood coagulation. Procoagulant platelets formed upon strong platelet stimulation, usually with thrombin plus collagen, are large "balloons" with a small (∼1 mm radius) "cap"-like convex region that is enriched with adhesive proteins. Spatial distribution of blood coagulation factors on the surface of procoagulant platelets was investigated using confocal microscopy. All of them, including factors IXa (FIXa), FXa/FX, FVa, FVIII, prothrombin, and PS-sensitive marker Annexin V were distributed nonhomogeneously: they were primarily localized in the "cap," where their mean concentration was by at least an order of magnitude, higher than on the "balloon." Assembly of intrinsic tenase on liposomes with various PS densities while keeping the PS content constant demonstrated that such enrichment can accelerate this reaction by 2 orders of magnitude. The mechanisms of such acceleration were investigated using a 3-dimensional computer simulation model of intrinsic tenase based on these data. Transmission electron microscopy and focal ion beam-scanning electron microscopy with Annexin V immunogold-labeling revealed a complex organization of the "caps." In platelet thrombi formed in whole blood on collagen under arterial shear conditions, ubiquitous "caps" with increased Annexin V, FX, and FXa binding were observed, indicating relevance of this mechanism for surface-attached platelets under physiological flow. These results reveal an essential heterogeneity in the surface distribution of major coagulation factors on the surface of procoagulant platelets and suggest its importance in promoting membrane-dependent coagulation reactions. (Blood. 2016;128(13):1745-1755

To cite this article: Obydennyy SI, Sveshnikova AN, Ataullakhanov FI, Panteleev MA. Dynamics of calcium spiking, mitochondrial collapse and phosphatidylserine exposure in platelet subpopulations during activation. J Thromb Haemost 2016; 14: 1867-81. Essentials• The sequence and logic of events leading to platelet procoagulant activity are poorly understood.• Confocal time-lapse microscopy was used to investigate activation of single adherent platelets.• Platelet transition to the procoagulant state followed cytosolic calcium oscillations.• Mitochondria did not collapse simultaneously and membrane potential loss could be reversible.Summary. Background: Activated platelets form two subpopulations, one of which is able to efficiently aggregate, and another that externalizes phosphatidylserine (PS) and thus accelerates membrane-dependent reactions of blood coagulation. The latter, procoagulant subpopulation is characterized by a high cytosolic calcium level and the loss of inner mitochondrial membrane potential, and there are conflicting opinions on their roles in its formation. Methods: We used confocal microscopy to investigate the dynamics of subpopulation formation by imaging single, fibrinogen-bound platelets with individual mitochondria in them upon loading with calcium-sensitive and mitochondrial potential-sensitive dyes. Stimulation was performed with thrombin or the proteaseactivated receptor (PAR) 1 agonist SFLLRN. Stochastic simulations with a computational systems biology model of PAR1 calcium signaling were employed for analysis. Results: Platelet activation resulted in a series of cytosolic calcium spikes and mitochondrial calcium uptake in all platelets. The frequency of spikes decreased with time for SFLLRN stimulation, but remained high for a long period of time for thrombin. In some platelets, uptake of calcium by mitochondria led to the mitochondrial permeability transition pore opening and inner mitochondrial membrane potential loss, which could be either reversible or irreversible. The latter resulted in an increase in the cytosolic calcium level and PS exposure. These platelets had higher cytosolic calcium levels before activation, and their mitochondria collapsed not simultaneously but one after another. Conclusions: These results support a model of procoagulant subpopulation development following a series of stochastic cytosolic calcium spikes that are accumulated by mitochondria, leading to a collapse, and suggest important roles of individual platelet reactivity and signal exchange between different mitochondria of a platelet.