Anastasia N. Sveshnikova

Dynamics of calcium spiking, mitochondrial collapse and phosphatidylserine exposure in platelet subpopulations during activation.

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.

Platelet Surface-Associated Activation and Secretion-Mediated Inhibition of Coagulation Factor XII

Coagulation factor XII (fXII) is important for arterial thrombosis, but its physiological activation mechanisms are unclear. In this study, we elucidated the role of platelets and platelet-derived material in fXII activation. FXII activation was only observed upon potent platelet stimulation (with thrombin, collagen-related peptide, or calcium ionophore, but not ADP) accompanied by phosphatidylserine exposure and was localised to the platelet surface. Platelets from three patients with grey platelet syndrome did not activate fXII, which suggests that platelet-associated fXII-activating material might be released from α-granules. FXII was preferentially bound by phosphotidylserine-positive platelets and annexin V abrogated platelet-dependent fXII activation; however, artificial phosphotidylserine/phosphatidylcholine microvesicles did not support fXII activation under the conditions herein. Confocal microscopy using DAPI as a poly-phosphate marker did not reveal poly-phosphates associated with an activated platelet surface. Experimental data for fXII activation indicates an auto-inhibition mechanism (k i/k a = 180 molecules/platelet). Unlike surface-associated fXII activation, platelet secretion inhibited activated fXII (fXIIa), particularly due to a released C1-inhibitor. Platelet surface-associated fXIIa formation triggered contact pathway-dependent clotting in recalcified plasma. Computer modelling suggests that fXIIa inactivation was greatly decreased in thrombi under high blood flow due to inhibitor washout. Combined, the surface-associated fXII activation and its inhibition in solution herein may be regarded as a flow-sensitive regulator that can shift the balance between surface-associated clotting and plasma-dependent inhibition, which may explain the role of fXII at high shear and why fXII is important for thrombosis but negligible in haemostasis.

Modulation and pre-amplification of PAR1 signaling by ADP acting via the P2Y12 receptor during platelet subpopulation formation

BACKGROUND Two major soluble blood platelet activators are thrombin and ADP. Of these two, only thrombin can induce mitochondrial collapse and programmed cell death leading to phosphatidylserine (PS) exposure required for blood clotting reactions acceleration. Thrombin can also greatly potentiate collagen-induced PS exposure. However, ADP acting through the P2Y12 receptor was shown to increase the PS-exposing (PS+) platelets fraction produced by thrombin or thrombin-plus-collagen via an unknown mechanism. METHODS We developed a comprehensive multicompartmental computational model of platelet PAR1-and-P2Y12 calcium signal transduction that included cytoplasmic signaling, dense tubular system and mitochondria. To test model predictions, flow cytometry experiments with washed, annexin V-labeled platelets were performed. RESULTS Stimulation of thrombin receptor PAR1 in the model induced cytoplasmic calcium oscillations, calcium uptake by mitochondria, opening of the permeability transition pore and collapse of the mitochondrial membrane potential. ADP stimulation of P2Y12 led to cAMP decrease that, in turn, caused changes in phospholipase C phosphorylation by protein kinase A, increase in cytoplasmic calcium level and, consequently, PS+ platelet formation. ADP addition before stimulation of PAR1 produced much greater increase of the PS+ fraction because cAMP concentration had time to go down prior to calcium oscillations; this prediction was also tested and confirmed experimentally. CONCLUSION These results suggest a mechanism of ADP-dependent PS exposure regulation and show a likely mode of action that could be important for the PS exposure regulation in thrombi, where ADP is released before thrombin formation.

Systems biology insights into the meaning of the platelet's dual‐receptor thrombin signaling

Essentials Roles of the two thrombin receptors in platelet signaling are poorly understood. Computational systems biology modeling was used together with continuous flow cytometry. Dual‐receptor system has wide‐range sensitivity to thrombin and optimal response dynamics. Procoagulant platelet formation is determined by donor‐specific activities of the two receptors.

The mechanisms and kinetics of initiation of blood coagulation by the extrinsic tenase complex

The system of hemostasis includes coagulation of blood plasma and formation of platelet aggregate. Plasma clotting is a cascade of proteolytic reactions, triggered by the contact of blood plasma with any tissue except the normal vessel endothelium. During the contact an enzymatic complex is formed of the soluble blood plasma protein, factor VIIa, and a membrane-anchored protein, tissue factor. This complex is called extrinsic tenase; it is the key initiator of blood coagulation. The main substrates of extrinsic tenase are blood plasma factors X and IX. During the reaction they undergo proteolytic cleavage and become active serine proteases, factors Xa and IXa, respectively. Factor Xa in complex with its cofactor factor Va catalyzes formation of the key coagulation enzyme, thrombin, which leads to fibrin polymerization and plasma gelation. Although all of the proteins that participate in this process have been known for a long time, several questions remain unanswered. As an example, what is the role of the reaction surface on which the complex is formed, what is the role of membrane-bound multimeres of factor X (Xa), and in what way does the activation of the factor VII proceed? Here, we review recent theoretical and experimental works focused on the biophysical mechanisms of extrinsic tenase functioning and discuss some of these problems.

Activation of the contact pathway of blood coagulation on the circulating microparticles may explain blood plasma coagulation induced by dilution

Recent studies have shown that the contact activation of blood coagulation can be initiated on the surface of circulating microparticles–particles formed as a result of the activation or apoptosis of blood cells or endothelial cells. In the present work, by means of a mathematical model, we investigated the mechanism of the activation of contact pathway of blood plasma coagulation. The model describes membrane-dependent reactions of the activation of factors XII and XI with account of the presence of blood plasma inhibitors. All reactions were described by ordinary differential equations integrated by an implicit multistep method. The current mathematical model is based on our previous model of factor XII activation on the platelet surface. The initial model is modified by the addition of factor XI, kallikrein, and blood plasma inhibitors. We show that the amidolytic activity of the contact pathway factors associated with the microparticles is proportional to the concentration of microparticles. In previous studies, an increase in the overall solution amidolytic activity after the dilution of plasma was observed. Computational analysis of the contact pathway activation in the diluted plasma shows that the increase in the activation appears from the dilution of blood plasma inhibitors. Thus, a well-known experimental phenomenon of the hypercoagulability of plasma after dilution can be explained by an increased activation of the blood plasma coagulation through the contact pathway on the circulating microparticles. In addition, the computational analysis reveals that a rapid stop of the contact pathway activation on the microparticles observed in the experiments could be explained by the rapid depletion of the free activation surface.

Physiological and pathophysiological aspects of blood platelet activation through CLEC-2 receptor

Platelet activating receptor CLEC-2 has been identified on platelet surface a decade ago. The only confirmed endogenous CLEC-2 agonist is podoplanin. Podoplanin is a transmembrane protein expressed by lymphatic endothelial cells, reticular fibroblastic cells in lymph nodes, kidney podocytes and by cells of certain tumors. Association of CLEC-2 with podoplanin is involved in processes of embryonic development (blood-lymph vessel separation and angiogenesis), maintaining of vascular integrity of small vessels during inflammation and prevention of blood-lymphatic mixing in high endothelial venules. However, CLEC-2 and podoplanin are contributing to tumor metastasis progression, Salmonella sepsis and deep-vein thrombosis. This makes CLEC-2 and podoplanin a perspective target for pharmacological treatment. Aspirin and Ibrutinib are considered to be perspective for abrogation of podoplanin-induced platelet activation via CLEC-2. The present review discusses already known pathological and physiological roles of CLEC-2 and possibilities of a targeted therapy for CLEC-2 associated diseases.

Extracellular vesicles of blood plasma: content, origin, and properties

Extracellular vesicles (EVs) are bilayer membrane fragments that are released by different cell types upon activation or death. The most well studied EVs are those of blood plasma. Two types of EVs are usually distinguished: exosomes (formed by the membranes of intracellular compartments, 50–100 nm in diameter) and ectosomes (also called microparticles or microvesicles, formed from plasma membrane, 100–1000 nm in diameter). The real picture is much more complicated and is still poorly understood. EVs are enriched by various proteins, mRNA and miRNA, and the EV lipid and protein composition can substantially differ from that of the parental cells, from which EV originate. The blood concentration of EVs greatly increases in many diseases and conditions. EVs have a wide spectrum of biological activities, from pro-coagulant to immunomodulating ones. This activity can be physiologically important and is believed to be absolutely important pathophysiologically. In recent studies, EVs are considered to be important not only as objects of basic research, but also as potential biomarkers, drug candidates, drug carriers, or therapeutic targets.