title: Identification of signal transduction pathways involved in the formation of platelet subpopulations upon activation.
abstract: Platelets are formed elements of blood. Upon activation, they externalize phosphatidylserine, thus accelerating membrane-dependent reactions of blood coagulation. Activated platelets form two subpopulations, only one of which expresses phosphatidylserine. This study aimed to identify signalling pathways responsible for this segregation. Gel-filtered platelets, intact or loaded with calcium-sensitive dyes, were activated and labelled with annexin V and antibodies, followed by flow cytometric analysis. Calcium Green and Fura Red dyes were compared, and only the latter was able to detect calcium level differences in the platelet subpopulations. Phosphatidylserine-positive platelets produced by thrombin had stably high intracellular calcium level; addition of convulxin increased and stabilized calcium level in the phosphatidylserine-negative subpopulation. PAR1 agonist SFLLRN also induced calcium rise and subpopulation formation, but the resulting platelets were not coated with alpha-granule proteins. Adenylatecyclase activator forskolin inhibited phosphatidylserine-positive platelets formation several-fold, while its inhibitor SQ22536 had no effect. This suggests that adenylatecyclase inactivation is necessary, but not rate-limiting, for subpopulation segregation. Inhibition of mitogen-activated protein kinase kinase (U0126) and glycoprotein IIb-IIIa (Monafram(®)) was without effect, whereas inhibitors of phosphatidylinositol 3-kinase (wortmannin) and Src tyrosine kinase (PP2) decreased the procoagulant subpopulation threefold. These data identify the principal signalling pathways controlling platelet heterogeneity.
authors: Centre for Theoretical Problems of Physicochemical Pharmacology, Russian Academy of Sciences, Moscow, Russia.
authors: Nikolai N
authors: NN
authors: Topalov
authors: Yana N
authors: YN
authors: Kotova
authors: Sergey A
authors: SA
authors: Vasil'ev
authors: Mikhail A
authors: MA
authors: Panteleev
mesh_terms: D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000378:metabolism; D000328:Adult; D017304:Annexin A5 / Q000378:metabolism; D001792:Blood Platelets / Q000166:cytology* / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D010718:Phosphatidylserines / Q000378:metabolism; D015539:Platelet Activation / Q000187:drug effects / Q000502:physiology*; D044463:Receptor, PAR-1 / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D010975:Platelet Aggregation Inhibitors
chemical_list: D000067956:Adenylyl Cyclase Inhibitors; D017304:Annexin A5; D004791:Enzyme Inhibitors; D010718:Phosphatidylserines; D044463:Receptor, PAR-1; D000262:Adenylyl Cyclases; D002118:Calcium; D010975:Platelet Aggregation Inhibitors