Identification of signal transduction pathways involved in the formation of platelet subpopulations upon activation

Abstract

Summary 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.

Two Types of Procoagulant Platelets Are Formed Upon Physiological Activation and Are Controlled by Integrin α IIb β 3

“…Interestingly, all researchers who reported inhibition of procoagulant activity by α IIb β 3 deficiency or antagonism [28][29][30][31] used a physiological range of platelet concentrations with a single exception, 32 and all those who reported no effect or stimulation by α IIb β 3 antagonists invariably used much lower platelet concentrations. 13,33,34 This discrepancy may, therefore, be explained by the finding that α IIb β 3 antagonism has no effect on the PS+/Ca+ platelets but strongly inhibits the PS+/ Ca-subpopulation ( Figure 3B). …”

“…13 Briefly, before gel filtration, resuspended platelets were incubated with 10 μmol/L Fura Red/AM for 30 minutes at room temperature in the presence of apyrase (0.1 U/mL) and prostaglandin E1 (1 μmol/L). The concentration of the calciumsensitive dye solvent, dimethylsulfoxide, did not exceed 0.1%; additional controls confirmed that the vehicle did not affect the results and that the loading procedure did not have any significant effect on the PS-positive platelet formation.…”

“…8,9 These PS-positive platelets formed by thrombin or proteaseactivated receptor agonists and collagen were always reported to have sustained increases of intracellular calcium, 2,10-13 which is believed to be indispensable for PS exposure. In addition to this, they are characterized by loss of membrane potential and opening of mitochondrial permeability transition pore, 14,15 calcein permeability, 16 inactivation of integrin α IIb β 3 , 12,17 and, in the case of thrombin, 13,17,18 retention of surface-associated α-granule proteins, such as fibrinogen or factor Va. This subpopulation was recently proposed to be called necrotic, 4 as these platelets seem to share several important similarities with necrotic cells.…”

Physiology and pathology of extracellular vesicules

“…В результате одна маленькая микровезику-ла по способности ускорять основные реакции свер-тывания крови сопоставима с полноценным тромбоцитом. При этом во время активации тромбо-цитов сильными стимуляторами [33][34][35][36] или во время иных процессов [37,38] (в частности, при хранении тромбоконцентратов [39]) идет интенсивная везику-ляция: свыше десятка везикул образуется на один прокоагулянтный тромбоцит (рис. 6) [40].…”

Activators, receptors and signal transduction pathways of blood platelets

“…Таким образом, коллаген, связываясь с GPVI, приводит практически ко всем видам ответов, таких как повышение концентрации внутриклеточного кальция, секреции гранул, изменению формы, а также к высшей степени активации тромбоцита -прокоагулянтной активности [28]. Вклад различных путей сигнализации в формирование гетерогенности тромбоцитов был проанализирован в [29].…”