Коагуляционные свойства мембранных микрочастиц тромбоцитов, эритроцитов, лейкоцитов и эндотелиальных клеток

Мембранные микрочастицы (МЧ) образуются при активации и/или повреждении клеток. Они обладают коагуляционной активностью (ускоряют свертывание крови), так как содержат на своей поверхности фосфатидилсерин субстрат для образования коагуляционных комплесов, а некоторые из них тканевой фактор (ТФ), главный инициатор коагуляционных реакций. Циркулирующие в кровотоке МЧ продуцируются клетками крови и эндотелиальными клетками сосудистой стенки, и их содержание может меняться при различных патологических состояниях. Сравнительные in vitro исследования показали, что наибольшей способностью ускорять свертывание плазмы крови обладают МЧ моноцитов и эндотелиальных клеток и более низкой МЧ тромбоцитов, эритроцитов и гранулоцитов (нейтрофиU лов). Эти различия обусловлены главным образом присутствием в МЧ моноцитов и эндотелиальных клеток активного ТФ и отсутствием его в МЧ тромбоцитов, эритроцитов и гранулоцитов. Вариации размера МЧ различного клеточного происхождения существенно не влияют на их коагуляционные свойства. Таким образом, на коагуляционный (протромботический) потенциал крови могут оказывать влияние в первую очередь МЧ, экспрессирующие ТФ, источником которых могут быть активированные и/или поврежденные моноциты и эндотелиальные клетки.Membrane microparticles (MPs) are formed upon cell activation and/or damage. They possess coagulation activity (accelerate blood clotting) since they contain on their surface posphatidylserine, substrate for assembling coagulation complexes, and some of them tissue factor (TF), major initiator of coagulation reactions. Circulating MPs are produced by blood cells and vessel wall endothelial cells and their amount could be changed in different pathological conditions. Comparative in vitro studies have shown that MPs derived from monocytes and endothelial cells have the highest and MPs derived from platelets, erythrocytes and granulocytes (neutrophils) much lower ability to accelerate blood plasma clotting. These differences are mainly determined by the presence of active TF in monocyte and endothelial MPs and its absence in platelet, erythrocyte and granulocyte MPs. Size variations of MPs of different cellular origin do not significantly affect their coagulation properties. Thus, coagulation (prothrombotic) blood potential could be influenced first of all by TF expressing MPs the source of which are activated and/or damaged monocytes and endothelial cells.

TNF-α Modulates P-Glycoprotein Expression and Contributes to Cellular Proliferation via Extracellular Vesicles

P-glycoprotein (Pgp/ABCB1) overexpression is associated with multidrug resistance (MDR) phenotype and, consequently, failure in cancer chemotherapy. However, molecules involved in cell death deregulation may also support MDR. Tumor necrosis factor-alpha (TNF-α) is an important cytokine that may trigger either death or tumor growth. Here, we examined the role of cancer cells in self-maintenance and promotion of cellular malignancy through the transport of Pgp and TNF-α molecules by extracellular vesicles (membrane microparticles (MP)). By using a classical MDR model in vitro, we identified a positive correlation between endogenous TNF-α and Pgp, which possibly favored a non-cytotoxic effect of recombinant TNF-α (rTNF-α). We also found a positive feedback involving rTNF-α incubation and TNF-α regulation. On the other hand, rTNF-α induced a reduction in Pgp expression levels and contributed to a reduced Pgp efflux function. Our results also showed that parental and MDR cells spontaneously released MP containing endogenous TNF-α and Pgp. However, these MP were unable to transfer their content to non-cancer recipient cells. Nevertheless, MP released from parental and MDR cells elevated the proliferation index of non-tumor cells. Collectively, our results suggest that Pgp and endogenous TNF-α positively regulate cancer cell malignancy and contribute to changes in normal cell behavior through MP.

Coagulation properties of erythrocyte derived membrane microparticles

Membrane microparticles (MP) produced upon cell activation and/or damage possess coagulation activity, i.e. ability to accelerate blood clotting. They contain on their surface phosphatidylserine (PS), a substrate for assembling coagulation enzymatic complexes, and some of them tissue factor (TF), the initiator of clotting cascade reactions. In this study coagulation properties of MP derived from erythrocytes have been investigated. These MP were obtained from donor's erythrocytes activated with ionophore A23187 as well as from outdated erythrocyte concentrates for transfusion. MP were counted by flow cytometry. Coagulation activity of MP was examined by modified plasma recalcification assay. Involvement of PS and TF in this reaction was assessed using PS blocker lactadherin and anti-TF antibodies. TF activity in MP was measured by its ability to activate factor X in a chromogenic assay. Size of MP was evaluated by dynamic light scattering. Properties of erythrocyte MP were compared with previously characterized (using the same methodological approaches) MP derived from platelets and monocytic THP-1 cells, lacking and containing TF, respectively. Erythrocyte MP accelerated plasma clotting, but less actively than MP from platelets and MP from THP-1 cells, which demonstrated maximal activity. Lactadherin completely inhibited coagulation activity of all MP. Anti-TF antibodies did not affect clotting parameters in the presence of platelet and erythrocyte MP, but slowed clotting in the presence of MP from THP-1 cells. TF activity was not detected in erythrocyte and platelet MP, unlike MP from THP-1 cells expressing active TF. MP derived from erythrocytes were smaller than MP from platelets and THP-1 cells, with average diameter about 200 nm and 400 nm respectively. Thus, MP from erythrocyte possess less ability to accelerate plasma clotting in comparison with MP from platelet and THP-1 cells. The data obtained suggest that lesser coagulation activity of erythrocyte MP in comparison with MP from THP-1 cells is due to the absence of TF in erythrocyte MP (in contrast to MP from THP-1 cells) and to their smaller size, and in comparison with MP from platelets (which as erythrocyte MP do not express TF) is due to their smaller size only.