Microparticles released by activated/apoptotic cells exhibit coagulation activity as they express phosphatidylserine and some of them – tissue factor. We compared procoagulant properties of microparticles from monocytes, granulocytes, platelets and endothelial cells and assessed the impact of tissue factor in observed differences. Microparticles were sedimented (20 000g, 30 min) from the supernatants of activated monocytes, monocytic THP-1 cells, granulocytes, platelets and endothelial cells. Coagulation activity of microparticles was examined using plasma recalcification assay. The size of microparticles was evaluated by dynamic light scattering. Tissue factor activity was measured by its ability to activate factor X. All microparticles significantly accelerated plasma coagulation with the shortest lag times for microparticles derived from monocytes, intermediate – for microparticles from THP-1 cells and endothelial cells, and the longest – for microparticles from granulocytes and platelets. Average diameters of microparticles ranged within 400–600 nm. The largest microparticles were produced by endothelial cells and granulocytes, smaller – by monocytes, and the smallest – by THP-1 cells and platelets. The highest tissue factor activity was detected in microparticles from monocytes, lower activity – in microparticles from endothelial cells and THP-1 cells, and no activity – in microparticles from platelets and granulocytes. Anti-tissue factor antibodies extended coagulation lag times for microparticles from monocytes, endothelial cells and THP-1 cells and equalized them with those for microparticles from platelets and granulocytes. Higher coagulation activity of microparticles from monocytes, THP-1 cells and endothelial cells in comparison with microparticles from platelets and granulocytes is determined mainly by the presence of active tissue factor.

Activity of tissue factor (TF) in membrane microparticles (MPs) produced in vitro by endothelial cells (ECs), monocytes, THP-1 monocytic cells, granulocytes, and platelets was investigated. ECs were isolated from human umbilical vein, and monocytes, granulocytes, and platelets - from the blood of healthy donors. ECs, monocytes, and THP-1 cells were activated by bacterial lipopolysaccharide, granulocytes - by lipopolysaccharide or phorbol myristate acetate, and platelets - by SFLLRN, thrombin receptor-activating peptide. MPs were sedimented from the culture medium or supernatant of activated cells at 20,000g for 30 min. Coagulation activity of MPs was analyzed in a modified recalcification assay by assessing their effects on coagulation of donor plasma depleted of endogenous MPs (by centrifuging at 20,000g for 90 min). MPs from all cell types accelerated plasma coagulation. Antibodies blocking TF activity prolonged coagulation lag-phase in the presence of MPs from ECs, monocytes, and THP-1 cells (by 2.7-, 2.0-, and 1.8-fold, respectively), but did not influence coagulation in the presence of MPs from granulocytes and platelets. In accordance with these data, TF activity measured by its ability to activate factor X was found in MPs from ECs, monocytes, and THP-1 cells, but not in MPs from granulocytes and platelets. The data obtained indicate that active TF is present in MPs produced in vitro by ECs, monocytes, and THP-1 cells, but not in MPs derived from granulocytes and platelets.

Reticulated platelets (RP) are young, functionally active platelet forms which are detected by RNA staining. Their content in the circulation reflects the intensity of bone marrow thrombocytopoesis. The aim of this study was to assess in healthy volunteers the relationship between RP percentage and platelet size and activity. RP were quantitated by thiazole orange staining using flow cytometry. Platelet size indexes included mean platelet volume (MPV), platelet large cell ratio (P-LCR) measured in a Coulter type hematological analyzer and forward scattering (FSC) measured in a flow cytometer. Platelet functional activity was evaluated by expression of activated glycoprotein (GP) IIb-IIIa (PAC-1 antibody binding) and P-selectin with the use of flow cytometry. Platelets were activated by thrombin receptor activating peptide (TRAP) (10 and 1 µM) and ADP (20 and 2.5 µM). The percentage of RP in healthy volunteers varied from 2.9% to 23.8% (mean ± SD ‒ 11.7 ± 4.7%, n = 99) and correlated with all platelet size indexes: MPV, P-LCR and FCS (r from 0.452 to 0.529, p < .001, n = 87–99). On average, RP were distributed at a ratio of 9:1 between 50% subpopulations of large and small platelets according to their FSC index. Expression of GP IIb-IIIa activated form correlated with RP percentage and platelet size indexes when platelets were activated by TRAP and ADP at both applied concentrations (r from 0.309 to 0.560, p from 0.014 to < 0.001, n = 50–62). P-selectin expression correlated with RP percentage and platelet size indexes when platelets were activated by 10 µM TRAP inducing maximum expression of this activation marker (r from 0.332 to 0.556, p from 0.008 to < 0.001, n = 65), but not by weaker agonists: 1 µM TRAP, 20 and 2.5 µM ADP (r < 0.3, n = 54–66). Thus, high RP content in healthy volunteers is associated with increased platelet size and activity in the whole platelet population.

Quantity of platelet adhesion molecules significantly varies in normal donors and cardiovascular patients and might be affected by platelet size and genetic variations. In this study, we assessed relationships of the content of glycoprotein (GP) IIb-IIIa and GPIb with mean platelet volume (MPV) and their genetic polymorphisms. MPV and GPIIb-IIIa and GPIb numbers were measured in 116 patients with acute coronary syndrome (ACS) at days 1, 3–5 and 8–12 after disease onset and in 32 healthy volunteers. GPIIb-IIIa and GPIb allelic variants were determined in ACS patients. Strong interactions of GPIIb-IIIa and GPIb numbers and MPV were observed in ACS patients and healthy volunteers. In patients, coefficients of correlation (r) were 0.642 and 0.510 (analysis of individual mean values) and in volunteers – 0.594 and 0.508 for GPIIb-IIIa and GPIb, respectively (everywhere P < 0.005). In ACS patients, correlations were highly significant at each tested time point. GPIIb-IIIa and GPIb genetic polymorphisms [GPIIIa Leu33Pro, GPIbα Thr145Met and GPIbα (−5)T/C (Kozak)] determined in ACS patients had no significant impact on their expression. Modest correlation was revealed between MPV and plasma thrombopoietin (TPO) measured at the first day of ACS (r = 0.279, P = 0.005). The data obtained indicated that GPIIb-IIIa and GPIb levels are mainly affected by platelet size (MPV) but not by their genetic variations. In some ACS patients, production of large platelets with high GPIIb-IIIa and GPIb contents might be stimulated by elevated TPO.

Backgrounds/Aims: Relationships of mean platelet volume (MPV) and thrombopoietin (TPO) with platelet turnover assessed by glycocalicin measurement were evaluated in thrombocytopenic patients. Methods: MPV, glycocalicin and platelet-associated IgG (PA-IgG) were measured in 107 patients with idiopathic thrombocytopenic purpura (ITP) and 19 patients with hypoproductive thrombocytopenia (HPT; aplastic anemia or leukemia), and TPO was measured in 53 ITP and 12 HPT patients. All the included ITP patients had PA-IgG ≥300% and glycocalicin ≥50% of control values, and HPT patients had PA-IgG <300% and glycocalicin <50% of control values. Results and Conclusions: MPV was higher in ITP than in HPT patients: 9.56 ± 1.69 and 7.59 ± 0.90 fl (p < 0.001). In the ITP group a direct correlation was detected between MPV and glycocalicin (r = 0.344, p < 0.001). This interaction was essentially expressed in patients with normal/increased glycocalicin (≥100% of control; r = 0.470, p < 0.001, n = 64). TPO was greatly enhanced in HPT in comparison with ITP patients (958 ± 659 and 11 ± 27 pg/ml, p < 0.001). In the ITP group a reverse correlation was detected between TPO and glycocalicin (r = -0.373, p = 0.006). © 2014 S. Karger AG, Basel

DNA aptamers (oligonucleotides) interacting with thrombin exosite I contain G-quadruplex, two T-T, and one T-G-T loops in their structure. They prevent exosite I binding with fibrinogen and thrombin...