Assessment of platelet functional activity in healthy individuals and patients receiving antiplatelet therapy. Possible inconsistencies between aggregation and flow cytometry tests.

Platelet functional activity was assessed in healthy volunteers (HV, n=92), patients with stable angina pectoris (SA, n=42) and acute coronary syndrome (ACS, n=73), treated with acetylsalicylic acid (ASA) + clopidogrel and ASA + ticagrelor, respectively. In all HV and patients we have compared parameters of platelet aggregation (maximum light transmission and velocity, Tmax and Vmax) and parameters, characterizing exposure of platelet activation markers, evaluated by flow cytometry. HV platelets were activated by 10 µM, 1 µM TRAP, and 20 µM, 5 µM, 2.5 µM ADP; patient platelets were activated by 10 µM TRAP and by 20 µM and 5 µM ADP. Strong and significant correlations between the aggregation and flow cytometry parameters (the r correlation coefficient from 0.4 up to >0.6) most frequently were registered in HV platelet during activation by 1 µM TRAP and in SA patients during platelet activation by 20 µM and 5 µM ADP. However, in many other cases these correlations were rather weak (r < 0.3) and sometimes statistically insignificant. In HV the differences in PAC-1 binding parameters between platelets activated by 10 µM TRAP (the strongest agonist) and all ADP concentrations were negligible (≤ 10%), while CD62P binding (at all ADP concentrations) and LTA parameters for (5 µM and 2.5 µM ADP) were significantly lower (by 40-60%). Antiplatelet therapy in patients decreased all parameters as compared to HV, but to varying extents. For 10 µM TRAP the MFI index for PAC-1 binding (40-50% decrease) and for both ADP concentrations the Tmax values (60-85% decrease) appeared to be the most sensitive in comparison with the other parameters that decreased to a lesser extent. The data obtained indicate a possibility of inconsistency between different LTA and flow cytometry parameters in assessing platelet activity and efficacy of antiplatelet drugs.

Use of Thrombodynamics for revealing the participation of platelet, erythrocyte, endothelial, and monocyte microparticles in coagulation activation and propagation.

BACKGROUND AND OBJECTIVE: For many pathological states, microparticles are supposed to be one of the causes of hypercoagulation. Although there are some indirect data about microparticles participation in coagulation activation and propagation, the integral hemostasis test Thrombodynamics allows to measure micropaticles participation in these two coagulation phases directly. Demonstrates microparticles participation in coagulation activation by influence on the appearance of coagulation centres in the plasma volume and the rate of clot growth from the surface with immobilized tissue factor.Methods: Microparticles were obtained from platelets and erythrocytes by stimulation with thrombin receptor-activating peptide (SFLLRN) and calcium ionophore (A23187), respectively, from monocytes, endothelial HUVEC culture and monocytic THP cell culture by stimulation with lipopolysaccharides. Microparticles were counted by flow cytometry and titrated in microparticle-depleted normal plasma in the Thrombodynamics test. RESULTS: Monocyte microparticles induced the appearance of clotting centres through the TF pathway at concentrations approximately 100-fold lower than platelet and erythrocyte microparticles, which activated plasma by the contact pathway. For endothelial microparticles, both activation pathways were essential, and their activity was intermediate. Monocyte microparticles induced plasma clotting by the appearance of hundreds of clots with an extremely slow growth rate, while erythrocyte microparticles induced the appearance of a few clots with a growth rate similar to that from surface covered with high-density tissue factor. Patterns of clotting induced by platelet and endothelial microparticles were intermediate. Platelet, erythrocyte and endothelial microparticles impacts on the rate of clot growth from the surface with tissue factor did not differ significantly within the 0-200·103/ul range of microparticles concentrations. However, at concentrations greater than 500·103/ul, erythrocyte microparticles increased the stationary clot growth rate to significantly higher levels than do platelet microparticles or artificial phospholipid vesicles consisting of phosphatidylcholine and phosphatidylserine. CONCLUSION: Microparticles of different origins demonstrated qualitatively different characteristics related to coagulation activation and propagation.

[Coagulation properties of erythrocyte derived membrane microparticles]. / Koaguliatsionnye svoistva membrannykh mikrochastits éritrotsitov.

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.

Maternal incompatibilities with fetal human platelet alloantigens -1a, -1b and -15 are the main causes of neonatal alloimmune thrombocytopenia in Russia.

AIM: Mechanisms underlying the development of neonatal alloimmune thrombocytopenia (NAIT) in in Russia have been studied. MATERIALS AND METHODS: Genetic polymorphisms of human platelet alloantigens (HPA) -1, -2, -3, -4, -5, and -15 were evaluated in 27 families having the newborns with NAIT. NAIT was diagnosed according to the following criteria: (1) newborn with thrombocytopenia; (2) mother with no thrombocytopenia and no increase of platelet associated IgG, (3) presence of antibodies reacting with paternal platelets in maternal plasma / serum. HPA genotyping revealed incompatibilities in 23 out of 27 tested families. In these 23 families HPA-1 conflicts were detected in 16 ones (70%). In 8 cases mothers were homozygous carriers of rare HPA-1b allele and in another 8 cases - of HPA-1a allele which cased incompatibilities with fetal HPA-1a and HPA-1b respectively. In 5 out of 23 families (22%) there were incompatibilities with fetal HPA-15 (HPA-15a, n=2 and HPA-15b, n=3), in 1 family - with HPA-5b (4%), and in 1 family - with HPA-3b (4%) alloantigens. CONCLUSION: In conclusion the main causes of NAIT in Russia were HPA-1a and -1b conflicts and HPA-15 conflicts were the second frequent ones.

Comparison of the size of membrane microparticles of different cellular origin by dynamic light scattering.

Size of membrane microparticles (MPs) from blood plasma and MPs produced in vitro by activated endothelial cells (ECs), monocytes, THP-1 monocytic cells, granulocytes, and platelets was evaluated by dynamic light scattering. MPs were sedimented from the culture media, cell supernatants, and plasma at 20 000 g for 30 min. Average diameters of all types of MPs ranged from 300 to 600 nm. Plasma MPs had the smallest size. Close sizes were registered for MPs from platelets and THP-1 cells. MPs from monocytes were larger, and MPs from granulocytes and ECs were the largest ones. The data obtained indicate that the size of membrane MPs depends on the type of their cell-producers.

Activity of Tissue Factor in Microparticles Produced in vitro by Endothelial Cells, Monocytes, Granulocytes, and Platelets.

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.

Platelet subpopulation bearing leukocyte specific antigen and tissue factor.

Platelets bearing leukocyte antigen CD45 were identified in the blood of patients with myocardial infarction (MI) and healthy donors by flow cytofluorimetry. Part of these platelets contained tissue factor (TF)-primary initiator of blood clotting. The number of CD45+ and CD45+/TF+ platelets in MI patients at the first day was comparable with their level in healthy donors, but was increased at 8-12 days after MI onset. At that time in some patients the amount of CD45+ and CD45+/TF+ platelets reached 5-6 and 2-3% of their total number. It is assumed that CD45+/TF+ platelets could be formed as a result of platelet interaction with leukocytes or leukocyte produced membrane microparticles.

Activation and damage of endothelial cells upon hypoxia/reoxygenation. Effect of extracellular pH.

Disturbances of blood flow upon vascular occlusions and spasms result in hypoxia and acidosis, while its subsequent restoration leads to reoxygenation and pH normalization (re-alkalization) in ischemic sites of the vascular bed. The effect of hypoxia/reoxygenation on activation and stimulation of apoptosis in cultured human endothelial cells was studied. The cells were subjected to hypoxia (2% O2, 5% CO2, 93% N(2)) for 24 h followed by reoxygenation (21% O2, 5% CO2, 74% N(2)) for 5 h. Reoxygenation was carried out at different pH-6.4 (preservation of acidosis after hypoxia), 7.0, and 7.4 (partial and complete re-alkalization, respectively). Hypoxia only slightly (by approximately 30%) increased the cell adhesion molecule ICAM-1 content on the cell surface, whereas reoxygenation more than doubled its expression. The reoxygenation effect depended on the medium acidity, and ICAM-1 increase was more pronounced at pH 7.0 compared to that at pH 6.4 and 7.4. Neither hypoxia nor reoxygenation induced expression of two other cell adhesion molecules, VCAM and E-selectin. Incubation of cells under hypoxic conditions but not reoxygenation stimulated secretion of von Willebrand factor and increased its concentration in the culture medium by more than 4 times. The percentage of cells containing apoptosis marker, activated caspase-3, was increased by approximately 1.5 times upon hypoxia as well as hypoxia/reoxygenation. Maximal values were achieved when reoxygenation was performed at pH 7.0. These data show that hypoxia/reoxygenation stimulate pro-inflammatory activation (ICAM-1 expression) and apoptosis (caspase-3 activation) of endothelial cells, and the extracellular pH influences both processes.