Flow cytometric analysis of microparticle phenotype and their role in thrombin generation

Abstract

Background: Microparticles may be generated from a number of cell types and are known to play a role in haemostasis by a variety of mechanisms. We investigated the role of platelet, red cell, and leucocyte-derived microparticles in the measurement of thrombin generation.Methods: Four parameters of thrombin generation (the endogenous thrombin potential (ETP), lag time, time to peak, peak height) and microparticle content was determined in 35 plasma samples from normal individuals pre and post filtration to remove microparticles. Immunofluorescent flow cytometry was used to identify and enumerate platelet, leucocyte, monocyte and red cell derived microparticles in plasma samples based on the expression of CD42b, CD45, CD15, and Glycophorin A respectively. Expression of phosphatidylserine and tissue factor by microparticles was determined by Annexin V and anti CD142 binding. The pre and post filtration results were compared.Results: There was a significant decrease in ETP and Peak Height, and an increase in the time to peak post filtration (P < 0.001). A significant decrease in the number of CD421, CD451, CD151, CD1421, and Annexin V1 microparticles was also observed. The change in CD42b1 microparticles correlated highly with the change in Annexin V1 microparticles (r 5 0.68). Whilst the change in ETP correlated best with the change in CD151 microparticles (r 5 0.45) and the change in time to peak correlated with the change in Annexin V binding (r 5 0.52) (P < 0.01).Conclusion: The presence of micropartcles in plasma significantly affects thrombin generation. V C 2010

Microparticle formation after co‐culture of human whole blood and umbilical artery in a novel in vitro model of flow

“…Compensation was performed using Comp Beads (BD) and photomultiplier voltages suitable for cellular analysis. The limit of detection of the instrument was shown to be below 0.1 lm as we demonstrated previously (34).…”

Platelet Microvesicles (Microparticles) in Cardiac Surgery

“…9 Platelets have rapid PS expression, allowing prompt promotion of coagulation at the sites of endothelial injury; 18 the removal of PS from the PMV surface decreases the rate of thrombin production, 22 providing evidence that the production of PMV is critical to physiological coagulation. The phenotype of a microvesicle alters its function; increasing expression of PS and TF enhances thrombin generation.…”

“…The phenotype of a microvesicle alters its function; increasing expression of PS and TF enhances thrombin generation. 22 Some PMV express anti-coagulant receptors, suggesting there may be a role for PMV in regulating hemostasis. 23 Procoagulant receptors are more densely expressed on the surface of PMV than the parent platelet by up to 100-fold.…”

“…27 However increased TF expression does not always correlate with increased coagulation, suggesting that other factors are similarly important. 22,28 Integrin α 11b β 3 , another receptor expressed on the outer PMV surface, has a role in binding PMV to fibrinogen to cause platelet adhesion. 29 Microvesicles taken from the pericardial cavity of patients undergoing cardiopulmonary bypass promote thrombin generation.…”

Fluorescent particles in the antibody solution result in false TF- and CD14-positive microparticles in flow cytometric analysis

“…Some reports have demonstrated discrepancies between TF-protein and TF activity levels, which have been explained by antibody binding to ''encrypted'' or degraded forms of inactive TF-protein (13,16).…”