Our program is focused on three different aspects of cell membrane microparticle (MP) research: Development of assays for analysis of MP in plasma and analysis of cell membrane microparticles in plasma of patients with different vascular pathologies. We have designed a three-color flow cytometry assay for analysis of MP in plasma and demonstrated the presence of four distinct populations of MP in plasma of healthy blood donors. MP of endothelial origin were identified as CD105+CD41-CD45-. The population of platelet-derived MP was recognized as CD41+CD105-CD45-, population of red blood cells MP was CD235a+CD105-CD45- and population of white blood cells MP was identified as CD45+CD41-CD105-. Using the newly developed assay we have analyzed endothelial cell membrane microparticles (EC MP) in blood of paroxysmal nocturnal hemoglobinuria (PNH) patients (n=9). Healthy blood donors (HD) (n=11), aplastic anemia (AA) patients (n=10) and sickle cell disease (SCD) patients (n=8) were used for comparison. EC MP were identified by antibodies to CD105 (endoglin) and highly specific CD144 (VE-cadherin). A subpopulation of phosphatidylserine-positive MP was detected using the annexin V-binding (ANB) assay. We found that a population of CD105+ANB EC MP was significantly (p<0.05) higher in SCD (680 ? 440 MP/ [micro]L) and PNH (340 ? 180 MP / [micro]L) patients when compared to AA (125 ? 65 MP / [micro]L) or HD (180 ? 50 MP / [micro]L). More pronounced differences were observed in a subpopulation of EC MP expressing a marker of inflammatory stimulation CD54 (CD105+CD54+). These MP were markedly elevated in SCD and PNH patients compared to AA or HD groups (p< 0.05), but no significant differences were found between SCD and PNH group, or between AA and HD group. Similarly EC MP expressing proteolysis sensitive antigen CD144 were increased in SCD and PNH group. CD54+ EC MP may indicate endothelial injury by the chronic inflammatory process present in hemolytic PNH and SCD patients, while elevated CD144+ EC MP may reflect acute endothelial injury. In addition to a possible pathophysiological role of circulating EC MP, analysis of different populations of EC MP may be valuable for understanding the mechanism of vessel wall injury in these patients. Study on mechanism of cell membrane microparticle release from blood and endothelial cells. Elevated plasma counts of endothelial microparticles (MP) have been demonstrated in various diseases with a vascular injury component. The mechanism of MP-release from endothelial cells is not known, however. We used flow cytometry to study the MP-release from cultured human umbilical vein endothelial cells (HUVEC) stimulated by various agonists. Overnight stimulation of HUVEC with LPS or TNFalpha, or 30 min stimulation with thrombin, phorbol-myristate-acetate, tissue plasminogen activator, or angiotensin-II did not cause a significant release of annexin V-binding MP. In contrast, induction of apoptosis with 5 [micro]M camptothecin, documented by annexin V-binding to the cell surface and DNA-fragmentation, led to a release of annexin V-binding MP (~80,000 MP/10exp3 cells). This MP-release was prevented by the caspase inhibitor Z-Val-Ala-Asp-fluoromethyl-ketone. Lower concentration of camptothecin (500 nM) induced comparable MP-release without significant increase in annexin V-binding to the cells or DNA-fragmentation. Analyzed MP were free of nucleic acids and 95% of MP were < 1 [micro]m in size (range 0.3 - 3.0 [micro]m). Double-labeling flow cytometry assay showed that all annexin V-binding MP expressed CD59 but only approximately 50% of these also expressed CD 105. In conclusion, HUVEC release different populations of annexin V-binding membrane MP in response to proapoptotic stimulation. MP-release from HUVEC after stimulation with camptothecin is caspase-dependent but does not correlate with the level of cell culture apoptosis and does not determine cell death. These observations offer a new insight into MP-release as a marker of endothelial stimulation and injury. Analysis of cell membrane microparticles in cellular blood products. We have started development of immunodetection and functional assays for analysis of cell membrane microparticles in platelet concentrates. The analysis is focused on prothrombotic and proinflammatory microparticles expressing tissue factor (CD142) and phosphatidyl serine or ICAM-1 (CD54) and other adhesion receptors, respectively.
