Release of annexin V-binding membrane microparticles from cultured human umbilical vein endothelial cells after treatment with camptothecin

Overnight stimulation of HUVEC with either LPS or TNFα, 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 μM camptothecin, documented by 60–70% desquamation of HUVEC culture, annexin V-binding to the cells and DNA-fragmentation, led to a release of annexin V-binding microparticles (~80,000 MP/103 cells). This microparticle-release was prevented by Z-Val-Ala-Asp-fluoromethyl-ketone (ZVAD). Lower concentration of camptothecin (500 nM) induced comparable microparticle-release without loss of the culture confluence and without increase in annexin V-binding to the cells or DNA-fragmentation. Analyzed microparticles were free of nucleic acids and 95% of microparticles were 0.3–1 μm in size. Double-labeling flow cytometry assay showed that all annexin V-binding Microparticles expressed CD59 but only approximately 50% of these also expressed CD105.Camptothecin treated HUVEC released different populations of annexin V-binding membrane microparticles at early stage after proapoptotic stimulation before detection of phosphatidylserine exposure on the cells or DNA fragmentation. The microparticle-release was ZVAD sensitive but was not enhanced at the executive phase of apoptosis. These observations offer a new insight into microparticle-release as a marker of endothelial stimulation and injury.Plasma membrane microparticles (MP) of endothelial origin have been identified in normal human blood [1-3] and increased counts of MP carrying endothelial antigens were documented in the blood of patients with lupus anticoagulant [1], acute coronary syndrome [4], thrombotic thrombocytopenic purpura [5] or in patients with exacerbated multiple sclerosis [6]. Similarly to blood platelets, endothelial cells release MP which express phosphatidylserine (PS) on their surface. PS-exposing MP in blood appear to act as the catalytic s