One micrometer beads

Microparticles were analyzed in the blood. Plasma (250 μlL aliquots) was centrifuged at 20,000 x g for 2.5 hours. The pellet was re-suspended in Hank’s buffered saline solution containing 20 mM HEPES and 5 mM glucose. It was re-suspended by vortexing the pellet for 1.5 minute in buffer at a volume of 40% of the initial plasma volume. Microparticles may be distinguished based upon protein, lipid and cholesterol composition. Multi-color fluorescence-activated cell sorting was performed to characterize the cellular source and activation state of the microparticles. An aliquot of microparticles was stained with antibodies to CD41a (Novus Biologicals) and CD31 (BD Biosciences) to identify whether they originated from platelets (CD41a⁺, CD31⁺) or endothelial cells (CD41a⁻CD31⁺). One micrometer beads (Molecular Probes) were used to gate the particles based on size. The number of microparticles from each cell type (platelet or endothelial) was measured in a 250 μl plasma sample. We measured CD62E levels (anti-E-selectin, Novus Biologicals) on the surface of the endothelial cell microparticles as an indicator of whether the cells that released the particles were activated.