MPs derived from cultured cells and platelets expose PCI on their surface.

<p>Jurkat cells and U937 cells were cultured in serum-free media (serum starvation, control) or treated with STS to induce apoptosis. MPs, isolated from the conditioned media of (A) Jurkat cells and (B) U937 cells, were stained with annexin V-EGFP and anti-PCI-IgG-AF633 and subjected to flow cytometry. MPs were defined as events with a size smaller than 1 μm, which show binding of annexin V. Data shown represent means and range of two independent experiments performed in duplicates. Washed platelets (n = 4) were activated with TRAP-6 (20 μM) or ionomycin (40 μM) to induce membrane blebbing and MP release. MP levels obtained from resting platelets served as control. MPs were analyzed by flow cytometry after staining with annexin V-EGFP, anti-PCI-IgG-AF633 and anti-CD62P-IgG-PE. (C) Stimulus dependent numbers of MPs (annexin V positive events). (D) Exposure of PCI on PS/CD62P double positive MPs after platelet activation with TRAP-6 (20 μM) or ionomycin (40 μM) in the absence or presence of pPCI (300 nM).