MP were harvested under sterile conditions from the supernatants of either young P1 ECs (eMPaPC) or RIN‐m5f β cells (βMP) submitted to a 70 nM aPC treatment during 24 hrs (Xigris®, Lilly). Floating apoptotic cells and debris were first discarded (800 g 15 min.) and MP washed in Hanks balanced salt solution (HBSS) and concentrated by a double‐centrifugation step (14,000 g, 1 hr). Washed MP were kept at 4°C for not more than 3 weeks. For control purposes, endothelial MP (eMPCTRL) were harvested from the supernatant of untreated ECs and isolated using the same procedure.
Total MP concentration was determined by prothrombinase assay as previously described. Briefly, MP captured onto insolubilized annexin A5 were incubated with blood clotting factors (FXa, FVa, FII) and CaCl2. Conversion of prothrombin to thrombin was revealed by a chromogenic substrate, using a spectrophotometric reader set at 405 nm. Results were expressed as nanomolar PhSer equivalent (nM PhSer eq.) by reference to a standard curve constructed using liposomes of known concentration and PhSer eq. proportion14 .
Total MP concentration was determined by prothrombinase assay as previously described. Briefly, MP captured onto insolubilized annexin A5 were incubated with blood clotting factors (FXa, FVa, FII) and CaCl2. Conversion of prothrombin to thrombin was revealed by a chromogenic substrate, using a spectrophotometric reader set at 405 nm. Results were expressed as nanomolar PhSer equivalent (nM PhSer eq.) by reference to a standard curve constructed using liposomes of known concentration and PhSer eq. proportion