2.5 Platelet microparticles (PMP):
Plasma concentrations of PMP (Siljander, 2011) were assayed in both stable CAS and during symptomatic crises, and compared with concentrations in control subjects.
Blood (8ml) was collected into citrate-containing cell preparation tubes (CPT Vacutainer, Becton Dickinson), centrifuged at room temperature at 1700g for 30 minutes and the supernatant diluted with an equal volume of phosphate buffered saline (PBS) and re-centrifuged multiple times (10mins @500g, 2mins@10000g and finally 45mins @17000g) to isolate a microparticle pellet, as previously described (Pope et al. , 2016). After re-suspension (1ml PBS containing 1% fetal calf serum, 2.5mM calcium), microparticles were incubated with Fc blocking agent for 10 minutes (0.1ml; Miltenyi Biotec). Aliquots (0.1ml) were then incubated with fluorescently labelled antibodies (BV421-Annexin V (5µl), APC-CD41 (20µl); Becton Dickinson) for 30 minutes at room temperature before addition of counting beads (25µl Accucount blank particles [8.0-12.9µm]; Spherotec) and analyzed via flow cytometry (BD FACS Canto II, Becton Dickinson). Sizing and specificity controls were used to identify populations of microparticles (1 μm) positive for Annexin V and platelet microparticles (CD41) staining.