Submicron cell-derived microparticles (MP) are important biological messengers. MP release is accompanied by Ca2+-dependent cytoskeleton degradation and phosphatidylserine (PS) exposure. Annexin V (AnV), which binds surface PS, has been long regarded as a marker of MP but it is unclear whether staining Ca2+ concentrations ([Ca2+]) could affect AnV binding to MP. In this study, we aimed to investigate this question.Methods
Fresh sodium citrated and heparinised platelet free plasma (PFP) were obtained from healthy donors (n=6). Platelets were isolated from citrated plasma and stimulated 30min with thrombin (0.5-1 U/mL). AnV + MP and platelet CD42b + MP (PMP) were quantified using an Apogee A50 flow cytometer (Hertfordshire, UK). AnV staining was performed in AnV binding buffer with increasing [Ca2+] (from 2.5 to 200 mM). The amount of AnV bind per MP was expressed as median fluorescence intensity (MFI).Results
The counts of AnV + MP were highly dependent on [Ca2+] for both anticoagulants with maximum MP levels detected at 25 mM CaCl2 (Table). Stimulation of platelets with thrombin induced shedding of PMP with increased PS exposure in a concentration-dependent manner. Thrombin stimulation of PFP containing only MP did not produce any changes in the counts of AnV + MP nor PMP.Conclusions
At commonly used [Ca2+] (2.5 mM), counts of AnV + MP are understimated in citrated plasma. Increasing [Ca2+] in the staining buffer allow initial recalcification and more accurate enumeration without affecting PS exposure. High Ca2+ influx might induce apoptosis, resulting in membrane disruption and PS externalization, reflected by MFI fluctuations. These results also indicate that thrombin induces platelet apoptosis accompanied by release of AnV + MP whereas PS exposure in pre-existent MPs is not affected by thrombin.