P578Calcium concentration affects annexin V binding but not phosphatidylserine externalization in microparticles

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Abstract

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.

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