Introduction:
In Australia, donor blood is processed into components including red blood cells (RBCs). RBCs are stored at 2-6oC for up to 42 days. During storage, extracellular vesicle (EV) concentration and procoagulant activity increases. This study investigated the role of EVs in this increased activity.
Methods:
RBC units (n=8) were sampled at expiry (day 42-44), centrifuged twice (3,000xg, 5 minutes), and RBC-supernatant (SN) was stored (-80°C). Aliquots underwent ultracentrifugation (UC; 110,000g, 2 hours, 4°C) to obtain EV-depleted-SN (with or without further filtration (0.02µm)) and UC-EV pellet was resuspended in filtered PBS. RBC-SN was also separated into EV and protein fractions using IZON 35nm qEV columns, concentrated and resuspended in filtered PBS.
The 6 samples (RBC-SN, UC-EV pellet, EV-depleted-SN +/- filtration, isolated EVs and isolated proteins) were assessed for nanoparticle size and concentration (Nanosight NS300), and procoagulant phospholipid (PPL) mediated coagulation (Amelung KC4A Microcoagulometer; STA Procoag PPL kit, Stago). Data were analysed using GraphPad Prism using one-way ANOVA.
Results:
Clotting times of RBC-SN (58.17±16.10secs) and UC-EV pellets (45.93±7.87secs) were not significantly different from normal pooled plasma control (40.93±0.3secs). However, clotting times in these fractions were significantly faster (p<0.0001) than those of the EV-depleted-SN +/- filtration (215.7±5.87secs; 216.6±11.23secs), and the PPL-depleted plasma control (184.9±2.8secs). Furthermore, clotting times of isolated EV (73.91±34.43secs) were significantly quicker than isolated protein samples (183.4±8.44secs) (p<0.0001).
Conclusion:
EVs present in RBC-SN contributed to PPL-mediated coagulation. This may have implications for adverse transfusion outcomes in patients susceptible to thromboembolic complications and warrants further investigation of the EV characteristics.