Microparticles from stored red blood cells promote a hypercoagulable state in a murine model of transfusion

Background

Red blood cell-derived microparticles are biologically active, submicron vesicles shed by erythrocytes during storage. Recent clinical studies have linked the duration of red blood cell storage with thromboembolic events in critically ill transfusion recipients. In the present study, we hypothesized that microparticles from aged packed red blood cell units promote a hypercoagulable state in a murine model of transfusion.

Methods

Microparticles were isolated from aged, murine packed red blood cell units via serial centrifugation. Healthy male C57BL/6 mice were transfused with microparticles or an equivalent volume of vehicle, and whole blood was harvested for analysis via rotational thromboelastometry. Serum was harvested from a separate set of mice after microparticles or saline injection, and analyzed for fibrinogen levels. Red blood cell-derived microparticles were analyzed for their ability to convert prothrombin to thrombin. Finally, mice were transfused with either red blood cell microparticles or saline vehicle, and a tail bleeding time assay was performed after an equilibration period of 2, 6, 12, or 24 hours.

Results

Mice injected with red blood cell-derived microparticles demonstrated an accelerated clot formation time (109.3?±?26.9 vs 141.6?±?28.2?sec) and increased α angle (68.8?±?5.0 degrees vs 62.8?±?4.7 degrees) compared with control (each P?<?.05). Clotting time and maximum clot firmness were not significantly different between the 2 groups. Red blood cell-derived microparticles exhibited a hundredfold greater conversion of prothrombin substrate to its active thrombin form (66.60?±?0.03 vs 0.70?±?0.01 peak OD; P?<?.0001). Additionally, serum fibrinogen levels were lower in microparticles-injected mice compared with saline vehicle, suggesting thrombin-mediated conversion to insoluble fibrin (14.0 vs 16.5?µg/mL, P?<?.05). In the tail bleeding time model, there was a more rapid cessation of bleeding at 2 hours posttransfusion (90.6 vs 123.7?sec) and 6 hours posttransfusion (87.1 vs 141.4?sec) in microparticles-injected mice as compared with saline vehicle (each P?<?.05). There was no difference in tail bleeding time at 12 or 24 hours.

Conclusion

Red blood cell-derived microparticles induce a transient hypercoagulable state through accelerated activation of clotting factors.