Y. Kim1, B. T. Xia1, A. D. Jung1, A. L. Chang1, R. M. Schuster1, R. A. Veile1, M. D. Goodman1, T. A. Pritts1 1University Of Cincinnati,Surgery,Cincinnati, OH, USA
Introduction: During storage, packed red blood cells undergo a series of physical, metabolic, and chemical changes collectively known as the red blood cell storage lesion. One key component of the red blood cell storage lesion is the accumulation of microparticles, which are submicron vesicles shed from erythrocytes as part of the aging process. Previous studies from our laboratory indicate that transfusion of these microparticles leads to lung injury, but the mechanism underlying this process is unknown. In the present study, we hypothesized that microparticles from aged packed red blood cell units induce procoagulant activity via accelerated thrombin generation.
Methods: Microparticles were isolated from leukoreduced, platelet-depleted, stored murine erythrocytes via ultracentrifugation. These microparticles were added to microplate wells supplemented with Factor Xa-Va mixture, calcium, thrombin inhibitors, and purified prothrombin and the activity of the generated thrombin substrate was measured. We also investigated the effects of microparticle transfusion on the murine pulmonary vasculature. In separate experiments, mice were transfused with erythrocyte-derived microparticles or an equivalent volume of vehicle. After 1, 4, 8, or 24 hours, lungs were harvested and analyzed under light microscopy or Martius Scarlet Blue (MSB) staining. Blood was harvested from a separate set of mice following injection with either microparticles or saline vehicle and tested for fibrinogen levels.
Results: Erythrocyte-derived microparticles demonstrated a nearly hundredfold greater conversion of prothrombin to thrombin than controls (66.60±0.03 vs 0.70±0.01 peak OD; p<0.0001). Fibrinogen levels were significantly lower in mice injected with microparticles compared with saline vehicle (14.0 vs 16.5 μg/mL, p<0.05), suggesting conversion of fibrinogen to fibrin. Mice injected with erythrocyte-derived microparticles exhibited a significantly greater number of congested pulmonary vessels at 1 hour (10.0 vs 6.5 vessels/HPF) and 4 hours (10.3 vs 6.4 vessels/HPF) as compared to control mice (p<0.05 each) and elevated pulmonary fibrin deposition at 24 hours as determined by MSB staining (3.5±1.5 vs zero deposits, p<0.0001).
Conclusion: Erythrocyte-derived microparticles directly propagate the conversion of prothrombin to thrombin in our murine model. Microparticle treatment in vivo led to decreased fibrinogen and increased pulmonary vessel congestion and fibrin deposition. Microparticles from aged packed red blood cell units may damage injure recipient lungs through accelerated thrombin conversion.
