A. D. Jung1, Y. Kim1, W. Abplanalp1, A. T. Makley1, M. D. Goodman1, T. A. Pritts1 1University Of Cincinnati,Surgery,Cincinnati, OH, USA
Introduction: Transfusion with packed red blood cells (pRBCs) remains essential for resuscitation after hemorrhage. During storage, pRBCs undergo a series of physical and biochemical changes that are collectively known as the red blood cell storage lesion. Previous work from our laboratory shows that this is associated with harmful effects in the transfusion recipient, including alterations in the coagulation cascade. Tissue factor, a constitutively expressed cell-surface glycoprotein, plays a key role in initiating the coagulation cascade, but the effect of transfusion of stored pRBCs on endothelial tissue factor expression is unknown. We hypothesized that aged pRBCs alter endothelial cell tissue factor expression.
Methods: Blood was harvested from murine donors, then separated into components. Leukoreduced and platelet-depleted pRBC units were stored in additive solution-3 at 4°C for 14 days. Microparticles were isolated from aged pRBC units by ultracentrifugation. In a simulated in vitro transfusion model, murine lung endothelial cells were incubated with fresh or 14-day-old erythrocytes suspended in media at 37°C for 1 h and endothelial cell tissue factor expression was analyzed via immunofluorescence. In a separate experiment, endothelial cells were exposed to aged vs fresh erythrocytes, then tissue factor expression was analyzed by Western blot. The third experiment involved exposing endothelial cells to media or isolated microparticles from aged pRBC units, and soluble tissue factor was determined using ELISA at 0.5, 6, 12, and 24h.
Results: Whole cell immunofluorescent analysis of endothelial cells exposed to aged pRBCs demonstrated significant decreases in tissue factor expression vs baseline (11.7±0.7 vs 16.9±0.6 RFI, p<0.05). Western Blot analysis also demonstrated a significant decrease in tissue factor expression as compared to baseline. Treatment of endothelial cells with microparticles isolated from stored PRBC units resulted in a significant attenuation in tissue factor concentration at 30 minutes (4.3±0.4 vs 6.2±0.8, p<0.05), 6 hours (3.63±0.3 vs 5.5±0.6, p<0.05), and 24 hours (2.8±0.6 vs 4.7±0.5, p<0.05) as determined by ELISA.
Conclusion: Treatment of endothelial cells with aged pRBCs attenuates expression of tissue factor. Additional experiments indicate that this process is driven by red blood cell microparticles. Together, these data provide further evidence that transfusion with stored pRBC units causes endothelial cell dysfunction.
