Microparticles from aged packed red blood cell units stimulate pulmonary microthrombus formation via P-selectin

Abstract

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 pulmonary thrombosis.

Materials and methods: Leukoreduced, platelet-depleted, murine packed red blood cells (pRBCS) were prepared then stored for up to 14 days. Microparticles were isolated from stored units via high-speed centrifugation. Mice were transfused with microparticles. The presence of pulmonary microthrombi was determined with light microscopy, Martius Scarlet Blue, and thrombocyte stains. In additional studies microparticles were labelled with CFSE prior to injection. Murine lung endothelial cells were cultured and P-selectin concentrations determined by ELISA. In subsequent studies, P-selectin was inhibited by PSI-697 injection prior to transfusion.

Results: We observed an increase in microthrombi formation in lung vasculature in mice receiving microparticles from stored packed red blood cell units as compared with controls. These microthrombi contained platelets, fibrin, and microparticles. Treatment of cultured lung endothelial cells with microparticles led to increased P-selectin in the media. Treatment of mice with a P-selectin inhibitor prior to microparticle infusion decreased microthrombi formation.

Conclusions: These data suggest that microparticles isolated from aged packed red blood cell units promote the development of pulmonary microthrombi in a murine model of transfusion. This pro-thrombotic event appears to be mediated by P-selectin.

Keywords: Endothelial activation; P-selectin; Pulmonary thrombus; RBC microparticles.

Figure 1.

(A) Representative photomicrographs demonstrating differences in murine lung vasculature between mice transfused with microparticles stored pRBC units (RBC MP) as compared to an equivalent volume of saline vehicle (control). (B) Quantification of the number of microthrombi observed per high-powered field at one and four hours after transfusion. *p<0.05 compared with control, n=8 at each time point.

Figure 2.

Representative photomicrographs from lung of mice transfused with red blood cell-derived microparticles (RBC MP) or saline vehicle (control). Immunohistochemical staining revealed the presence of platelet aggregates (top, dark brown) within these vessels. Martius Scarlet Blue (MSB) staining (bottom) revealed the presence of mature fibrin deposits (red).

Figure 3.

(A) Mice were injected with CFSE-labelled microparticles (green) isolated from murine packed red blood cell units. The presence of microparticles within pulmonary vasculature was confirmed through immunofluorescent microscopy. (B) Mice transfused with microparticles had the greatest number of microparticles per high-powered field at one hour, followed by a temporal decrease in quantity over 24 hours. *p<0.05 compared with one hour, n=8 at each time point. (C) Representative lung sections after staining with anti-thrombocyte stain (left) and immunofluorescence microscopy (right) demonstrated the presence of microparticles (green) within these microthrombi.

Figure 4.

Red blood cell derived microparticles promote the release of P-selectin from murine lung microvascular endothelial cells in a dose-response fashion. *p<0.05 compared with control, n=8.

Figure 5.

(A) Representative photomicrographs from lungs from mice treated with microparticles isolated from pRBC units (RBC MP) or normal saline (control) with or without the P-selectin inhibitor PSI-697 or vehicle (DMSO). (B) Quantification of microthrombi in lungs from mice treated with pRBC microparticles (MP) or normal saline (ctrl) with or without the P-selectin inhibitor PSI-697 or vehicle (DMSO). *p<0.05 vs control.