Microvesicles from stored red blood cells induce P-selectin and von Willebrand factor release from endothelial cells via a protein kinase C-dependent mechanism

Abstract

Introduction: The use of packed red blood cells (pRBCs) for resuscitation is limited by the red blood cell storage lesion, a series of biochemical and physiological changes that occur during the storage and aging of blood. Microvesicles (MVs) shed from pRBCs during this process are one component of the red blood cell storage lesion and lead to acute lung injury and pulmonary vascular microthrombi. We hypothesized that MVs from stored pRBCs lead to the release of P-selectin and von Willebrand factor (vWF) from endothelial cells and that this mechanism is mediated via activation of protein kinase C (PKC) or protein kinase A (PKA).

Methods: Leukoreduced, platelet-poor murine pRBCs were isolated from C57BL/6 8-12 week-old male mice via cardiac puncture, prepared via centrifugation using a Ficoll gradient, and stored for up to 14 days, the equivalent of 42 days of storage in humans. MVs were isolated from the stored pRBC units via sequential high-speed centrifugation. Murine lung endothelial cells (MLECs) were cultured and grown to confluence, then treated with MVs and either calphostin C, a PKC inhibitor (10 μg/mL), or PKI 14-22 amide, a PKA inhibitor (10 μM). The supernatant was collected after 1 h. P-selectin and vWF A2 concentrations were quantified via ELISA. Immunofluorescent staining for vWF was performed on MLECs. Statistical analysis was performed via unpaired t-test or ANOVA as indicated and reported as mean ± SD. Concentration is reported as pg/mL.

Results: MLECs treated with MVs isolated from stored pRBCs demonstrated increased release of P-selectin and vWF A2 in a dose-dependent fashion. MLECs treated with MVs prepared from stored as compared to fresh pRBCs demonstrated increased release of P-selectin (3751 ± 726 vs 359 ± 64 pg/mL, p < 0.0001) and vWF A2 (3141 ± 355 vs 977 ± 75 pg/mL, p < 0.0001) with increasing duration of storage. The treatment of MVs with calphostin C decreased the amount of P-selectin (1471 ± 444 vs 3751 ± 726 pg/mL, p < 0.0001) and VWF A2 (2401 ± 289 vs 3141 ± 355 pg/mL, p = 0.0017) released into the supernatant by MLECs compared to MVs alone. The treatment of MVs with PKI 14-22 increased the amount of P-selectin released compared to MVs alone (1999 ± 67 vs 1601 ± 135 pg/mL, p = 0.0018).

Conclusions: MVs from stored pRBCs stimulate the release of P-selectin and VWF A2 from endothelial cells. The effect of MVs increases with both dose of MVs and age of stored pRBCs from which they are formed. This mechanism is dependent on activation of PKC and inhibition of this enzyme represents a potentially significant strategy to modulate the inflammatory response to resuscitation with stored pRBCs.

Keywords: Endothelial cells; Microvesicles; P-selectin; Red blood cells; von Willebrand factor.

Figure 1.

Release of P-selectin and vWF into the supernatant by MLECs 1 hour after treatment with MVs isolated from aged pRBCs. (A) Release of P-selectin after treatment with MVs isolated from pRBCs stored for 1, 7, or 14 days. (B) Release of vWF A2 after treatment with MVs isolated from pRBCs stored for 1, 7, and 14 days. n=5 for all experiments. ** indicates p<0.01 as compared to indicated group. *** indicates p<0.001 as compared to indicated group. **** indicates p<0.0001 as compared to indicated group.

Figure 2.

Time course and dose dependency or P-selectin and vWF release into the supernatant by MLECs after treatment with MVs isolated from pRBCs stored for 15 days. (A) P-selectin in supernatant of MLECs after 30, 60, and 120 minutes. (B) Release of vWF A2 over time after treatment with NVs. (C) Release of P-selectin and (D) vWF into the supernatant by MLECs after treatment with 0.1 or 1 unit of MVs isolated from aged pRBCs for 1 hour. n≥5 for all groups. * indicates p<0.05 as compared to indicated group. **** indicates p<0.0001 as compared to indicated group.

Figure 3.

The effect of PKA activation and inhibition on the release of P-selectin and vWF by MLECs. (A) P-selectin concentrations in supernatant after 1 hour of treatment with 8-Br-cAMP. (B) vWF A2 concentrations after 1 hour of treatment with 8-Br-cAMP. (C) P-selectin concentrations after 1 hour of treatment with PKI 14–22 in addition to MVs as compared to MVs alone. n=5. (D) vWF A2 concentrations after 1 hour of treatment with PKI 14–22 with MVs as compared to MVs alone. n=5 for all experiments. * indicates p<0.05 as compared to indicated group. ** indicates p<0.01 as compared to indicated group. *** indicates p<0.001 as compared to indicated group. **** indicates p<0.0001 as compared to indicated group.

Figure 4.

The effect of PKC activation and inhibition on the release of P-selectin and vWF. (A) P selectin concentrations at 1, 2, and 4 hours after treatment of MLECs with PMA. (B) vWF A2 concentrations at 1, 2, and 4 hours after treatment of MLECs with PMA. (C) P selectin concentrations at 4 hours of treatment with media, PMA, or calphostin C with PMA. (D) vWF A2 concentrations 4 hours after treatment of MLECs with media, PMA, or calphostin C with PMA. n=5 for all experiments. * indicates p<0.05 as compared to indicated group. ** indicates p<0.01 as compared to indicated group. **** indicates p<0.0001 as compared to indicated group.

Figure 5.

The effect of PKC inhibition on the pRBC MV-induced release of P-selectin and vWF into the supernatant by MLECs. (A) P selectin concentrations 1 hour after treatment of MLECs with media, pRBC MVs, or MVs with the addition of calphostin C. (B) vWF A2 concentrations 1 hour after treatment of MLECs with media, pRBC MVs, or MVs with the addition of calphostin C. n≥5 for all groups. ** indicates p<0.01 as compared to indicated group. **** indicates p<0.0001 as compared to indicated group.