Procoagulant Activity of Red Blood Cell-Derived Microvesicles during Red Cell Storage

Abstract

Background: Red blood cells (RBCs) undergo structural and biochemical alterations during storage which are collectively called RBC storage lesion and cause a decrease in RBC recovery and survival. During storage, erythrocytes release an increasing number of microvesicles (MVs) that have key roles in biological processes. We aimed to investigate the procoagulant activity (PCA) of RBC-derived MVs during storage.

Methods: 20 packed RBCs were stored for up to 42 days. Samples were taken at seven different times and evaluated for the presence of RBC-MVs. MVs were separated, and following filtration flow cytometry was used to characterize RBC-MVs based on the expression of glycophorin A (Gly.A) and annexin V (AnnV) antigens. The coagulant activity of RBC-MVs was tested by clotting time (CT) and PCA assays. Results were compared before and after filtration.

Results: Flow cytometry revealed a 17.6-fold increase in RBC-MVs after 6 weeks of storage. Significant correlations were found between AnnV+ MVs and PCA (r = 0.96; p < 0.001), and CT (r = -0.77; p < 0.001) which was associated with increased PCA and shortened CT with RBC aging. Filtration of samples efficiently removed MVs (p < 0.001) and also reduced in vitro PCA of MVs (p < 0.001).

Conclusion: RBC-MVs are procoagulant (particularly AnnV+ MVs) Reduction of MVs from RBC concentrates may reduce the risk of transfusion-induced thrombotic complications.

Keywords: Filtration; Microvesicles; Procoagulant activity; Red blood cell storage.

Fig. 1

Flow cytometry plots of RBC-MVs in packed RBCs. FSC and SSC indicates size and granularity, respectively. The MV size gate was set by 1.0 μm beads. a Two gating regions are region R1 and R2 that represents MVs and 1.0 μm beads, respectively. Based on the FSC and SSC, RBC-MVs are located lower than 1.0 µm beads. b A histogram of logarithmic forward scatter versus count, showing the distribution of MVs in comparison to the beads. c Region RN1 represents region R1 events that were labeled with conjugated FITC-AnnV. d Region RN2 represents region R1 events that were labeled with conjugated PE anti-Gly.A and indicates RBC-derived MVs. e Region RN1 represents region R1 events that were labeled with conjugated FITC anti-CD42b, which indicates platelet-derived MVs. Non-stained events are demonstrated by the left peak in figure 1 c-e.

Fig. 2

Quantification of RBC-MVs in supernatant released by RBCs during storage of 20 different packed RBCs. a The number of total MV/μl that were counted based on the FSC and SSC. Plot revealed a statistically significant increase of MV number during storage (p < 0.0001 by ANOVA). *Significant difference (p < 0.001) between pre- and post-filtration MVs, by paired t-test. ^#Significant change (p < 0.001) over storage period, by ANOVA. b Total count of different MVs according to the expression of Gly.A, CD42b and AnnV that represents RBC, platelet and procoagulant MVs, respectively. Results are mean + SD.

Fig. 3

Assessment of CT on different days of storage and its correlation with the number of AnnV+ MVs in stored RBCs (n = 20). a CT changes during storage. The plot showed that CT decreased significantly in all units during storage (p < 0.001 by ANOVA). The minimum CT was observed at day 42. b Scatter plot showing the correlation between the number of AnnV+ MVs and CT. There was an inverse relationship between MVs and CT (r = −0.776, p < 0.001 by Pearson's test).

Fig. 4

Assessment of PCA due to PS-expressing MVs and its correlation with the number of AnnV+ MVs in stored RBCs (n = 20). a PCA of pre- and post-filtration samples during storage. The PCA increased significantly over time (^#p < 0.001; by ANOVA), and there were significant differences between PCA in pre- and post-filtration samples at all times (*p < 0.001; by paired t-test). b Correlation of AnnV+ MVs and PCA measurements. Change in AnnV+ MV number correlated best with the change in PCA (r = 0.96, p < 0.001 by Pearson's test). Data are presented as mean ± standard deviation; d0 = day 0; d42 = day 42 of storage.