Association of Circulating Procoagulant Microvesicles with Painful Vaso-Occlusive Crisis in Sickle Cell Disease

Abstract

Introduction: Thrombotic complication is one of the features of sickle cell disease (SCD), characterized by appearance of phosphatidylserine on the outer membrane of sickle-shaped red blood cells and most abundantly on membrane protrusions called microvesicles (MVs). However, the exact mechanism by which MVs may enhance coagulant activity in SCD patients has not been fully addressed. The aim of this study was to further investigate the procoagulant activity of circulating MVs in sickle cell crises.

Materials and methods: Subjects included in this cross-sectional study were 47 patients with SCD and 25 normal subjects with written informed consent obtained from all the participants. MV analysis was conducted by using CD61, CD235α, and Annexin-V monoclonal antibodies. The coagulant activity of MVs was determined by an ELISA-based procoagulant activity assay.

Results: The majority of MVs were originated from platelets (CD61+) and erythrocytes (CD235+). These MVs demonstrated significantly enhanced levels during the painful crisis when compared with the steady-state period (p < 0.001) and controls (p < 0.001). Also, the procoagulant activity of MVs was significantly higher in crisis compared to those of steady state (p < 0.001) and positively correlated with the number of Annexin-V+ MVs (p < 0.001). Significant correlations were found between erythrocyte-derived MVs with hemolysis marker (r = 0.51, p < 0.001) and the hemoglobin level (r = -0.63, p < 0.001).

Conclusion: The numbers of platelet- and erythrocyte-derived MVs are related to painful crisis, and their quantification in SCD may be helpful for identifying cases at increased risk of thrombotic complications.

Keywords: Coagulation; Crisis; Microvesicles; Sickle cell disease.

Fig. 1

Flow cytometry plots of MVs in SCD patients. FSC and SSC indicate, respectively, size and granularity. 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, MVs are located lower than 1.0 μm beads. b A histogram of logarithmic FSC vs. count, showing the distribution of MVs in comparison to the beads. c Region RN1 represents region R1 events that were labeled with conjugated FITC anti-CD235a and indicates erythrocyte-derived MVs. d Region RN2 represents region R1 events that were labeled with conjugated PE anti-CD42b and indicates platelet-derived MVs. e Region RN1 represents region R1 events that were labeled with conjugated FITC Annexin-V. Nonstained events are demonstrated by the left peak in c–e.

Fig. 2

Quantification of different MVs in SCD patients and controls. Total count of different MVs according to the expression of CD61, CD235a, and Annexin-V that represents platelet, erythrocyte, and procoagulant MVs, respectively. Plot revealed a statistically significant increase of MVs in painful crisis and steady state compared to controls (*p < 0.001 by ANOVA). Results are mean ± SD.

Fig. 3

Assessment of procoagulant activity due to PS-expressing MVs and its correlation with the number of Annexin-V+ MVs in SCD patients. a Procoagulant activity of collected samples in SCD patients compared to control. There was significant difference between procoagulant activity in painful crisis compared to steady state and control (*p < 0.001; by paired t test). b Correlation of Annexin-V+ MVs and procoagulant activity measurements. Change in the number of Annexin-V+ MVs correlated with the change in procoagulant activity in painful crisis (r = 0.86, p < 0.001 by Pearson's test) and steady state (r = 0.91, p < 0.001 by Pearson's test).