Red Blood Cells Microparticles Are Associated with Hemolysis Markers and Leg Ulceration in Patients with Sickle Cell Disease

Microparticles are sub-micron, unilamellar vesicles possessing protein and other materials derived from the plasma membrane of their parent cells and literature data suggest that they may have a role in the pathophysiology of SCD. Red blood cell microparticles (RMP) are derived from the red blood cells (RBC) as a result of shedding of unwanted surface features during reticulocyte maturation, apoptosis, and activation by endogenous triggers. Upon release, RMP carry trapped hemoglobin and heme along with them.

The contribution of RMP to hemolysis, endothelial cells lesions and clinical phenotypes of SCD is largely unknown. There are some data that suggest that, due to the repeated sickling-unsickling cycles and polymerization of hemoglobin S, patients with SCD are likely to generate more RMP in their blood than healthy subjects, and the generated RMP could contribute to the vascular lesions and vaso-occlusion. Hence, the current study aimed to study some aspects of MP in patients with SCD.

This study involved 118 adults with SCD (89 HbSS and 29 HbSC) in steady state, and on regular follow up and 39 healthy matched controls. Participants had their plasma RMP quantified by flow cytometry, plasma hemoglobin and heme by colorimetric assay kits and haptoglobin and hemopexin by ELISA kits. Since the identification of RMP is a procedure with several different approaches, in this work a very strict methodology was used (Splitter et al., 2016). RMP were measured by number per ml of blood. Automated analyzers were used to carry out hematologic and biochemical analyses and records of the SCD patients were examined for clinical events. Institutional review board approval was obtained and the study protocols followed the Declaration of Helsinki as revised in 2000.

We found that the SCD patients had higher levels of RMP than the controls (HbSS > HbSC > controls), 531,685.0±204,495.0 vs 171,034.0±54,144.0 vs 1,128.0±387.0/mL, respectively, P <0.0001). Similar patterns were observed with plasma hemoglobin (87.7±37.7 vs 60.19±19.7 vs 46.52±14.0mg/dL, P <0.0001), heme (62.5±32.4 vs 48.8±27.0 vs 35.2±16.4µM, P <0.0001), and HbF (14.6±8.2 vs 1.6±2.5 vs 0.29±0.2%, p<0.0001). On the contrary, a reverse order i.e HbSS < HbSC < controls was observed with haptoglobin (25.3±36.6 vs 135.5±160.0 vs 1205.0±553.0µg/mL, P <0.0001) and hemopexin (542.7±349.7 vs 669.0±296.3 vs 1029.0±146.6µg/mL, P <0.0001) and hemoglobin concentration (8.59±1.4 vs 11.3±1.4 vs 13.8±1.2g/dL, P <0.0001). When we tested the association between RMP and other parameters, we found that RMP strongly correlated positively with total bilirubin (r=0.49, p<0.0001), unconjugated bilirubin (r=0.509, P <0.0001), LDH (r=0.61,p<0.0001), reticulocyte counts (r=0.41, p<0.0001), plasma hemoglobin (r=0.46, P <0.0001), and heme (r=0.59, P <0.0001) in SCD patients. On the contrary, a modest negative correlation was observed between RMP and HbF (r= -0.31, P=0.0025), and hemopexin (r= -0.26, P=0.035). These data suggest that RMP is associated with hemolysis in the studied SCD cohorts.

We found no differences between SS patients with and without hydroxyurea n=68 & 50 respectively, (572,353.0±265,112.0 vs 400,000.0±127,511.0/ml, P=0.807). We also identified association between RMP and history of leg ulceration. SCD patients who had leg ulcer (n=17), had significantly higher levels of RMP compared with patients without leg ulceration (590,588±709,441/ml vs 422,000±1,809390/ml, P=0.0044). Similarly, they had higher LDH 461.5±186.9U/l vs 377.0±140.0U/l, P=0.04 and heme 82.6±45.5µM vs 55.8±27.7µM, P=0.0096.

In conclusion, these data suggest that RMP may be associated with hemolysis and leg ulceration in patients with SCD thus supporting the notion that RMP may have an important role in the pathophysiology of SCD. It is possible that RMP contribute to endothelial lesion and leg ulcer phenotype through the toxic injuries mediated by hemoglobin and heme trapped in them. Hence, therapies targeting RMP could be another strategy to combat SCD.