Evidence of Different Subspecies of Microparticles (MP) Derived from Red Blood Cells (RMP) and Comparison of Their Phenotypes and Procoagulant Activity.
Authors: Jy Wenche , Bang Jaehoon , Bidot Loreta , Lin Andrew , Jimenez Joaquin J. , Fontana Vincenzo , Ahn Eugene , Ahn Yeon-Soong 
Published: October 2019

Abstract
BACKGROUND: The potential roles of cell derived microparticles (MP) such as those derived from platelets (PMP), endothelium (EMP), leukocytes (LMP), and red cells (RMP) have been receiving increasing attention in disorders of hemostasis/thrombosis and inflammation and they are emerging as valuable biomarkers. However among these MP, little is known about RMP. Our recent clinical studies indicate that RMP play a role in hemostasis and thrombosis in patients with thrombocytopenia and in thrombocytosis. However, the phenotypes and procoagulant activity of their subspecies remain unknown. We report evidence for heterogeneity of RMP following differential centrifugation.
METHODS: RMP were prepared by exposure of washed RBC to the calcium ionophore, A23187, and the RBC were removed by low-speed centrifugation. The RMP were washed twice at 20,000xg for 15 min. Procoagulant activity of RMP was measured by the calibrated automated thrombogram (CAT) system (Hemker et al Pathophysiol Haemost Thromb.2002;32:249) using thrombin substrate Z-Gly-Gly-Arg-AMC on a fluorescence plate reader. The lag time and peak height (nM) of thrombin generation were recorded. Markers used for labeling RMP were PE-labeled anti-glycophorin (GlyP), FITC-anti-tissue factor (TF), FITC-annexin V (AnV), and/or FITC-lectin Ulex europeaus I (Ulex).
RESULTS: In thrombin generation assay, RMP induced a long lag time (24±3 min) but high thrombin peak (330±37 nM). These data were consistent with the flow cytometric finding that RMP carried very little TF (&lt;0.1%) but very high AnV binding (88±6%). By high speed centrifugation (15,000xg for 10 min), two populations of RMP were studied: the larger RMP in the pellet expressed GlyP, AnV and Ulex while the smaller or lighter RMP remaining in the supernatant, did not express GlyP and AnV but do express Ulex. The smaller RMP accounted for 30–40% of total Ulex+ RMP. These two subspecies (large and small) of RMP showed distinct thrombin generation profiles. The lag time and peak height of thrombin generation for large RMP (GlyP+/AnV+/Ulex+) was 23–28 min and 300–335 nM, respectively, which is close to values of whole RMP. On the other hand, the smaller RMP (Ulex+/GlyP−/AnV−) produced much longer lag time (31–38 min) and lower peak (60–75 nM), indicating that the majority of the procoagulant activity of RMP is associated with larger RMP.
SUMMARY: The present study demonstrates that RMP are rich in anionic phospholipids and effective in generating thrombin in vitro. We have identified 2 distinct subpopulations of RMP by differential centrifugation: One larger RMP express binding of anti-GlyP, AnV and Ulex, and carry the majority of procoagulant activity. The smaller RMP expressing only Ulex binding exhibit much weaker procoagulant activity. The roles of these two species of RMP remain to be elucidated. We speculate that smaller RMP may represent the nanovesicles described by Allen et al [Biochem J 188:881, 1980] and that Ulex may be a novel and convenient means for the study of these small vesicles.