Interaction of Red Cell Microparticles (RMP) with Platelets: Potential Role of RMP in Hemostasis and Thrombosis,

Abstract 3263 Introduction: We previously reported data indicating that RMP are well suited for use as hemostatic agent for treating bleeding disorders ([Jy et al, Hemophilia 17:4, 2011][1]). Previous studies have shown that RMP can contribute to RBC-related thrombotic complications such as sickle cell disease and PNH. Microparticles (MP) derived from platelets (PMP), endothelia (EMP), and leukocytes (LMP) are believed to play a role in hemostasis and thrombosis. They can adhere to blood cells and endothelia, facilitating prothrombotic and proinflammatory reactions. However, less is known about interaction of RMP with cells and their potential role in hemostasis and thrombosis. Here we report evidence of interaction of RMP with platelets resulting in enhanced platelet aggregation and increased size of adherent platelet aggregates induced by shear stress. Methods: (i) RMP were prepared by high-pressure extrusion of washed RBC. (ii) Platelet aggregation was performed in a Chrono-log aggregometer. PRP (490 μL) was mixed with 10 μL of RMP (1 × 108 /mL final conc.) for 5 min, then low-dose activating agent (ADP 0.2 μM, or arachidonic acid (AA) 0.3 mM) was added. (iii) Shear-induced platelet adhesion was measured in a cone-and-well device (Diamed Impact-R). Whole blood was pre-incubated with RMP as above for 10 min, then subjected to various shear rates (900, 1800, 2700 sec−1) for 1 min. The adherent platelets were then washed, stained, and quantitated by image analyzer. (iv) RMP-platelet interaction employed 2-color flow cytometry. RMP-platelet conjugates were identified by co-expression of α-CD41-FITC and α-glycophrin A-PE, in both the free platelet and micro-aggregated platelet populations. Results: (1) Platelet aggregation: Addition of RMP to PRP did not induce platelet aggregation. However, RMP enhanced platelet aggregation induced by low-dose ADP or AA. Low-dose ADP alone induced a transient increase of aggregation peaking at 25–35% followed by slow disaggregation to 0–5% at 10 min; but in presence of RMP, a similar rate (slope) of aggregation was seen but peaking at 50–60% and disaggregation was abolished. Using AA, the RMP also potentiated aggregation from 20–30% to 50–60%. These results were obtained with heparinized PRP. Interestingly, when citrated PRP was used, the RMP effect was negligible. (2) Shear-induced platelet adhesion: At 1800 sec−1 shear rate, which approximates venous blood flow, addition of RMP increases the adhered mean aggregate size from 47 to 53 μm2 (p<0.03) but decreased the number of adhering objects from 1380 to 1242 (p<0.01). At lower (900 sec−1) or higher (2700 sec−1) rate, the RMP effects disappeared. (3) Two-color flow cytomrtry showed that RMP do not conjugate with single platelets but do with platelet micro-aggregates induced by ADP. When platelet micro-aggregates are diluted with PBS (1:10), they usually disaggregate rapidly (t1/2 = 10–15 min) but in presence of RMP, the rate of dissociation was much slower (t1/2 = 30–40 min). Conclusions: These results reveal that RMP can interact with weakly activated platelets to enhance platelet adhesion and aggregation and stabilize platelet aggregates. Since these effects were seen with heparinized but not with citrated blood, calcium may be a cofactor for this interaction. We suggest that RMP-platelet interaction could play a role in hemostasis, and that therapeutic RMP may improve hemostatic abnormality in thrombocytopenia and platelet dysfunction partly by this mechanism, augmenting the limited platelet function. Disclosures: No relevant conflicts of interest to declare. [1]: #citation-1