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    Poster Networking

    Platelets and Megakaryocytes

    Poster Networking Session

    PB0865 - Endothelial Extracellular Vesicles Regulate In Situ Pulmonary Thrombosis in Sickle Cell Disease

    Monday, June 26, 2023
    18:30 – 19:30 ET
    Room: Exhibition
    Background: Sickle cell disease (SCD) is the most common hemolytic disorder affecting millions of people worldwide. Recent studies show that acute in situ pulmonary thrombosis (ISPT) is the underlying cause in ~20% of SCD patients hospitalized with respiratory failure. Although we recently discovered that adenosine diphosphate (ADP) promotes ISPT following acute-hemolysis in wild-type mice, P2Y12 receptor antagonists have shown no benefit to SCD patients in recent clinical trials. Importantly, intravascular ADP homeostasis is maintained by CD39. Though CD39 inhibits ADP-dependent platelet activation, its role in SCD is still unidentified.

    Aims: To determine whether circulating CD39-bearing endothelial extracellular vesicles (EVs) degrade excess ADP to prevent ISPT in SCD.

    Methods: To evaluate SCD platelet response to ADP in vivo we used intravital lung microscopy and a novel in vivo model of ADP-triggered thrombocytopenia in transgenic SCD mice. Additionally, both mouse and human SCD platelet ADP-dependent aggregation was examined using in vitro turbidimetric aggregation assay. Hemin, a major host-derived damage associated molecular pattern associated with SCD, was incubated with Human Lung Micro-Vascular Endothelial Cells (HMVEC-L). EVs were obtained from mouse and human plasma samples, and cell culture medium samples using size exclusion chromatography. Obtained EVs were subjected to nanoparticle tracking analysis. EVs CD39 levels and activity were determined using western blot and malachite green phosphate assays, respectively.

    Results: Platelet function tests clearly demonstrated that ADP-induced platelet aggregation and ISPT were impaired in SCD which was, further, significantly augmented by a specific CD39 inhibitor. Hemin triggered shedding of CD39-bearing EVs by HMVEC-L. Indeed, we discovered that isolated from mouse and human plasma endothelial cell-derived SCD EVs expressed higher CD39 levels and activity in comparison to control EVs.

    Conclusion(s): Our findings suggest that CD39-bearing endothelial EVs prevent ADP-mediated ISPT in SCD. Current study explains why P2Y12 blockers are not effective in SCD therapy.