PB0753 Circulating Levels of Podoplanin in Sickle Cell Disease

Background: Platelets are involved in hemostasis and innate immune processes.Dengue (DENV) and Zika (ZIKV) viruses’ encodes 3 structural and 7 nonstructural proteins.Nonstructural protein 1 (NS1) is mainly involved in the replication of viral genetic material.NS1 can be secreted into the extracellular microenvironment and has been associated with immune response evasion.Aims: The aim of this work was to evaluate the platelet response to NS1 protein of dengue and Zika viruses.Methods: Blood samples were obtained from healthy volunteers by venipuncture after signing an informed consent form, using 3.2% sodium citrate as an anticoagulant.Platelet-rich plasma (PRP) was obtained by slow centrifugation (100 g × 10 minutes).PRP was separated and resuspended in Tyrodes buffer.PRP (1X107 cells/ml) was stimulated with DENV and ZIKV NS1 proteins [2.5 ug/ml] for different times, 37°C.Platelet activity was assessed by flow cytometry: CD41-PECy7, CD62-PE, and PAC1-FITC.For Atomic Force Microscopy (AFM) a NT-MDT ® NTEGRA AFM and a 0.6 μm SNL-10 BRUKER ® cantilever and nitride-based silicone tip were used.For Quartz Crystal Microbalance assays a QCMopen, Novaetech ® was used.ADP 20 μM, collagen 20 uM, and epinephrine 100 μM were used as platelet activation known agonists.Results: We observed the highest expression of CD62P and PAC at 60 minutes of stimulation with both proteins.By AFM, plateletś pseudopod emission and increase in size were observed upon stimulation with the viral proteins.With the QCM assay, a significant increase in the quartz oscillation frequency difference was observed when injecting PRP on quartz coated with the NS1 protein of each of the viruses, in contrast to that observed when injecting PPP and wash buffer. Conclusion(s):There is platelet reactivity in response to stimulation with DENV and ZIKV NS1 proteins assessed by flow cytometry, atomic force microscopy and QCM.No differences in activation were observed between Zika and Dengue NS1 proteins.