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2017 Cardiovascular Research Day Abstract Book

101 Identification of a

101 Identification of a novel lipoprotein microRNA carrier in plasma Wanying Zhu 1 • Danielle L. Michell 1 • Ryan M. Allen 1 • Kasey C. Vickers 1 Vanderbilt University Medical Staff Recently, multiple groups have detected of microRNAs (miRNA) in plasma by high-throughput sequencing. Circulating sRNAs are likely protected from RNases in plasma through their association with lipid carriers. Our lab previously reported that lipoproteins (HDL and LDL) transport miRNAs in plasma within potential intercellular communication networks. Based on these previous studies, we sought to define the distribution of miRNAs across lipid carriers in plasma. Methods: To specifically precipitate carriers, whole plasma and pre-purified HDL (density-gradient ultracentrifugation) treated with lipid removal agents (LRA and Cleanascite) for the separation of lipid-associated miRNAs. Plasma samples from humans and mice were fractionated using sizeexclusion chromatography (FPLC, Superdex 200 Increase in triplicate). Each fraction was assessed for total cholesterol, triglycerides, protein, phospholipids, and markers for lipoproteins and extracellular vesicles by western blotting. Carrier sizes in each fraction were quantified using dynamic light scattering. Total RNA was isolated from whole fractions and separated contents, lipid pellets and supernatants, after centrifugation. Real-time PCR and high-throughput sequencing were used to quantify miRNAs. Results: Results demonstrated that Cleanascite efficiently removed lipids from plasma without damaging lipoproteins, as determined by the lack of phospholipids and apolipoproteins in the supernatants. Candidate miRNAs (miR-24-3p, miR-26a-5p, miR-146a-5p, miR-92a-3p, and miR- 223-3p) were detected in fractions corresponding to HDL and LDL based on the distribution of cholesterol and triglycerides. Strikingly, candidate miRNAs also entirely separated with lipids in fractions that contained low cholesterol and high phospholipid content with a mass corresponding to very small HDL. These fractions were found to contain apoA-I and spherical particles

102 The Ral-Exocyst Pathway Regulates Platelet Secretion Jinchao Zhang 1 and Sidney W. Whiteheart 1 1 Department of Molecular and Cellular Biochemistry, College of Medicine, University of Kentucky Postdoc In Platelets, Exocyst complex has been identified as an octamer consisting of Sec3, Sec5, Sec6, Sec8, Sec10, Sec 15, Exo70 and Exo84 in platelets. The complex plays an essential role in tethering vesicles to the plasma membrane and regulating dense core granule secretion in platelets. RalA/B (family members of GTPases) are regarded as the upstream regulator of the Exocyst complex, and RalA/B binds to Sec5 and Exo84 in a GTP-dependent manner. RalA/B, sharing over 88% identity in sequences, directly interact with Exo84 and Sec5 in the Exocyst complex. The role of RalA/B are interchangeable, but RalA has the higher ability to bind to the Exocyst components in GTP binding manner in cells. Several groups suggest that interrupting the association Sec3 and Exo70 with plasma membrane could disrupt exocytosis in other cells. However, it is little known how the role of the Exocyst complex in platelet secretion. Here we ask the question whether inhibitors of RalA/B could affect granule secretion or not in platelets. And what is the exact role of the Exocyst complex in granule secretion? How is the Exocyst complex regulating vesicles tethering in secretion process? Firstly, specific RalA/B inhibitors, RBC8 and BQU57, were used to investigate the role of RalA/B in granule secretions. From the biochemical perspective, these inhibitors affect RalA/B bound to their downstream effector (RLP76) due to the Ral-GDP bound forms. We also found the inhibitors of RalA/B affects platelet spreading (data are not shown here). At the same time, we also answer the questions of the localization of Rals and Exo70 (one of the Exocyst component) as well as their association with F-actin. 118

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