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2017 Cardiovascular Research Day Abstract Book

57 Fine Tuning Platelet

57 Fine Tuning Platelet Secretion to Modulate Hemostasis Smita Joshi, MS 1 • Irina Pokrovskaya, MS 2 • Brian Storrie, PhD 2 • Sidney W. Whiteheart, PhD 1 1Molecular and Cellular Biochemistry, University of Kentucky • 2 Department of Physiology and Biophysics, University of Arkansas for Medical Sciences Graduate Student Globally, occlusive thrombotic events: e.g., heart attacks and cerebral strokes, cause > 50% of total deaths attributed to the noninfectious disease. However, aggressive attempts to limit thrombosis cause bleeding, which can be equally catastrophic. What is needed is a strategy to limit clot formation, but not prevent it. Platelets play a critical role in controlling bleeding by sensing vascular damage and releasing a host of components to seal breaches. This secretion process is mediated by Soluble N-ethylmaleimide Sensitive Factor Attachment Protein Receptors (SNAREs) and their regulators. To drive secretion, vesicle (v)-SNARE on granules and target (t)-SNARE on the plasma membrane (PM) form a trans-bilayer complex that mediates membrane fusion. Syntaxin 11 and SNAP-23 form the functionally relevant t-SNARE heterodimer. For v-SNAREs, platelets contain Vesicle-Associated Membrane Protein (VAMP)-2, -3, -4, -5, -7, and -8. We focused on how the platelet VAMPs influence secretion and whether modulating secretion can modulate clot formation. To address this goal, we genetically titrated the different VAMPs to define their roles in exocytosis and hemostasis. We gathered global VAMP-3-/- and VAMP-8-/- animals. To overcome embryonic lethality of global VAMP-2 deletion, we generated platelet-specific VAMP-2/3-/- mice by using a tissue-specific promoter that facilitates expression of tetanus toxin that cleaves VAMP-2 and 3. We crossed these with VAMP-8-/- mice to create platelet-specific VAMP-2/3/8-/- mice. Structural analysis of wildtype and VAMP-deficient platelets showed that the α granule cargo solubilization/decondensation follows granule fusion. To define the structure of secretion, activation intermediates were fixed at various time points, post stimulation, and electron microscopy was performed. The data indicate that granule decondensation is time- and agonist concentration-dependent. Moreover, decondensation of granule cargo was VAMP dependent. Three dimensional EM analyses indicate that VAMP-8 plays a major role in compound, intra-granule fusion and also contributes to single, granule-PM fusion. Our structural data elucidate how platelet secretion occurs at the cellular level and explains the complex secretion kinetics previously reported in activated platelets. To further measure the functional importance of the VAMPs, ex vivo secretion assays were used to monitor the kinetics and the extent of release from all three platelet granules (dense, α, and lysosomes). Only VAMP-2/3/8-/- platelets showed a robust defect in secretion (~70% decrease), more than observed for VAMP-8-/- platelets (~50%). When we studied the effects of secretion on hemostasis, only VAMP-2/3/8-/- mice showed significantly increased tail-bleeding times and delayed arterial thrombosis. VAMP-8-/- animals did show a delay in thrombus formation but no overt bleeding diathesis. Our data show that small differences in secretion kinetics alter hemostasis, thus by modulating platelet secretion, we can control thrombus formation without inducing pathological bleeding. These data identify the secretory machinery as a viable target to control occlusive cardiovascular diseases. Our work is the first comprehensive study showing how by targeting secretion we can achieve the long-sought balance between occlusive thrombosis and spurious hemostasis. Additionally, by titrating amounts and types of VAMPs in platelets we have created a valuable set of animals to precisely analyze the role of platelet secretion in other vascular processes. 73

58 The XY sex chromosome complement augments Ang-II induced aortic arch aneurysms in female LDLr-/- mice Cassandra Woolley 1 • Yasir Al-Siraj 1 • Sean Thatcher, PhD 1 • Lisa Cassis, PhD 1 1Department of Pharmacology and Nutritional Sciences, University of Kentucky Undergraduate Objective: Sex difference in cardiovascular disease has been a subject of research efforts aiming to improve the efficacy and approach of therapeutics available. In humans, abdominal aortic aneurysms (AAAs) display sexual dimorphism, with higher risk for development in males but with females exhibiting more rapid AAA growth rates and AAA ruptures at smaller aneurysm sizes. Previous studies performed in our laboratory have demonstrated that testosterone increases incidence and rupture rate of angiotensin II (AngII)-induced AAA in hyperlipidemic mice. After establishing this relationship, the four-core mouse model was used to analyze the role of sex chromosomes separate from sex hormones in aneurysm pathology. Female XY LDLr-/- mice are more susceptible to AngII-induced AAA than female XX LDLr-/- mice. The XY chromosome complement demonstrated more severe pathology and higher rupture rate. However, studies examining the role of sex chromosomes in the aortic arch between XX and XY female mice have not been performed. The purpose of this preliminary study was to observe whether differences were present between female XX and XY mice in formation of the aortic arch aneurysm. Methods and Results: Female XX and XY LDLr-/- mice were placed on a Western diet (TD.88137) one week prior to implantation of a 28-day AngII osmotic pump. The pumps administered AngII (1,000 ng/kg/min) for 28 days to induce aneurysm formation. The body weight of the mice was recorded weekly. Baseline measurements of the aortic arch diameter were made at D0 using ultrasound and found to be significantly different between XX and XY females (XX, 1.4 ± 0.03; XY, 1.5 ± 0.05 mm; p

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