PROJECT ABSTRACT Portal hypertension (PHTN) is a common final pathway of multiple forms of chronic liver disease which accounts for significant morbidity and mortality among patients with liver disease. However, there is a paucity of therapies available to ameliorate PHTN. The overall objective of this proposal is to elucidate the contribution of neutrophils and platelets to the pathogenesis of PHTN, with the therapeutic goal of facilitating the design of therapies to decrease portal pressure. The pathophysiology of PHTN is complex and is regulated at multiple levels, including paracrine signaling within sinusoids, formation of microvascular thrombosis, and endothelial dysfunction. We have previously identified a novel but critical role of neutrophils in the pathogenesis of PHTN. We found that cyclic stretch imposed by congestive hepatopathy (CH) induces activation of mechanosensitive Piezo channels within liver sinusoidal endothelial cells (LSECs). Piezo channels activate mechanocrine signaling pathways which culminate in secretion of the neutrophil chemotactic cytokine CXCL1. CXCL1 induces infiltration of neutrophils into liver sinusoids. Neutrophils form complexes with platelets which lead to the formation of neutrophil extracellular traps, or NETs. We found that genetic and pharmacologic inhibition of NET formation significantly decreases portal pressures in murine models of CH and PHTN. Although platelets have been implicated in NET formation in certain settings, the mechanisms which recruit platelets and regulate their interactions with neutrophils to generate sinusoidal NETs require further investigation. Weibel-Palade bodies (WPBs) are endothelial organelles which generate extracellular vesicles (EVs) containing inflammatory and hemostatic factors. The impact of WPB-derived EVs on platelet recruitment to liver sinusoids has not been studied. We have formulated the central hypothesis that platelets activated by WPB-derived EVs interact with the CD11b/CD18 integrin receptor on neutrophils to stimulate NET formation and PHTN. We will test this hypothesis through the following independent but integrated specific aims which are both technically and conceptually innovative. First, we will test the hypothesis that Piezo channels serve as master mechanosensors whose activation regulates the generation of both neutrophil- and platelet- chemotactic factors which modulate portal pressures. We propose that Piezo activation generates platelet chemotactic factors within EVs derived from WPBs in LSECs. Finally, using murine models and clinically- relevant forms of platelet inhibition, we will test the hypothesis that interaction of the neutrophil integrin receptor CD11b/CD18 with the platelet glycoprotein receptor GPIbα drives NET formation. Our proposal is significant because it has the potential to elucidate novel therapeutic targets to better manage PHTN, a devastating and prevalent disease which is currently curable only with liver transplantation.