Project Summary Left ventricular assist device (LVAD) provides cardiac support for patients with end stage heart disease and has significantly improved the survival of these patients, but device-related bleeding remains common and are associated with poor clinical outcomes. Even latest (3rd) generation devices have been able to significant impact device thrombosis (e.g.MOMENTUM3 trial), however with some surprise they have not been able to resolve LVAD related bleeding which remains a significant source of morbidity and complicates clinical management, ultimately leading to poor clinical outcomes. The LVAD-associated bleeding is often called acquired von Willebrand syndrome (aVWS) that is believed to be caused by excessive cleavage of VWF multimers by ADAMTS-13. However, this tentative mechanism has not been experimentally validated and significant gaps remain. The loss of large VWF multimers is observed in nearly all patients, but only a fraction of patients experience significant bleeding. High shear stress is known to promote VWF cleavage and activation to bind platelets, but this shear-induced VWF activation has not been studied in patients on LVAD. We hypothesize that: 1) Plasma VWF multimers in LVAD patients are subjected to higher rates of cleavage and activation than healthy subjects and patients with end stage heart disease prior to LVAD implant 2) LVAD- induced high shear stress results in VWF dysfunctions responsible for impaired hemostasis and triggers downstream VWF mediated angiogenesis 3) These shear-induced structural changes of VWF can be detected in patients on LVAD and can predict bleeding propensity developed after LVAD implantation. We propose to test these hypotheses with three specific aims. First, develop predictive markers for LVAD-induced hemostatic complications by comprehensively analyzing the imbalance between VWF activity and cleavage in longitudinal plasma samples from 240 patients collected before and after LVAD implantation using flow cytometry, mass spectrometry, microfluidic chamber systems, and conventional VWF functional tests. Second, study shear- induced structural changes of the VWF A1 and A2 domains and their roles in regulating rates of VWF cleavage and activation using in vitro techniques and mouse models. Third study how VWF synergizes with extracellular vesicles to promote immature angiogenesis, which leads to bleeding-prone arteriovenous malformation. The objectives of this mechanistic study are to: 1) define shear-induced structural changes of VWF using state-of- art biophysical techniques and study how these changes influence VWF cleavage and activity, 2) study how high shear stress disrupts the inhibitory interaction between the A1 and A2 domains to alter the A1 interface with the platelet VWF receptor GP Ib and to unfold A2 for cleavage, and 3) investigate the role of VWF in angiogenesis. We have assembled a team of experts in related clinical and research fields from 3 institutions to conduct th...