Identification of AADACL1 As a Novel Regulator of Human Platelets Via Chemoproteomics

Abstract 381

A comprehensive knowledge of the platelet proteome is necessary for understanding important vascular pathologies such as myocardial infarction and stroke. To discover new signaling pathways in human platelets that may represent potential therapeutic targets, we took a combinatorial approach utilizing small library screening coupled with an innovative proteomics technique known as competitive activity based protein profiling (ABPP), a highly sensitive mass spectrometry (MS) technique that queries active enzymes from endogenous biological sources. Human platelets were pretreated with a novel carbamate library designed to interrogate enzymatically active proteins belonging to the serine hydrolase superfamily, which is estimated to represent 1% of the human proteome. Carbamate screening resulted in the identification of several novel inhibitors that reduced fibrinogen binding and P-selectin exposure on agonist-stimulated platelets. An endogenous protein target of the most selective inhibitor was identified by competitive ABPP as arylacetamide deacetylase-like 1 (AADACL1, also known as NCEH1), a lipid deacetylase implicated in cancer growth and atherosclerosis. Using highly selective second generation inhibitors of AADACL1 and MS-based metabolomics, we show that AADACL1 regulates platelet aggregation, Rap1 and PKC activation, lipid metabolism and fibrinogen binding. Elimination of AADACL1 protein levels via RNA interference in a megakaryocytic cell line that responds to platelet agonists also reduces inside-out signaling to αllbβ3. Furthermore, inhibition of AADACL1 reduces collagen-dependent thrombus growth under arterial shear conditions, suggesting that AADACL1 may be a relevant therapeutic target. Collectively, these data identify AADACL1 as a novel regulator of important signaling pathways in platelets and megakaryocytes and validate activity-based proteomics as a means to identify new classes of anti-platelet targets for thrombotic diseases.