The endothelial regulation of platelet activity is incompletely understood. We investigated how the presence of human umbilical vein endothelial cells (HUVEC) in whole-blood microfluidics affected platelet activation induced by collagen receptor glycoprotein VI (GPVI) and the PAR receptors for thrombin. On collagen/tissue factor surface, HUVEC potently suppressed platelet adhesion and Ca2+ rises upon high-shear blood flow at physiological temperature. HUVEC accordingly suppressed thrombus and fibrin formation. Similarly under stasis, platelet exposure to HUVEC (1-30 min) reduced Ca2+ responses to collagen-related peptide (CRP-XL, GPVI agonist) and thrombin (PAR agonist). Parallel samples of platelets from 3 donors, exposed to HUVEC, CRP-XL and/or thrombin, were analyzed by label-free phosphoproteome analysis. High-resolution mass spectrometry gave 5,463 platelet phosphopeptides, corresponding to 1,472 proteins, with good correlation between biological and technical replicates (R>0.86). Stringent filtering steps revealed 26 principal pathways (Reactome) and 143 kinase substrates (KEGG, PhosphoSitePlus). Repeated phosphoproteome analysis indicated a set of protein phosphorylation sites that was differentially (44) or similarly (110) regulated by HUVEC exposure or by agonist stimulation. The differential regulation was confirmed by stable-isotope analysis of platelets from 2 additional donors. Substrate analysis indicated preferential involvement of MAPK, CDK, DYRK, STK and PKC protein-kinase classes. Collectively, our results reveal a resetting of the protein phosphorylation profile in platelets exposed to endothelium or to conventional agonists, and to the endothelium-promoted activation of a multi-kinase network, beyond classical prostacyclin and nitric oxide actors, that may contribute to platelet inhibition.