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Here we describe novel approaches to find molecular pathways implicated on the platelet\u2013endothelium interaction. Using high\u2010shear whole\u2010blood microfluidics, employing coagulant or non\u2010coagulant conditions at physiological temperature, we observed that the presence of human umbilical vein endothelial cells (HUVEC) strongly suppressed platelet adhesion and activation, via the collagen receptor glycoprotein VI (GPVI) and the PAR receptors for thrombin. Real\u2010time monitoring of the cytosolic Ca\n                    <jats:sup>2+<\/jats:sup>\n                    rises in the platelets indicated no major improvement of inhibition by prostacyclin or nitric oxide. Similarly under stasis, exposure of isolated platelets to HUVEC reduced the Ca\n                    <jats:sup>2+<\/jats:sup>\n                    responses by collagen\u2010related peptide (CRP\u2010XL, GPVI agonist) and thrombin (PAR agonist). We then analyzed the label\u2010free phosphoproteome of platelets (three donors), exposed to HUVEC, CRP\u2010XL, and\/or thrombin. High\u2010resolution mass spectrometry gave 5463 phosphopeptides, corresponding to 1472 proteins, with good correlation between biological and technical replicates (\n                    <jats:italic>R<\/jats:italic>\n                    \u2009&gt;\u2009.86). Stringent filtering steps revealed 26 regulatory pathways (Reactome) and 143 regulated kinase substrates (PhosphoSitePlus), giving a set of protein phosphorylation sites that was differentially (44) or similarly (110) regulated by HUVEC or agonist exposure. The differential regulation was confirmed by stable\u2010isotope analysis of platelets from two additional donors. Substrate analysis indicated major roles of poorly studied protein kinase classes (MAPK, CDK, DYRK, STK, PKC members). Collectively, these results reveal a resetting of the protein phosphorylation profile in platelets exposed to endothelium or to conventional agonists and to endothelium\u2010promoted activity of a multi\u2010kinase network, beyond classical prostacyclin and nitric oxide actors, that may contribute to platelet inhibition.\n                  <\/jats:p>","DOI":"10.1096\/fj.202300360rr","type":"journal-article","created":{"date-parts":[[2024,2,9]],"date-time":"2024-02-09T09:14:05Z","timestamp":1707470045000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":13,"title":["Endothelium\u2010mediated regulation of platelet activation: Involvement of multiple protein kinases"],"prefix":"10.1096","volume":"38","author":[{"given":"Isabella","family":"Provenzale","sequence":"first","affiliation":[{"name":"Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM) Maastricht University  Maastricht The Netherlands"},{"name":"Institute for Cardiovascular and Metabolic Research (ICMR), School of Biological Sciences University of Reading  Reading UK"},{"name":"Leibniz\u2010Institut f\u00fcr Analytische Wissenschaften \u2013 ISAS \u2013 e.V.  Dortmund Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3199-6296","authenticated-orcid":false,"given":"Fiorella A.","family":"Solari","sequence":"additional","affiliation":[{"name":"Leibniz\u2010Institut f\u00fcr Analytische Wissenschaften \u2013 ISAS \u2013 e.V.  Dortmund Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4327-5472","authenticated-orcid":false,"given":"Claudia","family":"Sch\u00f6nichen","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM) Maastricht University  Maastricht The Netherlands"},{"name":"Center for Thrombosis and Haemostasis University Medical Center of the Johannes Gutenberg University Mainz  Mainz Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sanne L. N.","family":"Brouns","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM) Maastricht University  Maastricht The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Delia I.","family":"Fern\u00e1ndez","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM) Maastricht University  Maastricht The Netherlands"},{"name":"Center for Research in Molecular Medicine and Chronic Diseases (CIMUS) Universidade de Santiago de Compostela  Santiago de Compostela Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marijke J. E.","family":"Kuijpers","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM) Maastricht University  Maastricht The Netherlands"},{"name":"Thrombosis Expertise Center, Heart and Vascular Center, Maastricht University Medical Center+  Maastricht The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6532-2583","authenticated-orcid":false,"given":"Paola E. J.","family":"van der Meijden","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM) Maastricht University  Maastricht The Netherlands"},{"name":"Thrombosis Expertise Center, Heart and Vascular Center, Maastricht University Medical Center+  Maastricht The Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0372-5352","authenticated-orcid":false,"given":"Jonathan M.","family":"Gibbins","sequence":"additional","affiliation":[{"name":"Institute for Cardiovascular and Metabolic Research (ICMR), School of Biological Sciences University of Reading  Reading UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2388-5265","authenticated-orcid":false,"given":"Albert","family":"Sickmann","sequence":"additional","affiliation":[{"name":"Leibniz\u2010Institut f\u00fcr Analytische Wissenschaften \u2013 ISAS \u2013 e.V.  Dortmund Germany"},{"name":"Medizinische Fakult\u00e4t, Medizinische Proteom\u2010Center Ruhr\u2010Universit\u00e4t Bochum  Bochum Germany"},{"name":"Department of Chemistry, College of Physical Sciences University of Aberdeen  Aberdeen UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7537-1509","authenticated-orcid":false,"given":"Chris","family":"Jones","sequence":"additional","affiliation":[{"name":"Institute for Cardiovascular and Metabolic Research (ICMR), School of Biological Sciences University of Reading  Reading UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2848-5121","authenticated-orcid":false,"given":"Johan W. 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