Endothelium‐mediated regulation of platelet activation: Involvement of multiple protein kinases

• Published in: The FASEB journal 2024-02, Vol.38 (4), p.e23468-n/a
• Main Authors: Provenzale, Isabella, Solari, Fiorella A., Schönichen, Claudia, Brouns, Sanne L. N., Fernández, Delia I., Kuijpers, Marijke J. E., Meijden, Paola E. J., Gibbins, Jonathan M., Sickmann, Albert, Jones, Chris, Heemskerk, Johan W. M.
• Format: Article
• Language: English
• Subjects: Blood Platelets - metabolism; endothelial cells; Endothelial Cells - metabolism; Endothelium - metabolism; glycoprotein VI; Humans; inter‐cellular cross‐talk; Nitric Oxide - metabolism; phosphoproteome; Platelet Activation - physiology; Platelet Membrane Glycoproteins - metabolism; platelets; Prostaglandins I; protease‐activated receptors; Protein Kinases - metabolism; Thrombin - metabolism
• ISSN: 0892-6638; 1530-6860
• DOI: 10.1096/fj.202300360RR

The endothelial regulation of platelet activity is incompletely understood. Here we describe novel approaches to find molecular pathways implicated on the platelet–endothelium interaction. Using high‐shear whole‐blood microfluidics, employing coagulant or non‐coagulant 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‐time monitoring of the cytosolic Ca2+ 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 Ca2+ responses by collagen‐related peptide (CRP‐XL, GPVI agonist) and thrombin (PAR agonist). We then analyzed the label‐free phosphoproteome of platelets (three donors), exposed to HUVEC, CRP‐XL, and/or thrombin. High‐resolution mass spectrometry gave 5463 phosphopeptides, corresponding to 1472 proteins, with good correlation between biological and technical replicates (R > .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‐isotope 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‐promoted activity of a multi‐kinase network, beyond classical prostacyclin and nitric oxide actors, that may contribute to platelet inhibition. Novel regulation of a kinase network in platelets by endothelial cells and platelet agonists.