Quantitative Glycoproteomic Analysis Identifies Platelet-Induced Increase of Monocyte Adhesion via the Up-Regulation of Very Late Antigen 5
datasetposted on 07.08.2015, 00:00 by Jiqing Huang, Juergen Kast
Physiological stimuli, such as thrombin, or pathological stimuli, such as lysophosphatidic acid (LPA), activate platelets circulating in blood. Once activated, platelets bind to monocytes via P-selectin–PSGL-1 interactions but also release the stored contents of their granules. These platelet releasates, in addition to direct platelet binding, activate monocytes and facilitate their recruitment to atherosclerotic sites. Consequently, understanding the changes platelet releasates induce in monocyte membrane proteins is critical. We studied the glyco-proteome changes of THP-1 monocytic cells affected by LPA- or thrombin-induced platelet releasates. We employed lectin affinity chromatography combined with filter aided sample preparation to achieve high glyco- and membrane protein and protein sequence coverage. Using stable isotope labeling by amino acids in cell culture, we quantified 1715 proteins, including 852 membrane and 500 glycoproteins, identifying the up-regulation of multiple proteins involved in monocyte extracellular matrix binding and transendothelial migration. Flow cytometry indicated expression changes of integrin α5, integrin β1, PECAM-1, and PSGL-1. The observed increase in monocyte adhesion to fibronectin was determined to be mediated by the up-regulation of very late antigen 5 via a P-selectin–PSGL-1 independent mechanism. This novel aspect could be validated on CD14+ human primary monocytes, highlighting the benefits of the improved enrichment method regarding high membrane protein coverage and reliable quantification.
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PSGLmonocytic cellsexpression changesmembrane protein coverageatherosclerotic sitesTHPLPAtransendothelial migration500 glycoproteinsflow cytometrysample preparationchanges platelet releasates852 membranenovel aspectmonocyte membrane proteinslectin affinity chromatographycell cultureAntigen 5 Physiological stimulimembrane proteinplatelet releasatesmonocyte extracellular matrix bindingPECAMmonocyte adhesion1715 proteinsplatelet bindinglysophosphatidic acidprotein sequence coverageplatelets bindantigen 5enrichment methodMonocyte Adhesion