Comparative proteomics reveals unexpected quantitative phosphorylation differences linked to platelet activation state

There is a need to assess platelet activation in patients with thrombotic disorders. P-selectin and activated integrin αIIbβ3 are usually quantified by flow cytometry to measure platelet activation. Monitoring changes in vasodilator-stimulated phosphoprotein (VASP) phosphorylation is an established...

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Published in:	Scientific reports Vol. 9; no. 1; pp. 19009 - 16
Main Authors:	Schmidt, G. J., Reumiller, C. M., Ercan, H., Resch, U., Butt, E., Heber, S., Liutkevičiūte, Z., Basílio, J., Schmid, J. A., Assinger, A., Jilma, B., Zellner, M.
Format:	Journal Article
Language:	English
Published:	London Nature Publishing Group UK 12-12-2019 Nature Publishing Group
Subjects:	13/31 631/45/612/1246 692/308/53/2421 692/4017 82 82/29 82/58 Adult Blood Platelets - metabolism Blood Proteins - metabolism Cell Adhesion Molecules - metabolism Cyclic AMP-Dependent Protein Kinases - metabolism Electrophoresis, Gel, Two-Dimensional Female Humanities and Social Sciences Humans Hydrogen-Ion Concentration Inactivation Kinases Male Microfilament Proteins - metabolism Models, Biological multidisciplinary P-Selectin - metabolism Phosphoproteins - metabolism Phosphorylation Platelet Activation Protein-Serine-Threonine Kinases - metabolism Proteins Proteome - metabolism Proteomics Quality Control Science Science (multidisciplinary)
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Summary:	There is a need to assess platelet activation in patients with thrombotic disorders. P-selectin and activated integrin αIIbβ3 are usually quantified by flow cytometry to measure platelet activation. Monitoring changes in vasodilator-stimulated phosphoprotein (VASP) phosphorylation is an established method to determine the platelet-reactivity status. To study disruptions of platelet reactivity more comprehensively, we compared the human non-secretory platelet proteome after in-vitro -activation and –inhibition with their respective untreated controls using unbiased fluorescence two-dimensional differential in-gel electrophoresis. The non-secretory platelet proteome was more severely affected during inhibition than during activation. Strikingly, while VASP reached a 1.3-fold increase in phosphorylation levels in inhibited platelets, other protein kinase A targets showed several-fold stronger inhibition-induced phosphorylation levels, including LIM and SH3 domain protein 1 (6.7-fold), Src kinase-associated phosphoprotein 2 (4.6-fold), and Ras-related protein Rap1b (4.1-fold). Moreover, phosphorylation of integrin-linked protein kinase (ILK) and pleckstrin (PLEK) species was associated with P-selectin surface expression. The discrimination power between activation and inhibition was more pronounced for dephosphorylated ILK (3.79 Cohen’s d effect size) and phosphorylated PLEK (3.77) species than for P-selectin (2.35). These data reveal new insights into the quantitative changes of the platelet reactivity proteome and suggest powerful alternatives to characterise their activation and inactivation potential.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	2045-2322 2045-2322
DOI:	10.1038/s41598-019-55391-5