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

Publisher: Nature Publishing Group Country of Publication: England NLM ID: 101563288 Publication Model: Electronic Cited Medium: Internet ISSN: 2045-2322 (Electronic) Linking ISSN: 20452322 NLM ISO Abbreviation: Sci Rep Subsets: MEDLINE

Original Publication: London : Nature Publishing Group, copyright 2011-

Platelet Activation* ; Proteomics*; Adult ; Blood Platelets/metabolism ; Blood Proteins/metabolism ; Cell Adhesion Molecules/metabolism ; Cyclic AMP-Dependent Protein Kinases/metabolism ; Electrophoresis, Gel, Two-Dimensional ; Female ; Humans ; Hydrogen-Ion Concentration ; Male ; Microfilament Proteins/metabolism ; Models, Biological ; P-Selectin/metabolism ; Phosphoproteins/metabolism ; Phosphorylation ; Protein Serine-Threonine Kinases/metabolism ; Proteome/metabolism ; Quality Control

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.

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0 (Blood Proteins)
0 (Cell Adhesion Molecules)
0 (Microfilament Proteins)
0 (P-Selectin)
0 (Phosphoproteins)
0 (Proteome)
0 (SELP protein, human)
0 (platelet protein P47)
0 (vasodilator-stimulated phosphoprotein)
EC 2.7.1.- (integrin-linked kinase)
EC 2.7.11.1 (Protein Serine-Threonine Kinases)
EC 2.7.11.11 (Cyclic AMP-Dependent Protein Kinases)

Date Created: 20191214 Date Completed: 20201104 Latest Revision: 20211204

20231215

PMC6908631

10.1038/s41598-019-55391-5

31831789