Abstract
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.
Highlights
There is a need to assess platelet activation in patients with thrombotic disorders
To assure that our platelet proteome analysis was assessed from well-defined in-vitro activated and inhibited platelets, CD62P, a surface marker specific for platelet activation, was quantified by flow cytometry
A strong platelet activation (>90% CD62Ppositive platelets) was induced by thrombin receptor activating peptide-6 (TRAP-6) (15 μM) and a moderate response was provoked by ADP (5 μM, on average 50% CD62P-positive platelets), which is comparable to published data of platelet activation studies[27,28]
Summary
There is a need to assess platelet activation in patients with thrombotic disorders. P-selectin and activated integrin αIIbβ[3] are usually quantified by flow cytometry to measure platelet activation. 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. The aim of the present platelet proteome study was to reveal specific features of activated, as well as inhibited platelets, compared to their untreated controls within one biochemical method using fluorescence two-dimensional differential gel electrophoresis (2D-DIGE) This proteomics technique enables reliable quantitative results on differential protein expressions by displaying thousands of proteins, their isoforms and post-translational modifications at the same time. The generation of protein species may be of great interest in the proteome of anucleate platelets during activation and inhibition in the circulation
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