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. 2014 Jul 1;103(1):140-6.
doi: 10.1093/cvr/cvu113. Epub 2014 Apr 28.

Quantitative proteomics analysis reveals similar release profiles following specific PAR-1 or PAR-4 stimulation of platelets

Thijs C van Holten  1 , Onno B Bleijerveld  2 , Patrick Wijten  3 , Philip G de Groot  1 , Albert J R Heck  3 , Arjan D Barendrecht  1 , Tesy H Merkx  1 , Arjen Scholten  4 , Mark Roest  5
Affiliations

Quantitative proteomics analysis reveals similar release profiles following specific PAR-1 or PAR-4 stimulation of platelets

Thijs C van Holten et al. Cardiovasc Res. .

Abstract

Aims: Platelets are a natural source of growth factors, cytokines and chemokines, that regulate angiogenesis and inflammation. It has been suggested that differential release of pro- and anti-angiogenic growth factors from platelet α-granules by protease-activated receptors (PAR) 1 and 4 may be important for the regulation of angiogenesis. We aimed to compare the releasates of unstimulated platelets with PAR-1- and PAR-4-stimulated platelets.

Methods and results: The release of β-thromboglobulin, platelet factor (PF)-4, thrombospondin, platelet-derived growth factor (PDGF)-A/B, regulated and normal T-cell expressed and secreted (RANTES/CCL5), endostatin, CXCL12, and vascular endothelial growth factor (VEGF) was measured with enzyme-linked immunosorbent assay (ELISA). Mass spectrometry (MS)-based quantitative proteomics identified 93 proteins from platelets stimulated with PAR-1 and PAR-4. A strong correlation between the factors released after either stimulus was observed (Spearman's r 0.94, P < 0.001). Analysis with ELISA showed that stimulation with PAR-1 or PAR-4 lead to non-differential release of β-thromboglobulin, PF-4, thrombospondin, PDGF-A/B, RANTES/CCL5, endostatin, CXCL12, and VEGF. Release of thrombospondin was slightly lower after PAR-1 stimulation (7.2 μg/mL), compared with PAR-4 induced release (9.8 μg/mL; P < 0.05).

Conclusions: Both ELISA on established α-granule proteins and MS-based quantitative proteomics showed that the most abundant α-granule proteins are released in similar quantities from platelets after stimulation with either PAR-1 or PAR-4. Our findings provide evidence against the hypothesis that PAR-1 and PAR-4 stimulation of platelets trigger differential release of alpha-granule, but further studies are needed to draw conclusions for physiological conditions.

Keywords: Platelets; Protease-activated receptor; Quantitative proteomics; Releasate; Thrombosis.

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