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


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.
Laboratory for Clinical Chemistry and Haematology, UMC Utrecht, Utrecht, The Netherlands.
Thijs C
TC
van Holten
Laboratory of Experimental Cardiology, UMC Utrecht, Utrecht, The Netherlands Biomolecular Mass Spectrometry and Proteomics, Utrecht University, Utrecht, The Netherlands Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands Netherlands Proteomics Center, Utrecht, The Netherlands.
Onno B
OB
Bleijerveld
Biomolecular Mass Spectrometry and Proteomics, Utrecht University, Utrecht, The Netherlands Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands Netherlands Proteomics Center, Utrecht, The Netherlands.
Patrick
P
Wijten
Laboratory for Clinical Chemistry and Haematology, UMC Utrecht, Utrecht, The Netherlands.
Philip G
PG
de Groot
Biomolecular Mass Spectrometry and Proteomics, Utrecht University, Utrecht, The Netherlands Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands Netherlands Proteomics Center, Utrecht, The Netherlands.
Albert J R
AJ
Heck
Laboratory for Clinical Chemistry and Haematology, UMC Utrecht, Utrecht, The Netherlands.
Arjan D
AD
Barendrecht
Laboratory for Clinical Chemistry and Haematology, UMC Utrecht, Utrecht, The Netherlands.
Tesy H
TH
Merkx
Biomolecular Mass Spectrometry and Proteomics, Utrecht University, Utrecht, The Netherlands Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands Netherlands Proteomics Center, Utrecht, The Netherlands a.scholten@uu.nl mroest@umcutrecht.nl.
Arjen
A
Scholten
Laboratory for Clinical Chemistry and Haematology, UMC Utrecht, Utrecht, The Netherlands a.scholten@uu.nl mroest@umcutrecht.nl.
Mark
M
Roest
D051017:Apoptosis Regulatory Proteins / Q000097:blood*; D001792:Blood Platelets / Q000502:physiology*; D054377:Chemokine CXCL12 / Q000097:blood; D004797:Enzyme-Linked Immunosorbent Assay; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D018919:Neovascularization, Physiologic; D019007:P-Selectin / Q000097:blood; D015539:Platelet Activation / Q000502:physiology; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism; D040901:Proteomics; D044463:Receptor, PAR-1 / Q000097:blood*; D022142:Secretory Vesicles / Q000502:physiology; D042461:Vascular Endothelial Growth Factor A / Q000097:blood; D001620:beta-Thromboglobulin / Q000378:metabolism
D051017:Apoptosis Regulatory Proteins; C515255:CXCL12 protein, human; D054377:Chemokine CXCL12; D019007:P-Selectin; C061615:PPBP protein, human; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D044463:Receptor, PAR-1; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D001620:beta-Thromboglobulin; C101296:prostate apoptosis response-4 protein; D005340:Fibrinogen
Platelets; Protease-activated receptor; Quantitative proteomics; Releasate; Thrombosis