Abstract
Platelet protease-activated receptor 1 (PAR1) is a cell surface G-protein-coupled receptor (GPCR) that acts as a thrombin receptor promoting platelet aggregation. Targeting the PAR1 pathway by vorapaxar, a PAR1 antagonist, leads to a reduction in ischemic events in cardiovascular patients with a history of myocardial infarction or with peripheral arterial disease. In platelets, specialized microdomains highly enriched in cholesterol act as modulators of the activity of several GPCRs and play a pivotal role in the signaling pathway. However, their involvement in platelet PAR1 function remains incompletely characterized. In this context, we aimed to investigate whether activation of PAR1 in human platelets requires its localization in the membrane cholesterol-rich microdomains. Using confocal microscopy, biochemical isolation, and proteomics approaches, we found that PAR1 was not localized in cholesterol-rich microdomains in resting platelets, and only a small fraction of the receptor relocated to the microdomains following its activation. Vorapaxar treatment increased the level of PAR1 at the platelet surface, possibly by reducing its endocytosis, while its colocalization with cholesterol-rich microdomains remained weak. Consistent with a cholesterol-dependent activation of Akt and p38 MAP kinase in thrombin receptor-activating peptide (TRAP)-activated platelets, the proteomic data of cholesterol-rich microdomains isolated from TRAP-activated platelets showed the recruitment of proteins contributing to these signaling pathways. In conclusion, contrary to endothelial cells, we found that PAR1 was only weakly present in cholesterol-rich microdomains in human platelets but used these microdomains for efficient activation of downstream signaling pathways following TRAP activation.
Highlights
Protease-activated receptor 1 (PAR1) is the prototypical member of a family of G-protein-coupled receptors (GPCRs) that mediate cellular responses to thrombin, the strongest human platelet activator, and related proteases [1]
Our major findings are as follows: (i) in resting human platelets, PAR1 very weakly colocalized with cholesterol-rich microdomains; (ii) thrombin receptor-activating peptide (TRAP) stimulation increased the colocalization of PAR1 and cholesterol-rich microdomains, which remained, weak; (iii) cholesterol depletion affected Akt and p38 MAP kinase activation downstream of TRAP stimulation; (iv) cholesterol-rich microdomains contained proteins essential to PAR1 signaling pathway in platelets activated by TRAP
To investigate the role of platelet cholesterol-rich microdomains on signaling pathways initiated by PAR1, we defined four experimental groups: (i) control group; (ii) platelets stimulated by thrombin receptor-activating peptide (TRAP) (S7152, Sigma-Aldrich, Saint Louis, MI, USA); (iii) platelets treated by vorapaxar (ALSACHIM, Illkirch, France); (iv) platelets treated by vorapaxar and stimulated by TRAP
Summary
Protease-activated receptor 1 (PAR1) is the prototypical member of a family of G-protein-coupled receptors (GPCRs) that mediate cellular responses to thrombin, the strongest human platelet activator, and related proteases [1]. Thrombin irreversibly activates PAR1 by cleaving the amino-terminal exodomain of the receptor, which exposes a tethered peptide ligand that binds the heptahelical bundle of the receptor to induce G-protein activation via an intramolecular signaling mechanism [2]. PAR1 activation is based on a process named biased signaling, with a ligand-dependent preferential.
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