The protein disulphide isomerase inhibitor CxxCpep modulates oxidative burst and mitochondrial function in platelets

Gaspar, R. S., Mansilla, S., Vieira, V. A., da Silva, L. B., Gibbins, J. M. ORCID: https://orcid.org/0000-0002-0372-5352, Castro, L., Trostchansky, A. and de A. Paes, A. M. (2021) The protein disulphide isomerase inhibitor CxxCpep modulates oxidative burst and mitochondrial function in platelets. Free Radical Biology and Medicine, 172. pp. 668-674. ISSN 0891-5849 doi: 10.1016/j.freeradbiomed.2021.07.011

Abstract/Summary

Background: We have previously described CxxCpep, a peptide with anti-platelet properties that inhibits peri/epicellular protein disulphide isomerase (pecPDI) by forming a mixed disulfide bond with Cys400 within the pecPDI active site. Objectives: Here we sought to determine if pecPDI targeted by CxxCpep is relevant to redox mechanisms downstream of the collagen receptor GPVI in platelets. Methods and Results: Restriction of effects of CxxCpep to the platelet surface was confirmed by LC-MS/MS following cell fractionation. Platelet aggregation was measured in platelet-rich plasma (PRP) incubated with 30 μM CxxCpep or vehicle. CxxCpep inhibited collagen-induced platelet aggregation but exerted no effect in TRAP-6-stimulated platelets. PRP was incubated with DCFDA to measure oxidative burst upon platelet adhesion to collagen. Results showed that CxxCpep decreased oxidative burst in platelets adhered to immobilized collagen while the number of adherent cells was unaffected. Furthermore, flow cytometry studies using a FITC-maleimide showed that the GPVI agonist CRP stimulated an increase in free thiols on the platelet outer membrane, which was inhibited by CxxCpep. Finally, CxxCpep inhibited platelet mitochondrial respiration upon activation with collagen, but not with thrombin. Conclusions: Our data suggest that pecPDI is a potential modulator of GPVI-mediated redox regulation mechanisms and that CxxCpep can be further exploited as a template for new antiplatelet compounds.

Item Type	Article
URI	https://reading-pure-test.eprints-hosting.org/id/eprint/99182
Identification Number/DOI	10.1016/j.freeradbiomed.2021.07.011
Refereed	Yes
Divisions	Life Sciences > School of Biological Sciences > Biomedical Sciences Central Services
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