Endothelial protein disulfide isomerase A1 enhances membrane stiffness and platelet-endothelium interaction in hyperglycemia via SLC3A2 and LAMC1

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Gaspar, Renato S. ; Franca, Alefe Roger Silva ; Oliveira, Percillia Victoria Santos ; Diniz-Filho, Joel Felix Silva ; Teixeira, Livia ; Valadao, Iuri Cordeiro ; Debbas, Victor ; Santos, Clenilton Costa dos ; Massafera, Mariana Pereira ; Bustos, Silvina Odete ; Alencar, Luciana Magalhaes rebelo ; Ronsein, Graziella Eliza ; Laurindo, Francisco R. M.

• Document type: Journal article
• Source: Journal of Thrombosis and Haemostasis; v. 22, n. 11, p. 17-pg., 2024-10-24.

Abstract

Background: Diabetes carries an increased risk of cardiovascular disease and thromboembolic events. Upon endothelial dysfunction, platelets bind to endothelial cells to precipitate thrombus formation; however, it is unclear which surface proteins regulate platelet-endothelium interaction. We and others have shown that peri/epicellular protein disulfide isomerase A1 (pecPDI) influences the adhesion and migration of vascular cells. Objectives: We investigated whether pecPDI regulates adhesion-related molecules on the surface of endothelial cells and platelets that influence the binding of these cells in hyperglycemia. Methods: Immunofluorescence was used to assess platelet-endothelium interaction in vitro, cytoskeleton reorganization, and focal adhesions. Hydrogen peroxide production was assessed via Amplex Red assays (ThermoFisher Scientific). Cell biophysics was assessed using atomic force microscopy. Secreted proteins of interest were identified through proteomics (secretomics), and targets were knocked down using small interfering RNA. Protein disulfide isomerase A1 (PDI) contribution was assessed using whole-cell PDI or pecPDI inhibitors or small interfering RNA. Results: Platelets of healthy donors adhered more onto hyperglycemic human umbilical vein endothelial cells (HUVECs). Endothelial, but not platelet, pecPDI regulated this effect. Hyperglycemic HUVECs showed marked cytoskeleton reorganization, increased H2O2 production, and elongated focal adhesions. Indeed, hyperglycemic HUVECs were stiffer compared with normoglycemic cells. PDI and pecPDI inhibition reversed the abovementioned processes in hyperglycemic cells. A secretomics analysis revealed 8 proteins secreted in a PDI-dependent manner by hyperglycemic cells. Among these, we showed that genetic deletion of LAMC1 and SLC3A2 decreased platelet-endothelium interaction and did not potentiate the effects of PDI inhibitors. Conclusion: Endothelial pecPDI regulates platelet-endothelium interaction in hyperglycemia through adhesion-related proteins and alterations in endothelial membrane biophysics. (AU)

• FAPESP's process: 20/15944-8 - Investigation of peri/epicellular protein disulphide isomerase as a novel regulator of the platelet-endothelium interaction in normoglycaemia and Diabetes
• Grantee: Renato Simões Gaspar
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 13/07937-8 - Redoxome - Redox Processes in Biomedicine
• Grantee: Ohara Augusto
• Support Opportunities: Research Grants - Research, Innovation and Dissemination Centers - RIDC
• FAPESP's process: 22/05750-7 - Underlying mechanisms of cardiovascular outcomes in Diabetes: the role of glycated collagen
• Grantee: Renato Simões Gaspar
• Support Opportunities: Generation Project Research Grant
• FAPESP's process: 23/00995-4 - Development of metrics of quantitative proteomics capable of capturing the functional and compositional complexity of high-density lipoprotein
• Grantee: Graziella Eliza Ronsein
• Support Opportunities: Regular Research Grants
• FAPESP's process: 20/03838-9 - Protein Disulfide Isomerase A1 (PDIA1) as integrative pathway of cell differentiation and mitochondrial function
• Grantee: Lívia Teixeira
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 16/00696-3 - Proteomic as a tool to understand HDL function and composition
• Grantee: Graziella Eliza Ronsein
• Support Opportunities: Research Grants - Young Investigators Grants