A study conducted at the Center for Research on Redox Processes in Biomedicine (Redoxoma) sheds light on how high blood sugar (hyperglycemia), a key symptom of diabetes, can contribute to thrombosis. Published in the Journal of Thrombosis and Haemostasis, the findings offer valuable insights that could inform the development of preventive strategies for cardiovascular diseases in individuals with diabetes.
According to Renato Simões Gaspar, the first author of the study, cardiovascular events like heart attacks and strokes, which are common causes of death in Brazil and other Latin American countries, are often precipitated by arterial thrombosis. Hyperglycemia, along with other risk factors such as dyslipidemia and hypertension, is mainly associated with an increased risk of cardiovascular diseases. “Hyperglycemia appears to be significantly linked to cardiovascular dysfunction,” said Gaspar.
The research was supported by FAPESP and carried out during Gaspar’s postdoctoral research under the leadership of Francisco Laurindo, the last author of the article. Laurindo, a professor at the University of São Paulo’s Medical School (FM-USP) and a member of Redoxoma, collaborated with Gaspar in this investigation. Redoxoma is a Research, Innovation, and Dissemination Center (RIDC) established by FAPESP at the Institute of Chemistry (IQ-USP). Gaspar is a faculty member at the State University of Campinas (UNICAMP).
The study focuses on the relationship between prolonged hyperglycemia, diabetic ketoacidosis, and an increased risk of thrombosis. These conditions cause endothelial dysfunction, disrupting the blood vessel lining, which plays a pivotal role in platelet adhesion and thrombus formation. This mechanism underpins the heightened susceptibility to thrombosis seen in individuals with diabetes.
The research specifically explored the role of peri/epicellular protein disulfide isomerase A1 (pecPDI) in regulating platelet-endothelium interactions in hyperglycemia. The study revealed that pecPDI, through adhesion-related proteins and changes to endothelial membrane biophysics, is critical in mediating thrombosis in diabetic conditions. “We discovered that a pathway for PDI in endothelial cells mediates thrombosis in diabetes when hyperglycemia is present, involving a specific molecular mechanism that we identified,” said Laurindo.
PDI, an enzyme located in the endoplasmic reticulum, facilitates the formation of disulfide bridges in nascent proteins, ensuring that they fold into their correct three-dimensional structure. PecPDI, a form of PDI found in the extracellular space, is secreted or bound to the cell surface in various cells, including platelets and endothelial cells. Previous studies have shown that pecPDI regulates thrombosis across different models.
To delve deeper into the interaction between platelets and endothelial cells under hyperglycemic conditions, the researchers developed a model using human umbilical vein endothelial cells cultured under different glucose concentrations. This model generated both normoglycemic and hyperglycemic cells, allowing the team to assess the impact of PDI on platelet adhesion using PDI or pecPDI inhibitors. The results were striking: platelets adhered almost three times more to hyperglycemic cells than to normoglycemic ones. However, inhibiting PDI reversed this effect, suggesting that pecPDI plays a critical role in platelet-endothelium interactions during hyperglycemia.
To further understand these findings, the researchers examined biophysical changes such as cytoskeleton remodelling in endothelial cells. They discovered that hyperglycemic cells exhibited more structured actin filament fibres than normoglycemic cells. Additionally, they measured hydrogen peroxide production, as reactive oxygen species are key mediators of cytoskeleton reorganisation and cell adhesion—hyperglycemic cells produced twice as much hydrogen peroxide as their normoglycemic counterparts.
The team also explored whether cytoskeleton reorganisation affected cell membrane stiffness, influencing platelet adhesion. Using atomic force microscopy, they confirmed that hyperglycemic cells were stiffer than normoglycemic ones. The microscope images revealed elongations in the cells, accompanied by extracellular vesicles that appeared to detach from the elongations. This prompted the researchers to investigate the secretome—the collection of proteins secreted by cells into the extracellular space—to determine if these vesicles contained proteins that promoted platelet adhesion.
The researchers identified 947 proteins in the secretome of hyperglycemic cells, of which eight were involved in cellular adhesion. Using RNA interference to silence gene expression for three of these proteins, they found that two proteins, SLC3A2 and LAMC1, played key roles in modulating platelet adhesion. SLC3A2 is a membrane protein, while LAMC1 is a subunit of laminin 1, a critical extracellular matrix component.
The study’s overall conclusion is that hyperglycemia triggers the secretion of specific proteins that promote platelet adhesion. Inhibition of PDI and pecPDI prevented endothelial cells from secreting these proteins, offering a potential target for therapeutic intervention to reduce thrombosis risk in diabetic patients.
This research underscores the complex interplay between hyperglycemia, endothelial dysfunction, and thrombosis. Identifying the molecular mechanisms involved opens up new avenues for preventing cardiovascular complications in individuals with diabetes, potentially leading to more effective treatments for reducing the burden of cardiovascular diseases in diabetic populations.
More information: Renato S. Gaspar et al, Endothelial protein disulfide isomerase A1 enhances membrane stiffness and platelet-endothelium interaction in hyperglycemia via SLC3A2 and LAMC1, Journal of Thrombosis and Haemostasis. DOI: 10.1016/j.jtha.2024.08.001
Journal information: Journal of Thrombosis and Haemostasis Provided by Fundação de Amparo à Pesquisa do Estado de São Paulo
