Mechanistic insights into the thrombosis and insulin resistance pathways of type 2 diabetes

Patients with type 2 diabetes (T2D) are at increased risk of vascular thrombotic disease manifesting as heart attacks, strokes, and venous thromboembolism. The mechanistic links between thrombosis, insulin signaling, and adipose dysfunction remain poorly understood, hindering the development of effective therapies. Work from this thesis revealed a direct interaction between tissue factor (TF), the molecular trigger of blood clotting, and the insulin receptor (INSR), a key mediator of insulin signaling. We hypothesized that this interaction regulates functional crosstalk between the glucometabolic and coagulation pathways, contributing to the insulin resistance and thrombosis observed in patients with T2D. The physical interaction between TF and the INSR was shown using three techniques: 1. Proximity ligation assays to detect colocalization; 2. Enzyme linked immunosorbent assays to demonstrate direct binding; and 3. Microscale thermophoresis to quantify the affinity of the interaction. To assess its functional impacts, we conducted Factor Xa (FXa) generation assays on human melanoma A7-TF cells to measure the procoagulant activity of TF ± an anti-INSR antibody. Glucose uptake assays were performed on preadipocytes and mature adipocytes from wild type mice ± an anti-TF antibody. Additionally, AdipoQ-ER-Cre⁺⧸⁻ TFflox/flox mice and control mice were injected with tamoxifen to induce an adipocyte-specific excision of TF. These mice underwent an inferior vena cava (IVC) stenosis thrombosis model to evaluate the role of adipocyte TF in systemic coagulation. The binding assays showed that TF and the INSR colocalize on the surface of cells and bind directly with a Kd in the range of 1-20 nM. Anti-INSR antibodies significantly increased TF’s procoagulant activity via FXa generation assays. Similarly, treatment of the adipocytes with an anti-TF antibody significantly increased glucose uptake in response to insulin. Mice lacking adipocyte TF developed smaller clots in response to the IVC stenosis thrombosis model. These results demonstrate that TF and the INSR may coregulate crosstalk between the coagulation and glucometabolic pathways. Additionally, adipocyte TF may contribute to systemic coagulation. These are novel observations that could open new avenues for therapeutic development. Since vascular disease represents the most common causes of death among patients with T2D, effective treatments are urgently needed.