To Study the Role of Multidrug Resistance Protein 4 on Induction of Aspirin Resistance in Type 2 Diabetes Mellitus

Platelets play a crucial role in normal hemostasis. Platelets are also involved in the pathologic processes of cardiovascular diseases (CVD). People with diabetes mellitus (DM) have an increased risk of life-threatening CVD compared to the general population. Aspirin is one of the most important therapeutic agents used in primary or secondary prevention of CVD in diabetic patients. However, previous clinical trials have shown the lower effect of aspirin on prevention of cardiovascular disease, known as aspirin resistance, in diabetic patients compared with non-diabetic patients. The mechanism of aspirin resistance remains not fully understood. Recently, multidrug resistance protein 4 (MRP4) has been suggested to be associated with aspirin resistance in patients after coronary artery bypass grafting surgery that reveals MRP4 redistribution onto the plasma membrane and aspirin extrusion from platelets. Moreover, our previous study also indicates that MRP4 may export cAMP from platelets and enhance platelet activation. These evidence indicate that MRP4 may contribute to aspirin resistance. In addition, aldose reductase (AR), a central enzyme of the poly pathway, has been implicated in aberrant glucose metabolism and several diabetic complications. However, whether AR regulates MRP4 and lead to aspirin resistance remains obscure. Therefore, in this project, we will further clarify the role of MRP4 on aspirin resistance and the relationship of MRP4 and AR in the humanized mouse model of DM. Specific Aim 1 (first year): To elucidate the role of MRP4 in high glucose-mediated aspirin resistance in human platelet. In the first year, we will determine whether high glucose can reduce inhibitory activity of aspirin on platelet activation, and induce MRP4 distribution and aspirin extrusion, as detected by LC-MS/MS analysis, deconvolution microscope, and transmission electron microscope. In addition, clot retraction and PFA-100 will be used to validate that high glucose may reduce the effect of aspirin on platelet activation. Specific Aim 2 (second year): To determine the role of MRP4 in a mouse model of type 2 diabetes mellitus (DM). In the second year, a mouse model (LDLR- - mice) of type 2 DM will be used to confirm MRP4 overexpression and distribution in diabetic platelets. Aspirin resistance will be also validated in diabetic mouse platelets by the detection of aspirin extrusion and platelet aggregation. In addition, we will use the animal model of thrombosis in mesenteric microvessels induced by fluorescein sodium to analyze platelet activation in vivo. Specific Aim 3 (third year): To determine the relationship of MRP4 and aldose reductase (AR) in a humanized mouse model with overexpression of AR. In the third year, due to much lower levels of AR expression in mice, a AR transgenic mice (huAR-LDLR-/- mice) of type 2 DM will be used to mimic DM in human. We will first confirm whether AR overexpression promotes MRP4 distribution. We will also use pharmacological inhibitor of AR to confirm the effect of AR on platelet activation. In addition, we will also use the animal model of thrombosis in mesenteric microvessels and a stroke model to determine whether AR or MRP4 inhibitors may reverse these effects.