Project Summary/Abstract Heart disease and stroke are the No. 1 and 5 causes of deaths in the US. Type 1 diabetes mellitus (T1DM) significantly increases the risk for heart attacks and strokes. Intense glycemic control has been reported to reduce major cardiovascular events by >30%, suggesting that hyperglycemia is one of the major contributors to T1DM-associated heart attack and stroke risk escalation. However, how T1DM and hyperglycemia exacerbate such risk is unclear. Platelets are vascular first-responders that activate for hemostasis upon blood vessel damage; whereas pathogenic platelet activation leads to spurious thrombosis and acute vascular obstruction. T1DM and hyperglycemia lead to platelet hyperactivity and increased propensity to form thrombi. This proposals aims to understand how hyperglycemia causes platelet hyperactivity and thrombosis in T1DM, and to develop new therapeutic strategies to mitigate T1DM-associated heart attacks and strokes. Utilizing an integrated metabolism toolkit including state-of-the-art Stable Isotope Resolved Metabolomics (SIRM), we demonstrated critical role of altered platelet metabolism in thrombin-induced platelet activation. Specifically, thrombin stimulation alters platelet metabolism that is centered on glycogen metabolism, pentose phosphate pathway (PPP), and fructose 1,6-bisphosphate (F1,6BP), namely, “the glycogen-PPP-F1,6BP axis”, modulating energy, redox and calcium homeostasis in platelets and leading to their activation. Literature and our compelling preliminary data further reveal that hyperglycemia increases glycogen storage and its mobilization that generates ATP, PPP inhibition, reactive oxygen species, and intracellular calcium, all of which are in line with increased propensity for platelets to activate. Therefore, our overarching hypothesis is that hyperglycemia changes the glycogen-PPP-F1,6BP axis in platelets to drive platelet hyperactivity and thus thrombotic risk in T1DM. In Aim 1, we will delineate these hyperglycemia-induced changes in the glycogen-PPP-F1,6BP axis in platelets isolated from T1DM patients and normal healthy platelets subject to acute hyperglycemia in vitro. In Aim 2, we will determine how modulation of the glycogen-PPP-F1,6BP axis by pharmacological and genetic means suppresses hyperglycemia-induced platelet hyperactivity in vitro. In Aim 3, we will determine how modulation of the glycogen- PPP-F1,6BP axis by pharmacological and genetic means reduces hyperglycemia-exacerbated thrombosis and stroke in animal models. Our team is in a unique position to address our hypothesis, as we possess recognized expertise in metabolism and metabolomics (Qingjun Wang PhD and Matthew Gentry PhD), platelet biology (Sidney Whiteheart PhD), T1DM (Lisa Tannock MD), and stroke (Justin Fraser MD and Jill Roberts PhD), and we have strong partnership with the University of Kentucky Metabolomics, Redox Metabolism, and Rodent Surgery Cores. Upon completion of the proposed project, we will h...