Date of Award

2021

Degree Name

Biomedical Sciences

College

Joan C. Edwards School of Medicine

Type of Degree

Ph.D.

Document Type

Dissertation

First Advisor

Dr. Wei Li, Committee Chairperson

Second Advisor

Dr. Jung Han Kim

Third Advisor

Dr. Monica Valentovic

Fourth Advisor

Dr. Travis Salisbury

Fifth Advisor

Dr. Emine Koc

Abstract

Cardiovascular diseases (CVDs), such as ischemic heart disease and stroke, are the leading causes of death globally that disproportionally affects patients with type 2 diabetes mellitus (T2DM) at a 2-4-fold rate compared to non-diabetic patients. These CVDs result from a blood clot, also known as a thrombus, formed in arteries that can block blood flow resulting in tissue damage and death. Antiplatelet drugs, such as aspirin and clopidogrel, used to prevent thrombosis have been found to be less effective in diabetics and increase the risk of hemorrhagic events. The T2DM epidemic has made it imperative to find therapies that decrease thrombosis in diabetics without disrupting hemostasis. Previously, it was reported that thymidine phosphorylase (TYMP), a pyrimidine salvage pathway enzyme, deficiency attenuates thrombosis and platelet aggregation. Given that TYMP is elevated in T2DM patients’ serum, we hypothesize that thymidine phosphorylase plays a role in the high-risk of thrombosis in patients with T2DM. Therefore, we propose to investigate TYMP as a potential therapeutic target for preventing thrombosis in patients with T2DM. We fed 8-week-old Tymp-/- and wild type (WT) C57BL/6 male mice with either a 60% calorie from fat diet (HFD) or HFD augmented with tipiracil hydrochloride (TPI), a selective TYMP inhibitor, at a dose of 1 mg/kg/day for 16 weeks. Weekly weight, biweekly fasting plasma glucose, and pre-diet and post-diet intraperitoneal glucose tolerance tests (IPGTT) were measured to monitor progression of T2DM. After 16 weeks of feeding, mice were used for an in vivo FeCl3 injury-induced carotid artery injury thrombosis model using an intravital fluorescent microscope to assess thrombus formation. Age-matched WT and Tymp-/- male mice were also used in an in vivo thrombosis and tail bleeding assay to determine the efficacy of TPI compared to aspirin and clopidogrel in preventing thrombosis. Western blots assays of platelet signaling molecules, platelet aggregation assays, and flow cytometry were used to assess role of TYMP on platelet activation in vitro. We found that TYMP deficiency or inhibition decreased weight gain, glucose intolerance, thrombosis, and platelet activation compared to WT mice. WT mice fed the HFD augmented with TPI (WT + TPI) had increased lean mass, higher energy expenditure, and decreased fasting plasma glucose compared to both HFD-fed Tymp-/- and WT mice. In comparison to the control diet (CD) fed WT mice, HFD-feeding dramatically shortened time to thrombosis; however, HFD-feeding did not significantly affect thrombosis in the Tymp-/- mice when compared CD-fed Tymp-/- mice. Additionally, TYMP was increased in platelets isolated from HFD-fed WT mice compared to CD-fed WT mice. Treatment with TPI by either intraperitoneal injection, intravenous injection, or gavage feeding inhibited thrombosis without significantly prolonging hemostasis. These data show that TYMP deficiency and inhibition safely and effectively attenuates thrombosis and platelet aggregation. Furthermore, TYMP deficiency attenuates thrombosis equally between CDfed and HFD-fed mice, suggesting its potential for treating the high-risk of thrombosis in patients with T2DM. These data also show that TYMP plays a role in metabolic syndrome and T2DM progression, given that HFD-fed Tymp-/- and WT + TPI mice had decreased weight gain and glucose intolerance compared to HFD-fed WT mice. Taken together, these studies demonstrated that TYMP targeted therapy could be a promising novel treatment for the T2DM-associated high-risk of thrombosis.

Subject(s)

Cardiovascular system -- Diseases -- Research.

Diabetes -- Research.

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