Abstract 12759: Thymidine Phosphorylase Enhances SARS-CoV-2 Spike Protein and Platelet Factor 4 Complex Formation: A Potential Novel Mechanism That May Account for COVID-19-Associated Thrombosis

Abstract

Introduction: Scattered studies suggest that SARS-CoV-2 spike protein (SP) promotes platelet activation, though the exact mechanism is unknown. Thymidine phosphorylase (TYMP), which facilitates platelet activation and thrombosis, is significantly increased in the plasma and lungs of COVID-19 patients, suggesting that TYMP may play a role in the COVID-19 milieu. Hypothesis: We hypothesize that TYMP enhances SP/platelet factor 4 (PF4) complex formation and inhibition of TYMP attenuates SP-induced thrombosis and/or inflammation. Methods: BEAS-2B cells were treated with SP- or its receptor binding domain (RBD)-containing COS-7 cell lysate, or control cell lysate (p3.1) and TYMP and STAT3 expression were determined by western blot. The effect of SP on thrombosis was examined in K18-hACE2 and K18-hACE2/ Tymp -/- mice using the ferric chloride-induced carotid artery injury thrombosis model. SP-, TYMP-, or PF4-encoding plasmids were co-transfected into COS-7 and their protein interactions were assessed using co-IP, Blue Native-PAGE, and immunocytochemistry. Results: SP increased expression of TYMP and pY705-STAT3 in BEAS-2B. siRNA-mediated knockdown of TYMP reduced STAT3 activation. SP significantly enhanced thrombosis in the K18-hACE2 mice, which was inhibited by simultaneously treating mice with tipiracil, a selective TYMP inhibitor ( Figure ). K18-hACE2/ Tymp -/- mice were resistant to SP-enhanced thrombosis. For the first time, we found that SP, PF4, and TYMP form a complex whose formation is dose-dependently enhanced by TYMP. Conclusions: Our studies suggest that SARS-CoV-2 SP enhances TYMP expression and is prothrombotic and proinflammatory. TYMP inhibition, either via genetic knockout or by its chemical inhibitor, attenuates SP-enhanced thrombosis. TYMP promotes the formation of a PF4/SP complex, which provides a novel insight into COVID-19-associated thrombosis. TYMP could be a novel therapeutic target for COVID-19-associated sequelae.