Abstract
INTRODUCTION: Thymidine phosphorylase (TYMP), which facilitates platelet activation and thrombosis, is significantly increased in COVID-19 patients. We hypothesize that TYMP mediates SARS-CoV-2 spike protein (SP)-induced thrombosis. MATERIALS AND METHODS: Plasmids encoding wildtype SP or empty vector (p3.1) were transfected into COS-7 cells, and cell lysates were prepared as a reservoir for SP or p3.1 (control), respectively. K18-hACE2(TG) and K18-hACE2(TG)/Tymp(-/-) mice were treated with a single dose of SP or p3.1 by intraperitoneal injection and then subjected to thrombosis studies three days later. The role of SP on inflammatory signaling activation was assessed in BEAS-2B cells. RESULTS: SARS-CoV-2 SP increased the expression of TYMP, resulting in the activation of STAT3 and NF-κB in BEAS-2B cells. A siRNA-mediated knockdown of TYMP attenuated SP-enhanced activation of STAT3. SP significantly promoted arterial thrombosis in K18-hACE2(TG) mice. SP-accelerated thrombosis was attenuated by inhibition or genetic ablation of TYMP. SP treatment did not influence ADP- or collagen-induced platelet aggregation but significantly increased platelet adhesion to fibrinogen. SP treatment also significantly shortened activated partial thromboplastin time, which was reversed and even prolonged by TYMP deficiency. Additionally, SP binds to platelet factor 4 (PF4) and TYMP. TYMP does not bind PF4 but enhances the formation of the SP/PF4 complex, which may augment the procoagulant and prothrombotic effect of PF4. CONCLUSIONS: We conclude that SP is prothrombotic and upregulates TYMP expression, and TYMP inhibition or knockout mitigates SP-enhanced thrombosis. These findings suggest that inhibition of TYMP may be a novel therapeutic strategy for COVID-19-associated thrombosis.