OC 19.1 - Targeting thymidine phosphorylase proves to be an effective strategy in mitigating SARS-CoV-2 Spike Protein-enhanced thrombosis

Background: COVID-19 and some COVID-19 vaccines increase the risk of thrombosis with unknown mechanisms. Accumulated studies suggest that the SARS-CoV-2 spike protein (SP) promotes platelet activation. Thymidine phosphorylase (TYMP), which facilitates platelet activation and thrombosis, is significantly increased in COVID-19 patients.

Aims: To test the hypothesis that TYMP mediates SP-induced inflammation and thrombosis.

Methods: Wildtype SARS-CoV-2 SP was generated by overexpressing the corresponding plasmid in COS-7 cells, and the lysate of empty plasmid transfected cells served as control (p3.1). K18-hACE2 and K18-hACE2/Tymp-/- mice were treated with SP or p3.1 and then subjected to the in vivo thrombosis assay using the FeCl3-induced carotid artery injury thrombosis model. SP- or p3.1-enhanced inflammation was assessed in BEAS-2B cells. Gene transfection was conducted with COS-7 cells, and protein interactions were assessed by co-immunoprecipitation.

Results: SP significantly enhanced thrombosis in K18-hACE2 mice, which was attenuated in the K18-hACE2/Tymp-/- mice and inhibited by simultaneously treating mice with tipiracil, a potent TYMP inhibitor (Figure). Treating K18-hACE2TG mice with SP did not affect platelet aggregation in response to ADP and collagen. SP significantly increased the expression of TYMP and activation of STAT3 and NF-kB in BEAS-2B. In silico analysis suggested that TYMP binds to SP, which was validated by co-immunoprecipitation. Additionally, we found that SARS-CoV-2 SP binds platelet factor 4 (PF4), and the SP/PF4 binding was enhanced by TYMP in a dose-dependent manner. Treating whole blood harvested from K18-hACE2TG and K18-hACE2/Tymp-/- mice with PF4 alone did not affect platelet adhesion to the collagen-coated surface; however, treating them with the SP/PF4 complex significantly enhanced K18-hACE2TG platelet adhesion, which was significantly mitigated by TYMP knockout.

Conclusion(s): Our study demonstrated that TYMP plays an important role in SP-enhanced thrombosis and coagulation. TYMP promotes the formation of a PF4/SP complex, providing a novel insight into COVID-19-associated thrombosis. TYMP could be a novel therapeutic target for COVID-19-associated thrombophilia.