Gut metabolite TMAO and its structural analogs bind to fibrinogen thereby enhancing clot formation: a rationale for atherosclerosis risk.

Elevated levels of the gut-derived metabolite trimethylamine N-oxide (TMAO) are associated with atherosclerosis and thrombotic disorders, yet the molecular basis of its contribution to these pathologies remains poorly understood. Atherosclerosis initiates with endothelial damage and progresses through lipid deposition, foam cell formation, plaque development, rupture, and ultimately fibrin-rich thrombus formation, which may embolize. In this study, we investigate how TMAO modulates fibrinogen structure and consequently influences fibrin clot formation and stability. Using biophysical experiments with in-silico docking and molecular dynamics simulations, we show that TMAO binds specifically to the β1 calcium-binding site of fibrinogen. This interaction alters fibrinogen's dynamics and enhances its propensity to form fibrin clots. Clots generated in the presence of TMAO are more resistant to proteolytic degradation, indicating stability. Our analyses show that β-nodules of native fibrinogen may exist in two conformational states: one harboring high-affinity holes and another containing low-affinity holes. TMAO binding appears to shift this equilibrium toward the high-affinity state, promoting protofibril assembly. Structurally similar N-oxides from antidepressants showed comparable fibrinogen binding and accelerated fibrin assembly. Overall, our study identifies the β1 calcium-binding site as a promising therapeutic target for regulating clot stability and mitigating cardiovascular complications driven by gut metabolites and drug-derived N-oxides.