Inhibitory mechanism of thrombin (FIIa) by garlic-derived H2S: persulfidation cleavage of the Cys42-Cys58 disulfide bond triggers FIIa cascade inactivation.

Thrombin (FIIa) is a pivotal serine protease in the coagulation cascade. Its aberrant activation drives pathological thrombosis, myocardial infarction, and stroke. Although garlic-derived sulfur compounds are known for cardiovascular protection, their redox-dependent regulation of key coagulation proteases remains largely undefined. In this study, we used a multi-scale approach combining enzymatic assays, structural characterization, molecular dynamics, and functional validation to elucidate how garlic-derived hydrogen sulfide (H2S) inhibits FIIa. Real-time activity assays demonstrated that garlic-derived H2S donors, including glutathione (GSH)-activated diallyl trisulfide (DATS), diallylthiosulfinate (Allicin), and diallyl disulfide (DADS), inhibited FIIa in an H2S-release-dependent manner, confirming H2S as the key bioactive FIIa inhibitor. Structural analyses showed that H2S binding induced global conformational rigidification of FIIa, with decreased β-sheet and increased α-helix contents. Mechanistically, direct evidence from matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS/MS) confirmed that H2S cleaved the Cys42-Cys58 disulfide bond (SCys42-SCys58) and specifically persulfidated Cys42-SH to Cys42-SSH. Computational modeling suggested this SCys42-SCys58 cleavage may arrest the catalytic canyon's "self-chewing" motion, disrupt the oxyanion hole, decouple Na+-mediated allostery, and induce rigidity, potentially locking FIIa in an autoinhibited state. Functionally, in vivo assays verified the anticoagulant efficacy of garlic-derived H2S. Based on experimental and computational data, we proposed two interconnected mechanisms: the modeling-derived "Na+-coordination-gated allosteric catalytic canyon self-chewing cycle" governing FIIa-mediated fibrinogen cleavage, and the experimentally supported "FIIa cascade inactivation via SCys42-SCys58 persulfidation cleavage" underlying H2S-dependent FIIa inhibition. This work identified a potential novel paradigm for redox-dependent regulation of the coagulation cascade, advanced natural product-based enzyme inhibition mechanisms, and provided a foundation for developing next-generation redox-targeted anticoagulants.