Omics-guided discovery and functional profiling of Da-29, a bioactive serine protease from Deinagkistrodon acutus venom, with antithrombotic activity.

BACKGROUND

Snake venom serine proteases (SVSPs) are critical toxic components that interfere with the hemostatic balance and serve as a key candidate library for anticoagulant drug development. Despite the diversity of SVSPs in Deinagkistrodon acutus venom, systematic studies of low-abundance SVSPs with thrombin-like activity profiles are limited. Hence, this research aimed to isolate and characterize a bioactive SVSP candidate from D. acutus venom using integrated multi-omics and to assess its antithrombotic effects and bleeding risk.

METHODS

PacBio full-length transcriptome sequencing combined with label-free quantitative proteomics was used to screen SVSP candidates with matched transcription-translation associations. The target protein Da-29 was purified using gel filtration and reverse-phase chromatography, and its primary structure was confirmed using transcriptome sequencing and liquid chromatography-tandem mass spectrometry. In vitro enzymatic activity and fibrinogen hydrolysis specificity, as well as in vivo antithrombotic efficacy and bleeding risk, were evaluated using rodent thrombosis and bleeding models.

RESULTS

Da-29 was observed to be a 260-amino-acid SVSP containing a conserved catalytic triad and an S1 substrate-binding pocket. It exhibited significant amidase and fibrinogen-clotting activities and selectively hydrolyzed fibrinogen Aα and Bβ chains. Furthermore, Da-29 exerted dose-associated antithrombotic effects in vivo, extending the coagulation time, reducing the fibrinogen/TXB2 levels, and upregulating 6-keto-PGF1α, without causing considerable bleeding risks at effective doses.

CONCLUSION

Da-29 exhibits antithrombotic activity associated with fibrinogen degradation and linked to concurrent alterations in TXB2 and 6-keto-PGF1α levels, establishing its potential as an antithrombotic experimental lead. This integrated omics strategy can effectively identify low- abundance bioactive molecules from snake venom, offering a reproducible paradigm for the discovery of functional proteins in natural products.