Functional characterization of secreted Schistosoma mansoni serpin SmSPI reveals factor XIIa inhibition as evidenced by biochemical and human plasma assays and supported by AI-assisted structural modeling

Schistosoma mansoni persists in host vasculature for years without inducing thrombosis or excessive inflammation, suggesting active interference with host serine proteases involved in hemostasis and immunity. To explore the mechanisms behind these evasion strategies, we investigated S. mansoni Serine Protease Inhibitor (serpin), SmSPI, previously described as a circulating antigen. Our gene expression profiling revealed SmSPI to be the most highly expressed parasite serpin in intravascular life stages, including adult worms and eggs. This indicates its constant accessibility to host proteolytic pathways at multiple host-parasite interfaces (in the bloodstream, liver, and intestine), which was confirmed by immunolocalization experiments. To identify potential host targets of SmSPI, we used ColabFold-based structural prediction to screen 34 candidate host serine proteases involved in immune-, digestive-, and hemostasis-related processes. The resulting in silico predictions were validated by enzymatic activity assays, which demonstrated that recombinant SmSPI effectively inhibited several host proteases, including cathepsin G, urokinase, and pancreatic elastase, with weaker inhibition observed against chymotrypsin. Importantly, the SmSPI also exhibited significant inhibition of Factor XIIa (FXIIa), a key initiator of the contact activation system in the intrinsic coagulation pathway. To confirm the functional relevance of this interaction, we performed activated partial thromboplastin time (aPTT)-based assays in human plasma. Plasma was preincubated with rSmSPI, which resulted in a significant prolongation of thrombin generation time. Furthermore, this effect was dependent on the concentration of FXII present in human plasma, confirming the specificity of this inhibition. These results establish SmSPI as a multifunctional host-interacting serpin with immunomodulatory and targeted anticoagulant properties. Notably, its ability to inhibit FXIIa identifies SmSPI as the first helminth-derived inhibitor of the contact activation system, making the protein a promising candidate for future anticoagulant therapies designed to limit thrombosis while preserving normal hemostasis.