Divalent cations and SERPIN gene expression modulate horseshoe crab coagulation under LPS induction

Horseshoe crabs (Tachypleus gigas) have a unique immune system used in enzyme research, particularly in coagulation studies. The coagulation process in these crabs involves serine protease enzymes. However, how these enzymes and their inhibitors regulate clotting under lipopolysaccharide (LPS) induction is unclear. This study aimed to examine the effects of divalent cations and pH on the gelation and proteolytic activity of trypsin-like serine protease in wild and captive horseshoe crab blood. It also compared the protein profiles of Tachypleus amoebocyte lysate (TAL) and measured SERPIN gene expression during LPS induction. The methods used included the gel clotting method to assess gelation, SDS-PAGE for protein profiling, LC-MS/MS for protein identification, and RT-PCR for gene expression analysis. Results showed that wild horseshoe crab lysate had higher clotting levels compared to captive crabs. Among 35 divalent cation combinations, 0.15 M MgCl2 + 0.15 M CaCl2 resulted in higher clotting in wild TAL. Fresh TAL had higher proteolytic activity, and four major proteins were detected. SERPIN gene expression increased fivefold in July compared to June. Overall, captivity reduced TAL quality and clotting efficiency.