Effective inhibition of thrombin by Rhipicephalus microplus serpin-15 (RmS-15) obtained in the yeast Pichia pastoris

The cattle tick (Rhipicephalus microplus) affects cattle industries in tropical and subtropical countries because it is the vector of babesiosis and anaplasmosis which constitutes a threat to the health of cattle. During blooding feeding, ticks secrete saliva containing a complex of bioactive molecules into the injured site to evade host's defensive responses. Serine protease inhibitors (serpins) are important anti-haemostatic molecules present in tick saliva that are necessary for a successful blood feeding. Several serpin sequences have been reported in R. microplus but there is a gap of information about their functions during host-parasite interactions. In this study, the RmS-15 expressed in the yeast Pichia pastoris was characterised using kinetic assays and in vitro analysis. The inhibitory enzymatic assays conducted showed that RmS-15 is a physiological inhibitor of thrombin with a stoichiometric inhibition (SI) of 1.5 and high inhibition affinity with ka=9.3±0.5×104M(-1)s(-1). RmS-15 delayed the clotting of plasma in a dose-dependent manner as determined in a recalcification time assay. Significant elevated ELISA titres were observed in tick resistant and susceptible cattle on day 28 after the tick infestation (p<0.001). This data suggests direct contact of RmS-15 with the immune system of the host at the tick-feeding site. The present study contributed to the understanding of the biological functions of R. microplus serpins during host-parasite interactions which contributes to the design of future innovative methods for tick control.