Amblyomma americanum tick saliva serine protease inhibitor 6 is a cross-class inhibitor of serine proteases and papain-like cysteine proteases that delays plasma clotting and inhibits platelet aggregation

We previously demonstrated that Amblyomma americanum tick serine protease inhibitor 6 (AamS6) was secreted into the host during tick feeding and that both its mRNA and protein were ubiquitously and highly expressed during the first 3 days of tick feeding. This study demonstrates that AamS6 is a cross-class inhibitor of both serine- and papain-like cysteine proteases that has apparent antihaemostatic functions. Consistent with the typical inhibitory serpin characteristics, enzyme kinetics analyses revealed that Pichia pastoris-expressed recombinant (r) AamS6 reduced initial velocities of substrate hydrolysis (V0) and/or maximum enzyme velocity (Vmax) of trypsin, chymotrypsin, elastase, chymase, and papain in a doseresponse manner. We speculate that rAamS6 inhibited plasmin in a temporary fashion in that while rAamS6 reduced V0 of plasmin by up to approximate to 53%, it had no effect on Vmax. Our data also suggest that rAmS6 has minimal or no apparent effect on V0 or Vmax of thrombin, factor Xa, and kallikrein. We speculate that AamS6 is apparently involved in facilitating blood meal feeding in that various amounts of rAamS6 reduced platelet aggregation by up to approximate to 47% and delayed plasma clotting time in the recalcification time assay by up to approximate to 210 s. AamS6 is most likely not involved with the tick's evasion of the host's complement defense mechanism, in that rAamS6 did not interfere with the complement activation pathway. Findings in this study are discussed in the context of expanding our understanding of tick proteins that control bloodmeal feeding and hence tick-borne disease transmission by ticks.