Conserved Amblyomma americanum tick Serpin19, an inhibitor of blood clotting factors Xa and XIa, trypsin and plasmin, has anti-haemostatic functions

Abstract

Tick saliva serine protease inhibitors (serpins) facilitate tick blood meal feeding through inhibition of protease mediators of host defense pathways. We previously identified a highly conserved Amblyomma americanum serpin (AAS) 19 that is characterized by its reactive center loop being 100% conserved in ixodid ticks. In this study, biochemical characterization reveals that the ubiquitously transcribed AAS19 is an anti-coagulant protein, inhibiting the activity of five of the eight serine protease blood clotting factors. Pichia pastoris-expressed recombinant (r) AAS19 inhibits the enzyme activity of trypsin, plasmin and blood clotting factors (f) Xa and XIa, with stoichiometry of inhibition estimated at 5.1, 9.4, 23.8 and 28, respectively. Similar to typical inhibitory serpins, rAAS19 forms irreversible complexes with trypsin, fXa and fXIa. At a higher molar excess of rAAS19, fXIIa is inhibited by 82.5%, and thrombin (fIIa), fIXa, chymotrypsin and tryptase are inhibited moderately by 14 – 29%. In anti-hemostatic functional assays, rAAS19 inhibits thrombin but not ADP and cathepsin G activated platelet aggregation, delays clotting in recalcification and thrombin time assays by up to 250 s, and up to 40 s in the activated partial thromboplastin time assay. Given AAS19 high cross-tick species conservation, and specific reactivity of rAAS19 with antibodies to A. americanum tick saliva proteins, we conclude that rAAS19 is a potential candidate for development of a universal tick vaccine.

Target validation of highly conserved Amblyomma americanum tick saliva serine protease inhibitor 19

“…In another study we showed that AAS19 mRNA was expressed in multiple tick organs, and that native AAS19 was an immunogenic tick saliva protein as indicated by antibodies to replete fed tick saliva proteins specifically binding yeast expressed rAAS19. Further, we showed that rAAS19 has anti-haemostatic functions and that it is an inhibitor of trypsin-like proteases including five of the eight serine proteases in the blood clotting cascade (Kim et al, 2015a). These earlier findings collectively suggested the potential for AAS19 to regulate important functions in tick feeding success.…”

“…Of significant interest to us, the antibody response to rAAS19 also reacted with epitopes on glycosylated and non-glycosylated forms of native AAS19 as revealed by western blotting analysis data. The observation that native AAS19 protein is present in multiple tick organs and the fact that ticks inject this protein into the host during feeding (Kim et al, 2015a) suggests that this protein may function inside the tick and at the tick-feeding site. This observation is not unique to AAS19.…”

“…Expression and purification of the immunizing antigen, rAAS19, in Pichia pastoris (X-33) was previously described in Kim et al, (2015a). Two immunizations were administered at days 0 and 30.…”

“…Given that the immunizing antigen was glycosylated (Kim et al, 2015a), we investigated if the observed antibody response was directed toward glycans on rAAS19 by including three controls: yeast expressed deglycosylated and non-deglycosylated (Kim et al, 2015a) and bacteria expressed rAAS19 (described below). Samples were loaded and resolved on a 10% SDS-PAGE gel and transferred onto a PVDF membrane.…”

“…Through this pipeline we identified highly conserved A. americanum tick saliva serine protease inhibitor (serpin) (AAS) 19 characterized by 100% conservation of the functional domain reactive center loop in ixodid ticks (Porter et al, 2015). Kim et al (2015a) recently showed that AAS19 mRNA is expressed in most tick tissues, that the native protein is injected into the host during tick feeding, and that this protein has anti-haemostic functions with broad inhibitor activity against trypsin, plasmin and five of the eight serine proteases (factors [f] Xa and XIa, strongly, fIIa [thrombin], factors IXa, and XIIa, moderately) in the mammalian blood clotting cascade. In this study the goal was to validate the significance of AAS19 in A. americanum tick feeding success and assess its anti-tick vaccine efficacy.…”