Identification and anticoagulant activity of a novel Kunitz-type protein HA11 from the salivary gland of the tick Hyalomma asiaticum

Abstract

Kunitz/bovine pancreatic trypsin inhibitor proteins are abundant in the salivary glands of ticks and perform multiple functions in blood feeding, including inhibiting blood coagulation, regulating host blood supply and disrupting host angiogenesis. In this study, we identified a novel gene designated HA11 (Hyalomma asiaticum 11 kDa protein) from the salivary gland of the tick H. asiaticum. HA11 is encoded by a gene with an open reading frame of 306 bp that is translated into a deduced 101 amino acid 11 kDa protein that shares 27% sequence identity with a Kunitz-like protease inhibitor precursor in Amblyomma variegatum. Bioinformatic analysis confirmed HA11 as a member of the Kunitz-type family of inhibitors. Real time-PCR detected HA11 mRNA transcripts in tick larvae and nymphae stages, with levels highest in salivary gland tissue, and transcription was induced by blood feeding. HA11 anticoagulant activity was demonstrated by its ability to delay normal clotting of rabbit plasma in an activated partial thromboplastin time assay. Furthermore, RNA interference confirmed that HA11 influences H. asiaticum development and blood feeding, and the recombinant protein exerted low hemolytic activity. These results suggest HA11 is a novel Kunitz-type anticoagulant protein involved in tick blood feeding that may have potential as an anticoagulant drug or vaccine.

Gut contents, digestive half-lives and feeding state prediction in the soil predatory mite Pergamasus longicornis (Mesostigmata: Parasitidae)

“…This supports the view that mesostigmatids employ extracorporeal digestion (like pseudoscorpions—Vachon ( 1934 ) and predatory beetles—Evans ( 1965 )) and possibly also use emulsifiers (see Vonk 1962 ; Collatz and Mommsen 1974 ) or anticoagulants (known to occur in the saliva and intestines of closely related ticks—Nuttal and Strickland 1908 ; Pavolvsky and Chodukin 1929 ; Zhang et al. 2017 ). Extra-corporeal digestion is already suspected in other mites (see Hamilton et al.…”

Tick-Borne Encephalitis Virus Infection Alters the Sialome of Ixodes ricinus Ticks During the Earliest Stages of Feeding

“…In some animals, however, these inhibitors evolved to serve as venom (Mourao and Schwartz, 2013). For arachnids, Kunitz-domain-containing proteins either interfere with ion channel activity (Chen et al, 2012;Dai et al, 2012;Valdes and Moal, 2014;Santibañez-Lopez et al, 2018) or inhibit critical host proteins (Decrem et al, 2008;Bronsoms et al, 2011;Andreotti et al, 2012;Louw et al, 2013;Zhang et al, 2017). Tick saliva is essentially a form of venom evolved to facilitate bloodfeeding, and although Ixodes spp.…”

“…Kunitz-domain serine protease inhibitors are known to inhibit coagulation (Dai et al, 2012;Chen et al, 2013;Louw et al, 2013;Assumpção et al, 2015;Zhang et al, 2017), angiogenesis (Drewes et al, 2012;Soares et al, 2016), and may reduce inflammation (Bronsoms et al, 2011). Their primary purpose is to ensure successful feeding by inhibiting blood coagulation.…”

Peptidase inhibitors in tick physiology

“…In turn, the thrombin inhibitors variegin and amblin from Amblyomma variegatum (Fabricius, 1794) (Koh et al ., , ) and Amblyomma hebraeum (Koch, 1844) (Lai et al ., ), respectively, are being studied as potential anti‐hemostatic drugs. Another Kunitz‐like peptidase inhibitor with anti‐hemostatic property is HA11, from Hyalomma asiaticum (Schulze & Schlottke, 1930) (Zhang et al ., ), which is induced during larval and nymphal blood feeding phases; recombinant HA11 delays normal plasma clotting in aPTT assays.…”