A blood meal-induced Ixodes scapularis tick saliva serpin inhibits trypsin and thrombin, and interferes with platelet aggregation and blood clotting

Abstract

Ixodes scapularis is a medically important tick species that transmits causative agents of important human tick-borne diseases including borreliosis, anaplasmosis and babesiosis. An understanding of how this tick feeds is needed prior to the development of novel methods to protect the human population against tick-borne disease infections. This study characterizes a blood meal-induced I. scapularis (Ixsc) tick saliva serine protease inhibitor (serpin (S)), in-house referred to as IxscS-1E1. The hypothesis that ticks use serpins to evade the host's defense response to tick feeding is based on the assumption that tick serpins inhibit functions of protease mediators of the host's anti-tick defense response. Thus, it is significant that consistent with hallmark characteristics of inhibitory serpins, Pichia pastoris-expressed recombinant IxscS-1E1 (rIxscS-1E1) can trap thrombin and trypsin in SDS- and heat-stable complexes, and reduce the activity of the two proteases in a dose-responsive manner. Additionally, rIxscS-1E1 also inhibited, but did not apparently form detectable complexes with, cathepsin G and factor Xa. Our data also show that rIxscS-1E1 may not inhibit chymotrypsin, kallikrein, chymase, plasmin, elastase and papain even at a much higher rIxscS-1E1 concentration. Native IxscS-1E1 potentially plays a role(s) in facilitating I. scapularis tick evasion of the host's hemostatic defense as revealed by the ability of rIxscS-1E1 to inhibit adenosine diphosphate (ADP)- and thrombin-activated platelet aggregation, and delay activated partial prothrombin time (APTT) and thrombin time (TT) plasma clotting in a dose-responsive manner. We conclude that native IxscS-1E1 is part of the tick saliva protein complex that mediates its anti-hemostatic, and potentially inflammatory, functions by inhibiting the actions of thrombin, trypsin and other yet unknown trypsin-like proteases at the tick-host interface.

Ixodes scapularistick saliva proteins sequentially secreted every 24h during blood feeding

“…For instance serpin EEC19556.1 in SCA (Fig 3B1) is 98% identical to AID54718.1, an inhibitor of trypsin and thrombin that also inhibited blood clotting and platelet aggregation [43]. Similarly I. ricinus serpin ABI94056, the homolog of I. scapularis serpin EEC14235.1 in this study (Fig 3B1 SCB) is an immunosuppressant, anti-inflammatory, and anti-hemostatic serpin [78][79][80].…”

“…Ticks were considered mated once males were detached from the females. Routinely, ticks were fed on rabbits as previously described [43]. Mated I. scapularis ticks were restricted to feed onto the outer part of the ear of New Zealand rabbits with orthopedic stockinet's glued with Kamar adhesive (Kamar Products Inc., Zionsville, IN, USA).…”

“…Additionally one protein was identified in I. scapularis nymphs as an immunogenic protein that bound to human serum from exposure to tick bites [198]. The remaining proteins were found in saliva proteomes of R. microplus (n = 28, [50]), H. longicornis (n = 22, [52]), D. andersoni (n = 2, [51]), O. moubata (n = 5, [53], sequencing of I. scapularis tick saliva by Edman degradation (n = 4) [48], and others were verified as secreted in western blotting studies [42,43,[199][200][201][202][203][204].…”

“…al., [40] and Lewis et. al., [41] used antibodies to 24-48 h tick saliva proteins [42,43] to immunoscreen phage display cDNA expression libraries to identify 24-48 h Amblyomma americanum and 24 h I. scapularis immunogenic tick saliva proteins. Similar immunoscreening approaches were used to identify immunodominant I. scapularis tick saliva proteins [44][45][46][47].…”

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

“…Accordingly, inhibitory tick serpins have been found and characterized in tick saliva, including A. americanum salivary serpin (AAS) 6 (Mulenga et al, 2007; Chalaire et al, 2011), a cross-class inhibitor of papain and trypsin-like proteases able to inhibit blood clotting and complement activation (Mulenga et al, 2013). A blood meal-induced salivary I. scapularis serpin has been shown to act upon thrombin and platelet aggregation (Ibelli et al, 2014). In related studies, an inhibitor of pro-inflammation proteases, elastase, cathepsin G and chymase was found in I. ricinus (Prevot et al, 2006, 2009; Chmelar et al, 2011).…”