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- and thrombin-activated platelet aggregation, and delay activated partial prothrombin time and thrombin time 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.

Keywords: Ixodes scapularis; Serine protease inhibitors; Tick anti-blood clotting function; Tick anti-platelet aggregation function; Tick-feeding physiology.

Fig 1

Cloning of the open reading frame (ORF) for Ixodes scapularis serpin (IxscS-1E1). ORF PCR primers were designed from the sequence of the ORF assembled from two sequence fragments (GenBank Accession numbers ISCW023621 and EW88858). Translated amino acid sequences of the cloned (IxscS-1E1(C)) and assembled (IxscS-1E1 (A)) ORFs were aligned using MacVector software. The signal peptide, putative N-glycosylation sites and the reactive center loop are noted. Asterisks (*) denote amino acid differences between the ORFs. Identical amino acid residues are boxed and shaded gray.

Fig. 2

Expression, deglycosylation and western blotting analyses of recombinant Ixodes scapularis serpin (rIxscS-1E1). Affinity purified rIxscS-1E1 was subjected to SDS-PAGE with silver staining (A). Untreated (D−) and treated with deglycosylation enzyme mix (D+) rIxscS-1E1 was subjected to routine western blotting analysis using (B) 1:100 dilution pre-immune sera, or (C) immune sera to replete-fed I. scapularis tick saliva proteins.

Fig. 3

Inhibitor function profiling of recombinant Ixodes scapularis serpin (rIxscS-1E1). Various amounts of affinity purified rIxscS-1E1 (indicated) were pre-incubated with (A) 0.54 μM thrombin, (B) 6.68 μM trypsin, (C) 0.267 μ,M cathepsin G, (D) 0.0217 μM factor Xafor 10min at 37°C. Following incubation, peptide substrates were added and hydrolysis continuously monitored every 20 s up to 30min at A_405nm using the VersaMax microplate reader. Substrate hydrolysis data was acquired using the SoftMax Pro software set to the default kinetics parameters to estimate end point maximum enzyme velocity (V_max) or residual enzyme activity (REA). V_max or REA at different rIxscS-1E1 concentrations were expressed as percentages of V_max in the absence of rIxscS-1E1. Subtracting the percentage of REA from the assumed 100% enzyme activity in the absence of rIxscS-1E1 calculated the percentage of enzyme inhibition levels summarized as the mean ± S.E.M. of two assays.

Fig 4

Formation of SDS- and heat-stable complexes between recombinant Ixodes scapularis serpin (rIxscS-1E1) and target protease. A single reaction of rIxscS-1E1 and (A) thrombin or (B) trypsin was incubated at room temperature and sampled at 0, 5, 30, 60 min and overnight (ON). Overnight and the 60 min aliquots were not run for trypsin (B). Aliquots were subjected to western blotting analysis using antibodies to thrombin and trypsin as described in Section 2.8. Complexes between rIxcsS-1E1 and thrombin (A) or trypsin (B) are indicated by arrows. L, molecular size ladder; TH, thrombin; S, rIxscS-1E1.

Fig. 5

The effect of recombinant Ixodes scapularis serpin (rIxscS-1E1) on platelet aggregation function. Various amounts, of rIxscS-1E1 (as indicated in A-D) were co-incubated with cattle whole blood for 10 min at 37°C as described in Section 2.10. Addition of 20 μM adenosine diphosphate (AD ) or 0.5U/μL of thrombin triggered platelet aggregation. Platelet aggregation was monitored over 8 min using the whole blood platelet aggregometer as described in Section 2.10. (A B) The observed electrical resistance as an index for platelet aggregation. (C, D) The percentage of inhibition of ADP- and thrombin-triggered platelet aggregation, respectively. Experiments were duplicated.

Fig. 6

The effect of recombinant Ixodes scapularis serpin (rIxscS-1E1) on plasma clotting time in the activated partial thromboplastin time (APTT, A), and thrombin time (TT, B) assays. Indicated amounts of rIxscS-1E1 were pre-incubated with (A) citrated human reference plasma or (B) the TT reagent at 37°C for 10 min. Following activation of plasma clotting, the time to clot formation was determined using the KC1 DELTA coagulometer as described in Section 2.10.