Anti-hemostatic Effects of a Serpin from the Saliva of the Tick Ixodes ricinus

Abstract

Serpins (serine protease inhibitors) are a large family of structurally related proteins found in a wide variety of organisms, including hematophagous arthropods. Protein analyses revealed that Iris, previously described as an immunomodulator secreted in the tick saliva, is related to the leukocyte elastase inhibitor and possesses serpin motifs, including the reactive center loop (RCL), which is involved in the interaction between serpins and serine proteases. Only serine proteases were inhibited by purified recombinant Iris (rIris), whereas mutants L339A and A332P were found devoid of any protease inhibitory activity. The highest K a was observed with human leukocyteelastase, suggesting that elastase-like proteases are the natural targets of Iris. In addition, mutation M340R completely changed both Iris substrate specificity and affinity. This likely identified Met-340 as amino acid P1 in the RCL. The effects of rIris and its mutants were also tested on primary hemostasis, blood clotting, and fibrinolysis. rIris increased platelet adhesion, the contact phase-activated pathway of coagulation, and fibrinolysis times in a dose-dependent manner, whereas rIris mutant L339A affected only platelet adhesion. Taken together, these results indicate that Iris disrupts coagulation and fibrinolysis via the anti-proteolytic RCL domain. One or more other domains could be responsible for primary hemostasis inhibition. To our knowledge, this is the first ectoparasite serpin that interferes with both hemostasis and the immune response.Ticks are blood-sucking arthropods that infest a large variety of vertebrate hosts (mammals, birds, reptiles, and amphibians) in many parts of the world (1). To complete their blood meal, blood-sucking arthropods express a wide range of anti-hemostatic molecules in their saliva, including vasodilators, inhibitors of the platelet aggregation, and anti-coagulants (2). Tick saliva and salivary gland extracts are also known to modulate the host's defense mechanisms (3-6). Both anti-hemostatic and immunosuppressive compounds were identified, isolated, and characterized from soft and hard ticks. These compounds include histamine-binding proteins, tissue factor pathway inhibitor-like proteins, anti-thrombin-like proteins, and anticomplement factors (7-16).These last years, several laboratories reported the construction and screening of cDNA libraries from tick salivary glands. Thus, Das et al. (17) found 14 Ixodes scapularis immunodominant antigens, whereas Leboulle et al. (18) identified 27 mRNA, the expression of which is specifically induced or up-regulated during the Ixodes ricinus blood meal. Finally, Ribeiro and coworkers explored the sialome of the tick I. scapularis (19,20) and uncovered a large variety of putative bioactive agents. These studies all identified some serine protease inhibitors, containing serpin, kunitz, kazal, or ␣-macroglobulin motifs (21).To date, ϳ500 serpins have been identified in a large variety of species, including animals, viruses, and plants. On average, serpins are 35...

Protective immunity against Ixodes ricinus induced by a salivary serpin

“…The specificity of these signals was indicated by the detection of a discrete band at the expected molecular weight of 48 kDa when recombinant Iris was loaded onto the gel (lanes 5 and 6). The difference between the natural and recombinant proteins could be explained by the presence of V5 epitope and His tag on the C-terminus of rIris [27]. An additional band was also detected at 54 kDa in salivary gland extracts (lane 3).…”

“…This activity was monitored by testing its ability to inhibit the pancreatic porcine elastase (PPE) activity [27]. As reported previously, rIris inhibits elastase activity by around 70%, while the same amount of ovalbumin has no effect.…”

“…Accordingly, we have named the protein Iris for "Ixodes ricinus immunosuppressor". We have also shown that Iris is a specific elastase inhibitor that interferes with the contact phase coagulation pathway and fibrinolysis, and disrupts platelet adhesion [27]. To our knowledge, this is the first ectoparasite serpin that interferes with both haemostasis and the immune response.…”

“…We tested whether antisera neutralized the antiproteasic activity of rIris on porcine pancreatic elastase (PPE) as described previously [27]. Briefly, rIris was pre-incubated with dilutions of immune and control sera for 5 min at 37 • C in Tris buffer.…”

“…Recombinant Iris was produced on a baculovirus system according to Prevot et al [27]. The membrane was revealed with the rIris rabbit antiserum or pre-immune serum from the same rabbit as negative control (1:1000 dilution on PBS-0.1% Tween 20-0.5% Instagel) at room temperature for 1 h, and washed three times with PBS-Tween.…”