Research Article: In Vitro Mode of Action and Anti-thrombotic Activity of Boophilin, a Multifunctional Kunitz Protease Inhibitor from the Midgut of a Tick Vector of Babesiosis, Rhipicephalus microplus

Date Published: January 8, 2016

Publisher: Public Library of Science

Author(s): Teresa C. Assumpção, Dongying Ma, Daniella M. Mizurini, R. Manjunatha Kini, José M. C. Ribeiro, Michail Kotsyfakis, Robson Q. Monteiro, Ivo M. B. Francischetti, Joseph M. Vinetz.

Abstract: BackgroundHematophagous mosquitos and ticks avoid host hemostatic system through expression of enzyme inhibitors targeting proteolytic reactions of the coagulation and complement cascades. While most inhibitors characterized to date were found in the salivary glands, relatively few others have been identified in the midgut. Among those, Boophilin is a 2-Kunitz multifunctional inhibitor targeting thrombin, elastase, and kallikrein. However, the kinetics of Boophilin interaction with these enzymes, how it modulates platelet function, and whether it inhibits thrombosis in vivo have not been determined.Methodology/Principal FindingsBoophilin was expressed in HEK293 cells and purified to homogeneity. Using amidolytic assays and surface plasmon resonance experiments, we have demonstrated that Boophilin behaves as a classical, non-competitive inhibitor of thrombin with respect to small chromogenic substrates by a mechanism dependent on both exosite-1 and catalytic site. Inhibition is accompanied by blockade of platelet aggregation, fibrin formation, and clot-bound thrombin in vitro. Notably, we also identified Boophilin as a non-competitive inhibitor of FXIa, preventing FIX activation. In addition, Boophilin inhibits kallikrein activity and the reciprocal activation, indicating that it targets the contact pathway. Furthermore, Boophilin abrogates cathepsin G- and plasmin-induced platelet aggregation and partially affects elastase-mediated cleavage of Tissue Factor Pathway Inhibitor (TFPI). Finally, Boophilin inhibits carotid artery occlusion in vivo triggered by FeCl3, and promotes bleeding according to the mice tail transection method.Conclusion/SignificanceThrough inhibition of several enzymes involved in proteolytic cascades and cell activation, Boophilin plays a major role in keeping the midgut microenvironment at low hemostatic and inflammatory tonus. This response allows ticks to successfully digest a blood meal which is critical for metabolism and egg development. Boophilin is the first tick midgut FXIa anticoagulant also found to inhibit thrombosis.

Partial Text: Hematophagous insects and ticks are strict blood feeders, which express protease inhibitors in different tissues and organs. These molecules belong to the Kunitz, Kazal, Serpin, Cathepsin, and Trypsin Inhibitor-Like (TIL)-domains family of enzyme inhibitors, among others, and prevent hemostasis, inflammation and immune responses. Most of these inhibitors have been identified in the salivary glands of hematophagous animals. They display unique specificity and high-affinity binding for coagulation factors, or components of the complement cascade. Protease inhibitors also affect immune cells, endothelial cells and platelet functions [1–10].

Fig 1A shows the alignment for Boophilin sequence employed for this study and elsewhere [3,21], and the 2 variants deposited in the databases: Boophilin-G2 (gi 74844209) and Boophilin-H2 (gi74935652). Fig 1B shows a phylogenetic tree constructed with the sequences of several Kunitz protease inhibitors from other species. Boophilin clades with orthologous from Dermacentor sp (gi 194246037), Hyalomma sp (gi 217034827), and Ixodes sp (gi 241999004) indicating that it is belongs to an expanded family of proteins.

During feeding, blood remains in fluid phase in the mouthparts and digestive tracts of hematophagous arthropods. Protease inhibitors are critical in this step, since it interferes with several pro-hemostatic enzymes involved in clot formation and inflammation. Most inhibitors reported to date have been discovered in the salivary glands [1–10]. Usually, these molecules exhibit high-affinity binding and specificity towards components of the coagulation cascade, and complement system or interfere with different cell types involved in inflammatory and immune responses [4]. In contrast, fewer inhibitors have been comparatively found in the midgut of these animals, including hemalin from Haemaphysalis longicornis [12] and infestin family members from Triatoma infestans which target FXIIa, elastase and thrombin [15–17]. Thrombin inhibitors have also been identified in Ornithodorus moubatta (ornithodorin) [11], T. brasiliensis (brasiliensin) [12], R. hemaphysaloides (rhipilin-2)[19], Dipetalogaster maxima (dipetalogastin) [13] and Rhodnius prolixus (rhodniin) [14]. More recently, Boophilin from the midgut of Riphicephalus microplus was characterized as a multifunctional protease inhibitor affecting thrombin, plasmin and elastase, with residual activity towards kallikrein and FVIIa [4,21]. Boophilin inhibits thrombin in a non-canonical manner, despite possessing a canonical reactive site loop [20]. The importance of Boophilin has been demonstrated by silencing Boophilin gene, which resulted in 20% reduction in egg weight production [21]. This effect might be explained by inhibition of midgut proteases initiating and sustaining inflammatory reactions, which are presumably detrimental to tick and mosquito survival [42,43].



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