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

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.

RNA-seq analyses of the midgut from blood- and serum-fed Ixodes ricinus ticks

“…The genes represented by contigs Ir-119569 and Ir-110707 each contain two BPTI/Kunitz domains and are related to boophilin , a Kunitz-type inhibitor from the midgut of the cattle tick R. microplus 57. It was recently suggested that boophilin inhibits proteolytic enzymes of host origin and thereby controls undesired blood coagulation or complement activation within the tick gut lumen58. We assume that the tick midgut serine protease inhibitors that are strikingly up-regulated in fully engorged females may function against exogenous serine proteases, either to control haemostasis or inhibit premature digestion of blood-meal stored in the midgut lumen.…”

The immunosuppressive functions of two novel tick serpins, HlSerpin‐a and HlSerpin‐b, from Haemaphysalis longicornis

“…Although multiple tick serpins have been reported to regulate the host immune process, including blood coagulation and immune cell activation, the underling mechanisms, especially how tick serpins suppress cytokine production from immune cells, remain obscure. Tick serpin Ipis‐1 was reported to inhibit the proliferations of PBMCs and T‐cells by direct binding to these cells .…”

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

“…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.…”