Dual inhibition of factor XIIa and factor XIa as a therapeutic approach for safe thromboprotection.

Clinical practice shows that a critical unmet need in the field of medical device-associated thrombosis prevention is the availability of an anticoagulant therapy without hemorrhagic risk. In the quest for new drugs that are at least as effective as those currently available, while avoiding bleeding complications, molecules that target nearly every step of the coagulation pathway have been developed. Among these molecules, inhibitors of factor XII (FXII) or factor XI (FXI) are promising alternatives as deficiencies in these factors protect against thrombosis without causing spontaneous hemorrhage, as revealed by epidemiological and preclinical data. Ixodes ricinus-contact phase inhibitor (Ir-CPI), a new anticoagulant candidate with an innovative mechanism of action could be this ideal anticoagulant agent for safe prevention from clotting on medical devices. This protein, which selectively binds to FXIIa, FXIa, and plasma kallikrein and inhibits the reciprocal activation of FXII, prekallikrein, and FXI in human plasma, was shown to prevent thrombosis in an ovine cardiopulmonary bypass system associated with cardiac surgeries. Furthermore, as opposed to unfractionated heparin, Ir-CPI appears to be devoid of bleeding risk. This review outlines the rationale for targeting upstream coagulation factors in order to prevent medical device-associated thrombosis; examines the novel approaches under development; and focuses on Ir-CPI, which shows promising properties in the field of thrombosis prevention.

Anticoagulation With an Inhibitor of Factors XIa and XIIa During Cardiopulmonary Bypass.

BACKGROUND: Exposure of blood to polyanionic artificial surfaces, for example, during cardiopulmonary bypass (CPB), induces a highly procoagulant condition requiring strong anticoagulation. Unfractionated heparin (UFH) is currently used during CPB but can lead to serious bleeding complications or development of a hypercoagulable state culminating in life-threatening thrombosis, highlighting the need for safer antithrombotics. Ixodes ricinus contact phase inhibitor (Ir-CPI) is a protein expressed by I. ricinus ticks, which specifically inhibits both factors XIIa and XIa, 2 factors contributing to thrombotic disease while playing a limited role in hemostasis. OBJECTIVES: This study assessed the antithrombotic activity of Ir-CPI in animal contact phase-initiated thrombosis models, including CPB. The safety of Ir-CPI also was evaluated. METHODS: The authors evaluated the antithrombotic activity of Ir-CPI by using in vitro catheter-induced clotting assays and rabbit experimental models of catheter occlusion and arteriovenous shunt. During CPB with cardiac surgery in sheep, the clinical applicability of Ir-CPI was investigated and its efficacy compared to that of UFH using an uncoated system suitable for adult therapy. Taking advantage of the similar hemostatic properties of pigs and humans, the authors performed pig liver bleeding assays to evaluate the safety of Ir-CPI. RESULTS: Ir-CPI prevented clotting in catheter and arteriovenous shunt rabbit models. During CPB, Ir-CPI was as efficient as UFH in preventing clot formation within the extracorporeal circuit and maintained physiological parameters during and post-surgery. Unlike UFH, Ir-CPI did not promote bleeding. CONCLUSIONS: Preclinical animal models used in this study showed that Ir-CPI is an effective and safe antithrombotic agent that provides a clinically relevant approach to thrombosis prevention in bypass systems, including highly thrombogenic CPB.

Ir-CPI, a coagulation contact phase inhibitor from the tick Ixodes ricinus, inhibits thrombus formation without impairing hemostasis.

Blood coagulation starts immediately after damage to the vascular endothelium. This system is essential for minimizing blood loss from an injured blood vessel but also contributes to vascular thrombosis. Although it has long been thought that the intrinsic coagulation pathway is not important for clotting in vivo, recent data obtained with genetically altered mice indicate that contact phase proteins seem to be essential for thrombus formation. We show that recombinant Ixodes ricinus contact phase inhibitor (Ir-CPI), a Kunitz-type protein expressed by the salivary glands of the tick Ixodes ricinus, specifically interacts with activated human contact phase factors (FXIIa, FXIa, and kallikrein) and prolongs the activated partial thromboplastin time (aPTT) in vitro. The effects of Ir-CPI were also examined in vivo using both venous and arterial thrombosis models. Intravenous administration of Ir-CPI in rats and mice caused a dose-dependent reduction in venous thrombus formation and revealed a defect in the formation of arterial occlusive thrombi. Moreover, mice injected with Ir-CPI are protected against collagen- and epinephrine-induced thromboembolism. Remarkably, the effective antithrombotic dose of Ir-CPI did not promote bleeding or impair blood coagulation parameters. To conclude, our results show that a contact phase inhibitor is an effective and safe antithrombotic agent in vivo.

Direct protein detection with a nano-interdigitated array gate MOSFET.

A new protein sensor is demonstrated by replacing the gate of a metal oxide semiconductor field effect transistor (MOSFET) with a nano-interdigitated array (nIDA). The sensor is able to detect the binding reaction of a typical antibody Ixodes ricinus immunosuppressor (anti-Iris) protein at a concentration lower than 1 ng/ml. The sensor exhibits a high selectivity and reproducible specific detection. We provide a simple model that describes the behavior of the sensor and explains the origin of its high sensitivity. The simulated and experimental results indicate that the drain current of nIDA-gate MOSFET sensor is significantly increased with the successive binding of the thiol layer, Iris and anti-Iris protein layers. It is found that the sensor detection limit can be improved by well optimizing the geometrical parameters of nIDA-gate MOSFET. This nanobiosensor, with real-time and label-free capabilities, can easily be used for the detection of other proteins, DNA, virus and cancer markers. Moreover, an on-chip associated electronics nearby the sensor can be integrated since its fabrication is compatible with complementary metal oxide semiconductor (CMOS) technology.

Exosites mediate the anti-inflammatory effects of a multifunctional serpin from the saliva of the tick Ixodes ricinus.

Serine protease inhibitors (serpins) are a structurally related but functionally diverse family of ubiquitous proteins. We previously described Ixodes ricinus immunosuppressor (Iris) as a serpin from the saliva of the tick I. ricinus displaying high affinity for human leukocyte elastase. Iris also displays pleotropic effects because it interferes with both the immune response and hemostasis of the host. It thus inhibits lymphocyte proliferation and the secretion of interferon-gamma or tumor necrosis factor-alpha by peripheral blood mononuclear cells, and also platelet adhesion, coagulation and fibrinolysis. Its ability to interfere with coagulation and fibrinolysis, but not platelet adhesion, depends on the integrity of its antiproteolytic reactive center loop domain. Here, we dissect the mechanisms underlying the interaction of recombinant Iris with peripheral blood mononuclear cells. We show that Iris binds to monocytes/macrophages and inhibits their ability to secrete tumor necrosis factor-alpha. Recombinant Iris also has a protective role in endotoxemic shock. The anti-inflammatory ability of Iris does not depend on its antiprotease activity. Moreover, we pinpoint the exosites involved in this activity.

Ir-LBP, an ixodes ricinus tick salivary LTB4-binding lipocalin, interferes with host neutrophil function.

BACKGROUND: During their blood meal, ticks secrete a wide variety of proteins that can interfere with their host's defense mechanisms. Among these proteins, lipocalins play a major role in the modulation of the inflammatory response. METHODOLOGY/PRINCIPAL FINDINGS: We previously identified 14 new lipocalin genes in the tick Ixodes ricinus. One of them codes for a protein that specifically binds leukotriene B4 with a very high affinity (Kd: +/-1 nM), similar to that of the neutrophil transmembrane receptor BLT1. By in silico approaches, we modeled the 3D structure of the protein and the binding of LTB4 into the ligand pocket. This protein, called Ir-LBP, inhibits neutrophil chemotaxis in vitro and delays LTB4-induced apoptosis. Ir-LBP also inhibits the host inflammatory response in vivo by decreasing the number and activation of neutrophils located at the tick bite site. Thus, Ir-LBP participates in the tick's ability to interfere with proper neutrophil function in inflammation. CONCLUSIONS/SIGNIFICANCE: These elements suggest that Ir-LBP is a "scavenger" of LTB4, which, in combination with other factors, such as histamine-binding proteins or proteins inhibiting the classical or alternative complement pathways, permits the tick to properly manage its blood meal. Moreover, with regard to its properties, Ir-LBP could possibly be used as a therapeutic tool for illnesses associated with an increased LTB4 production.

Ixodes ricinus tick lipocalins: identification, cloning, phylogenetic analysis and biochemical characterization.

BACKGROUND: During their blood meal, ticks secrete a wide variety of proteins that interfere with their host's defense mechanisms. Among these proteins, lipocalins play a major role in the modulation of the inflammatory response. METHODOLOGY/PRINCIPAL FINDINGS: Screening a cDNA library in association with RT-PCR and RACE methodologies allowed us to identify 14 new lipocalin genes in the salivary glands of the Ixodes ricinus hard tick. A computational in-depth structural analysis confirmed that LIRs belong to the lipocalin family. These proteins were called LIR for "Lipocalin from I. ricinus" and numbered from 1 to 14 (LIR1 to LIR14). According to their percentage identity/similarity, LIR proteins may be assigned to 6 distinct phylogenetic groups. The mature proteins have calculated pM and pI varying from 21.8 kDa to 37.2 kDa and from 4.45 to 9.57 respectively. In a western blot analysis, all recombinant LIRs appeared as a series of thin bands at 50-70 kDa, suggesting extensive glycosylation, which was experimentally confirmed by treatment with N-glycosidase F. In addition, the in vivo expression analysis of LIRs in I. ricinus, examined by RT-PCR, showed homogeneous expression profiles for certain phylogenetic groups and relatively heterogeneous profiles for other groups. Finally, we demonstrated that LIR6 codes for a protein that specifically binds leukotriene B4. CONCLUSIONS/SIGNIFICANCE: This work confirms that, regarding their biochemical properties, expression profile, and sequence signature, lipocalins in Ixodes hard tick genus, and more specifically in the Ixodes ricinus species, are segregated into distinct phylogenetic groups suggesting potential distinct function. This was particularly demonstrated by the ability of LIR6 to scavenge leukotriene B4. The other LIRs did not bind any of the ligands tested, such as 5-hydroxytryptamine, ADP, norepinephrine, platelet activating factor, prostaglandins D2 and E2, and finally leukotrienes B4 and C4.

A family of putative metalloproteases in the salivary glands of the tick Ixodes ricinus.

Ticks are obligate blood-feeding arachnids. During their long-lasting blood meal, they have to counteract the protective barriers and defense mechanisms of their host. These include tissue integrity, pain, hemostasis, and the inflammatory and immune reactions. Here, we describe a multigene family coding for five putative salivary metalloproteases induced during the blood meal of Ixodes ricinus. The evolutionary divergence inside the family was driven by positive Darwinian selection. This came together with individual variation of expression, functional heterogeneity, and antigenic diversification. Inhibition of the expression of some of these genes by RNA interference prevented completion of the tick blood meal and affected the ability of the tick saliva to interfere with host fibrinolysis. This family of proteins could therefore participate in the inhibition of wound healing after the tick bite, thereby facilitating the completion of the blood meal.

Variability and action mechanism of a family of anticomplement proteins in Ixodes ricinus.

BACKGROUND: Ticks are blood feeding arachnids that characteristically take a long blood meal. They must therefore counteract host defence mechanisms such as hemostasis, inflammation and the immune response. This is achieved by expressing batteries of salivary proteins coded by multigene families. METHODOLOGY/PRINCIPAL FINDINGS: We report the in-depth analysis of a tick multigene family and describe five new anticomplement proteins in Ixodes ricinus. Compared to previously described Ixodes anticomplement proteins, these segregated into a new phylogenetic group or subfamily. These proteins have a novel action mechanism as they specifically bind to properdin, leading to the inhibition of C3 convertase and the alternative complement pathway. An excess of non-synonymous over synonymous changes indicated that coding sequences had undergone diversifying selection. Diversification was not associated with structural, biochemical or functional diversity, adaptation to host species or stage specificity but rather to differences in antigenicity. CONCLUSIONS/SIGNIFICANCE: Anticomplement proteins from I. ricinus are the first inhibitors that specifically target a positive regulator of complement, properdin. They may provide new tools for the investigation of role of properdin in physiological and pathophysiological mechanisms. They may also be useful in disorders affecting the alternative complement pathway. Looking for and detecting the different selection pressures involved will help in understanding the evolution of multigene families and hematophagy in arthropods.

Distantly related lipocalins share two conserved clusters of hydrophobic residues: use in homology modeling.

BACKGROUND: Lipocalins are widely distributed in nature and are found in bacteria, plants, arthropoda and vertebra. In hematophagous arthropods, they are implicated in the successful accomplishment of the blood meal, interfering with platelet aggregation, blood coagulation and inflammation and in the transmission of disease parasites such as Trypanosoma cruzi and Borrelia burgdorferi. The pairwise sequence identity is low among this family, often below 30%, despite a well conserved tertiary structure. Under the 30% identity threshold, alignment methods do not correctly assign and align proteins. The only safe way to assign a sequence to that family is by experimental determination. However, these procedures are long and costly and cannot always be applied. A way to circumvent the experimental approach is sequence and structure analyze. To further help in that task, the residues implicated in the stabilisation of the lipocalin fold were determined. This was done by analyzing the conserved interactions for ten lipocalins having a maximum pairwise identity of 28% and various functions. RESULTS: It was determined that two hydrophobic clusters of residues are conserved by analysing the ten lipocalin structures and sequences. One cluster is internal to the barrel, involving all strands and the 310 helix. The other is external, involving four strands and the helix lying parallel to the barrel surface. These clusters are also present in RaHBP2, a unusual "outlier" lipocalin from tick Rhipicephalus appendiculatus. This information was used to assess assignment of LIR2 a protein from Ixodes ricinus and to build a 3D model that helps to predict function. FTIR data support the lipocalin fold for this protein. CONCLUSION: By sequence and structural analyzes, two conserved clusters of hydrophobic residues in interactions have been identified in lipocalins. Since the residues implicated are not conserved for function, they should provide the minimal subset necessary to confer the lipocalin fold. This information has been used to assign LIR2 to lipocalins and to investigate its structure/function relationship. This study could be applied to other protein families with low pairwise similarity, such as the structurally related fatty acid binding proteins or avidins.

The impact of gene knock-down and vaccination against salivary metalloproteases on blood feeding and egg laying by Ixodes ricinus.

Two cDNAs coding homologous putative metalloproteases (Metis 1 and Metis 2, expected molecular weights of 55.6 and 56.0kDa, respectively) were identified from the hard tick Ixodes ricinus. The expression of Metis genes was induced in salivary glands during tick blood meal. RNA interference was used to assess the role of both Metis 1 and Metis 2 in tick feeding. It was found that salivary gland extracts lacking Metis 1-2 had a restricted ability to interfere with fibrinolysis. RNAi against Metis 1-2 also induced a high mortality rate. An immune reaction was raised in repeatedly bitten animals against Metis 1 and 2. Vaccination of hosts with the recombinant Metis 1 protein produced in a eukaryotic system partially interfered with completion of the blood meal. Although vaccination did not alter the survival rate or feeding time of ticks, their weight gain and oviposition rate were reduced. This will affect their reproductive fitness in the field. We believe this is the first report of an anti-tick vaccine trial using a metalloprotease derived from I. ricinus.

Anti-hemostatic effects of a serpin from the saliva of the tick Ixodes ricinus.

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 Ka was observed with human leukocyte-elastase, 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.

Characterization of Borrelia lusitaniae isolates collected in Tunisia and Morocco.

Borrelia lusitaniae is a species within the complex Borrelia burgdorferi sensu lato and is infrequently isolated in Europe. In contrast, this species is by far the most predominant in North Africa and in Portugal. In this study, we analyzed the genetic diversity, at several loci, of a large population of isolates from free-living Ixodes ricinus ticks collected in Tunisia and Morocco. We found a moderate diversity of the whole genome by using pulsed-field gel electrophoresis as well as in the ospA gene sequences, compared to a high level of strain homogeneity in the small noncoding ribosomal spacer. In contrast, a high diversity of this locus has been previously reported for Portuguese isolates. We hypothesize that B. lusitaniae strains isolated in North Africa constitute a clone of Portuguese origin.

PCR-reverse line blot typing method underscores the genomic heterogeneity of Borrelia valaisiana species and suggests its potential involvement in Lyme disease.

Detection of the Borrelia burgdorferi sensu lato complex in biological samples is currently done by conventional immunological and molecular biological methods. To improve on the accuracy of these methods and to simplify the procedure for testing large numbers of samples, a solid-phase sandwich hybridization system readily applicable to the detection of PCR products has been designed. This colorimetric detection system relies on the use of polybiotinylated detection probes and of specific capture oligonucleotides covalently linked at allocated positions on nylon membrane strips. From a phylogenetic analysis on a great number of ospA gene sequences, we have designed and synthesized a set of PCR primers specific to the five Borrelia burgdorferi sensu lato genospecies present in Europe and a subset of probes (capture and detection probes) specific to these five genospecies (B. burgdorferi sensu stricto, B. garinii, B. afzelii, B. valaisiana, and B. lusitaniae). This combined PCR hybridization system was evaluated with a large number of various B. burgdorferi isolates and clinical specimens. These analyses clearly showed that the system could be used as a typing method to distinguish five genospecies belonging to the B. burgdorferi sensu lato complex. In addition, the study showed that B. valaisiana strains might be more heterologous than suspected up to now and clustered into three genomic groups.

OspA heterogeneity of Borrelia valaisiana confirmed by phenotypic and genotypic analyses.

BACKGROUND: Although European Borrelia burgdorferi sensu lato isolates have been divided into five genospecies, specific tools for the serotype characterization of only three genospecies are available. Monoclonals antibodies (mAbs) H3TS, D6 and I17.3 identify B. burgdorferi sensu stricto (ss.), B. garinii and B. afzelii respectively, but no mAbs are available to identify B. valaisiana. In the same way, specific primers exist to amplify the OspA gene of B. burgdorferi ss., B. garinii and B. afzelii. The aim of the study was to develop species-specific mAb and PCR primers for the phenotypic and genetic identification of B. valaisiana. RESULTS: This study describes a mAb that targets OspA of B. valaisiana and primers targeting the OspA gene of this species. As the monoclonal antibody A116k did not react with strains NE231, M7, M53 and Frank and no amplification was observed with strains NE231, M7 and M53, the existence of two subgroups among European B. valaisiana species was confirmed. CONCLUSIONS: The association of both monoclonal antibody A116k and primers Bval 1F and Bval 1R allows to specific identification of the B. valaisiana isolates belonging to subgroup 1.

Isolation of Ixodes ricinus salivary gland mRNA encoding factors induced during blood feeding.

In tick salivary glands, genes induced during blood feeding result in the expression of new proteins secreted into tick saliva. These proteins are potentially involved in modulation of vertebrate host immune and hemostatic responses. In this study, subtractive and full-length cDNA libraries were constructed by use of mRNA extracted from salivary glands of unfed and 5-day engorged Ixodes ricinus. Sequences from these 2 libraries were compared with European Molecular Biology Laboratory (EMBL)/GenBank databases, which led to their classification into 2 major groups. The first group comprises cDNAs that failed to match or showed low homology to genes of known function. The second group includes sequences that showed high homology to genes of known function--for example, anticoagulants, inhibitors of platelet aggregation, and immunomodulatory proteins. Analyses of corresponding proteins suggest that they may be secreted by salivary gland cells. To study the properties of the recombinant proteins, selected cDNAs were expressed in mammalian or bacterial systems.

Characterization of a novel salivary immunosuppressive protein from Ixodes ricinus ticks.

In tick salivary glands, several genes are induced during the feeding process, leading to the expression of new proteins. These proteins are typically secreted in tick saliva and are potentially involved in the modulation of the host immune and hemostatic responses. In a previous study, the construction and the analysis of a subtractive library led to the identification of Ixodes ricinus immunosuppressor (Iris), a novel protein, differentially expressed in I. ricinus salivary glands during the blood meal. In the present study, the data strongly suggest that this protein is secreted by tick salivary glands into the saliva. In addition, Iris is also found to modulate T lymphocyte and macrophage responsiveness by inducing a Th2 type response and by inhibiting the production of pro-inflammatory cytokines. In conclusion, these results suggest that Iris is an immunosuppressor, which might play an important role in the modulation of host immune response.