Tick-Derived Peptide Blocks Potassium Channel TREK-1.

Ticks transmit a variety of pathogens, including rickettsia and viruses, when they feed on blood, afflicting humans and other animals. Bioactive components acting on inflammation, coagulation, and the immune system were reported to facilitate ticks' ability to suck blood and transmit tick-borne diseases. In this study, a novel peptide, IstTx, from an Ixodes scapularis cDNA library was analyzed. The peptide IstTx, obtained by recombinant expression and purification, selectively inhibited a potassium channel, TREK-1, in a dose-dependent manner, with an IC50 of 23.46 ± 0.22 µM. The peptide IstTx exhibited different characteristics from fluoxetine, and the possible interaction of the peptide IstTx binding to the channel was explored by molecular docking. Notably, extracellular acidification raised its inhibitory efficacy on the TREK-1 channel. Our results found that the tick-derived peptide IstTx blocked the TREK-1 channel and provided a novel tool acting on the potassium channel.

Structural basis of chemokine recognition by the class A3 tick evasin EVA-ACA1001.

Ticks produce chemokine-binding proteins, known as evasins, in their saliva to subvert the host's immune response. Evasins bind to chemokines and thereby inhibit the activation of their cognate chemokine receptors, thus suppressing leukocyte recruitment and inflammation. We recently described subclass A3 evasins, which, like other class A evasins, exclusively target CC chemokines but appear to use a different binding site architecture to control target selectivity among CC chemokines. We now describe the structural basis of chemokine recognition by the class A3 evasin EVA-ACA1001. EVA-ACA1001 binds to almost all human CC chemokines and inhibits receptor activation. Truncation mutants of EVA-ACA1001 showed that, unlike class A1 evasins, both the N- and C-termini of EVA-ACA1001 play minimal roles in chemokine binding. To understand the structural basis of its broad chemokine recognition, we determined the crystal structure of EVA-ACA1001 in complex with the human chemokine CCL16. EVA-ACA1001 forms backbone-backbone interactions with the CC motif of CCL16, a conserved feature of all class A evasin-chemokine complexes. A hydrophobic pocket in EVA-ACA1001, formed by several aromatic side chains and the unique disulfide bond of class A3 evasins, accommodates the residue immediately following the CC motif (the "CC + 1 residue") of CCL16. This interaction is shared with EVA-AAM1001, the only other class A3 evasins characterized to date, suggesting it may represent a common mechanism that accounts for the broad recognition of CC chemokines by class A3 evasins.

A fluorescently-tagged tick kinin neuropeptide triggers peristalsis and labels tick midgut muscles.

Ticks are blood-feeding arthropods that require heme for their successful reproduction. During feeding they also acquire pathogens that are subsequently transmitted to humans, wildlife and/or livestock. Understanding the regulation of tick midgut is important for blood meal digestion, heme and nutrient absorption processes and for aspects of pathogen biology in the host. We previously demonstrated the activity of tick kinins on the cognate G protein-coupled receptor. Herein we uncovered the physiological role of the kinin receptor in the tick midgut. A fluorescently-labeled kinin peptide with the endogenous kinin 8 sequence (TMR-RK8), identical in the ticks Rhipicephalus microplus and R. sanguineus, activated and labeled the recombinant R. microplus receptor expressed in CHO-K1 cells. When applied to the live midgut the TMR-RK8 labeled the kinin receptor in muscles while the labeled peptide with the scrambled-sequence of kinin 8 (TMR-Scrambled) did not. The unlabeled kinin 8 peptide competed TMR-RK8, decreasing confocal microscopy signal intensity, indicating TMR-RK8 specificity to muscles. TMR-RK8 was active, inducing significant midgut peristalsis that was video-recorded and evaluated with video tracking software. The TMR-Scrambled peptide used as a negative control did not elicit peristalsis. The myotropic function of kinins in eliciting tick midgut peristalsis was established.

Crimean-Congo haemorrhagic fever virus uses LDLR to bind and enter host cells.

Climate change and population densities accelerated transmission of highly pathogenic viruses to humans, including the Crimean-Congo haemorrhagic fever virus (CCHFV). Here we report that the Low Density Lipoprotein Receptor (LDLR) is a critical receptor for CCHFV cell entry, playing a vital role in CCHFV infection in cell culture and blood vessel organoids. The interaction between CCHFV and LDLR is highly specific, with other members of the LDLR protein family failing to bind to or neutralize the virus. Biosensor experiments demonstrate that LDLR specifically binds the surface glycoproteins of CCHFV. Importantly, mice lacking LDLR exhibit a delay in CCHFV-induced disease. Furthermore, we identified the presence of Apolipoprotein E (ApoE) on CCHFV particles. Our findings highlight the essential role of LDLR in CCHFV infection, irrespective of ApoE presence, when the virus is produced in tick cells. This discovery holds profound implications for the development of future therapies against CCHFV.

The diverse functions of Mu-class Glutathione S-transferase HrGSTm1 during the development of Hyalomma rufipes with a focus on the detoxification metabolism of cyhalothrin.

BACKGROUND: Glutathione S-transferases (GSTs) are a superfamily of multifunctional enzymes in living organisms with metabolic and detoxification functions, which can detoxify exogenous and endogenous compounds and thereby reduce the damage caused by toxic substances to the body. Ticks are obligate blood-sucking ectoparasites that can transmit various pathogens, and the characterization of tick-derived GSTs may help improve current understanding of the molecular mechanism of tick resistance to insecticides. In this study, a novel GST gene, named HrGSTm1, was identified from Hyalomma rufipes. METHODS: Sequence analysis was performed by using bioinformatics techniques. A prokaryotic expression system was used to obtain the recombinant expression protein rHrGSTm1. Detection of spatiotemporal expression patterns of target genes and their response to the toxicity of cyhalothrin on female H. rufipes was performed by using a quantitative PCR platform. The optimal enzymological parameters of rHrGSTm1 using glutathione as substrate were calculated. The antioxidant capacity of the recombinant protein was evaluated by DPPH• (1,1-Diphenyl-2-picrylhydrazyl radical 2,2-Diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl). Knockdown of the HrGSTm1 genes through RNA interference was used to analyze their effects on the physiological parameters of ticks. The changes in HrGSTm1 messenger RNA expression patterns under cypermethrin stress were analyzed. RESULTS: The complementary DNA sequence of HrGSTm1 contained a 672-bp open reading frame, which potentially encoded 223 amino acids. The predicted molecular weight was 25.62 kDa, and the isoelectric point 8.22. HrGSTm1 is a Mu-class GST, belonging to the cytoplasmic GSTs with no signal peptide observed. The Vmax and Km of rHrGSTm1 were 3.367 ± 0.81 uM and 2.208 ± 0.76 uM, respectively, and its activities were dependent on different temperatures and pH conditions; the scavenging rate of rHrGSTm1 to DPPH• reached 76.4% at 1.25 mg/ml. Variable expressions of HrGSTm1 were observed under various treatment periods and in different tissues, with the highest appearing in eggs (analysis of variance [ANOVA], F(2, 9) = 279.9, P < 0.0001) and Malpighian tubules (ANOVA, F(3, 12) = 290.5, P < 0.0001). After knockdown of HrGSTm1, compared with the control group, the mortality in the treatment group was increased by 16.7%, the average oviposition rate decreased by 33.9%, the average engorged body weight decreased by 287.38 mg and egg weight decreased by 127.46 mg, although only the engorged body weight was significantly different (t-test, t(44) = 2.886, P = 0.006). After exposure to three sublethal concentrations (LC05, LC10, LC50) of cyhalothrin, the expression level of HrGSTm1 in the midgut, ovary and salivary gland was upregulated, whereas in Malpighian tubules, it showed a trend of upregulation at first and then downregulation, implying different functions during the detoxification in different tissues. CONCLUSIONS: In this study, a novel GST of the Mu-class was successfully isolated from H. rufipes and systematically subjected to bioinformatic analysis and recombination identification. The variation trend of HrGSTm1 expression level in different tissues suggests that the gene has different detoxification functions in different tissues. The potential function of this gene was analyzed to provide basic research for further investigation of its detoxification mechanism.

Tick-borne flavivirus NS5 antagonizes interferon signaling by inhibiting the catalytic activity of TYK2.

The mechanisms utilized by different flaviviruses to evade antiviral functions of interferons are varied and incompletely understood. Using virological approaches, biochemical assays, and mass spectrometry analyses, we report here that the NS5 protein of tick-borne encephalitis virus (TBEV) and Louping Ill virus (LIV), two related tick-borne flaviviruses, antagonize JAK-STAT signaling through interactions with the tyrosine kinase 2 (TYK2). Co-immunoprecipitation (co-IP) experiments, yeast gap-repair assays, computational protein-protein docking and functional studies identify a stretch of 10 residues of the RNA dependent RNA polymerase domain of tick-borne flavivirus NS5, but not mosquito-borne NS5, that is critical for interactions with the TYK2 kinase domain. Additional co-IP assays performed with several TYK2 orthologs reveal that the interaction is conserved across mammalian species. In vitro kinase assays show that TBEV and LIV NS5 reduce the catalytic activity of TYK2. Our results thus illustrate a novel mechanism by which viruses suppress the interferon response.

ATP-sensitive inward rectifier potassium channels regulate secretion of pro-feeding salivary proteins in the lone star tick (Amblyomma americanum).

Understanding the physiological and molecular regulation of tick feeding is necessary for developing intervention strategies to curb disease transmission by ticks. Pharmacological activation of ATP-gated inward rectifier potassium (KATP) channels reduced fluid secretion from isolated salivary gland and blood feeding in the lone star tick, Amblyomma americanum, yet the temporal expression pattern of KATP channel proteins remained unknown. KATP channels were highly expressed in type II and III acini in off-host stage and early feeding phase ticks, yet expression was reduced in later stages of feeding. We next assessed KATP channel regulation of the secreted proteome of tick saliva. LC-MS/MS analysis identified 40 differentially secreted tick saliva proteins after exposure to KATP activators or inhibitors. Secretion of previously validated tick saliva proteins that promote tick feeding, AV422, AAS27, and AAS41 were significantly reduced by upwards of 8 log units in ticks exposed to KATP channel activators when compared to untreated ticks. Importantly, activation of KATP channels inhibited tick feeding and vice versa for KATP channel inhibitors. Data indicate KATP channels regulate tick feeding biology by controlling secretion of pro-feeding proteins that are essential during early feeding phases, which provides insights into physiological and molecular regulation of tick feeding behavior.

Engineering broad-spectrum inhibitors of inflammatory chemokines from subclass A3 tick evasins.

Chemokines are key regulators of leukocyte trafficking and attractive targets for anti-inflammatory therapy. Evasins are chemokine-binding proteins from tick saliva, whose application as anti-inflammatory therapeutics will require manipulation of their chemokine target selectivity. Here we describe subclass A3 evasins, which are unique to the tick genus Amblyomma and distinguished from "classical" class A1 evasins by an additional disulfide bond near the chemokine recognition interface. The A3 evasin EVA-AAM1001 (EVA-A) bound to CC chemokines and inhibited their receptor activation. Unlike A1 evasins, EVA-A was not highly dependent on N- and C-terminal regions to differentiate chemokine targets. Structures of chemokine-bound EVA-A revealed a deep hydrophobic pocket, unique to A3 evasins, that interacts with the residue immediately following the CC motif of the chemokine. Mutations to this pocket altered the chemokine selectivity of EVA-A. Thus, class A3 evasins provide a suitable platform for engineering proteins with applications in research, diagnosis or anti-inflammatory therapy.

The Lyme disease spirochete, Borrelia burgdorferi, as a model vector-borne pathogen: insights on regulation of gene and protein expression.

The Lyme disease spirochete persists in nature through cycles between ticks and vertebrates. Although the spirochete interacts with numerous, distinct tissues and environmental conditions during its infectious cycle, Borrelia burgdorferi appears to possess a limited ability to sense its external environment. This apparent paradox is being resolved through detailed investigations of the molecular mechanisms through which B. burgdorferi controls production of virulence-associated factors such as the Erp outer surface proteins. The results have led to development of a model for how B. burgdorferi controls expression of its diverse proteins, wherein physiological and metabolic states that are unique to specific points in the infectious cycle trigger changes in gene and protein expression levels.

Type I interferon shapes brain distribution and tropism of tick-borne flavivirus.

Viral tropism within the brain and the role(s) of vertebrate immune response to neurotropic flaviviruses infection is largely understudied. We combine multimodal imaging (cm-nm scale) with single nuclei RNA-sequencing to study Langat virus in wildtype and interferon alpha/beta receptor knockout (Ifnar-/-) mice to visualize viral pathogenesis and define molecular mechanisms. Whole brain viral infection is imaged by Optical Projection Tomography coregistered to ex vivo MRI. Infection is limited to grey matter of sensory systems in wildtype mice, but extends into white matter, meninges and choroid plexus in Ifnar-/- mice. Cells in wildtype display strong type I and II IFN responses, likely due to Ifnb expressing astrocytes, infiltration of macrophages and Ifng-expressing CD8+ NK cells, whereas in Ifnar-/-, the absence of this response contributes to a shift in cellular tropism towards non-activated resident microglia. Multimodal imaging-transcriptomics exemplifies a powerful way to characterize mechanisms of viral pathogenesis and tropism.

[Structural Motifs and Spatial Structures of Helicase (NS3) and RNA-dependent RNA-polymerase (NS5) of a Flavi-like Kindia tick virus (unclassified Flaviviridae)].

INTRODUCTION: Kindia tick virus (KITV) is a novel segmented unclassified flavi-like virus of the Flaviviridae family. This virus is associated with ixodes ticks and is potentially pathogenic to humans. The main goal of this work was to search for structural motifs of viral polypeptides and to develop a 3D-structure for viral proteins of the flavi-like KITV. MATERIALS AND METHODS: The complete genome sequences for KITV, Zika, dengue, Japanese encephalitis, West Nile and yellow fever viruses were retrieved from GenBank. Bioinformatics analysis was performed using the different software packages. RESULTS: Analysis of the KITV structural proteins showed that they have no analogues among currently known viral proteins. Spatial models of NS3 and NS5 KITV proteins have been obtained. These models had a high level of topological similarity to the tick-borne encephalitis and dengue viral proteins. The methyltransferase and RNA-dependent RNA-polymerase domains were found in the NS5 KITV. The latter was represented by fingers, palm and thumb subdomains, and motifs A-F. The helicase domain and its main structural motifs IVI were identified in NS3 KITV. However, the protease domain typical of NS3 flaviviruses was not detected. The highly conserved amino acid motives were detected in the NS3 and NS5 KITV. Also, eight amino acid substitutions characteristic of KITV/2018/1 and KITV/2018/2 were detected, five of them being localized in alpha-helix and three in loops of nonstructural proteins. CONCLUSION: Nonstructural proteins of KITV have structural and functional similarities with unsegmented flaviviruses. This confirms their possible evolutionary and taxonomic relationships.

Localization of secreted ferritin (FER2) in the embryos of the tick Haemaphysalis longicornis.

Despite the absence of a blood meal, embryogenesis involves many processes that require nutrients and other essential elements, including iron. Due to the lack of an external source of these nutrients, these requirements are acquired maternally. Because of the toxic nature of iron, they are transferred through iron transport molecules such as secreted ferritin (FER2). Here we tried to follow the trail of the FER2 through indirect immunofluorescence, and we observed an apparent shift of FER2 from the germ layer at the early part of development to the appendages during the late stage of embryogenesis. FER2 is also found in the middle part of the legs of the embryo. The apparent movement not only sheds light on iron processing events during embryogenesis but also indirectly guides organogenesis in the tick.

A Deeper Insight into the Tick Salivary Protein Families under the Light of Alphafold2 and Dali: Introducing the TickSialoFam 2.0 Database.

Hard ticks feed for several days or weeks on their hosts and their saliva contains thousands of polypeptides belonging to dozens of families, as identified by salivary transcriptomes. Comparison of the coding sequences to protein databases helps to identify putative secreted proteins and their potential functions, directing and focusing future studies, usually done with recombinant proteins that are tested in different bioassays. However, many families of putative secreted peptides have a unique character, not providing significant matches to known sequences. The availability of the Alphafold2 program, which provides in silico predictions of the 3D polypeptide structure, coupled with the Dali program which uses the atomic coordinates of a structural model to search the Protein Data Bank (PDB) allows another layer of investigation to annotate and ascribe a functional role to proteins having so far being characterized as "unique". In this study, we analyzed the classification of tick salivary proteins under the light of the Alphafold2/Dali programs, detecting novel protein families and gaining new insights relating the structure and function of tick salivary proteins.

Evaluating EcxR for Its Possible Role in Ehrlichia chaffeensis Gene Regulation.

Ehrlichia chaffeensis, a tick-transmitted intraphagosomal bacterium, is the causative agent of human monocytic ehrlichiosis. The pathogen also infects several other vertebrate hosts. E. chaffeensis has a biphasic developmental cycle during its growth in vertebrate monocytes/macrophages and invertebrate tick cells. Host- and vector-specific differences in the gene expression from many genes of E. chaffeensis are well documented. It is unclear how the organism regulates gene expression during its developmental cycle and for its adaptation to vertebrate and tick host cell environments. We previously mapped promoters of several E. chaffeensis genes which are recognized by its only two sigma factors: σ32 and σ70. In the current study, we investigated in assessing five predicted E. chaffeensis transcription regulators; EcxR, CtrA, MerR, HU and Tr1 for their possible roles in regulating the pathogen gene expression. Promoter segments of three genes each transcribed with the RNA polymerase containing σ70 (HU, P28-Omp14 and P28-Omp19) and σ32 (ClpB, DnaK and GroES/L) were evaluated by employing multiple independent molecular methods. We report that EcxR binds to all six promoters tested. Promoter-specific binding of EcxR to several gene promoters results in varying levels of gene expression enhancement. This is the first detailed molecular characterization of transcription regulators where we identified EcxR as a gene regulator having multiple promoter-specific interactions.

A Novel MicroRNA and the Target Gene TAB2 Can Regulate the Process of Sucking Blood in and the Spawn Rate of Hyalomma asiaticum (Acari: Ixodidae) Ticks.

Ticks are blood-sucking parasites that are harmful to humans and animals. MicroRNAs are a class of conserved small noncoding RNAs that play regulatory roles in the expression of many genes at the posttranscriptional level. Here, a novel miRNA (nov-miR-17) was identified from a small RNA data library of Hyalomma asiaticum by next-generation sequencing. PCR was used to obtain precursor nov-miR-17 by RACE using mature loop primers. The secondary structure was predicted with UNAFold. The interaction of nov-miR-17 with its target gene TAB2 was predicted using RNAhybrid software and identified in vitro by luciferase assays. Moreover, the interaction was confirmed in vivo by phenotype rescue experiments in which dsTAB2 was used for RNA interference (RNAi) and an antagomir of nov-miR-17 was used for miRNA silencing. The expression levels of nov-miR-17 and TAB2 in ticks at different developmental stages and the expression of nov-miR-17 in different tissues were analyzed by real-time qPCR. All data were analyzed using GraphPad Prism version 5. Results: The results showed that TAB2 was a target gene of nov-miR-17. When the blood-sucking process of larval, nymph and adult ticks was prolonged, the expression of nov-miR-17 was decreased, and TAB2 expression was increased. However, the level of nov-miR-17 in the midgut of engorged ticks was highest at all stages. Therefore, nov-miR-17 plays an important role in the blood-sucking process. The overexpression of nov-miR-17 indicated that this miRNA affected the engorged weight (P < 0.001) and spawn rate (P < 0.001) of female ticks. RNAi of TAB2 also had the same effect. dsRNA not only impacted the weight (P < 0.01) but also reduced the spawn rate (P < 0.001) of the ticks. Furthermore, significant recovery was observed in nov-miR-17-silenced ticks after TAB2 silencing by RNAi. nov-miR-17 silencing by antagomir not only impacted the engorged weight of the female ticks (P < 0.001) but also the number of days that the females needed to progress from engorgement to spawning (P < 0.001). The study showed that nov-miR-17, as a new miRNA, plays an important role along with its target gene TAB2 in the blood-sucking and spawning processes in female ticks.

Serpins in Tick Physiology and Tick-Host Interaction.

Tick saliva has been extensively studied in the context of tick-host interactions because it is involved in host homeostasis modulation and microbial pathogen transmission to the host. Accumulated knowledge about the tick saliva composition at the molecular level has revealed that serine protease inhibitors play a key role in the tick-host interaction. Serpins are one highly expressed group of protease inhibitors in tick salivary glands, their expression can be induced during tick blood-feeding, and they have many biological functions at the tick-host interface. Indeed, tick serpins have an important role in inhibiting host hemostatic processes and in the modulation of the innate and adaptive immune responses of their vertebrate hosts. Tick serpins have also been studied as potential candidates for therapeutic use and vaccine development. In this review, we critically summarize the current state of knowledge about the biological role of tick serpins in shaping tick-host interactions with emphasis on the mechanisms by which they modulate host immunity. Their potential use in drug and vaccine development is also discussed.

Structure-guided engineering of tick evasins for targeting chemokines in inflammatory diseases.

As natural chemokine inhibitors, evasin proteins produced in tick saliva are potential therapeutic agents for numerous inflammatory diseases. Engineering evasins to block the desired chemokines and avoid off-target side effects requires structural understanding of their target selectivity. Structures of the class A evasin EVA-P974 bound to human CC chemokine ligands 7 and 17 (CCL7 and CCL17) and to a CCL8-CCL7 chimera reveal that the specificity of class A evasins for chemokines of the CC subfamily is defined by conserved, rigid backbone-backbone interactions, whereas the preference for a subset of CC chemokines is controlled by side-chain interactions at four hotspots in flexible structural elements. Hotspot mutations alter target preference, enabling inhibition of selected chemokines. The structure of an engineered EVA-P974 bound to CCL2 reveals an underlying molecular mechanism of EVA-P974 target preference. These results provide a structure-based framework for engineering evasins as targeted antiinflammatory therapeutics.

Mast Cells and Basophils in the Defense against Ectoparasites: Efficient Degradation of Parasite Anticoagulants by the Connective Tissue Mast Cell Chymases.

Ticks, lice, flees, mosquitos, leeches and vampire bats need to prevent the host's blood coagulation during their feeding process. This is primarily achieved by injecting potent anticoagulant proteins. Basophils frequently accumulate at the site of tick feeding. However, this occurs only after the second encounter with the parasite involving an adaptive immune response and IgE. To study the potential role of basophils and mast cells in the defense against ticks and other ectoparasites, we produced anticoagulant proteins from three blood-feeding animals; tick, mosquito, and leech. We tested these anticoagulant proteins for their sensitivity to inactivation by a panel of hematopoietic serine proteases. The majority of the connective tissue mast cell proteases tested, originating from humans, dogs, rats, hamsters, and opossums, efficiently cleaved these anticoagulant proteins. Interestingly, the mucosal mast cell proteases that contain closely similar cleavage specificity, had little effect on these anticoagulant proteins. Ticks have been shown to produce serpins, serine protease inhibitors, upon a blood meal that efficiently inhibit the human mast cell chymase and cathepsin G, indicating that ticks have developed a strategy to inactivate these proteases. We show here that one of these tick serpins (IRS-2) shows broad activity against the majority of the mast cell chymotryptic enzymes and the neutrophil proteases from human to opossum. However, it had no effect on the mast cell tryptases or the basophil specific protease mMCP-8. The production of anticoagulants, proteases and anti-proteases by the parasite and the host presents a fascinating example of an arms race between the blood-feeding animals and the mammalian immune system with an apparent and potent role of the connective tissue mast cell chymases in the host defense.

The unusual cell wall of the Lyme disease spirochaete Borrelia burgdorferi is shaped by a tick sugar.

Peptidoglycan-a mesh sac of glycans that are linked by peptides-is the main component of bacterial cell walls. Peptidoglycan provides structural strength, protects cells from osmotic pressure and contributes to shape. All bacterial glycans are repeating disaccharides of N-acetylglucosamine (GlcNAc) ß-(1-4)-linked to N-acetylmuramic acid (MurNAc). Borrelia burgdorferi, the tick-borne Lyme disease pathogen, produces glycan chains in which MurNAc is occasionally replaced with an unknown sugar. Nuclear magnetic resonance, liquid chromatography-mass spectroscopy and genetic analyses show that B. burgdorferi produces glycans that contain GlcNAc-GlcNAc. This unusual disaccharide is chitobiose, a component of its chitinous tick vector. Mutant bacteria that are auxotrophic for chitobiose have altered morphology, reduced motility and cell envelope defects that probably result from producing peptidoglycan that is stiffer than that in wild-type bacteria. We propose that the peptidoglycan of B. burgdorferi probably evolved by adaptation to obligate parasitization of a tick vector, resulting in a biophysical cell-wall alteration to withstand the atypical torque associated with twisting motility.

Efficacy and safety of next-generation tick transcriptome-derived direct thrombin inhibitors.

Despite their limitations, unfractionated heparin (UFH) and bivalirudin remain standard-of-care parenteral anticoagulants for percutaneous coronary intervention (PCI). We discovered novel direct thrombin inhibitors (DTIs) from tick salivary transcriptomes and optimised their pharmacologic activity. The most potent, ultravariegin, inhibits thrombin with a Ki of 4.0 pM, 445-fold better than bivalirudin. Unexpectedly, despite their greater antithrombotic effect, variegin/ultravariegin demonstrated less bleeding, achieving a 3-to-7-fold wider therapeutic index in rodent thrombosis and bleeding models. When used in combination with aspirin and ticagrelor in a porcine model, variegin/ultravariegin reduced stent thrombosis compared with antiplatelet therapy alone but achieved a 5-to-7-fold lower bleeding time than UFH/bivalirudin. Moreover, two antibodies screened from a naïve human antibody library effectively reversed the anticoagulant activity of ultravariegin, demonstrating proof-of-principle for antidote reversal. Variegin and ultravariegin are promising translational candidates for next-generation DTIs that may reduce peri-PCI bleeding in the presence of antiplatelet therapy.