The Tick-Derived Anticoagulant Madanin Is Processed by Thrombin and Factor Xa

Figueiredo, Ana C.; Sanctis, Daniele de; Pereira, Pedro José Barbosa

doi:10.1371/journal.pone.0071866

Cited by 19 publications

(27 citation statements)

References 47 publications

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“…While unmodified variants of the hyalomin family have been shown to exhibit thrombin inhibitory activity (24,25), based on our prior work on madanin-1 we predict that each is likely to be sulfated based on the presence of conserved tyrosine residues flanked by a highly acidic amino acid sequence--a quintessential motif for posttranslational sulfation (26). To date MadL1, MadL2 (14,27), And82, and And310 (23) have only been characterized as putative thrombin inhibitors; however, based on the sequence alignment of these proteins ( Fig. 1) we predicted that they are likely to be thrombin inhibitory proteins based on the highly conserved tyrosine sulfation motif that is also present in the hyalomins and madanin-1.…”

Section: Significancementioning

confidence: 95%

“…With the desired library of homogenously sulfated proteins in hand, we next investigated the inhibitory activity against α-thrombin using a well-established kinetic assay using the chromogenic substrate Tos-Gly-Pro-Arg-p-nitroanilide (SI Appendix, Fig. S112) (27). With the exception of And82 and And310, each of the synthetic proteins exhibited potent inhibition of α-thrombin, with the incorporation of the tyrosine sulfate posttranslational modifications leading to dramatic improvements in inhibitor potency ( Fig.…”

Section: Synthesis Of a Tick-derived Sulfopeptide Library Via Diselenmentioning

confidence: 99%

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Rapid assembly and profiling of an anticoagulant sulfoprotein library

Watson

Ripoll‐Rozada

Lee

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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Hematophagous organisms produce a suite of salivary proteins which interact with the host’s coagulation machinery to facilitate the acquisition and digestion of a bloodmeal. Many of these biomolecules inhibit the central blood-clotting serine proteinase thrombin that is also the target of several clinically approved anticoagulants. Here a bioinformatics approach is used to identify seven tick proteins with putative thrombin inhibitory activity that we predict to be posttranslationally sulfated at two conserved tyrosine residues. To corroborate the biological role of these molecules and investigate the effects of amino acid sequence and sulfation modifications on thrombin inhibition and anticoagulant activity, a library of 34 homogeneously sulfated protein variants were rapidly assembled using one-pot diselenide-selenoester ligation (DSL)-deselenization chemistry. Downstream functional characterization validated the thrombin-directed activity of all target molecules and revealed that posttranslational sulfation of specific tyrosine residues crucially modulates potency. Importantly, access to this homogeneously modified protein library not only enabled the determination of key structure–activity relationships and the identification of potent anticoagulant leads, but also revealed subtleties in the mechanism of thrombin inhibition, between and within the families, that would be impossible to predict from the amino acid sequence alone. The synthetic platform described here therefore serves as a highly valuable tool for the generation and thorough characterization of libraries of related peptide and/or protein molecules (with or without modifications) for the identification of lead candidates for medicinal chemistry programs.

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Section: Significancementioning

confidence: 95%

Section: Synthesis Of a Tick-derived Sulfopeptide Library Via Diselenmentioning

confidence: 99%

Rapid assembly and profiling of an anticoagulant sulfoprotein library

Watson

Ripoll‐Rozada

Lee

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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“…106 Short peptides with no cysteines and the ability to bind to thrombin in an extended conformation are found not only in ticks but also in mosquitoes and flies, displaying high diversity in sequences and binding modes. These molecules include madanins, 107,108 chimadanin, 109 and hemathrins 110 (from ticks), anophelin [111][112][113] (from mosquito), and thrombostasin 114 and TTI 115,116 (from flies). Madanins, chimadanin, and hemathrins differ from variegin in having most of their acidic residues near the N-termini and having multiple sulfotyrosine residues to facilitate binding to thrombin exosite-II.…”

Section: Inhibitors Of Thrombinmentioning

confidence: 99%

Toxins Are an Excellent Source of Therapeutic Agents against Cardiovascular Diseases

Koh

Modahl

Kulkarni

et al. 2018

Semin Thromb Hemost

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Venomous and hematophagous animals use their venom or saliva for survival, to obtain food, and for self-defense. Venom and saliva from these animals are cocktails of bioactive molecules primarily composed of proteins and peptides. These molecules are called toxins because they cause unwanted consequences on prey. They exhibit unique, diverse, and specific biological activities that perturb normal physiological processes of their prey and host. However, the potential of toxins as inspirations for the development of therapeutic agents or pharmacological tools has also long been recognized. In addition to their small size, the exquisite selectivity and structural stability of toxins make them attractive as starting molecule in the development of therapeutic and diagnostic agents. Drug discovery and development from venomous and hematophagous animals against cardiovascular diseases have been particularly successful. Some of the notable success include antihypertensive (captopril and enalapril) and antiplatelet agents (tirofiban and eptifibatide), as well as anticoagulants (lepirudin and bivalirudin). Highlighted in this review are many venom or saliva-derived cardiovascular-active proteins and peptides of therapeutic interest, including those that are currently in preclinical stages and those that have been approved by FDA and currently in the market. The authors attempt to summarize their structure, function, mechanism of action, and development with respect to cardiovascular diseases.

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“…A second class of small, tick-derived thrombin inhibitors has been described from Haemaphysalis longicornis [ 14 , 15 ]. These peptides, known as madanins 1 and 2, were shown to inhibit coagulation and thrombin-mediated cleavage of macromolecular substrates, but did not inhibit hydrolysis of chromogenic substrates, and were suggested to interact only at an exosite [ 15 ].…”

Section: Introductionmentioning

confidence: 99%

“…These peptides, known as madanins 1 and 2, were shown to inhibit coagulation and thrombin-mediated cleavage of macromolecular substrates, but did not inhibit hydrolysis of chromogenic substrates, and were suggested to interact only at an exosite [ 15 ]. In a subsequent study, madanins were found to inhibit chromogenic substrate cleavage at subphysiological salt concentrations, and to be cleaved by thrombin and FXa at multiple sites, suggesting interaction with the active site [ 14 ]. Unlike variegin, the cleavage products did not inhibit thrombin, and provided no information on possible exosite interactions.…”

Section: Introductionmentioning

confidence: 99%

Identification and Mechanistic Analysis of a Novel Tick-Derived Inhibitor of Thrombin

et al. 2015

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A group of peptides from the salivary gland of the tick Hyalomma marginatum rufipes, a vector of Crimean Congo hemorrhagic fever show weak similarity to the madanins, a group of thrombin-inhibitory peptides from a second tick species, Haemaphysalis longicornis. We have evaluated the anti-serine protease activity of one of these H. marginatum peptides that has been given the name hyalomin-1. Hyalomin-1 was found to be a selective inhibitor of thrombin, blocking coagulation of plasma and inhibiting S2238 hydrolysis in a competitive manner with an inhibition constant (Ki) of 12 nM at an ionic strength of 150 mM. It also blocks the thrombin-mediated activation of coagulation factor XI, thrombin-mediated platelet aggregation, and the activation of coagulation factor V by thrombin. Hyalomin-1 is cleaved at a canonical thrombin cleavage site but the cleaved products do not inhibit coagulation. However, the C-terminal cleavage product showed non-competitive inhibition of S2238 hydrolysis. A peptide combining the N-terminal parts of the molecule with the cleavage region did not interact strongly with thrombin, but a 24-residue fragment containing the cleavage region and the C-terminal fragment inhibited the enzyme in a competitive manner and also inhibited coagulation of plasma. These results suggest that the peptide acts by binding to the active site as well as exosite I or the autolysis loop of thrombin. Injection of 2.5 mg/kg of hyalomin-1 increased arterial occlusion time in a mouse model of thrombosis, suggesting this peptide could be a candidate for clinical use as an antithrombotic.

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The Tick-Derived Anticoagulant Madanin Is Processed by Thrombin and Factor Xa

Cited by 19 publications

References 47 publications

Rapid assembly and profiling of an anticoagulant sulfoprotein library

Rapid assembly and profiling of an anticoagulant sulfoprotein library

Toxins Are an Excellent Source of Therapeutic Agents against Cardiovascular Diseases

Identification and Mechanistic Analysis of a Novel Tick-Derived Inhibitor of Thrombin

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