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

Jablonka W, Kotsyfakis M, Mizurini DM, Monteiro RQ, Lukszo J, Drake SK, Ribeiro JM, Andersen JF - PLoS ONE (2015)

Bottom Line: Hyalomin-1 is cleaved at a canonical thrombin cleavage site but the cleaved products do not inhibit coagulation.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Malaria and Vector Research, NIAID, National Institutes of Health, Rockville, Maryland, United States of America.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Alignment of hyalomins from H. marginatum rufipes with the madanins from H. longicornis and similar sequences from D. andersoni.Signal peptide sequences have been removed, and regions of conservation are highlighted in black. Accession numbers from the Genbank database are: GI:307006449, GI:307006483, GI:307006445 and GI:307006427 for hyalomins 1–4, GI:30025562 and GI:30025564 for madanins 1 and 2, GI:67906166 and GI:67968369 for madanin-like peptides 1 and 2. D. ander 76, 310, 82 and 240 sequences are found in Francischetti et al, 2009 support material.
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pone.0133991.g001: Alignment of hyalomins from H. marginatum rufipes with the madanins from H. longicornis and similar sequences from D. andersoni.Signal peptide sequences have been removed, and regions of conservation are highlighted in black. Accession numbers from the Genbank database are: GI:307006449, GI:307006483, GI:307006445 and GI:307006427 for hyalomins 1–4, GI:30025562 and GI:30025564 for madanins 1 and 2, GI:67906166 and GI:67968369 for madanin-like peptides 1 and 2. D. ander 76, 310, 82 and 240 sequences are found in Francischetti et al, 2009 support material.

Mentions: The salivary gland transcriptome from the hard tick Hyalomma marginatum rufipes contains four sequences encoding peptides, designated as hyalomins-1-4, that show weak similarity to the thrombin inhibitors madanin 1 and 2 from Haemaphysalis longicornis (Fig 1) [16]. The peptides in this group (including four from a third tick species, Dermacentor andersoni) [22] contain similar numbers of amino acids, but differ in length at either their N- or C-termini, leaving an approximately 50-residue shared central core region having low overall identity (Fig 1). The madanins have an extended N-terminal region compared with peptides from the other genera while the hyalomins and the D. andersoni peptides are extended at the C-terminus (Fig 1) [15]. Chimadanin, a second form of thrombin inhibitor from H. longicornis is extended at both the N and C-termini (Fig 1) [23]. The central core of these peptides contains a weakly conserved acidic region that lies N-terminal to a Pro-Arg-Leu motif and forms a putative serine protease cleavage site (Fig 1).


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

Jablonka W, Kotsyfakis M, Mizurini DM, Monteiro RQ, Lukszo J, Drake SK, Ribeiro JM, Andersen JF - PLoS ONE (2015)

Alignment of hyalomins from H. marginatum rufipes with the madanins from H. longicornis and similar sequences from D. andersoni.Signal peptide sequences have been removed, and regions of conservation are highlighted in black. Accession numbers from the Genbank database are: GI:307006449, GI:307006483, GI:307006445 and GI:307006427 for hyalomins 1–4, GI:30025562 and GI:30025564 for madanins 1 and 2, GI:67906166 and GI:67968369 for madanin-like peptides 1 and 2. D. ander 76, 310, 82 and 240 sequences are found in Francischetti et al, 2009 support material.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4526366&req=5

pone.0133991.g001: Alignment of hyalomins from H. marginatum rufipes with the madanins from H. longicornis and similar sequences from D. andersoni.Signal peptide sequences have been removed, and regions of conservation are highlighted in black. Accession numbers from the Genbank database are: GI:307006449, GI:307006483, GI:307006445 and GI:307006427 for hyalomins 1–4, GI:30025562 and GI:30025564 for madanins 1 and 2, GI:67906166 and GI:67968369 for madanin-like peptides 1 and 2. D. ander 76, 310, 82 and 240 sequences are found in Francischetti et al, 2009 support material.
Mentions: The salivary gland transcriptome from the hard tick Hyalomma marginatum rufipes contains four sequences encoding peptides, designated as hyalomins-1-4, that show weak similarity to the thrombin inhibitors madanin 1 and 2 from Haemaphysalis longicornis (Fig 1) [16]. The peptides in this group (including four from a third tick species, Dermacentor andersoni) [22] contain similar numbers of amino acids, but differ in length at either their N- or C-termini, leaving an approximately 50-residue shared central core region having low overall identity (Fig 1). The madanins have an extended N-terminal region compared with peptides from the other genera while the hyalomins and the D. andersoni peptides are extended at the C-terminus (Fig 1) [15]. Chimadanin, a second form of thrombin inhibitor from H. longicornis is extended at both the N and C-termini (Fig 1) [23]. The central core of these peptides contains a weakly conserved acidic region that lies N-terminal to a Pro-Arg-Leu motif and forms a putative serine protease cleavage site (Fig 1).

Bottom Line: Hyalomin-1 is cleaved at a canonical thrombin cleavage site but the cleaved products do not inhibit coagulation.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Malaria and Vector Research, NIAID, National Institutes of Health, Rockville, Maryland, United States of America.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus