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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

Hyalomin-1 is a competitive fast binding classical inhibitor of thrombin that is also cleaved by thrombin.(A) Kinetics of S2238 hydrolysis by thrombin in the presence of increasing concentrations of hyalomin-1 at a sodium chloride concentration of 150 mM. Inhibitor concentrations: 0 nM (filled circles), 10 nM (filled squares), 50 nM (filled triangles), 100 nM (filled inverted triangles), 200 nM (filled diamonds), 400 nM (open circles), 600 nM (open squares). (B) Experiment performed as in panel A but at a sodium chloride concentration of 50 mM. Hyalomin-1 concentrations: 0 nM (filled circles), 1.25 nM (filled squares), 2.5 nM (filled triangles), 5 nM (filled inverted triangles), 10 nM (filled diamonds), 25 nM (open circles), 50 nM (open squares), 100 nM (open triangles). (C) Progress curves of thrombin activity in the presence of hyalomin-1: 0 nM (filled circles), 50 nM (filled squares), 100 nM (filled triangles), 200 nM (filled inverted triangles), 400 nM (filled diamonds), 600 nM (open circles). (D) Measurement of thrombin binding to immobilized hyalomin-1 by SPR. Sensograms were obtained by injection of thrombin at concentrations of 50 nM (1), 25 nM (2), 12.5 nM (3), 6.25 nM (4), 3.125 nM (5) and 1.563 nM (6). Kinetic constants are indicated in the text. (E) Mass spectral analysis of hyalomin-1 cleavage products after incubation with thrombin for 2 h at 37°C. The mass values on the graph correspond to cleavage at the Arg41-Leu42 peptide bond (peptides 01–41 and 42–59 in Fig 5A). (F) Effect of incubation time at 37°C on the inhibition of thrombin (0.5 nM) by hyalomin-1 (400 nM) in the presence of 50 μM S2238. The activity of thrombin in the absence of hyalomin-1 is shown as filled circles, while activity in the presence of hyalomin-1 is shown as filled squares.
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pone.0133991.g003: Hyalomin-1 is a competitive fast binding classical inhibitor of thrombin that is also cleaved by thrombin.(A) Kinetics of S2238 hydrolysis by thrombin in the presence of increasing concentrations of hyalomin-1 at a sodium chloride concentration of 150 mM. Inhibitor concentrations: 0 nM (filled circles), 10 nM (filled squares), 50 nM (filled triangles), 100 nM (filled inverted triangles), 200 nM (filled diamonds), 400 nM (open circles), 600 nM (open squares). (B) Experiment performed as in panel A but at a sodium chloride concentration of 50 mM. Hyalomin-1 concentrations: 0 nM (filled circles), 1.25 nM (filled squares), 2.5 nM (filled triangles), 5 nM (filled inverted triangles), 10 nM (filled diamonds), 25 nM (open circles), 50 nM (open squares), 100 nM (open triangles). (C) Progress curves of thrombin activity in the presence of hyalomin-1: 0 nM (filled circles), 50 nM (filled squares), 100 nM (filled triangles), 200 nM (filled inverted triangles), 400 nM (filled diamonds), 600 nM (open circles). (D) Measurement of thrombin binding to immobilized hyalomin-1 by SPR. Sensograms were obtained by injection of thrombin at concentrations of 50 nM (1), 25 nM (2), 12.5 nM (3), 6.25 nM (4), 3.125 nM (5) and 1.563 nM (6). Kinetic constants are indicated in the text. (E) Mass spectral analysis of hyalomin-1 cleavage products after incubation with thrombin for 2 h at 37°C. The mass values on the graph correspond to cleavage at the Arg41-Leu42 peptide bond (peptides 01–41 and 42–59 in Fig 5A). (F) Effect of incubation time at 37°C on the inhibition of thrombin (0.5 nM) by hyalomin-1 (400 nM) in the presence of 50 μM S2238. The activity of thrombin in the absence of hyalomin-1 is shown as filled circles, while activity in the presence of hyalomin-1 is shown as filled squares.

Mentions: Kinetic analyses showed hyalomin-1 to be a competitive inhibitor of S2238 hydrolysis by thrombin (Fig 3A and 3B) with inhibition constants (Ki) of 11.9 ± 1.8 nM and 0.4 ± 0.1 nM at salt concentrations of 150 and 50 mM, respectively, as compared to reported values of 56 nM for madanin-1 and 32 nM for madanin-2 at an ionic strength of 50 mM (14). The linear progress curves obtained immediately after thrombin addition indicated rapid equilibrium binding of hyalomin-1, making it a “fast binding” inhibitor (Fig 3C). Binding to thrombin was also evaluated by surface plasmon resonance (SPR) using hyalomin-1 immobilized through a biotin moiety added to the N-terminal lysine residue of the peptide (Fig 3D). Analysis of the concentration-dependent kinetics of thrombin binding to this surface produced a second order association rate constant (ka) for thrombin binding of 1 x 106 M-1s-1 and a dissociation rate constant (kd) of 0.02 s-1. The calculated dissociation equilibrium constant (KD) of 19 nM was consistent with the Ki obtained from steady-state kinetic results.


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)

Hyalomin-1 is a competitive fast binding classical inhibitor of thrombin that is also cleaved by thrombin.(A) Kinetics of S2238 hydrolysis by thrombin in the presence of increasing concentrations of hyalomin-1 at a sodium chloride concentration of 150 mM. Inhibitor concentrations: 0 nM (filled circles), 10 nM (filled squares), 50 nM (filled triangles), 100 nM (filled inverted triangles), 200 nM (filled diamonds), 400 nM (open circles), 600 nM (open squares). (B) Experiment performed as in panel A but at a sodium chloride concentration of 50 mM. Hyalomin-1 concentrations: 0 nM (filled circles), 1.25 nM (filled squares), 2.5 nM (filled triangles), 5 nM (filled inverted triangles), 10 nM (filled diamonds), 25 nM (open circles), 50 nM (open squares), 100 nM (open triangles). (C) Progress curves of thrombin activity in the presence of hyalomin-1: 0 nM (filled circles), 50 nM (filled squares), 100 nM (filled triangles), 200 nM (filled inverted triangles), 400 nM (filled diamonds), 600 nM (open circles). (D) Measurement of thrombin binding to immobilized hyalomin-1 by SPR. Sensograms were obtained by injection of thrombin at concentrations of 50 nM (1), 25 nM (2), 12.5 nM (3), 6.25 nM (4), 3.125 nM (5) and 1.563 nM (6). Kinetic constants are indicated in the text. (E) Mass spectral analysis of hyalomin-1 cleavage products after incubation with thrombin for 2 h at 37°C. The mass values on the graph correspond to cleavage at the Arg41-Leu42 peptide bond (peptides 01–41 and 42–59 in Fig 5A). (F) Effect of incubation time at 37°C on the inhibition of thrombin (0.5 nM) by hyalomin-1 (400 nM) in the presence of 50 μM S2238. The activity of thrombin in the absence of hyalomin-1 is shown as filled circles, while activity in the presence of hyalomin-1 is shown as filled squares.
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Related In: Results  -  Collection

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

pone.0133991.g003: Hyalomin-1 is a competitive fast binding classical inhibitor of thrombin that is also cleaved by thrombin.(A) Kinetics of S2238 hydrolysis by thrombin in the presence of increasing concentrations of hyalomin-1 at a sodium chloride concentration of 150 mM. Inhibitor concentrations: 0 nM (filled circles), 10 nM (filled squares), 50 nM (filled triangles), 100 nM (filled inverted triangles), 200 nM (filled diamonds), 400 nM (open circles), 600 nM (open squares). (B) Experiment performed as in panel A but at a sodium chloride concentration of 50 mM. Hyalomin-1 concentrations: 0 nM (filled circles), 1.25 nM (filled squares), 2.5 nM (filled triangles), 5 nM (filled inverted triangles), 10 nM (filled diamonds), 25 nM (open circles), 50 nM (open squares), 100 nM (open triangles). (C) Progress curves of thrombin activity in the presence of hyalomin-1: 0 nM (filled circles), 50 nM (filled squares), 100 nM (filled triangles), 200 nM (filled inverted triangles), 400 nM (filled diamonds), 600 nM (open circles). (D) Measurement of thrombin binding to immobilized hyalomin-1 by SPR. Sensograms were obtained by injection of thrombin at concentrations of 50 nM (1), 25 nM (2), 12.5 nM (3), 6.25 nM (4), 3.125 nM (5) and 1.563 nM (6). Kinetic constants are indicated in the text. (E) Mass spectral analysis of hyalomin-1 cleavage products after incubation with thrombin for 2 h at 37°C. The mass values on the graph correspond to cleavage at the Arg41-Leu42 peptide bond (peptides 01–41 and 42–59 in Fig 5A). (F) Effect of incubation time at 37°C on the inhibition of thrombin (0.5 nM) by hyalomin-1 (400 nM) in the presence of 50 μM S2238. The activity of thrombin in the absence of hyalomin-1 is shown as filled circles, while activity in the presence of hyalomin-1 is shown as filled squares.
Mentions: Kinetic analyses showed hyalomin-1 to be a competitive inhibitor of S2238 hydrolysis by thrombin (Fig 3A and 3B) with inhibition constants (Ki) of 11.9 ± 1.8 nM and 0.4 ± 0.1 nM at salt concentrations of 150 and 50 mM, respectively, as compared to reported values of 56 nM for madanin-1 and 32 nM for madanin-2 at an ionic strength of 50 mM (14). The linear progress curves obtained immediately after thrombin addition indicated rapid equilibrium binding of hyalomin-1, making it a “fast binding” inhibitor (Fig 3C). Binding to thrombin was also evaluated by surface plasmon resonance (SPR) using hyalomin-1 immobilized through a biotin moiety added to the N-terminal lysine residue of the peptide (Fig 3D). Analysis of the concentration-dependent kinetics of thrombin binding to this surface produced a second order association rate constant (ka) for thrombin binding of 1 x 106 M-1s-1 and a dissociation rate constant (kd) of 0.02 s-1. The calculated dissociation equilibrium constant (KD) of 19 nM was consistent with the Ki obtained from steady-state kinetic results.

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