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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

Activity of hyalomin-1 cleavage products and truncated forms.(A) Alignment of full length hyalomin-1 (01–59) with its thrombin cleavage products (01–41 and 42–59) as well as two other truncated peptides (36–59 and 13–44) that include the Pro-Arg-Leu thrombin cleavage site. Asterisks denote tyrosine sulfation of the sulfated form of the 13–44 peptide (B) SPR analysis of thrombin binding with immobilized 01–41 peptide. (C) Inhibition of coagulation of plasma by hyalomin-1 and derivative peptides. Clotting time in the PT (white bars) and APTT (hatched bars) assays are normalized to a value of 1 in the absence of peptide. (D) Steady state kinetic analysis of inhibition of thrombin-catalyzed hydrolysis of S2238 by the 42–59 peptide in buffer containing 150 mM NaCl. Inhibitor concentrations: 0 μM (filled circles), 0.313 μM (filled squares), 0.625 μM (filled triangles), 1.25 μM (filled inverted triangles), 2.5 μM (filled diamonds), 5 μM (open circles), 10 μM (open squares) (E) Experiment in panel D performed in buffer containing 50 mM NaCl. (F) Kinetics of thrombin-catalyzed hydrolysis of S2238 in the presence of hyalomin-1 (36–59) in buffer containing 150 mM NaCl. Inhibitor concentrations: 0 μM (filled circles), 0.13 μM (filled squares), 0.26 μM (filled triangles), 0.51 μM (filled inverted triangles), 1 μM (filled diamonds), 2 μM (open circles), 4.1 μM (open squares), 8.2 μM (open triangles) (G) The experiment of panel F performed in buffer containing 50 mM NaCl. (H) Coagulation time of human plasma incubated with hyalomin-1 (36–59) as measured using the APTT (solid line) and PT (dashed line) assay procedures. (I) Fibrinogen clotting time by thrombin at increasing concentrations of hyalomin-1 (36–59) as indicated by time (s) taken to reach an absorbance value of 0.01 at 650 nm. (J) and (K) Mass spectral analysis of hyalomin-1 (36–59) incubated with thrombin (J) or alone (K).
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pone.0133991.g005: Activity of hyalomin-1 cleavage products and truncated forms.(A) Alignment of full length hyalomin-1 (01–59) with its thrombin cleavage products (01–41 and 42–59) as well as two other truncated peptides (36–59 and 13–44) that include the Pro-Arg-Leu thrombin cleavage site. Asterisks denote tyrosine sulfation of the sulfated form of the 13–44 peptide (B) SPR analysis of thrombin binding with immobilized 01–41 peptide. (C) Inhibition of coagulation of plasma by hyalomin-1 and derivative peptides. Clotting time in the PT (white bars) and APTT (hatched bars) assays are normalized to a value of 1 in the absence of peptide. (D) Steady state kinetic analysis of inhibition of thrombin-catalyzed hydrolysis of S2238 by the 42–59 peptide in buffer containing 150 mM NaCl. Inhibitor concentrations: 0 μM (filled circles), 0.313 μM (filled squares), 0.625 μM (filled triangles), 1.25 μM (filled inverted triangles), 2.5 μM (filled diamonds), 5 μM (open circles), 10 μM (open squares) (E) Experiment in panel D performed in buffer containing 50 mM NaCl. (F) Kinetics of thrombin-catalyzed hydrolysis of S2238 in the presence of hyalomin-1 (36–59) in buffer containing 150 mM NaCl. Inhibitor concentrations: 0 μM (filled circles), 0.13 μM (filled squares), 0.26 μM (filled triangles), 0.51 μM (filled inverted triangles), 1 μM (filled diamonds), 2 μM (open circles), 4.1 μM (open squares), 8.2 μM (open triangles) (G) The experiment of panel F performed in buffer containing 50 mM NaCl. (H) Coagulation time of human plasma incubated with hyalomin-1 (36–59) as measured using the APTT (solid line) and PT (dashed line) assay procedures. (I) Fibrinogen clotting time by thrombin at increasing concentrations of hyalomin-1 (36–59) as indicated by time (s) taken to reach an absorbance value of 0.01 at 650 nm. (J) and (K) Mass spectral analysis of hyalomin-1 (36–59) incubated with thrombin (J) or alone (K).

Mentions: (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.


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)

Activity of hyalomin-1 cleavage products and truncated forms.(A) Alignment of full length hyalomin-1 (01–59) with its thrombin cleavage products (01–41 and 42–59) as well as two other truncated peptides (36–59 and 13–44) that include the Pro-Arg-Leu thrombin cleavage site. Asterisks denote tyrosine sulfation of the sulfated form of the 13–44 peptide (B) SPR analysis of thrombin binding with immobilized 01–41 peptide. (C) Inhibition of coagulation of plasma by hyalomin-1 and derivative peptides. Clotting time in the PT (white bars) and APTT (hatched bars) assays are normalized to a value of 1 in the absence of peptide. (D) Steady state kinetic analysis of inhibition of thrombin-catalyzed hydrolysis of S2238 by the 42–59 peptide in buffer containing 150 mM NaCl. Inhibitor concentrations: 0 μM (filled circles), 0.313 μM (filled squares), 0.625 μM (filled triangles), 1.25 μM (filled inverted triangles), 2.5 μM (filled diamonds), 5 μM (open circles), 10 μM (open squares) (E) Experiment in panel D performed in buffer containing 50 mM NaCl. (F) Kinetics of thrombin-catalyzed hydrolysis of S2238 in the presence of hyalomin-1 (36–59) in buffer containing 150 mM NaCl. Inhibitor concentrations: 0 μM (filled circles), 0.13 μM (filled squares), 0.26 μM (filled triangles), 0.51 μM (filled inverted triangles), 1 μM (filled diamonds), 2 μM (open circles), 4.1 μM (open squares), 8.2 μM (open triangles) (G) The experiment of panel F performed in buffer containing 50 mM NaCl. (H) Coagulation time of human plasma incubated with hyalomin-1 (36–59) as measured using the APTT (solid line) and PT (dashed line) assay procedures. (I) Fibrinogen clotting time by thrombin at increasing concentrations of hyalomin-1 (36–59) as indicated by time (s) taken to reach an absorbance value of 0.01 at 650 nm. (J) and (K) Mass spectral analysis of hyalomin-1 (36–59) incubated with thrombin (J) or alone (K).
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Related In: Results  -  Collection

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pone.0133991.g005: Activity of hyalomin-1 cleavage products and truncated forms.(A) Alignment of full length hyalomin-1 (01–59) with its thrombin cleavage products (01–41 and 42–59) as well as two other truncated peptides (36–59 and 13–44) that include the Pro-Arg-Leu thrombin cleavage site. Asterisks denote tyrosine sulfation of the sulfated form of the 13–44 peptide (B) SPR analysis of thrombin binding with immobilized 01–41 peptide. (C) Inhibition of coagulation of plasma by hyalomin-1 and derivative peptides. Clotting time in the PT (white bars) and APTT (hatched bars) assays are normalized to a value of 1 in the absence of peptide. (D) Steady state kinetic analysis of inhibition of thrombin-catalyzed hydrolysis of S2238 by the 42–59 peptide in buffer containing 150 mM NaCl. Inhibitor concentrations: 0 μM (filled circles), 0.313 μM (filled squares), 0.625 μM (filled triangles), 1.25 μM (filled inverted triangles), 2.5 μM (filled diamonds), 5 μM (open circles), 10 μM (open squares) (E) Experiment in panel D performed in buffer containing 50 mM NaCl. (F) Kinetics of thrombin-catalyzed hydrolysis of S2238 in the presence of hyalomin-1 (36–59) in buffer containing 150 mM NaCl. Inhibitor concentrations: 0 μM (filled circles), 0.13 μM (filled squares), 0.26 μM (filled triangles), 0.51 μM (filled inverted triangles), 1 μM (filled diamonds), 2 μM (open circles), 4.1 μM (open squares), 8.2 μM (open triangles) (G) The experiment of panel F performed in buffer containing 50 mM NaCl. (H) Coagulation time of human plasma incubated with hyalomin-1 (36–59) as measured using the APTT (solid line) and PT (dashed line) assay procedures. (I) Fibrinogen clotting time by thrombin at increasing concentrations of hyalomin-1 (36–59) as indicated by time (s) taken to reach an absorbance value of 0.01 at 650 nm. (J) and (K) Mass spectral analysis of hyalomin-1 (36–59) incubated with thrombin (J) or alone (K).
Mentions: (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.

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