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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 does not inhibit or interact with γ-thrombin.(A) Steady state kinetic analysis of γ-thrombin in the absence (filled circles) or presence (filled squares) of 600 nM hyalomin-1. (B) γ-Thrombin (black circles) and α-thrombin (black squares)-catalyzed hydrolysis S2238 (50 μM) in the presence of hyalomin-1. (C) Binding of γ-thrombin and α-thrombin to immobilized hyalomin-1 measured by SPR. Buffer alone (1), 50 nM γ-thrombin (2), 100 nM γ-thrombin (3), 50 nM α-thrombin (4), 100 nM α-thrombin (5).
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pone.0133991.g004: Hyalomin-1 does not inhibit or interact with γ-thrombin.(A) Steady state kinetic analysis of γ-thrombin in the absence (filled circles) or presence (filled squares) of 600 nM hyalomin-1. (B) γ-Thrombin (black circles) and α-thrombin (black squares)-catalyzed hydrolysis S2238 (50 μM) in the presence of hyalomin-1. (C) Binding of γ-thrombin and α-thrombin to immobilized hyalomin-1 measured by SPR. Buffer alone (1), 50 nM γ-thrombin (2), 100 nM γ-thrombin (3), 50 nM α-thrombin (4), 100 nM α-thrombin (5).

Mentions: As is the case with other peptide inhibitors of thrombin, the binding of hyalomin-1 is most likely stabilized by exosite interactions. γ-Thrombin is generated through cleavages of the B chain of α-thrombin at Arg75 and Lys149E that disrupt the structures of the autolysis loop in the vicinity of the catalytic site, and exosite I, the fibrinogen binding site. These changes produce an enzyme form that hydrolyzes S2238 normally, but is incapable of cleaving fibrinogen due to the loss of exosite surfaces [25]. We determined kinetic parameters for γ-thrombin cleavage of S2238 and found them to be essentially identical to those seen with α-thrombin (Fig 4A). However, hyalomin-1 did not inhibit hydrolysis of the chromogenic substrate by γ-thrombin at peptide concentrations of up to 600 nM (Fig 4A), while α-thrombin was strongly inhibited under this same concentration and conditions (Fig 4B). γ-Thrombin also showed no detectable binding to immobilized hyalomin-1 in SPR experiments, while α-thrombin exhibited high levels of binding to the same surface (Fig 4C). These results suggest that disruption of the thrombin structure in the vicinity of the autolysis loop and exosite I abrogated hyalomin-1 binding, thereby implicating these areas as potential binding sites for the peptide.


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 does not inhibit or interact with γ-thrombin.(A) Steady state kinetic analysis of γ-thrombin in the absence (filled circles) or presence (filled squares) of 600 nM hyalomin-1. (B) γ-Thrombin (black circles) and α-thrombin (black squares)-catalyzed hydrolysis S2238 (50 μM) in the presence of hyalomin-1. (C) Binding of γ-thrombin and α-thrombin to immobilized hyalomin-1 measured by SPR. Buffer alone (1), 50 nM γ-thrombin (2), 100 nM γ-thrombin (3), 50 nM α-thrombin (4), 100 nM α-thrombin (5).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0133991.g004: Hyalomin-1 does not inhibit or interact with γ-thrombin.(A) Steady state kinetic analysis of γ-thrombin in the absence (filled circles) or presence (filled squares) of 600 nM hyalomin-1. (B) γ-Thrombin (black circles) and α-thrombin (black squares)-catalyzed hydrolysis S2238 (50 μM) in the presence of hyalomin-1. (C) Binding of γ-thrombin and α-thrombin to immobilized hyalomin-1 measured by SPR. Buffer alone (1), 50 nM γ-thrombin (2), 100 nM γ-thrombin (3), 50 nM α-thrombin (4), 100 nM α-thrombin (5).
Mentions: As is the case with other peptide inhibitors of thrombin, the binding of hyalomin-1 is most likely stabilized by exosite interactions. γ-Thrombin is generated through cleavages of the B chain of α-thrombin at Arg75 and Lys149E that disrupt the structures of the autolysis loop in the vicinity of the catalytic site, and exosite I, the fibrinogen binding site. These changes produce an enzyme form that hydrolyzes S2238 normally, but is incapable of cleaving fibrinogen due to the loss of exosite surfaces [25]. We determined kinetic parameters for γ-thrombin cleavage of S2238 and found them to be essentially identical to those seen with α-thrombin (Fig 4A). However, hyalomin-1 did not inhibit hydrolysis of the chromogenic substrate by γ-thrombin at peptide concentrations of up to 600 nM (Fig 4A), while α-thrombin was strongly inhibited under this same concentration and conditions (Fig 4B). γ-Thrombin also showed no detectable binding to immobilized hyalomin-1 in SPR experiments, while α-thrombin exhibited high levels of binding to the same surface (Fig 4C). These results suggest that disruption of the thrombin structure in the vicinity of the autolysis loop and exosite I abrogated hyalomin-1 binding, thereby implicating these areas as potential binding sites for the peptide.

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