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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

Sulfation of hyalomin-1 (13–44) truncated form results in a modest increase in potency.(A) Conversion of fibrinogen to fibrin by thrombin in the presence of different hyalomin-1 truncated forms (5 μM) as indicated by time in seconds for increase in absorbance of 0.01 at 650 nm. Bars represent mean with SE. Full hyalomin-1 (01–59) completely inhibited fibrinogen clotting during the time assayed. (B) Same assay as in A but at different concentrations of hyalomin-1 (13–44) sulfated. (C) APTT and (D) PT assay procedures for coagulation time of human plasma in the presence of different concentrations of hyalomin -1 (13–44) sulfated (dashed lines), or hyalomin-1 (13–44) solid lines.
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pone.0133991.g006: Sulfation of hyalomin-1 (13–44) truncated form results in a modest increase in potency.(A) Conversion of fibrinogen to fibrin by thrombin in the presence of different hyalomin-1 truncated forms (5 μM) as indicated by time in seconds for increase in absorbance of 0.01 at 650 nm. Bars represent mean with SE. Full hyalomin-1 (01–59) completely inhibited fibrinogen clotting during the time assayed. (B) Same assay as in A but at different concentrations of hyalomin-1 (13–44) sulfated. (C) APTT and (D) PT assay procedures for coagulation time of human plasma in the presence of different concentrations of hyalomin -1 (13–44) sulfated (dashed lines), or hyalomin-1 (13–44) solid lines.

Mentions: Finally, we tested a 13–44 peptide variant that contains the conserved acidic region N-terminal to the cleavage site as well as the cleavage region including the P1´-P3´ residues (Fig 5A). This peptide did not inhibit coagulation of plasma (Fig 5C) or cleavage of S2238 at concentrations up to 5 μM (data not shown), but was hydrolyzed by thrombin at the predicted site after 2 hr at a peptide concentration of 25 μM and a thrombin concentration of 1 μM (data not shown). This indicates that the region upstream of the cleavage site interacts only weakly with thrombin, but does interact with the active site. Two potential tyrosine sulfation sites [26] are present in this peptide at positions 17 and 20 (in 1–59 numbering) (Fig 5A), leading us to synthesize a variant of the 13–44 peptide with sulfotyrosine replacing tyrosine at these positions. The sulfated peptide only weakly inhibited the cleavage of fibrinogen and coagulation of plasma (Fig 6), but did not inhibit hydrolysis of S2238 even at elevated concentrations (data not shown). However, the peptide was hydrolyzed by thrombin at the predicted cleavage site (data not shown), indicating that it must interact weakly at the thrombin active site, The reduction in potency of hyalomin-1 at high salt concentrations and the lack of salt sensitivity of inhibition by the 36–59 variant suggests that ionic interactions of the highly charged region upstream of the cleavage site may enhance affinity of the full length peptide at lower salt concentrations, but the region C-terminal to the cleavage site remains essential to this interaction, and little inhibition occurs in its absence. Any sulfation of tyrosine occurring in the salivary gland may assist the interaction of the peptide with the enzyme, but the effect of this modification does not appear to be large.


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)

Sulfation of hyalomin-1 (13–44) truncated form results in a modest increase in potency.(A) Conversion of fibrinogen to fibrin by thrombin in the presence of different hyalomin-1 truncated forms (5 μM) as indicated by time in seconds for increase in absorbance of 0.01 at 650 nm. Bars represent mean with SE. Full hyalomin-1 (01–59) completely inhibited fibrinogen clotting during the time assayed. (B) Same assay as in A but at different concentrations of hyalomin-1 (13–44) sulfated. (C) APTT and (D) PT assay procedures for coagulation time of human plasma in the presence of different concentrations of hyalomin -1 (13–44) sulfated (dashed lines), or hyalomin-1 (13–44) solid lines.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0133991.g006: Sulfation of hyalomin-1 (13–44) truncated form results in a modest increase in potency.(A) Conversion of fibrinogen to fibrin by thrombin in the presence of different hyalomin-1 truncated forms (5 μM) as indicated by time in seconds for increase in absorbance of 0.01 at 650 nm. Bars represent mean with SE. Full hyalomin-1 (01–59) completely inhibited fibrinogen clotting during the time assayed. (B) Same assay as in A but at different concentrations of hyalomin-1 (13–44) sulfated. (C) APTT and (D) PT assay procedures for coagulation time of human plasma in the presence of different concentrations of hyalomin -1 (13–44) sulfated (dashed lines), or hyalomin-1 (13–44) solid lines.
Mentions: Finally, we tested a 13–44 peptide variant that contains the conserved acidic region N-terminal to the cleavage site as well as the cleavage region including the P1´-P3´ residues (Fig 5A). This peptide did not inhibit coagulation of plasma (Fig 5C) or cleavage of S2238 at concentrations up to 5 μM (data not shown), but was hydrolyzed by thrombin at the predicted site after 2 hr at a peptide concentration of 25 μM and a thrombin concentration of 1 μM (data not shown). This indicates that the region upstream of the cleavage site interacts only weakly with thrombin, but does interact with the active site. Two potential tyrosine sulfation sites [26] are present in this peptide at positions 17 and 20 (in 1–59 numbering) (Fig 5A), leading us to synthesize a variant of the 13–44 peptide with sulfotyrosine replacing tyrosine at these positions. The sulfated peptide only weakly inhibited the cleavage of fibrinogen and coagulation of plasma (Fig 6), but did not inhibit hydrolysis of S2238 even at elevated concentrations (data not shown). However, the peptide was hydrolyzed by thrombin at the predicted cleavage site (data not shown), indicating that it must interact weakly at the thrombin active site, The reduction in potency of hyalomin-1 at high salt concentrations and the lack of salt sensitivity of inhibition by the 36–59 variant suggests that ionic interactions of the highly charged region upstream of the cleavage site may enhance affinity of the full length peptide at lower salt concentrations, but the region C-terminal to the cleavage site remains essential to this interaction, and little inhibition occurs in its absence. Any sulfation of tyrosine occurring in the salivary gland may assist the interaction of the peptide with the enzyme, but the effect of this modification does not appear to be large.

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