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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 specific thrombin inhibitor.(A) The activity of 16 serine proteases in the presence of hyalomin-1 (1 μM) relative to their activity in the absence of inhibitor. (B) Coagulation time of human plasma incubated with hyalomin-1 as measured using the APTT (solid line), and PT (dashed line) assay procedures. (C) Conversion of fibrinogen to fibrin by thrombin in the presence of increasing concentrations of hyalomin-1 as indicated by seconds for increase in absorbance to 0.01 at 650 nm. (D) Aggregation of washed platelets induced by thrombin in the presence of various concentrations of hyalomin-1, as measured by an increase in transmittance in an aggregometer. (E) Polyphosphate-activated cleavage of FXI by thrombin in presence of hyalomin-1. FXIa was measured by hydrolysis of the chromogenic substrate S2236. (F) FV cleavage by thrombin in the presence and absence of hyalomin-1 as measured by SDS-PAGE. Lane 1 –FV alone after 60 min incubation. Lane 2 –FV and thrombin after 10 minutes incubation. Lane 3 –FV, thrombin and hyalomin-1 after 10 minutes incubation. Lane 4 –FV and thrombin after 60 minutes incubation. Lane 5 –thrombin alone.
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pone.0133991.g002: Hyalomin-1 is a specific thrombin inhibitor.(A) The activity of 16 serine proteases in the presence of hyalomin-1 (1 μM) relative to their activity in the absence of inhibitor. (B) Coagulation time of human plasma incubated with hyalomin-1 as measured using the APTT (solid line), and PT (dashed line) assay procedures. (C) Conversion of fibrinogen to fibrin by thrombin in the presence of increasing concentrations of hyalomin-1 as indicated by seconds for increase in absorbance to 0.01 at 650 nm. (D) Aggregation of washed platelets induced by thrombin in the presence of various concentrations of hyalomin-1, as measured by an increase in transmittance in an aggregometer. (E) Polyphosphate-activated cleavage of FXI by thrombin in presence of hyalomin-1. FXIa was measured by hydrolysis of the chromogenic substrate S2236. (F) FV cleavage by thrombin in the presence and absence of hyalomin-1 as measured by SDS-PAGE. Lane 1 –FV alone after 60 min incubation. Lane 2 –FV and thrombin after 10 minutes incubation. Lane 3 –FV, thrombin and hyalomin-1 after 10 minutes incubation. Lane 4 –FV and thrombin after 60 minutes incubation. Lane 5 –thrombin alone.

Mentions: Hyalomin-1 was synthesized without its putative signal sequence and tested for inhibition of eight coagulation proteases along with eight additional important serine proteases having functions not related to coagulation (Fig 2A). In each case, the effect of the peptide on the rate of hydrolysis of an appropriate fluorogenic substrate was evaluated. Only thrombin (α-thrombin throughout manuscript unless otherwise indicated) was significantly inhibited in these tests, losing 72% of its control activity in the presence of the peptide (Fig 2A). In a manner consistent with these observations, the coagulation time of recalcified human plasma in the APTT and PT assays was prolonged by 3.4 and 3.5-fold, respectively, at a concentration of 2 μM hyalomin-1 when compared to untreated controls (Fig 2B). The thrombin-catalyzed formation of fibrin from purified fibrinogen was also inhibited by hyalomin-1. The time required to convert purified fibrinogen to a fibrin gel, as indicated by time in seconds to an increase in the optical density at 650 nm, increased 11.8-fold at a hyalomin-1 concentration of 500 nM compared to a peptide-free control (Fig 2C). Together, these data indicate that hyalomin-1 is a specific inhibitor of thrombin capable of significantly delaying the formation of a fibrin clot in whole plasma.


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 specific thrombin inhibitor.(A) The activity of 16 serine proteases in the presence of hyalomin-1 (1 μM) relative to their activity in the absence of inhibitor. (B) Coagulation time of human plasma incubated with hyalomin-1 as measured using the APTT (solid line), and PT (dashed line) assay procedures. (C) Conversion of fibrinogen to fibrin by thrombin in the presence of increasing concentrations of hyalomin-1 as indicated by seconds for increase in absorbance to 0.01 at 650 nm. (D) Aggregation of washed platelets induced by thrombin in the presence of various concentrations of hyalomin-1, as measured by an increase in transmittance in an aggregometer. (E) Polyphosphate-activated cleavage of FXI by thrombin in presence of hyalomin-1. FXIa was measured by hydrolysis of the chromogenic substrate S2236. (F) FV cleavage by thrombin in the presence and absence of hyalomin-1 as measured by SDS-PAGE. Lane 1 –FV alone after 60 min incubation. Lane 2 –FV and thrombin after 10 minutes incubation. Lane 3 –FV, thrombin and hyalomin-1 after 10 minutes incubation. Lane 4 –FV and thrombin after 60 minutes incubation. Lane 5 –thrombin alone.
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pone.0133991.g002: Hyalomin-1 is a specific thrombin inhibitor.(A) The activity of 16 serine proteases in the presence of hyalomin-1 (1 μM) relative to their activity in the absence of inhibitor. (B) Coagulation time of human plasma incubated with hyalomin-1 as measured using the APTT (solid line), and PT (dashed line) assay procedures. (C) Conversion of fibrinogen to fibrin by thrombin in the presence of increasing concentrations of hyalomin-1 as indicated by seconds for increase in absorbance to 0.01 at 650 nm. (D) Aggregation of washed platelets induced by thrombin in the presence of various concentrations of hyalomin-1, as measured by an increase in transmittance in an aggregometer. (E) Polyphosphate-activated cleavage of FXI by thrombin in presence of hyalomin-1. FXIa was measured by hydrolysis of the chromogenic substrate S2236. (F) FV cleavage by thrombin in the presence and absence of hyalomin-1 as measured by SDS-PAGE. Lane 1 –FV alone after 60 min incubation. Lane 2 –FV and thrombin after 10 minutes incubation. Lane 3 –FV, thrombin and hyalomin-1 after 10 minutes incubation. Lane 4 –FV and thrombin after 60 minutes incubation. Lane 5 –thrombin alone.
Mentions: Hyalomin-1 was synthesized without its putative signal sequence and tested for inhibition of eight coagulation proteases along with eight additional important serine proteases having functions not related to coagulation (Fig 2A). In each case, the effect of the peptide on the rate of hydrolysis of an appropriate fluorogenic substrate was evaluated. Only thrombin (α-thrombin throughout manuscript unless otherwise indicated) was significantly inhibited in these tests, losing 72% of its control activity in the presence of the peptide (Fig 2A). In a manner consistent with these observations, the coagulation time of recalcified human plasma in the APTT and PT assays was prolonged by 3.4 and 3.5-fold, respectively, at a concentration of 2 μM hyalomin-1 when compared to untreated controls (Fig 2B). The thrombin-catalyzed formation of fibrin from purified fibrinogen was also inhibited by hyalomin-1. The time required to convert purified fibrinogen to a fibrin gel, as indicated by time in seconds to an increase in the optical density at 650 nm, increased 11.8-fold at a hyalomin-1 concentration of 500 nM compared to a peptide-free control (Fig 2C). Together, these data indicate that hyalomin-1 is a specific inhibitor of thrombin capable of significantly delaying the formation of a fibrin clot in whole plasma.

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