THROMBOSIS RESEARCH 67; 721-730,1992 0049-3848/92 $5.00 + .OOPrinted in the USA. Copyright (c) 1992 Pergamon Press Ltd. Alt rights reserved.
INHIBITION OF
PLASMA KALLIKREIN. KININASE AND KININ-LIKE PREPARATIONS FROM THE MEDICINAL LEECHES
I.P.Baskova, S.Khalil, Laboratory of Blood Coagulation, Medical and Biological Chemistry,
ACTIVITIES
OF
V.F.Nartikova*, T.S.Paskhina* Moscow State University and *Institute of Russian Academy of Medical Sciences, Moscow
(Received 25.4.1992; accepted in revised form 8.8.1992 by Editor O.N. Ulutin)
ABSTRACT The medicinal leech salivary gland secretion deprived of hirudin antithrombin activity inhibits amidolytic (substrate S-2302) and kininogenase (substrate kininogen) activities of plasma kallikrein, the main component of the intrinsic mechanism of bloed coagulation. It therefore possesses high anticoagulant properties. Kininase (substrate bradykinin) activity of leech saliva and extracts from the medicinal leeches, as wel1 as kinin-like effects one is of extracts heated at 100°C have been detected. The last correlated with the hyperalgetic property of the heated extract. An annlgetic effect was observed with the unheated extract but nat with leech saliva after intranasal administration to rats.
Medicinal leeches have been used for therapy of different diseases since the leeching is provided by The positive effect of the antiquity. Though many substances of the leech saliva (1,2,3,4). biologically active authors ascribe propertjes of the leech saliva exclusively to hirudin (5) and the recombinant hirudin as antithrombotic remedy (ô), it is advertise into consideration that the leech saliva deprived of to take necessary hirudin antithrombin activity preserves practically the same antithromlbotic pot.ency as the native saJ iva possessing anti thrombin activity (7). saliva laech paper we describe the capacity of thr medicinal this In deprived of hirudin activity to inhihjt plasma kallikrein, the key component compo#nents of the intrinsic mechanism of blood coagulation. Interaction with the kallikrein-kinin system has been shown to inact ivat,c bradykinin by of leech saliva and extracts from the medicinal leeches.
-____-_-_Km words : kininase
medicinal leech activity, analgetic
saliva; effect.
inhibitor
721
of
blood
plasma
kallikrein,
722
PLASMA KALLIKREIN INHIBITION
Vol. 67, No. 6
MATERIALS AND METHODS the leech Hirudo saliva (diluted 5 times by saline) from The native and the saliva deprived of hirudin antithrombin activity were medicinalis used (1). Extracts from the leeches were prepared by the method described in (8). A highly purified preparation of human plasma kallikrein was produced according to the method described in (9). The specific D-Pro-Phe-Arg-pNA amidase activity of the enzyme was equal to 60 umoles/min per mg.protein, its bradykinin kininogenase activity corresponded to the release of 240 ug of from substrate plasma per min per 1 mg enzyme protein. Glandular kallikrein was obtained from human urine by the method described in (10). Blood plasma recalcification time was determined as follows: 1.0 ml of bovine citrate plasma was incubated for 5 min at 37’C with 0.05, 0.10 or 0.20 control the leech saliva or with the same amounts of saline in ml of experiments. Coagulation time of 0.1 ml of the mixture was determined after the addition of 0.1 ml of 26 mM CaCl Recalcification time in presence of dextran sulfate (30 mg per 1 ml of wa e’er) was determined by the method (13). Amidase activity was determined by Svendsen method at 405 nm (12). Kininase activity was measured as the velocity of bradykini.n inactivation. The amount of bradykinin was determined by a biological test on an isolated rat uterine horn, using synthetic bradykinin as the standard (13). Kininogenase activity of human blood plasma kallikrein was determined by a biological method (13), as the amount of bradykinin released from kininogen in a unit of time. The heated human blood plasma (“substrate plasma”) was used as the source of kininogen (9). The analgetic effect of leech extracts administered int.ranasal ly was determined in rats (body mass 150- 160 g) by a tai l---flick test, as the prolongation of time of rat’s tail flick from the water bath heated to 56’C (34). Protein concentration was determined by Lowry method (15). Bradykinin is the product of “Reanal” (Hungary), dextran sulfate - of “Fluks” (Switzerland). Chromogenic substrates S-2302 (D-Pro-Phe-Arg-pNA, and S-2266 (D-ValLeu-Arg-pNA) are from “Kabi Diagnostica” (Sweden).
RESULTS _-Inhibition of the II^--------.-__--_ intrinsic mechanism of blood coapulation: Activation of the -intrinsic mechanism of blood plasmic hemostasis was stimulat.ed by CaCl2, Prolongation of plasma coagulat.ion time in the presence of the l.eech sal. iva deprived of hirudin antithrombin activity was observed. 0.05 ml of the leech saliva stimulated an increase of recalcification time by 220’10%; 0.10 ml by 370~15% and 0.20 ml - by 1150’20% as compared wi th t.he cont.rol . The inhibitory effect of the leech sal i.va on the acl. ivation of the contact phase of blood coagulation was estimated. A prolongat.ion of the amounts of t.he leech coagulation time of bovine plasma (0.2 ml) by increasing saliva deprived of antithrombin activity in presence of 0.1 ml dextran sulfate and 0.2 ml of 25 mM CaCl was observed (Fig. 1). In the fol lowing experiment we observed the inhibition of pl íisma ka1 likrein amidase activity (substrate S-2302) during the activation of the intrinsic mechani sm of coagulation in presence of leech saliva. 0.1 ml of bovine citric plasma was incubated 3 min at 37°C with 0.1 ml of leech sal iva (or sal.ine in control experi.ments) and 0.2 ml of dextran sulfate (30 mg per 1 ml of water). To this mixture were added 1.6 ml of 0.05 M Tris-HCl buffer, pH 7.9 and 0.2 ml of chromogenic substrat.e S-2302.
Vol. 67, No. 6
PLASMA KALLIKREIN INHIBITION
I
1
10
'30 volume
723
i
I '50 70 of saliva (yl)
FIC . 1. Prolongation of recalcification time of plasma activated sulfate in presence of increasing amounts of leech saliva deprived antithrombin activity.
by of
dextran hirudin
The absorption was determined at 405 nm at 37’C. Fig. 2 shows the drastic decrease in blood plasma kallikrein generation during the activation of contact stage of the intrinsic mechanism of blood coagulation. This inhibitory effect is proportional to the amount of the leech saliva addled. These experiments did not give a direct answer to the question as to whether the leech saliva inhibits blood plasma kallikrein directly or indirectly. To solve this question the influence of leech saliva and extract from the medicinal leeches on the amidolytic and kininogenase activity of human plasma kallikrein was examined. Inhibition of amidolytic and kininogenase activities of a highly puirif ied preparation of human plasma kallikrein: The inhibition of amidolytic activity of human plasma kallikrein by the leech saliva was investigated using a highly purified preparation of the enzyme. Its incubation with the leech saliva during 10 min at 37’C was followed by a decrease in amidolytic activity (substrate 5-2302) (Fig. 3). In Fig. 4 the dependence of reaction velocity (Ago ) on the period of incubati on at 37’C at different amounts of the leech sa 7.lva is represented. 90 ul of the s.aliva fully blocks amidolytic activity of plasma kallikrein. Kinetic analysis shows that this irrevefsible_fnhibition follows the pseudofirst kinetics order: n=0.92, k2-322 L min activity of human suppresses kininogenase The leech saliva also of highly purified plasma kallikrein Tpe capacity plasma kallikrein. M solution in 0.05 M TrisHCl buffer, pH=8.0 preparation (1.6 10 and EDTA) to release bradykinin from kininogen containing 8-oxychinoline
724
PLASMA KALLIKREIN INHIBITION
Vol. 67, No. 6
1 of saliva
0,IO
ml of saliva
0,15 ml of saliva
period of inoubation (min) 2. Inhibition of kallikrein amidolytic activity (substrate S-2302) of FIG. plasma activated by dextran sulfate in presence of the leech saliva deprived of hirudin antithrombin activity. decreased from 140 pg of bradykinin per 1 ml in control to 40 /ug of bradykinin per 1 ml after 20 min of incubation with 100 pl of the leech saliva. Instead of kallikrein saline solution was used in control experiment.s. We have not observed any changes in the amount of bradykinin: i.t means that the leech saliva does not have its own kininogenase activity. Incubation of leech saliva with glandular kallikrein prepared from human urine does not influence the amidolytic activity of the enzyme assayed with the substrate S-2266. Kininase activity of the leech saliva and extracts form Hirudo medicinalis: Kininase activity of the leech saliva, an extract from the leech head regi on which contains the salivary gland and their secretion and extract from whole leeches were investigated. 100,nI of bradykinin solution (200 ng per 1 ml of 0.004 M oxalic acid) and 100 ~1 of the medicinal leech preparation in 1 ml of 0.05 M Tris-HCl buffer, pH 8.0 were incubated at 37’C. After 2, 5, 10 and 15 min 0.2 ml aliyuots were added to 1.8 ml of 0.004 M oxalic acid (t=+4OC). The control mixture containing 100 ~1 of the same bradykinin solution and
Vol. 67, No. 6
PLASMA KALLIKREIN INHIBITION
volume
725
of
saliva
FIG. 3. Inhibition of amidolytlc acl.ivity of a highly purified human plasma kallikrein (substrate S-2302) by the leech preliminary 10 min preincubation.
(, .l) /upreparation of saliva aftel-
buffer (pH=8.0) was also incubated for 2. 5, Ifl and 0.9 ml of 0.05 M Tris-HCl 15 min. The quantity of bradykinin inactivated by the leech preparations W EGOROVA, T.P., kinetic parameters of kininogens. Biokhimiya
10. RABINOVICH, physico-chemical Biokhimiya urine.
S.E., and 55,
RETS, E.V. and PASKHINA, T.S. interaction between human high and 51, 463-471, 1986.
LOBAREVA, L.S. and enzymatic properties 1675-1689, 1990.
PASKHINA, T.S. Purification of tissue kallikrein
of from
Some low
some human
and GOZIN, D. Improved methods to assay contact activation. 11. SOULIER, J.P. (Eds.) G.Haberland and U.Hanberg Current Concepts in Kinin Research, In: Oxfordnd New-York, Pergamon Press, 1979, pp. 271-280. SVENDSEN, L., BLOMBACK, B., BLOMBACK, M. and 12. substrates for determination of trypsin, chromogenic like enzymes. Thromb. Res. 1, 267-268, 1972. GULIKOVA, PASKHINA, T.S., 13. Determination of SHMYREVA, R.F. Modern Methods in methods. In: 1968, pp. 205-232. Medicina,
OLSSON, thrombin
P.I. and
Synthetic thrombin
M.C. and SUROVIKINA, O.M., EGOROVA, T.P., bradikynin by biological and chromatographic MO~COW, (Ed.). Biochemistry, V.N.Orehovich
730
PLASMA KALLIKREIN INHIBITION
Vol. 67, No. 6
14. D'AMOJJR, F.E. and SMJTH, D.C. A method for determining loss of pain sensation. J. Pharmacol. Exp. Ther., 72, 74-79, 1941. 15. LOWRY, O.H., ROSENBROIJGH,N.Y., FARR, A.L. and RANDALL, F.J. Protein measurement with the forin phenol reagent. J. Biel. Chem., 193, 266-273, 1951. 16. FRITZ, H., GEBHARDT, M., WEISTER, R. and FINK, E. Trypsin-plasmin inhibitors from leeches. Isolation, amino acid composition, inhibitory characteristics. In: Res. Conf. on Proteinase Inhibitors, H.Fritz, M.Tschesche (Eds.) Berlin, Walter de Gruyter, 1971, pp. 271-280. 17. SEEMULLER, IJ., MEIER, M., OHESSON, K., MULLER, H.-P. and FRITZ, H. Isolation and characterization of a low molecular weight inhibitor (of chymotrypsin and human granulocytic elastase and cathepsin G) from leeches. Hoppe-Seyler's Z. Physiol. Chem., 358, 1105-1117, 1977.
INHIBITION OF
PLASMA KALLIKREIN. KININASE AND KININ-LIKE PREPARATIONS FROM THE MEDICINAL LEECHES
I.P.Baskova, S.Khalil, Laboratory of Blood Coagulation, Medical and Biological Chemistry,
ACTIVITIES
OF
V.F.Nartikova*, T.S.Paskhina* Moscow State University and *Institute of Russian Academy of Medical Sciences, Moscow
(Received 25.4.1992; accepted in revised form 8.8.1992 by Editor O.N. Ulutin)
ABSTRACT The medicinal leech salivary gland secretion deprived of hirudin antithrombin activity inhibits amidolytic (substrate S-2302) and kininogenase (substrate kininogen) activities of plasma kallikrein, the main component of the intrinsic mechanism of bloed coagulation. It therefore possesses high anticoagulant properties. Kininase (substrate bradykinin) activity of leech saliva and extracts from the medicinal leeches, as wel1 as kinin-like effects one is of extracts heated at 100°C have been detected. The last correlated with the hyperalgetic property of the heated extract. An annlgetic effect was observed with the unheated extract but nat with leech saliva after intranasal administration to rats.
Medicinal leeches have been used for therapy of different diseases since the leeching is provided by The positive effect of the antiquity. Though many substances of the leech saliva (1,2,3,4). biologically active authors ascribe propertjes of the leech saliva exclusively to hirudin (5) and the recombinant hirudin as antithrombotic remedy (ô), it is advertise into consideration that the leech saliva deprived of to take necessary hirudin antithrombin activity preserves practically the same antithromlbotic pot.ency as the native saJ iva possessing anti thrombin activity (7). saliva laech paper we describe the capacity of thr medicinal this In deprived of hirudin activity to inhihjt plasma kallikrein, the key component compo#nents of the intrinsic mechanism of blood coagulation. Interaction with the kallikrein-kinin system has been shown to inact ivat,c bradykinin by of leech saliva and extracts from the medicinal leeches.
-____-_-_Km words : kininase
medicinal leech activity, analgetic
saliva; effect.
inhibitor
721
of
blood
plasma
kallikrein,
722
PLASMA KALLIKREIN INHIBITION
Vol. 67, No. 6
MATERIALS AND METHODS the leech Hirudo saliva (diluted 5 times by saline) from The native and the saliva deprived of hirudin antithrombin activity were medicinalis used (1). Extracts from the leeches were prepared by the method described in (8). A highly purified preparation of human plasma kallikrein was produced according to the method described in (9). The specific D-Pro-Phe-Arg-pNA amidase activity of the enzyme was equal to 60 umoles/min per mg.protein, its bradykinin kininogenase activity corresponded to the release of 240 ug of from substrate plasma per min per 1 mg enzyme protein. Glandular kallikrein was obtained from human urine by the method described in (10). Blood plasma recalcification time was determined as follows: 1.0 ml of bovine citrate plasma was incubated for 5 min at 37’C with 0.05, 0.10 or 0.20 control the leech saliva or with the same amounts of saline in ml of experiments. Coagulation time of 0.1 ml of the mixture was determined after the addition of 0.1 ml of 26 mM CaCl Recalcification time in presence of dextran sulfate (30 mg per 1 ml of wa e’er) was determined by the method (13). Amidase activity was determined by Svendsen method at 405 nm (12). Kininase activity was measured as the velocity of bradykini.n inactivation. The amount of bradykinin was determined by a biological test on an isolated rat uterine horn, using synthetic bradykinin as the standard (13). Kininogenase activity of human blood plasma kallikrein was determined by a biological method (13), as the amount of bradykinin released from kininogen in a unit of time. The heated human blood plasma (“substrate plasma”) was used as the source of kininogen (9). The analgetic effect of leech extracts administered int.ranasal ly was determined in rats (body mass 150- 160 g) by a tai l---flick test, as the prolongation of time of rat’s tail flick from the water bath heated to 56’C (34). Protein concentration was determined by Lowry method (15). Bradykinin is the product of “Reanal” (Hungary), dextran sulfate - of “Fluks” (Switzerland). Chromogenic substrates S-2302 (D-Pro-Phe-Arg-pNA, and S-2266 (D-ValLeu-Arg-pNA) are from “Kabi Diagnostica” (Sweden).
RESULTS _-Inhibition of the II^--------.-__--_ intrinsic mechanism of blood coapulation: Activation of the -intrinsic mechanism of blood plasmic hemostasis was stimulat.ed by CaCl2, Prolongation of plasma coagulat.ion time in the presence of the l.eech sal. iva deprived of hirudin antithrombin activity was observed. 0.05 ml of the leech saliva stimulated an increase of recalcification time by 220’10%; 0.10 ml by 370~15% and 0.20 ml - by 1150’20% as compared wi th t.he cont.rol . The inhibitory effect of the leech sal i.va on the acl. ivation of the contact phase of blood coagulation was estimated. A prolongat.ion of the amounts of t.he leech coagulation time of bovine plasma (0.2 ml) by increasing saliva deprived of antithrombin activity in presence of 0.1 ml dextran sulfate and 0.2 ml of 25 mM CaCl was observed (Fig. 1). In the fol lowing experiment we observed the inhibition of pl íisma ka1 likrein amidase activity (substrate S-2302) during the activation of the intrinsic mechani sm of coagulation in presence of leech saliva. 0.1 ml of bovine citric plasma was incubated 3 min at 37°C with 0.1 ml of leech sal iva (or sal.ine in control experi.ments) and 0.2 ml of dextran sulfate (30 mg per 1 ml of water). To this mixture were added 1.6 ml of 0.05 M Tris-HCl buffer, pH 7.9 and 0.2 ml of chromogenic substrat.e S-2302.
Vol. 67, No. 6
PLASMA KALLIKREIN INHIBITION
I
1
10
'30 volume
723
i
I '50 70 of saliva (yl)
FIC . 1. Prolongation of recalcification time of plasma activated sulfate in presence of increasing amounts of leech saliva deprived antithrombin activity.
by of
dextran hirudin
The absorption was determined at 405 nm at 37’C. Fig. 2 shows the drastic decrease in blood plasma kallikrein generation during the activation of contact stage of the intrinsic mechanism of blood coagulation. This inhibitory effect is proportional to the amount of the leech saliva addled. These experiments did not give a direct answer to the question as to whether the leech saliva inhibits blood plasma kallikrein directly or indirectly. To solve this question the influence of leech saliva and extract from the medicinal leeches on the amidolytic and kininogenase activity of human plasma kallikrein was examined. Inhibition of amidolytic and kininogenase activities of a highly puirif ied preparation of human plasma kallikrein: The inhibition of amidolytic activity of human plasma kallikrein by the leech saliva was investigated using a highly purified preparation of the enzyme. Its incubation with the leech saliva during 10 min at 37’C was followed by a decrease in amidolytic activity (substrate 5-2302) (Fig. 3). In Fig. 4 the dependence of reaction velocity (Ago ) on the period of incubati on at 37’C at different amounts of the leech sa 7.lva is represented. 90 ul of the s.aliva fully blocks amidolytic activity of plasma kallikrein. Kinetic analysis shows that this irrevefsible_fnhibition follows the pseudofirst kinetics order: n=0.92, k2-322 L min activity of human suppresses kininogenase The leech saliva also of highly purified plasma kallikrein Tpe capacity plasma kallikrein. M solution in 0.05 M TrisHCl buffer, pH=8.0 preparation (1.6 10 and EDTA) to release bradykinin from kininogen containing 8-oxychinoline
724
PLASMA KALLIKREIN INHIBITION
Vol. 67, No. 6
1 of saliva
0,IO
ml of saliva
0,15 ml of saliva
period of inoubation (min) 2. Inhibition of kallikrein amidolytic activity (substrate S-2302) of FIG. plasma activated by dextran sulfate in presence of the leech saliva deprived of hirudin antithrombin activity. decreased from 140 pg of bradykinin per 1 ml in control to 40 /ug of bradykinin per 1 ml after 20 min of incubation with 100 pl of the leech saliva. Instead of kallikrein saline solution was used in control experiment.s. We have not observed any changes in the amount of bradykinin: i.t means that the leech saliva does not have its own kininogenase activity. Incubation of leech saliva with glandular kallikrein prepared from human urine does not influence the amidolytic activity of the enzyme assayed with the substrate S-2266. Kininase activity of the leech saliva and extracts form Hirudo medicinalis: Kininase activity of the leech saliva, an extract from the leech head regi on which contains the salivary gland and their secretion and extract from whole leeches were investigated. 100,nI of bradykinin solution (200 ng per 1 ml of 0.004 M oxalic acid) and 100 ~1 of the medicinal leech preparation in 1 ml of 0.05 M Tris-HCl buffer, pH 8.0 were incubated at 37’C. After 2, 5, 10 and 15 min 0.2 ml aliyuots were added to 1.8 ml of 0.004 M oxalic acid (t=+4OC). The control mixture containing 100 ~1 of the same bradykinin solution and
Vol. 67, No. 6
PLASMA KALLIKREIN INHIBITION
volume
725
of
saliva
FIG. 3. Inhibition of amidolytlc acl.ivity of a highly purified human plasma kallikrein (substrate S-2302) by the leech preliminary 10 min preincubation.
(, .l) /upreparation of saliva aftel-
buffer (pH=8.0) was also incubated for 2. 5, Ifl and 0.9 ml of 0.05 M Tris-HCl 15 min. The quantity of bradykinin inactivated by the leech preparations W EGOROVA, T.P., kinetic parameters of kininogens. Biokhimiya
10. RABINOVICH, physico-chemical Biokhimiya urine.
S.E., and 55,
RETS, E.V. and PASKHINA, T.S. interaction between human high and 51, 463-471, 1986.
LOBAREVA, L.S. and enzymatic properties 1675-1689, 1990.
PASKHINA, T.S. Purification of tissue kallikrein
of from
Some low
some human
and GOZIN, D. Improved methods to assay contact activation. 11. SOULIER, J.P. (Eds.) G.Haberland and U.Hanberg Current Concepts in Kinin Research, In: Oxfordnd New-York, Pergamon Press, 1979, pp. 271-280. SVENDSEN, L., BLOMBACK, B., BLOMBACK, M. and 12. substrates for determination of trypsin, chromogenic like enzymes. Thromb. Res. 1, 267-268, 1972. GULIKOVA, PASKHINA, T.S., 13. Determination of SHMYREVA, R.F. Modern Methods in methods. In: 1968, pp. 205-232. Medicina,
OLSSON, thrombin
P.I. and
Synthetic thrombin
M.C. and SUROVIKINA, O.M., EGOROVA, T.P., bradikynin by biological and chromatographic MO~COW, (Ed.). Biochemistry, V.N.Orehovich
730
PLASMA KALLIKREIN INHIBITION
Vol. 67, No. 6
14. D'AMOJJR, F.E. and SMJTH, D.C. A method for determining loss of pain sensation. J. Pharmacol. Exp. Ther., 72, 74-79, 1941. 15. LOWRY, O.H., ROSENBROIJGH,N.Y., FARR, A.L. and RANDALL, F.J. Protein measurement with the forin phenol reagent. J. Biel. Chem., 193, 266-273, 1951. 16. FRITZ, H., GEBHARDT, M., WEISTER, R. and FINK, E. Trypsin-plasmin inhibitors from leeches. Isolation, amino acid composition, inhibitory characteristics. In: Res. Conf. on Proteinase Inhibitors, H.Fritz, M.Tschesche (Eds.) Berlin, Walter de Gruyter, 1971, pp. 271-280. 17. SEEMULLER, IJ., MEIER, M., OHESSON, K., MULLER, H.-P. and FRITZ, H. Isolation and characterization of a low molecular weight inhibitor (of chymotrypsin and human granulocytic elastase and cathepsin G) from leeches. Hoppe-Seyler's Z. Physiol. Chem., 358, 1105-1117, 1977.
