European Journal of Pharmacology, 216 (1992) 401-405 © 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
401
EJP 52499
Inhibition by heparin of platelet activation induced by neutrophil-derived cathepsin G Virgilio E v a n g e l i s t a , P a o l a P i c c a r d o n i , N o r m a M a u g e r i , G i o v a n n i D e G a e t a n o a n d C h i a r a C e r l e t t i Giulio Bizzozero Laboratory of Platelet and Leucocyte Pharmacology, Istituto di Ricerche Farmacologiche Mario Negri, Consorzio Mario Negri Sud, 66030 Santa Maria Imbaro, Italy Received 24 October 1991, revised MS received 6 March 1992, accepted 24 March 1992
Heparin is the most widely used anticoagulant drug for prevention and treatment of thrombosis. However, inhibition of blood coagulation might not fully explain the antithrombotic activity of this drug. The present study shows that different heparin preparations (50 nM) completely prevent human platelet aggregation, serotonin release and thromboxane B 2 production induced by purified neutrophil-derived cathepsin G (E.C. No. 3.4.21.20). This inhibitory effect was not related to the anticoagulant property of the compounds, since a heparin preparation with an inactivated active site for antithrombin III was also effective. Heparins inhibited the protease activity of the enzyme over the same range of concentrations. Since the effect of cathepsin G on platelets requires an intact proteolytic active site, the inhibitory effect of the drugs on cathepsin G-induced platelet activation may be explained by a blockade of protease activity. Heparins were also shown to reduce platelet activation induced by cathepsin G released from activated polymorphonuclear leucocytes in mixed cell suspensions. As polymorphonuclear leucocytes might contribute to both arterial and venous thrombosis through platelet activation induced by the release of cathepsin G, this novel property of heparin could be used to optimize its antithrombotic efficacy. Heparin; Platelets; Neutrophils; Cathepsin G
I. Introduction
Recent in vitro studies demonstrated that polymorphonuclear leucocytes (PMN) activated by several agonists can induce platelet activation (Chignard et al., 1986; Del Maschio et al., 1990). Cathepsin G, a neutral serine protease, contained together with elastase in the azurophilic granules of PMN, is a very potent platelet agonist (Selak et al., 1988; Ferrer-Lopez et al., 1990; Evangelista et al., 1991) with a specific binding site on the surface of platelets (Selak and Smith, 1990). PMNinduced platelet activation via cathepsin G can be prevented by antiproteinases, which inhibit the proteinase activity of cathepsin G (Ferrer-Lopez et al., 1990). Several epidemiological (Ernst et al., 1987) and in vivo (Nash et al., 1988; Mehta et al., 1989; Thomas et al., 1988) observations suggest that PMN could play a role in the pathophysiology of both arterial and venous
Correspondence to: V. Evangelista, Giulio Bizzozero Laboratory of Platelet and Leucocyte Pharmacology, Consorzio Mario Negri Sud, 66030 Santa Maria Imbaro, Italy. Tel. 39.872.5701, fax 39.872.578 240.
thrombosis. PMN-induced platelet activation could be a mechanism by which PMN contribute to vascular occlusion. Heparin is a widely used antithrombotic drug for the prevention and treatment of venous and arterial thrombosis. The rationale for the use of heparin is based upon its ability to inhibit blood coagulation by enhancing the inhibition of thrombin by antithrombin III. However, whether anticoagulation is totally responsible for the antithrombotic activity of heparin is questionable. The antithrombotic activity could partly be due to effects on the endothelium a n d / o r on fibrinolysis (Lane and Lindahl, 1989). Standard heparin was reported to inhibit the proteinase activity of cathepsin G (Marossy, 1981; Redini et al., 1988). Recently, Ferrer et al. (1990) reported that standard and low-molecular weight heparin prevent the effect of this protease on platelets. In this study we investigated the effect of four heparins with different physico-chemical characteristics (namely molecular weight and degree of sulphation, absence of antithrombin III active site) on platelet activation induced by purified cathepsin G or by PMN challenged with the chemotactic peptide formyl-MetLeu-Phe (fMLP) in mixed cell suspensions.
402
2. Materials and methods
2.1. Platelet and P M N isolation
Human platelets were obtained from healthy volunteers who had not received any medication for at least two weeks, and were washed as described previously (Evangelista et al., 1991) in the presence of 1 /zM prostaglandin (PG) E 1. PMN were isolated by dextran sedimentation, followed by Ficoll-Hypaque gradient and hypotonic lysis of erytrocytes (Evangelista et al., 1991). After they had been washed, cells were resuspended in HEPES-Tyrode buffer containing 1 mM Ca 2÷ and maintained at room temperature during the experiment. 2.2. Experimental conditions
Platelets (108/ml) were preincubated for 2 min at 37°C under constant stirring (1000 rpm) in an aggregometer in the presence of fibrinogen (0.38 mg/ml). Cathepsin G from human neutrophils (Calbiochem; specific activity: 2 units/mg) (0.2/xM) was then added to platelets and changes in light transmission were recorded for 3 min as an index of platelet aggregation. This concentration of cathepsin G corresponds to the amount released by 5 x 10 6 PMN activated by 1 /zM fMLP in the presence of 2.5 izg/ml cytochalasin B (Evangelista et al., 1991). Samples were immediately centrifuged (14000 rpm) in an Eppendorf centrifuge and supernatants were stored at -20°C for serotonin and thromboxane (Tx) B 2 assay. In the experiments in which PMN were used, platelets and PMN (20/1) were preincubated together in mixed cell suspensions for 2 min at 37°C under constant stirring (1000 rpm) in the presence of fibrinogen (0.38 mg/ml) and cytochalasin B (2.5 tzg/ml) with or without heparins and then exposed to the chemotactic peptide fMLP (1 /xM). In this mixed system platelet activation is measured as aggregation.
Phe-p-nitroanilide (Sigma) by monitoring the rate of release of nitroaniline at 410 nm as described (Nakajima et al., 1979). Briefly, 5 tzl of a 100 mM solution of the specific synthetic substrate in N-methylpirrolidinone (0.5 mM final concentration) was added to 985 p.1 HEPES-Tyrode buffer with or without different concentrations of heparins. The cleavage of the substrate was monitored by following the release of nitroaniline at 410 nm for 5 min after addition of 10/zl of 20 /zM solution of cathepsin G dissolved in saline (0.2 /xM final concentration). 2.5. Heparins
The heparins used were an unfractionated preparation from pig intestinal mucosa (V), and high- and Aggregalion
100-, 0000-
\ O' Serotonin release
1001
.-*- v
001 001 4oi 0 Tx B2
100.
2.3. TxB e and serotonin measurement
TxB 2 was measured by radioimmunoassay, using a specific antiserum (kindly provided by Prof. C. Patrono, Catholic University, Rome, Italy), according to Evangelista et al. (1991). Serotonin released by activated platelets was measured by HPLC as described (Buczko et al., 1984) and is expressed as a percentage of the total content measured in the supernatant of 108 sonicated platelets. The total content of serotonin was 0.34 + 0.1 nmol/108 platelets (mean + S.D., n = 9). 2.4. Cathepsin G catalytic activity measurement
Cathepsin G catalytic activity was measured with the specific chromogenic substrate N-succinyl-Ala-Ala-Pro-
40. 200
o
1'o
3'o
Heparln (nM)
so
Fig. L Effect of bcparins on platelet activation induced by purified cathepsin G. Platelet activation induced by 200 nM of purified cathepsin G (see Materials and methods) was measured as aggregation, serotonin release and TxB z production, in the presence or absence of different concentrations of heparins. Data (means_+ S.E.M., n = 4) are expressed as % of control values, corresponding to 86.5_+4.6 (% of light transmission), 63.5_+5 (% of total content), 96.3 + 30 ( p m o l / m l ) for platelet aggregation, serotonin release and TxB 2 production, respectively.
403 Cathepsln
G
Aggregation
120.1 T ~
~
'
~
5
0
nM (3.4:1=2.5) 100
tN
•
II
•
vl
ov ~ VII
80
.~
,lO, 20. 0
25 nM (34.1:L'9.7)
10
50 Heparln(nM)
100
Fig. 3. Effect of heparins on platelet activation induced by fMLPactivated PMN in mixed cell suspensions. In the h.'xed cell system platelet activation was measured as aggregation and expressed as % of control (corresponding to 72.7 _+7.2% of light transmission). * P < 0.05, ** P
401
EJP 52499
Inhibition by heparin of platelet activation induced by neutrophil-derived cathepsin G Virgilio E v a n g e l i s t a , P a o l a P i c c a r d o n i , N o r m a M a u g e r i , G i o v a n n i D e G a e t a n o a n d C h i a r a C e r l e t t i Giulio Bizzozero Laboratory of Platelet and Leucocyte Pharmacology, Istituto di Ricerche Farmacologiche Mario Negri, Consorzio Mario Negri Sud, 66030 Santa Maria Imbaro, Italy Received 24 October 1991, revised MS received 6 March 1992, accepted 24 March 1992
Heparin is the most widely used anticoagulant drug for prevention and treatment of thrombosis. However, inhibition of blood coagulation might not fully explain the antithrombotic activity of this drug. The present study shows that different heparin preparations (50 nM) completely prevent human platelet aggregation, serotonin release and thromboxane B 2 production induced by purified neutrophil-derived cathepsin G (E.C. No. 3.4.21.20). This inhibitory effect was not related to the anticoagulant property of the compounds, since a heparin preparation with an inactivated active site for antithrombin III was also effective. Heparins inhibited the protease activity of the enzyme over the same range of concentrations. Since the effect of cathepsin G on platelets requires an intact proteolytic active site, the inhibitory effect of the drugs on cathepsin G-induced platelet activation may be explained by a blockade of protease activity. Heparins were also shown to reduce platelet activation induced by cathepsin G released from activated polymorphonuclear leucocytes in mixed cell suspensions. As polymorphonuclear leucocytes might contribute to both arterial and venous thrombosis through platelet activation induced by the release of cathepsin G, this novel property of heparin could be used to optimize its antithrombotic efficacy. Heparin; Platelets; Neutrophils; Cathepsin G
I. Introduction
Recent in vitro studies demonstrated that polymorphonuclear leucocytes (PMN) activated by several agonists can induce platelet activation (Chignard et al., 1986; Del Maschio et al., 1990). Cathepsin G, a neutral serine protease, contained together with elastase in the azurophilic granules of PMN, is a very potent platelet agonist (Selak et al., 1988; Ferrer-Lopez et al., 1990; Evangelista et al., 1991) with a specific binding site on the surface of platelets (Selak and Smith, 1990). PMNinduced platelet activation via cathepsin G can be prevented by antiproteinases, which inhibit the proteinase activity of cathepsin G (Ferrer-Lopez et al., 1990). Several epidemiological (Ernst et al., 1987) and in vivo (Nash et al., 1988; Mehta et al., 1989; Thomas et al., 1988) observations suggest that PMN could play a role in the pathophysiology of both arterial and venous
Correspondence to: V. Evangelista, Giulio Bizzozero Laboratory of Platelet and Leucocyte Pharmacology, Consorzio Mario Negri Sud, 66030 Santa Maria Imbaro, Italy. Tel. 39.872.5701, fax 39.872.578 240.
thrombosis. PMN-induced platelet activation could be a mechanism by which PMN contribute to vascular occlusion. Heparin is a widely used antithrombotic drug for the prevention and treatment of venous and arterial thrombosis. The rationale for the use of heparin is based upon its ability to inhibit blood coagulation by enhancing the inhibition of thrombin by antithrombin III. However, whether anticoagulation is totally responsible for the antithrombotic activity of heparin is questionable. The antithrombotic activity could partly be due to effects on the endothelium a n d / o r on fibrinolysis (Lane and Lindahl, 1989). Standard heparin was reported to inhibit the proteinase activity of cathepsin G (Marossy, 1981; Redini et al., 1988). Recently, Ferrer et al. (1990) reported that standard and low-molecular weight heparin prevent the effect of this protease on platelets. In this study we investigated the effect of four heparins with different physico-chemical characteristics (namely molecular weight and degree of sulphation, absence of antithrombin III active site) on platelet activation induced by purified cathepsin G or by PMN challenged with the chemotactic peptide formyl-MetLeu-Phe (fMLP) in mixed cell suspensions.
402
2. Materials and methods
2.1. Platelet and P M N isolation
Human platelets were obtained from healthy volunteers who had not received any medication for at least two weeks, and were washed as described previously (Evangelista et al., 1991) in the presence of 1 /zM prostaglandin (PG) E 1. PMN were isolated by dextran sedimentation, followed by Ficoll-Hypaque gradient and hypotonic lysis of erytrocytes (Evangelista et al., 1991). After they had been washed, cells were resuspended in HEPES-Tyrode buffer containing 1 mM Ca 2÷ and maintained at room temperature during the experiment. 2.2. Experimental conditions
Platelets (108/ml) were preincubated for 2 min at 37°C under constant stirring (1000 rpm) in an aggregometer in the presence of fibrinogen (0.38 mg/ml). Cathepsin G from human neutrophils (Calbiochem; specific activity: 2 units/mg) (0.2/xM) was then added to platelets and changes in light transmission were recorded for 3 min as an index of platelet aggregation. This concentration of cathepsin G corresponds to the amount released by 5 x 10 6 PMN activated by 1 /zM fMLP in the presence of 2.5 izg/ml cytochalasin B (Evangelista et al., 1991). Samples were immediately centrifuged (14000 rpm) in an Eppendorf centrifuge and supernatants were stored at -20°C for serotonin and thromboxane (Tx) B 2 assay. In the experiments in which PMN were used, platelets and PMN (20/1) were preincubated together in mixed cell suspensions for 2 min at 37°C under constant stirring (1000 rpm) in the presence of fibrinogen (0.38 mg/ml) and cytochalasin B (2.5 tzg/ml) with or without heparins and then exposed to the chemotactic peptide fMLP (1 /xM). In this mixed system platelet activation is measured as aggregation.
Phe-p-nitroanilide (Sigma) by monitoring the rate of release of nitroaniline at 410 nm as described (Nakajima et al., 1979). Briefly, 5 tzl of a 100 mM solution of the specific synthetic substrate in N-methylpirrolidinone (0.5 mM final concentration) was added to 985 p.1 HEPES-Tyrode buffer with or without different concentrations of heparins. The cleavage of the substrate was monitored by following the release of nitroaniline at 410 nm for 5 min after addition of 10/zl of 20 /zM solution of cathepsin G dissolved in saline (0.2 /xM final concentration). 2.5. Heparins
The heparins used were an unfractionated preparation from pig intestinal mucosa (V), and high- and Aggregalion
100-, 0000-
\ O' Serotonin release
1001
.-*- v
001 001 4oi 0 Tx B2
100.
2.3. TxB e and serotonin measurement
TxB 2 was measured by radioimmunoassay, using a specific antiserum (kindly provided by Prof. C. Patrono, Catholic University, Rome, Italy), according to Evangelista et al. (1991). Serotonin released by activated platelets was measured by HPLC as described (Buczko et al., 1984) and is expressed as a percentage of the total content measured in the supernatant of 108 sonicated platelets. The total content of serotonin was 0.34 + 0.1 nmol/108 platelets (mean + S.D., n = 9). 2.4. Cathepsin G catalytic activity measurement
Cathepsin G catalytic activity was measured with the specific chromogenic substrate N-succinyl-Ala-Ala-Pro-
40. 200
o
1'o
3'o
Heparln (nM)
so
Fig. L Effect of bcparins on platelet activation induced by purified cathepsin G. Platelet activation induced by 200 nM of purified cathepsin G (see Materials and methods) was measured as aggregation, serotonin release and TxB z production, in the presence or absence of different concentrations of heparins. Data (means_+ S.E.M., n = 4) are expressed as % of control values, corresponding to 86.5_+4.6 (% of light transmission), 63.5_+5 (% of total content), 96.3 + 30 ( p m o l / m l ) for platelet aggregation, serotonin release and TxB 2 production, respectively.
403 Cathepsln
G
Aggregation
120.1 T ~
~
'
~
5
0
nM (3.4:1=2.5) 100
tN
•
II
•
vl
ov ~ VII
80
.~
,lO, 20. 0
25 nM (34.1:L'9.7)
10
50 Heparln(nM)
100
Fig. 3. Effect of heparins on platelet activation induced by fMLPactivated PMN in mixed cell suspensions. In the h.'xed cell system platelet activation was measured as aggregation and expressed as % of control (corresponding to 72.7 _+7.2% of light transmission). * P < 0.05, ** P
