97
European Journal of Pharmacology, 198 (1991) 97-100
© 1991 Elsevier Science Publishers B.V. 0014-2999/91/$03.50 ADONIS 001429999100412U EJP 20830 Short communication
Polymorphonuclear leukocyte-dependent modulation of platelet function: effect of cloricromene Alberto Z a t t a , Marco Prosdocimi, Gianfranco Bazzoni 1, Elisabetta D e j a n a 1 and Aldo Del Maschio 1 Fidia Research Laboratories, Via Ponte della Fabbrica3 / A, 35031-Abano Terme, Italy and 1Laboratory of VascularBiology, Istituto di Ricerche Farmacologiche Mario Negri, Via Eritrea 62, 20157-Milano, Italy
Received 12 March 1991,accepted 2 April 1991
Cloricromene inhibits the activation of platelets and polymorphonuc!ear leukocytes (PMN) induced by specific stimuli. We report here that cloricromene caused a concentration-dependent reduction of platelet aggregation,/3-thromboglobulin (/3-TG) release and cytoplasmic Ca2÷ movements induced by PMN stimulated with N-formyl-L-methionyl-L-leucyl-L-phenylalaaine (fMLP), and enhanced the inhibitory action of unstimulated PMN on platelet responses to the stable endoperoxide analog U46619. These effects may be of pharmacological interest in view of the multiple interactions between platelets and leukocytes that occur in thrombotic arid inflammatory diseases. Platelet-polymorphonuclear leukocyte interaction; Cloricromene
1. Introduction
2. Materials and methods
Thrombosis and inflammation are multicellular processes involving biochemical interactions between platelets and polymorphonuclear leukocytes (PMN) (Marcus, 1990). Therefore, pharnmcological interventions directed against both cell types and acting on the mechanisms of intercellular communication may represent novel therapeutic approaches. Cloricromene (8monochloro-3-fl-diethylaminoethyl-4-methyl-7-etoxycarbonyl methoxy coumarin) is a coumarin derivative that, besides preventing platelet activation (Prosdocimi et al., 1986), inhibits several PMN functions (Bertocchi et al., 1989). We evaluated the effect of cloricromene on two mechanisms whereby PMN modulate platelet responses in vitro: activation of platelet functions by N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-stimulated PMN (Chignard et al., 1986; Del Maschio et al., 1990) and their inhibition by resting PMN (Zatta et al., 1990). Cloricromene inhibited platelet responses triggered by stimulated PMN and amplified the ability of resting PMN to limit platelet activation, suggesting that this experimental drug may have a protective role in thrombotic and inflammatory conditions.
2.1. Chemicals
Correspondence to: M. Prosdocimi, Fidia Research Laboratories, Via Ponte della Fabbrica 3/A, 35031-AbanoTerme, Italy.
All materials for the preparation of platelets and PMN were purchased frem Sigma Chemical Co. (St. Louis, USA), Pharmacia Fine Chemicals (Uppsala, Sweden) and Nycomed 'AS (Oslo, Norway) as reported previously (Zatta et al., 1990). fMLP was from Sigma and was dissolved in dimethyl sulfoxide to a final concentration of 50 mM, stored at - 2 0 °C and diluted in isotonic saline just before use; [15 S] hydroxy-ll [epoxy methano] prosta-[5Z, 13E]-dienoic acid (U46619), purchased from Upjohn Co. (Kalamazoo, USA), was dissolved in ethanol and diluted in Tris 15 mM (pH 7.4); cathepsin G was a gift from Dr. M. Chignard (Institut Pasteur, Paris, France); aequorin (extracted from the photocytes of the jellyfish Aequorea aequorea) was purchased from Dr. J. Blinks (Mayo Clinic, Rochester, USA);/3-thromboglobu|in (fl-TG) radioimmunoassay kit was purchased from Amersham (Buckinghamshire, UK); cloricromene was from Fidia (Abano Terme, Italy). 2.2. Experimental procedures
Venous blood was obtainea from healthy volunteers who had not received any medication for at least two weeks. Washed platelets and PMN were prepared as described previously (Zatta et al., 1990) and resus-
pended in HEPES-Tyrode buffer. Aliquots of washed platelets (3 x 10S/ml), PMN (107/ml) or a mixture of ~he two ceils were preincubated, with or without cloricromene (for 3 min at 37 °C under constant stirring), before addition of fMLP (in the presence of 2.5/~g/ml ojto~halasin B) or U46619. Platelet aggregation was quantified 3 rain after addition of stimuli by measuring the decrease in platelet count with a platelet analyzer (Baker 8!0 Instrument; A. De Mori, Milano, Italy) as reported l~reviously (Zatta et al., 1990). In some experiments, the effect of purified human cathepsin G on platelet aggregation was evaluated under the same experimental conditions. For the study of cytoplasmic Ca 2. movements, platelets were washed and loaded with aequorin as described (Johnson et al., 1985). Cytoplasmic Ca 2+ was measured ha a platelet-ionized calcium aggregometer (PICA, Chrono-Log) under the experimental conditions reported previously (Del Maschio et al., 1990). At the end of the ir~cubation time, samples were immediately centrifuged arid supernatants collected for the determination of/3-TG release by radioimmunoassay and for the measurement of lysozyme release. Lysozyme concentration was determined spectrophotometrically by measuring the lysis of Micrococcus luteus (Boehringer, Mannheim, FRG) and comparing the resuits with those obtained with known amounts of lysozyme.
platelet-PMN suspensions (platelet/PMN ratio of 30: 1) induced platelet aggregation, cytoplasmic Ca 2+ movements and fl-TG release in a concentration-dependent manner as reported previously (Chignard et al., 1986; Del Maschio et al., 1990). At the highest fMLP concentration, platelet activation was maximal: indeed, platelet aggregation reached 86 5: 3%, intracellular Ca 2+ level rose to 3.2 _+0.3 /~M and fl-TG re° lease averaged 2.6 _+ 0.1/~g/ml. Preincubation of cellular suspensions with cloricromene (10-200 /~M) for 3 min at 3 7 ° C inhibited these parameters of platelet activation in a concentration-dependent manner; cloricromene concentrations giving half-maximal inhibition of platelet responses were in the range of 80-120/~M (fig. 1A). Release of lysozyme, a specific marker of PMN activation, was reduced in a concentration-dependent manner by cloricromene; the half-maximal inhibitory concentration averaged 180/~M (fig. 1A). Cathepsin G, a serine proteinase released by activated PMN, is a potent stimulus for platelets (Selak et al., 1988). In our experimental system, platelet aggregation and fl-TG release induced by 100 nM exogenous cathepsin G, a concentration reached in the surrounding space of activated PMN, were inhibited by cloricromene (10-200 /zM) in a concentration-dependent way (fig. 1B).
3.2. Effect of cloricromene on PMN-mediated platelet inhibition 3. Results
As reported previously (Zatta et al., 1990), unstimulated PMN inhibited plat,elet aggregation and fl-TG release after exposure of cellular suspensions (platelet/PMN ratio of 30: 1) to a specific platelet stimulus, the stable endoperoxide analog U46619. Platelet inhibition was almost complete when cellular suspensions
3.1. Effect of cloricromene on PMN-mediated p!atelet activation Activation of cytochalasin B-pretreated PMN with the chemotactic peptide fMLP (10-9-10 -6 M) in mixed A
B
1001
100=
75
75
50
50
25
25
2 O
o "6
0
"
0
I
50
i
I
100
i
l
150
i
I
200
0
0
50
100
150
200
Cloricromene ( I~M ) Cloricrornene ( I~M ) Fig. 1. Cloricromene inhibition of platelet aggregation (0)~ jS-TG release (e), cytoplasmic Ca2+ movements ( [ ] ) and PMN lysozyme release ( I ) in mixed suspensions of human platelets (3 x 10S/ml) and PMN (107/ml) stimulated 10.6 M fMLP (A). Effect of cloricromene on aggregation (c) and/~-TG release (e) from platelets exposed to 100 nM cathepsin G (B). Data (means + S.E.), derived from five separate experiments (in duplicate), are expressed as a percent of values obtained in the absence of cloricromene.
99 ~00
A
8
A
B
A
50
~
25
0 100
4
2
200
300
400
500
600
U46619 ( nM )
0 100
200
300
400
500
600
U46519 ( nM )
Fig. 2. Aggregation (A) and fl-TG release (B) from U46619-stimulated platelets t3x]0S/ml) incubated with control buffer (o), 5 /zM cloricromene (e), PMN (]07/mi; 121)or a combination of cloricromenc and PMN (ll). Data (means+S.E.) are derived from five separate experiments performed in duplicate.
were stimulated with U46619 at concentrations lower than or equal to 300 nM. PMN-dependent inhibition was, however, overcome by higher concentrations of U46619 (up to 600 nM). Cloricromene (5 /zM), in the absence of PMN, inhibited platelet aggregation induced by U46619 in concentrations that caused threshold aggregation (the lowest concentration of stimulus causing at leas~ a 75% decrease in platelet count), while at higher stimulus concentrations it was ineffective. However, in the presence of PMN, cloricromene (5/zM) almost completely inhibited platelet aggregation induced by U46619 in concentrations up to 450 nM. Inhibition was still present with the highest U466!9 concentration (fig. 2A). A 2-fold higher cloricromene concentration (10/~M) was per se ineffective against maximal platelet stimulation (600 nM of U46619; not shown) but, in the presence of PMN, it caused an almost complete inhibition (84 _+ 9% of inhibition). The. PMN-dependent inhibition of U46619-induced release of fl-TG was similarly enhanced by cloricromene (fig. 2B).
4. Discussion
Evaluation of platelet responses in the presence of autologous PMN may represent an in vitro model to investigate the multicellular processes involved in thrombosis and inflammation (Marcus, 1990). Stimulated PMN induce platelet activation by the release of secretory products, the most relevant one being cathepsin G (Selak et al., 1988). However, it has recently been demonstrated that unstimulated PMN may exert an inhibitory effect on platelet responsiveness to specific platelet agonists (Salvemini et al., 1989; Zatta et al., 1990). Multicellular in vitro systems may
help to better define the effectiveness and mode of action of new antithrombotic and antiinflammatory drugs. We investigated the effect of cloricromene, a new antiplatelet (Prosdocimi et al., 1986) and leukocyte inhibitory agent (Bertocchi et al., 1989), on aggregation, /3-TG release and cytoplasmic Ca 2÷ movements in washed platelets incubated with autologous PMN at a physiological platelet/PMN ratio. Platelet responses were indirectly triggered through PMN stimulation with the chemotactic peptide fMLP. In the present study we present evidence that cloricromene, besides directly acting as an antiplatelet agent, may limit platelet activation induced by fMLP-stimulated PMN. Cloricromene inhibited all parameters of the platelet response induced by fMLP-stimulated PMN (fig. IA) and by purified cathepsin G (fig. 1B), suggesting that the PMN-dependent inhibition is probably due to an interference of the drug with the cathepsin G-mediated mechanism of platelet stimulation. Cloricromene was more effective on cathepsin G-induced platelet activation, probably because other PMN-derived products, such as oxygen free radicals and elastase, may contribute to a complete platelet activation. Data suggest that, in this experimental condition, platelets were more sensitive to cloricromene, since lower concentrations of the drug were required to inhibit platelet responses than those required to inhibit PMN activation as evaluated by lysozyme release (fig. 1A). A proposed mechanism of action of cloricromene is that it increases cGMP levels, counterbalancing cytoplasmic Ca 2÷ availability (Hakim et al., 1988). It is noteworthy that aspirin, a widely used anti-platelet drug, is totally ineffective in this experimental system when tested at concentrations 6-fold greater than the half-maximal inhibitory concentration of cloricromene (Chignard et al., 1986; Del Maschio et al., 1990).
Interestingly, higher concentrations of cloricromene (more than one order of m a g n i t u d e ) were required to inhibit P M N - m e d i a t e d platelet activation than the platelet stimulation induced by U46619, suggesting that the inhibitory, capacity of cloricromene on P M N - m e d i ated platelet activation is lower than its ability to interfere directly with U46619-induced platelet responses. This might be explained by the fact that the drug is less effective on cathepsin G- than on U46619activated platelets (fig. 1). F u r t h e r m o r e , in P M N - m e d i ated platelet activation, different stimuli can interplay to make cloricromeme inhibition more difficult. We e x a m i n e d the effect of cioricromene on the aggregation a n d / 3 - T G release of platelets activated by the stable endoperoxide analog U46619 in the presence of resting PMN. We found that cloricromene and PMN, tested alone, inhibited platelet activation, but their inhibition was overcome by U46619 concentrations greater than the threshold aggregatory concentration. Hoxvever, w h e n platelets were incubated with PMN, cloricromene inhibition was still a p p a r e n t even at the highest concentrations of U46619 (fig. 2). It is tempting to speculate that additive effect on intraplatelet c G M P levels might possibly explain the m e c h a n i s m underlying the interaction between cloricromene and PMN. Indeed, cloricromene inhibits platelet c G M P phosphodiesterase activity (Hakim et al., 1988) and P M N - d e r i v e d nitric oxide inhibits platelet activation by stimulating guanylate cyclase (Wright et ai., 1989). We propose that cloricromene may exert t h e r a p e u tic effects by modalating the interplay between the cci!ular c o m p o n e n t s involved in different pathological p~ocesses. The ability of cloricromene to d a m p e n platelet responses induced by stimulated P M N may become relevant in conditions of phagocytic activation, such as at sites of inflammation. Moreover, the enh a n c e m e n t by cloricromene of the P M N - d e p e n d e n t inhibition of platelet activation may be useful in thrombotic diseases.
References Bertocchi, F., F. Breviario, P. Proserpio, J. Ming Wang, P. Ghezzi, R.A. Travagli, M. Prosdocimi and E. Dejana, 1989, In vitro inhibition of human polymorphonuclear cell function by cloricromene, Naunyn-Schmiedeb. Arch. Pharmacol. 339, 697. Chignard, M., M.A. Selak and J.B. Smith, 1986, Direct evidence for the existence of a neutrophil-derived platelet acivator (neutrophilin), Proc. Natl. Acad. Sci. U.S.A. 83, 8609. Del Maschio, A., V. Evangelista, G. Rajtar, Z.M. Chen, C. Cerletti and G. De Gaetano, 1990, Platelet activation by polymorphonuclear leukocytes exposed to chemotactic agents, Am. J. Physiol. 258, H870. Hakim, G., D. Fiorentini, A. Falasca, M. Prosdocimi and C.A. Rossi, 1988, Effect of AD6 (8-monochloro-3-beta-diethylamino-ethyl-4methyl-7-ethoxycarbonyimethoxycoumarin) on cyclic nucleotide phosphodiesterases in human platelets, Experientia 44, 226. Johnson, P.C., J.A. Ware, P.B. Cliveden, M. Smith, A.M. Dvorak and E.W. Salzman, 1985, Measurement of ionized calcium in blood platelets with the photoprotein aequorin. Comparison with quin 2, J. Biol. Chem. 260, 2069. Marcus, A.J., 1990, Thrombosis and inflammation as multicellular processes: pathophysiologic significance of transcellular metabolism, Blood 76, 1903. Prosdocimi, M., A, Zatta, A. Gorio, A. Zanetti and E. Dejana, 1986, Action of AD6 (8-mGnochloro-3-beta-diethylaminoethyi-4methyl-7-ethoxycarbonylmethoxycoumarin) on human platelets in vitro, Naunyn-Schmiedeb. Arch. Pharmacol. 332, 305. Salvemini, D., G. De Nucci, R.G. Gryglewski and J.R. Vane, 1989, Human neutrophils and mononuclear cells inhibit platelet aggregation by releasing a nitric oxide-like factor, Proc. Natl. Acad. Sci. U.S.A. 86, 6328. Selak, M.A., M. Chignard and J.B. Smith, 1988, Cathepsin G is a strong platelet agonist released by neutrophils, Biochem. J. 251, 293. Wright, C.D., A. Mulsch, R. Busse and H. Osswald, 1989, Generation of nitric oxide by human neutrophils, Biochem. Byophys. Res. Commun. 160, 813. Zatta, A., M. Prosdocimi, V. Bertel~, G. Bazzoni and A. Del Maschio, 1990, Inhibition of platelet function by polymorphonuclear leukocytes, J. Lab. Clin. Med. 116, 651.
European Journal of Pharmacology, 198 (1991) 97-100
© 1991 Elsevier Science Publishers B.V. 0014-2999/91/$03.50 ADONIS 001429999100412U EJP 20830 Short communication
Polymorphonuclear leukocyte-dependent modulation of platelet function: effect of cloricromene Alberto Z a t t a , Marco Prosdocimi, Gianfranco Bazzoni 1, Elisabetta D e j a n a 1 and Aldo Del Maschio 1 Fidia Research Laboratories, Via Ponte della Fabbrica3 / A, 35031-Abano Terme, Italy and 1Laboratory of VascularBiology, Istituto di Ricerche Farmacologiche Mario Negri, Via Eritrea 62, 20157-Milano, Italy
Received 12 March 1991,accepted 2 April 1991
Cloricromene inhibits the activation of platelets and polymorphonuc!ear leukocytes (PMN) induced by specific stimuli. We report here that cloricromene caused a concentration-dependent reduction of platelet aggregation,/3-thromboglobulin (/3-TG) release and cytoplasmic Ca2÷ movements induced by PMN stimulated with N-formyl-L-methionyl-L-leucyl-L-phenylalaaine (fMLP), and enhanced the inhibitory action of unstimulated PMN on platelet responses to the stable endoperoxide analog U46619. These effects may be of pharmacological interest in view of the multiple interactions between platelets and leukocytes that occur in thrombotic arid inflammatory diseases. Platelet-polymorphonuclear leukocyte interaction; Cloricromene
1. Introduction
2. Materials and methods
Thrombosis and inflammation are multicellular processes involving biochemical interactions between platelets and polymorphonuclear leukocytes (PMN) (Marcus, 1990). Therefore, pharnmcological interventions directed against both cell types and acting on the mechanisms of intercellular communication may represent novel therapeutic approaches. Cloricromene (8monochloro-3-fl-diethylaminoethyl-4-methyl-7-etoxycarbonyl methoxy coumarin) is a coumarin derivative that, besides preventing platelet activation (Prosdocimi et al., 1986), inhibits several PMN functions (Bertocchi et al., 1989). We evaluated the effect of cloricromene on two mechanisms whereby PMN modulate platelet responses in vitro: activation of platelet functions by N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-stimulated PMN (Chignard et al., 1986; Del Maschio et al., 1990) and their inhibition by resting PMN (Zatta et al., 1990). Cloricromene inhibited platelet responses triggered by stimulated PMN and amplified the ability of resting PMN to limit platelet activation, suggesting that this experimental drug may have a protective role in thrombotic and inflammatory conditions.
2.1. Chemicals
Correspondence to: M. Prosdocimi, Fidia Research Laboratories, Via Ponte della Fabbrica 3/A, 35031-AbanoTerme, Italy.
All materials for the preparation of platelets and PMN were purchased frem Sigma Chemical Co. (St. Louis, USA), Pharmacia Fine Chemicals (Uppsala, Sweden) and Nycomed 'AS (Oslo, Norway) as reported previously (Zatta et al., 1990). fMLP was from Sigma and was dissolved in dimethyl sulfoxide to a final concentration of 50 mM, stored at - 2 0 °C and diluted in isotonic saline just before use; [15 S] hydroxy-ll [epoxy methano] prosta-[5Z, 13E]-dienoic acid (U46619), purchased from Upjohn Co. (Kalamazoo, USA), was dissolved in ethanol and diluted in Tris 15 mM (pH 7.4); cathepsin G was a gift from Dr. M. Chignard (Institut Pasteur, Paris, France); aequorin (extracted from the photocytes of the jellyfish Aequorea aequorea) was purchased from Dr. J. Blinks (Mayo Clinic, Rochester, USA);/3-thromboglobu|in (fl-TG) radioimmunoassay kit was purchased from Amersham (Buckinghamshire, UK); cloricromene was from Fidia (Abano Terme, Italy). 2.2. Experimental procedures
Venous blood was obtainea from healthy volunteers who had not received any medication for at least two weeks. Washed platelets and PMN were prepared as described previously (Zatta et al., 1990) and resus-
pended in HEPES-Tyrode buffer. Aliquots of washed platelets (3 x 10S/ml), PMN (107/ml) or a mixture of ~he two ceils were preincubated, with or without cloricromene (for 3 min at 37 °C under constant stirring), before addition of fMLP (in the presence of 2.5/~g/ml ojto~halasin B) or U46619. Platelet aggregation was quantified 3 rain after addition of stimuli by measuring the decrease in platelet count with a platelet analyzer (Baker 8!0 Instrument; A. De Mori, Milano, Italy) as reported l~reviously (Zatta et al., 1990). In some experiments, the effect of purified human cathepsin G on platelet aggregation was evaluated under the same experimental conditions. For the study of cytoplasmic Ca 2. movements, platelets were washed and loaded with aequorin as described (Johnson et al., 1985). Cytoplasmic Ca 2+ was measured ha a platelet-ionized calcium aggregometer (PICA, Chrono-Log) under the experimental conditions reported previously (Del Maschio et al., 1990). At the end of the ir~cubation time, samples were immediately centrifuged arid supernatants collected for the determination of/3-TG release by radioimmunoassay and for the measurement of lysozyme release. Lysozyme concentration was determined spectrophotometrically by measuring the lysis of Micrococcus luteus (Boehringer, Mannheim, FRG) and comparing the resuits with those obtained with known amounts of lysozyme.
platelet-PMN suspensions (platelet/PMN ratio of 30: 1) induced platelet aggregation, cytoplasmic Ca 2+ movements and fl-TG release in a concentration-dependent manner as reported previously (Chignard et al., 1986; Del Maschio et al., 1990). At the highest fMLP concentration, platelet activation was maximal: indeed, platelet aggregation reached 86 5: 3%, intracellular Ca 2+ level rose to 3.2 _+0.3 /~M and fl-TG re° lease averaged 2.6 _+ 0.1/~g/ml. Preincubation of cellular suspensions with cloricromene (10-200 /~M) for 3 min at 3 7 ° C inhibited these parameters of platelet activation in a concentration-dependent manner; cloricromene concentrations giving half-maximal inhibition of platelet responses were in the range of 80-120/~M (fig. 1A). Release of lysozyme, a specific marker of PMN activation, was reduced in a concentration-dependent manner by cloricromene; the half-maximal inhibitory concentration averaged 180/~M (fig. 1A). Cathepsin G, a serine proteinase released by activated PMN, is a potent stimulus for platelets (Selak et al., 1988). In our experimental system, platelet aggregation and fl-TG release induced by 100 nM exogenous cathepsin G, a concentration reached in the surrounding space of activated PMN, were inhibited by cloricromene (10-200 /zM) in a concentration-dependent way (fig. 1B).
3.2. Effect of cloricromene on PMN-mediated platelet inhibition 3. Results
As reported previously (Zatta et al., 1990), unstimulated PMN inhibited plat,elet aggregation and fl-TG release after exposure of cellular suspensions (platelet/PMN ratio of 30: 1) to a specific platelet stimulus, the stable endoperoxide analog U46619. Platelet inhibition was almost complete when cellular suspensions
3.1. Effect of cloricromene on PMN-mediated p!atelet activation Activation of cytochalasin B-pretreated PMN with the chemotactic peptide fMLP (10-9-10 -6 M) in mixed A
B
1001
100=
75
75
50
50
25
25
2 O
o "6
0
"
0
I
50
i
I
100
i
l
150
i
I
200
0
0
50
100
150
200
Cloricromene ( I~M ) Cloricrornene ( I~M ) Fig. 1. Cloricromene inhibition of platelet aggregation (0)~ jS-TG release (e), cytoplasmic Ca2+ movements ( [ ] ) and PMN lysozyme release ( I ) in mixed suspensions of human platelets (3 x 10S/ml) and PMN (107/ml) stimulated 10.6 M fMLP (A). Effect of cloricromene on aggregation (c) and/~-TG release (e) from platelets exposed to 100 nM cathepsin G (B). Data (means + S.E.), derived from five separate experiments (in duplicate), are expressed as a percent of values obtained in the absence of cloricromene.
99 ~00
A
8
A
B
A
50
~
25
0 100
4
2
200
300
400
500
600
U46619 ( nM )
0 100
200
300
400
500
600
U46519 ( nM )
Fig. 2. Aggregation (A) and fl-TG release (B) from U46619-stimulated platelets t3x]0S/ml) incubated with control buffer (o), 5 /zM cloricromene (e), PMN (]07/mi; 121)or a combination of cloricromenc and PMN (ll). Data (means+S.E.) are derived from five separate experiments performed in duplicate.
were stimulated with U46619 at concentrations lower than or equal to 300 nM. PMN-dependent inhibition was, however, overcome by higher concentrations of U46619 (up to 600 nM). Cloricromene (5 /zM), in the absence of PMN, inhibited platelet aggregation induced by U46619 in concentrations that caused threshold aggregation (the lowest concentration of stimulus causing at leas~ a 75% decrease in platelet count), while at higher stimulus concentrations it was ineffective. However, in the presence of PMN, cloricromene (5/zM) almost completely inhibited platelet aggregation induced by U46619 in concentrations up to 450 nM. Inhibition was still present with the highest U466!9 concentration (fig. 2A). A 2-fold higher cloricromene concentration (10/~M) was per se ineffective against maximal platelet stimulation (600 nM of U46619; not shown) but, in the presence of PMN, it caused an almost complete inhibition (84 _+ 9% of inhibition). The. PMN-dependent inhibition of U46619-induced release of fl-TG was similarly enhanced by cloricromene (fig. 2B).
4. Discussion
Evaluation of platelet responses in the presence of autologous PMN may represent an in vitro model to investigate the multicellular processes involved in thrombosis and inflammation (Marcus, 1990). Stimulated PMN induce platelet activation by the release of secretory products, the most relevant one being cathepsin G (Selak et al., 1988). However, it has recently been demonstrated that unstimulated PMN may exert an inhibitory effect on platelet responsiveness to specific platelet agonists (Salvemini et al., 1989; Zatta et al., 1990). Multicellular in vitro systems may
help to better define the effectiveness and mode of action of new antithrombotic and antiinflammatory drugs. We investigated the effect of cloricromene, a new antiplatelet (Prosdocimi et al., 1986) and leukocyte inhibitory agent (Bertocchi et al., 1989), on aggregation, /3-TG release and cytoplasmic Ca 2÷ movements in washed platelets incubated with autologous PMN at a physiological platelet/PMN ratio. Platelet responses were indirectly triggered through PMN stimulation with the chemotactic peptide fMLP. In the present study we present evidence that cloricromene, besides directly acting as an antiplatelet agent, may limit platelet activation induced by fMLP-stimulated PMN. Cloricromene inhibited all parameters of the platelet response induced by fMLP-stimulated PMN (fig. IA) and by purified cathepsin G (fig. 1B), suggesting that the PMN-dependent inhibition is probably due to an interference of the drug with the cathepsin G-mediated mechanism of platelet stimulation. Cloricromene was more effective on cathepsin G-induced platelet activation, probably because other PMN-derived products, such as oxygen free radicals and elastase, may contribute to a complete platelet activation. Data suggest that, in this experimental condition, platelets were more sensitive to cloricromene, since lower concentrations of the drug were required to inhibit platelet responses than those required to inhibit PMN activation as evaluated by lysozyme release (fig. 1A). A proposed mechanism of action of cloricromene is that it increases cGMP levels, counterbalancing cytoplasmic Ca 2÷ availability (Hakim et al., 1988). It is noteworthy that aspirin, a widely used anti-platelet drug, is totally ineffective in this experimental system when tested at concentrations 6-fold greater than the half-maximal inhibitory concentration of cloricromene (Chignard et al., 1986; Del Maschio et al., 1990).
Interestingly, higher concentrations of cloricromene (more than one order of m a g n i t u d e ) were required to inhibit P M N - m e d i a t e d platelet activation than the platelet stimulation induced by U46619, suggesting that the inhibitory, capacity of cloricromene on P M N - m e d i ated platelet activation is lower than its ability to interfere directly with U46619-induced platelet responses. This might be explained by the fact that the drug is less effective on cathepsin G- than on U46619activated platelets (fig. 1). F u r t h e r m o r e , in P M N - m e d i ated platelet activation, different stimuli can interplay to make cloricromeme inhibition more difficult. We e x a m i n e d the effect of cioricromene on the aggregation a n d / 3 - T G release of platelets activated by the stable endoperoxide analog U46619 in the presence of resting PMN. We found that cloricromene and PMN, tested alone, inhibited platelet activation, but their inhibition was overcome by U46619 concentrations greater than the threshold aggregatory concentration. Hoxvever, w h e n platelets were incubated with PMN, cloricromene inhibition was still a p p a r e n t even at the highest concentrations of U46619 (fig. 2). It is tempting to speculate that additive effect on intraplatelet c G M P levels might possibly explain the m e c h a n i s m underlying the interaction between cloricromene and PMN. Indeed, cloricromene inhibits platelet c G M P phosphodiesterase activity (Hakim et al., 1988) and P M N - d e r i v e d nitric oxide inhibits platelet activation by stimulating guanylate cyclase (Wright et ai., 1989). We propose that cloricromene may exert t h e r a p e u tic effects by modalating the interplay between the cci!ular c o m p o n e n t s involved in different pathological p~ocesses. The ability of cloricromene to d a m p e n platelet responses induced by stimulated P M N may become relevant in conditions of phagocytic activation, such as at sites of inflammation. Moreover, the enh a n c e m e n t by cloricromene of the P M N - d e p e n d e n t inhibition of platelet activation may be useful in thrombotic diseases.
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