THROMBOSIS RESEARCH 65; 559-570,1992 0049-3848/92 $5.00 + .OO Printed in the USA. Copyright (c) 1992 Pergamon Press Ltd. All rights reserved.
TICLOPIDINE AND PLATELET FUNCTION IN HEALTHY VOLUNTEERS Aldona Dembinska-Kiec l*), Irene Virgolini 2, F. Rauscha 3, and H. Sinzinger 2 Copernicus Medical Academy, Department of Pharmacology, N. Cracow, Poland 1, Wilhelm Auerswald-Atherosclerosis Research Atherosclerosis Research Group (ATK) of Group (ASF) Vienna 2, the Austrian Academy of Sciences Vienna 2, and Department of Cardiology, University of Vienna 3, Austria (Received
11.12.1991;
accepted
in revised form 21.12.1991
by Editor H.A. Vinazzer)
ABSTRACT with 500 mg The influence of a 4-weeks therapy ticlopidine daily on platelet function parameters was examined in 10 male healthy volunteers aged 20-33 years in order to extend the knowledge on the antiplatelet activity of this substance. Ticlopidine significantly affected ex-vivo platelet aggregation 0.01) (P < induced by ADP and increased platelet sensitivity to the antiaggregatory action of PG12. Generation of TXB2 from endogenous substrate during spontaneous clotting of blood (serum-TXBZ), conversion of exogenous radiolabelled arachidonic acid into TXB2 and MDA-formation in isolated platelets were unaffected by the treatment. The TXB2-level in plasma of volunteers, however, was the drug. The decreased, after administration of (P-thrombodiminished a-granule content liberation globulin: p < 0,Ol; PDGF: p < 0,Ol; PF4 not significant) indicates that ticlopidine induces a decrease in platelet activity. The beneficial effect on release reaction is not associated with a decrease in TXA2formation. Our results demonstrate that ticlopidine activity, especially the PDGFinhibits platelet release. These results confirm the value of this drug in the prevention of atherosclerosis and its thromboembolic complications. ____________________~~~~~~~~
Key words: Ticlopidine, platelet platelet derived growth factor
function,
thromboxane
B2,
*) Dr. Aldona Dembinska-Kiec is a visiting professor in the of Vienna, Austria Department of Pharmacology, University 559
560
TICLOPIDINE AND PLATELET FUNCTION
Vol. 65, Nos. 45
INTRODUCTION Ticlopidine (5-(O-chlorobenzyl)-4,5,6,7_tetrahydrothieno[3,2-cl-pyridine hydrochloride) is a well known antiplatelet agent, its mechanism of action, however, is not yet fully understood (for review see 1 2). The most important way of action seems to be the inhibition of ADP-mediated fibrinogen binding to the platelet membrane glycoprotein IIb/IIIa complex (3, 4). Ticlopidine has been shown to be of clinical benefit in patients with thromboembolic complications connected with atherosclerosis. In well controlled studies (CATS 151, TASS [63, STIMS [73, STAI [81) a consistent benefit Erom the intake of ticlopidine was reported with a reduction in fatal and nonfatal events in coronary, cerebral and peripheral vessels. This study was undertaken to examine the platelet functional response during ticlopidine treatment healthy volunteers, and in particular to evaluate whether ticlopidine administration influences the platelet derived growth factor (PDGF)-release from platelets. MATERIAL AND METHODS Volunteers: Ten healthy male volunteers, aged 20-33 a (26 f 3 a), normocholesterolemics with a body mass index in the normal range were included into this study. They were all non-smokers without any risk factor for the development of atherosclerosis. It was assured that they had not been taking drugs since at least 4 weeks prior study begin. Ticlopidine was administered twice daily (500 mg/day) during 4 weeks. Before the first dose, and 12 hours after the last ingestion of the drug, blood for the following tests (*) was drawn from the cubital vein after an at least la-hours overnight fast and at least 30 minutes at rest. The study was performed according to the Helsinkatdeclaration. (*I 1. Spontaneous platelet aggregation: -Anticoagulated blood (3,8 % sodium-citrate 1:9 v/v) was sedimented at room temperature (22°C) for 10 minutes. Platelet rich plasma (PRP) was prepared using a 10 minutes centrifugation at 150 g. Platelet poor plasma (PPP) was obtained by a further centrifugation step at 1000 g. PRP was adjusted with PPP to a final platelet count of 250 x lOE+3 cells/ul. 600 ul samples of adjusted PRP were added to a cuvette under constant stirring at aggregometer. Spontaneous aggregation Born-type 37°C in a response was assessed by quantification of the maximal amplitude. All the aggregation measurements were performed within 60 minutes after the blood had been drawn. 2 ADP-induced platelet aggregation: -1. Adjusted PRP-samples (600 ~1) were transferred into a cuvette. 100 ul of ADP-(Boehringer Mannheim, Germany; 1 nM end concentration in PRP) were added to induce aggregation. The aggre-
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TICLOPIDINE AND PLATELET FUNCTION
561
gation response was quantified using the slope a ("1 and the maximum amplitude (Tmax; %) at the response curve. 3. Platelet sensitivity -_(PS) to PG12: One minute prior to induction of aggregation by ADP, PG12 (The Upjohn Company, Kalamazoo, Mi, USA) (0.5-5.0 ng/ml PRP dissolved in Tris-HCl buffer (pH 7.4) was added. Aggregation generated in the presence of the PGI2-solvent served as a control. The platelet sensitivity to the antiaggregatory properties of PG12 was expressed as the dose (ng) of PGI2/ml PRP necessary to reduce the in-vitro induced platelet aggregation by 50% (IC50). 4. Serum thromboxane BJ (TXB2): -Native blood (2 ml) drawn into prewarmed syringes was allowed to clot under standardized conditions at 37°C for 60 minutes. Thereafter, the samples were centrifuged at 1000 g for 10 minutes twice in order to remove blood cells. The serum was then stored at -70°C for not longer than 2 weeks before measurement of TXB2 by a specific RIA (Amersham, Buckinghamshire, UK). The separation of free and antibody-bound ligand was performed using charcoal. 5. Malondialdehyde (MDA)-photometry: PRP was prepared as described above from blood anticoagulated with acid citrate dextrose. 0.5 ml of PRP was centrifuged (700 g, 25 minutes, 22°C) to get the platelet pellet which was buffer (pH 7.2) containing 0.9% sodium washed twice with chloride, 2% sodium-EDTA and 50 mM Tris-HCl (20:1:2). The pellet was resuspended in 0.5 ml 10 mM Tris-HCl-buffer (pH 7.4) and incubated with 5 U/100 ul thrombin (Topostatin; Hofmann La Roche, Basle, Switzerland) for 30 minutes at 37OC shaking. The reaction was stopped on ice after addition of 0.4 ml of trichloroacetic acid. After homogenization by means of ultrasound, the protein precipitate was separated by centrifugation (1500 g 10 minutes at 4OC). The resulting supernatant was used for MDAdetermination by the thiobarbituric acid method. Results are presented in nmol/lOE+9 platelets. 6. Radiothinlayer-chromatoqraphy (RTLC): Anticoagulated blood (7:3 v/v with acid citrate dextrose) was used for separation of PRP as described above. 0.5 ml of PRP was centrifuged (700 g, 25 minutes, 22'C) and PPP was removed with a vacuum pump. The resulting platelet pellet was resuspended in 0.5 ml 50 mM Tris-buffer (pH 7.4) and incubated with 0.25 uCi [14Cl arachidonic acid (AA; Amersham, Buckinghamshire, UK) for 5 minutes at 37'C shaking. The reaction was stopped by acidification down to pH 3 with 1N HCl. After centrifugation (5000 g, 10 minutes, 4OC) the supernatant was extracted with ethyl acetate. The organic phase was dried under nitrogen, dissolved in 100 ul of absolute ethanol and stored at -2OOC. After chromatography on silica gel columns (Merck, Darmstadt,
562
TICLOPIDINE AND PLATELET FUNCTION
Vol. 65, Nos. 415
Germany), the labelled AA-metabolites were analysed with a TLClinear scanner (Berthold, Wildbad, Germany). Radiolabelled standards (New England Nuclear, Dreieich, Germany) were used for comparison. B2 (TXB2): -7. Plasma thromboxane Blood (9 volumes) was carefully drawn from a non-occluded vein by an 1,2 mm diameter needle into (1 volume) 2% sodium EDTA and aspisol (acetylsalicylic acid, final concentration: 1 mg/ml; Bayer, Leverkusen, Germany) in precooled syringes. After a short sedimentation at 4'C, the samples were centrifuged (4°C) at 1500 g for separation of cells. The resulting PPP was transferred into Eppendorf-vials and stored there at -70°C for not longer than 2 weeks before the measurement of TXB2 was done by a specific RIA essentially as described above. The separation of free and antibody-bound ligand, however, was performed using a goat antirabbit gammaglobulin double antibody (OTOP 15/16; Behring, Marburg, Germany). 8 8-thromboglobulin (P-TG): --1. Blood was carefully drawn from a cubital vein without stasis under the usual precautions into the commercially available sampling tubes (Amersham, Buckinghamshire, UK). Plasma concentrations of this a-granule protein were determined using the commercial radioimmunoassay procedure. 9. Platelet factor 4 (PF4): The blood samples were drawn as described for 8-TG. The plasma PF4-concentrations were determined using the RIA of Abbott (North Chicago, Illinois, USA). 10. Platelet migration: from a non-occluded cubital vein and Blood was withdrawn anticoagulated with acid citrate dextrose (9:l v/v). PRP and PPP were prepared as described above. PRP was adjusted with PPP to a final platelet count of 250.103+3/ul using the technique of Brecher and Cronkite. A petri-dish was filled with 10 ml of Gibco-medium. A 10 cm capillary with an inner diameter of 0.8 mm was filled with PRP and was consequently placed with one end into the center of the dish. The petri-dish was closed and incubated for 24 hours at 37'C under continuous supply of 95% oxygen and 5% carbondioxyde. 100 U of penicillin and 50 mg streptomycin were added. After 24 hours an area in which the platelets migrated was marked with a pencil and quantified using a plan-compensation-polarimeter (Zeiss, Jena, Germany). The migration area is given as the mean of 2 estimations always done in parallel (9). For control purpose of morphology of migrated platelets scanning electron microscopy was done fixing the material after 24 hours with 2% phosphate buffered (pH 7.4) morphology of the the glutaraldehyde. After preparation, platelets was studied using a scanning electron microscope (Siemens-Elmiskop, Germany).
Vol. 65, Nos. 45
TICLOPIDINE AND PLATELET FUNCTION
563
11. Wu-test for circulating aggregates: Blood was anticoagulated with 2% EDTA/formaldehyde and with 2% EDTA alone. PRP was prepared as described above and the number of platelets was determined in a Biirker-Turk chamber. The platelet count ratio between these differently anticoagulated blood-samples expressed the amount of the circulating platelet aggregates (modified after Wu and Hoak, 10). 12. Fibrinogen: Fibrinogen was determined in anticoagulated (l:lO, sodium citrate) plasma samples. Plasma samples were collected in EDTA/aspisol/PGI2-mixture (4'C). After repeated (2 x) centrifugation (3000 g, 20 minutes, 4°C) the supernatant was stored at -70°C for less than 4 weeks until RIA. 13. Platelet derived growth factor (PDGF): PDGF was derived from isolation and purification as described earlier (11). An antibody was raised in rabbits after coupling to Freunds incomplete adjuvant. For RIA 1251 was used as a tracer. A double-antibody has been used for the separation of free and antibody-bound ligand. RIA was performed modified according to Sturzebecher (12). The other laboratory parameters (hematocrit, leukocytes) were determined according to routine laboratory procedure. Statistical methodology: The data are presented as mean + SEM. Statistical analysis was performed using the Student's t-test for paired data. RESULTS The administration of ticlopidine for 4 weeks resulted in the significant inhibition of ADP-induced platelet aggregation (Tab. 1) as demonstrated by a decreased initial slope (p < 0.01) and a lower maximum amplitude (p < 0.01). The spontaneous aggregation, which appeared in 2 of the tested volunteers was no longer observed after the treatment with ticlopidine. The transformation of exogenous radioactive arachidonic acid substrate to TXB2, TXB2-generation during clotting (serum TXB2) as well as the MDA-production remained unaffected by ticlopidine. In contrast, however, the plasma TXB2-level was significantly (p < 0.01) decreased by the administration of ticlopidine. Platelet sensitivity testing revealed a sensitization to the antiaggregatory action of PG12 (p < 0.05). Platelet migration showed signs of lower migratory activity, however, not reaching the level of significance. The number of circulating platelet aggregates (WU-index) was unaffected by the I-weeks ticlopidine therapy. The release of platelet proteins was decreased significantly for PDGF (p < 0.01) and for P-thromboglobulin (p < 0.011, but not significantly for platelet factor 4. Fibrinogen
564
TICLOPIDINE AND PLATELET FUNCTION
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TABLE 1 Platelet function tests
a
("1 _& Tmax (%I PS ng/ml TXB2-serum
62.3 f 2.59
53.7 f 1.80
13.8
0.01
< 70
57.7 f 2.67
39.1 + 3.80
32.2
0.01
< 60
0.70f 0.04
0.67f. 0.03
6.9
0.05
< 1.0
214.1 + 4.65
208.5 f 3.63
2.6
n.s.
180-250
w/ml
TXB2-conv. 31.0 f (%I MDA 3.71f nM/lOE+9 pl. TXBZ-plasma 10.7 f pg/ml PDGF-lib. 67.5 f ng/ml P-TG 16.6 f ng/ml PF4 6.6 f
1.65
31.0 + 1.91
0
n.s.
20-35
0.26
3.53+ 0.22
4.9
n.s.
TICLOPIDINE AND PLATELET FUNCTION IN HEALTHY VOLUNTEERS Aldona Dembinska-Kiec l*), Irene Virgolini 2, F. Rauscha 3, and H. Sinzinger 2 Copernicus Medical Academy, Department of Pharmacology, N. Cracow, Poland 1, Wilhelm Auerswald-Atherosclerosis Research Atherosclerosis Research Group (ATK) of Group (ASF) Vienna 2, the Austrian Academy of Sciences Vienna 2, and Department of Cardiology, University of Vienna 3, Austria (Received
11.12.1991;
accepted
in revised form 21.12.1991
by Editor H.A. Vinazzer)
ABSTRACT with 500 mg The influence of a 4-weeks therapy ticlopidine daily on platelet function parameters was examined in 10 male healthy volunteers aged 20-33 years in order to extend the knowledge on the antiplatelet activity of this substance. Ticlopidine significantly affected ex-vivo platelet aggregation 0.01) (P < induced by ADP and increased platelet sensitivity to the antiaggregatory action of PG12. Generation of TXB2 from endogenous substrate during spontaneous clotting of blood (serum-TXBZ), conversion of exogenous radiolabelled arachidonic acid into TXB2 and MDA-formation in isolated platelets were unaffected by the treatment. The TXB2-level in plasma of volunteers, however, was the drug. The decreased, after administration of (P-thrombodiminished a-granule content liberation globulin: p < 0,Ol; PDGF: p < 0,Ol; PF4 not significant) indicates that ticlopidine induces a decrease in platelet activity. The beneficial effect on release reaction is not associated with a decrease in TXA2formation. Our results demonstrate that ticlopidine activity, especially the PDGFinhibits platelet release. These results confirm the value of this drug in the prevention of atherosclerosis and its thromboembolic complications. ____________________~~~~~~~~
Key words: Ticlopidine, platelet platelet derived growth factor
function,
thromboxane
B2,
*) Dr. Aldona Dembinska-Kiec is a visiting professor in the of Vienna, Austria Department of Pharmacology, University 559
560
TICLOPIDINE AND PLATELET FUNCTION
Vol. 65, Nos. 45
INTRODUCTION Ticlopidine (5-(O-chlorobenzyl)-4,5,6,7_tetrahydrothieno[3,2-cl-pyridine hydrochloride) is a well known antiplatelet agent, its mechanism of action, however, is not yet fully understood (for review see 1 2). The most important way of action seems to be the inhibition of ADP-mediated fibrinogen binding to the platelet membrane glycoprotein IIb/IIIa complex (3, 4). Ticlopidine has been shown to be of clinical benefit in patients with thromboembolic complications connected with atherosclerosis. In well controlled studies (CATS 151, TASS [63, STIMS [73, STAI [81) a consistent benefit Erom the intake of ticlopidine was reported with a reduction in fatal and nonfatal events in coronary, cerebral and peripheral vessels. This study was undertaken to examine the platelet functional response during ticlopidine treatment healthy volunteers, and in particular to evaluate whether ticlopidine administration influences the platelet derived growth factor (PDGF)-release from platelets. MATERIAL AND METHODS Volunteers: Ten healthy male volunteers, aged 20-33 a (26 f 3 a), normocholesterolemics with a body mass index in the normal range were included into this study. They were all non-smokers without any risk factor for the development of atherosclerosis. It was assured that they had not been taking drugs since at least 4 weeks prior study begin. Ticlopidine was administered twice daily (500 mg/day) during 4 weeks. Before the first dose, and 12 hours after the last ingestion of the drug, blood for the following tests (*) was drawn from the cubital vein after an at least la-hours overnight fast and at least 30 minutes at rest. The study was performed according to the Helsinkatdeclaration. (*I 1. Spontaneous platelet aggregation: -Anticoagulated blood (3,8 % sodium-citrate 1:9 v/v) was sedimented at room temperature (22°C) for 10 minutes. Platelet rich plasma (PRP) was prepared using a 10 minutes centrifugation at 150 g. Platelet poor plasma (PPP) was obtained by a further centrifugation step at 1000 g. PRP was adjusted with PPP to a final platelet count of 250 x lOE+3 cells/ul. 600 ul samples of adjusted PRP were added to a cuvette under constant stirring at aggregometer. Spontaneous aggregation Born-type 37°C in a response was assessed by quantification of the maximal amplitude. All the aggregation measurements were performed within 60 minutes after the blood had been drawn. 2 ADP-induced platelet aggregation: -1. Adjusted PRP-samples (600 ~1) were transferred into a cuvette. 100 ul of ADP-(Boehringer Mannheim, Germany; 1 nM end concentration in PRP) were added to induce aggregation. The aggre-
Vol. 65, Nos. 4/5
TICLOPIDINE AND PLATELET FUNCTION
561
gation response was quantified using the slope a ("1 and the maximum amplitude (Tmax; %) at the response curve. 3. Platelet sensitivity -_(PS) to PG12: One minute prior to induction of aggregation by ADP, PG12 (The Upjohn Company, Kalamazoo, Mi, USA) (0.5-5.0 ng/ml PRP dissolved in Tris-HCl buffer (pH 7.4) was added. Aggregation generated in the presence of the PGI2-solvent served as a control. The platelet sensitivity to the antiaggregatory properties of PG12 was expressed as the dose (ng) of PGI2/ml PRP necessary to reduce the in-vitro induced platelet aggregation by 50% (IC50). 4. Serum thromboxane BJ (TXB2): -Native blood (2 ml) drawn into prewarmed syringes was allowed to clot under standardized conditions at 37°C for 60 minutes. Thereafter, the samples were centrifuged at 1000 g for 10 minutes twice in order to remove blood cells. The serum was then stored at -70°C for not longer than 2 weeks before measurement of TXB2 by a specific RIA (Amersham, Buckinghamshire, UK). The separation of free and antibody-bound ligand was performed using charcoal. 5. Malondialdehyde (MDA)-photometry: PRP was prepared as described above from blood anticoagulated with acid citrate dextrose. 0.5 ml of PRP was centrifuged (700 g, 25 minutes, 22°C) to get the platelet pellet which was buffer (pH 7.2) containing 0.9% sodium washed twice with chloride, 2% sodium-EDTA and 50 mM Tris-HCl (20:1:2). The pellet was resuspended in 0.5 ml 10 mM Tris-HCl-buffer (pH 7.4) and incubated with 5 U/100 ul thrombin (Topostatin; Hofmann La Roche, Basle, Switzerland) for 30 minutes at 37OC shaking. The reaction was stopped on ice after addition of 0.4 ml of trichloroacetic acid. After homogenization by means of ultrasound, the protein precipitate was separated by centrifugation (1500 g 10 minutes at 4OC). The resulting supernatant was used for MDAdetermination by the thiobarbituric acid method. Results are presented in nmol/lOE+9 platelets. 6. Radiothinlayer-chromatoqraphy (RTLC): Anticoagulated blood (7:3 v/v with acid citrate dextrose) was used for separation of PRP as described above. 0.5 ml of PRP was centrifuged (700 g, 25 minutes, 22'C) and PPP was removed with a vacuum pump. The resulting platelet pellet was resuspended in 0.5 ml 50 mM Tris-buffer (pH 7.4) and incubated with 0.25 uCi [14Cl arachidonic acid (AA; Amersham, Buckinghamshire, UK) for 5 minutes at 37'C shaking. The reaction was stopped by acidification down to pH 3 with 1N HCl. After centrifugation (5000 g, 10 minutes, 4OC) the supernatant was extracted with ethyl acetate. The organic phase was dried under nitrogen, dissolved in 100 ul of absolute ethanol and stored at -2OOC. After chromatography on silica gel columns (Merck, Darmstadt,
562
TICLOPIDINE AND PLATELET FUNCTION
Vol. 65, Nos. 415
Germany), the labelled AA-metabolites were analysed with a TLClinear scanner (Berthold, Wildbad, Germany). Radiolabelled standards (New England Nuclear, Dreieich, Germany) were used for comparison. B2 (TXB2): -7. Plasma thromboxane Blood (9 volumes) was carefully drawn from a non-occluded vein by an 1,2 mm diameter needle into (1 volume) 2% sodium EDTA and aspisol (acetylsalicylic acid, final concentration: 1 mg/ml; Bayer, Leverkusen, Germany) in precooled syringes. After a short sedimentation at 4'C, the samples were centrifuged (4°C) at 1500 g for separation of cells. The resulting PPP was transferred into Eppendorf-vials and stored there at -70°C for not longer than 2 weeks before the measurement of TXB2 was done by a specific RIA essentially as described above. The separation of free and antibody-bound ligand, however, was performed using a goat antirabbit gammaglobulin double antibody (OTOP 15/16; Behring, Marburg, Germany). 8 8-thromboglobulin (P-TG): --1. Blood was carefully drawn from a cubital vein without stasis under the usual precautions into the commercially available sampling tubes (Amersham, Buckinghamshire, UK). Plasma concentrations of this a-granule protein were determined using the commercial radioimmunoassay procedure. 9. Platelet factor 4 (PF4): The blood samples were drawn as described for 8-TG. The plasma PF4-concentrations were determined using the RIA of Abbott (North Chicago, Illinois, USA). 10. Platelet migration: from a non-occluded cubital vein and Blood was withdrawn anticoagulated with acid citrate dextrose (9:l v/v). PRP and PPP were prepared as described above. PRP was adjusted with PPP to a final platelet count of 250.103+3/ul using the technique of Brecher and Cronkite. A petri-dish was filled with 10 ml of Gibco-medium. A 10 cm capillary with an inner diameter of 0.8 mm was filled with PRP and was consequently placed with one end into the center of the dish. The petri-dish was closed and incubated for 24 hours at 37'C under continuous supply of 95% oxygen and 5% carbondioxyde. 100 U of penicillin and 50 mg streptomycin were added. After 24 hours an area in which the platelets migrated was marked with a pencil and quantified using a plan-compensation-polarimeter (Zeiss, Jena, Germany). The migration area is given as the mean of 2 estimations always done in parallel (9). For control purpose of morphology of migrated platelets scanning electron microscopy was done fixing the material after 24 hours with 2% phosphate buffered (pH 7.4) morphology of the the glutaraldehyde. After preparation, platelets was studied using a scanning electron microscope (Siemens-Elmiskop, Germany).
Vol. 65, Nos. 45
TICLOPIDINE AND PLATELET FUNCTION
563
11. Wu-test for circulating aggregates: Blood was anticoagulated with 2% EDTA/formaldehyde and with 2% EDTA alone. PRP was prepared as described above and the number of platelets was determined in a Biirker-Turk chamber. The platelet count ratio between these differently anticoagulated blood-samples expressed the amount of the circulating platelet aggregates (modified after Wu and Hoak, 10). 12. Fibrinogen: Fibrinogen was determined in anticoagulated (l:lO, sodium citrate) plasma samples. Plasma samples were collected in EDTA/aspisol/PGI2-mixture (4'C). After repeated (2 x) centrifugation (3000 g, 20 minutes, 4°C) the supernatant was stored at -70°C for less than 4 weeks until RIA. 13. Platelet derived growth factor (PDGF): PDGF was derived from isolation and purification as described earlier (11). An antibody was raised in rabbits after coupling to Freunds incomplete adjuvant. For RIA 1251 was used as a tracer. A double-antibody has been used for the separation of free and antibody-bound ligand. RIA was performed modified according to Sturzebecher (12). The other laboratory parameters (hematocrit, leukocytes) were determined according to routine laboratory procedure. Statistical methodology: The data are presented as mean + SEM. Statistical analysis was performed using the Student's t-test for paired data. RESULTS The administration of ticlopidine for 4 weeks resulted in the significant inhibition of ADP-induced platelet aggregation (Tab. 1) as demonstrated by a decreased initial slope (p < 0.01) and a lower maximum amplitude (p < 0.01). The spontaneous aggregation, which appeared in 2 of the tested volunteers was no longer observed after the treatment with ticlopidine. The transformation of exogenous radioactive arachidonic acid substrate to TXB2, TXB2-generation during clotting (serum TXB2) as well as the MDA-production remained unaffected by ticlopidine. In contrast, however, the plasma TXB2-level was significantly (p < 0.01) decreased by the administration of ticlopidine. Platelet sensitivity testing revealed a sensitization to the antiaggregatory action of PG12 (p < 0.05). Platelet migration showed signs of lower migratory activity, however, not reaching the level of significance. The number of circulating platelet aggregates (WU-index) was unaffected by the I-weeks ticlopidine therapy. The release of platelet proteins was decreased significantly for PDGF (p < 0.01) and for P-thromboglobulin (p < 0.011, but not significantly for platelet factor 4. Fibrinogen
564
TICLOPIDINE AND PLATELET FUNCTION
Vol. 65, Nos. 415
TABLE 1 Platelet function tests
a
("1 _& Tmax (%I PS ng/ml TXB2-serum
62.3 f 2.59
53.7 f 1.80
13.8
0.01
< 70
57.7 f 2.67
39.1 + 3.80
32.2
0.01
< 60
0.70f 0.04
0.67f. 0.03
6.9
0.05
< 1.0
214.1 + 4.65
208.5 f 3.63
2.6
n.s.
180-250
w/ml
TXB2-conv. 31.0 f (%I MDA 3.71f nM/lOE+9 pl. TXBZ-plasma 10.7 f pg/ml PDGF-lib. 67.5 f ng/ml P-TG 16.6 f ng/ml PF4 6.6 f
1.65
31.0 + 1.91
0
n.s.
20-35
0.26
3.53+ 0.22
4.9
n.s.
