Haemostasis 6 : 127-136 (1977)
Ditazole and Platelets I. Effect of Ditazole on Human Platelet Function in vitro
G.
de
G aetano, M .C .T onolli, M .P. Bertoni and M .C . R oncaglioni1 Laboratory for Haemostasis and Thrombosis Research, Istituto di Ricerche Farmacologiche ‘Mario Negri’, Milan
Key Words. Platelet aggregation • Ditazole • Clot retraction • Thrombelastography • Gel-filtered platelets • Serotonin Abstract. Ditazole (4,5-diphenyl-2-bis-(2-hydroxyethyl)-aminoxazol) has been shown to be a strong in vitro inhibitor of human platelet aggregation brought about by release reaction inducers; in contrast, it did not significantly affect primary ADP-induced aggre gation. Ditazole strongly inhibited the release of platelet-bound 14C-serotonin under the influence of Thrombofax, whereas it did not interfere with the transport and storage of serotonin in nonstimulated platelets. The effect of ditazole was not potentiated by acetylsalicylic acid. Ditazole also inhibited ADP-reptilase clot retraction and modified thrombininduced clot formation. The inhibition of platelet aggregation exerted by ditazole in plasma could be removed following gel filtration of platelets on Sepharose 2-B gel. This would indicate that ditazole does not act on platelets by a ‘hit and run’ mechanism.
Introduction Ditazole (4,5-diphenyl-2-bis-(2-hydroxyethyl)-aminoxazol) is a new drug shown by C a p r in o et al. [3] to inhibit human and rabbit platelet aggregation and to prolong bleeding time in mice. Ditazole is also endowed with mod erate anti-inflammatory, analgesic and antipyretic properties [2], The inhibitory effect of this compound on some platelet functions in vitro has been confirmed by other investigators, including our group [4, 12]. The
Received: August 6, 1976; accepted by editor J.V ermylen: November 25, 1976.
Downloaded by: Lund University Libraries 130.235.66.10 - 1/1/2019 5:56:58 PM
1 Mrs. A my C rook, Miss A nna M ancini and Mrs. G raziella Scalvini kindly helped us in preparing this manuscript.
128
de
G aetano/T onolli/B ertoni/R oncaguoni
present study reports on further investigations performed to better charac terize the in vitro effect of ditazole on human platelets.
Materials and Methods Ditazole (Serono, Rome, Italy) and other compounds tested in parallel (acetylsalicylic acid, ASA: Bayer, Milan, Italy; indomethacin, Merck, Sharp & Dohme, Rome, Italy; pyrimido-pyrimidine compounds VK 744 and SH 869, Karl Thomae, Biberach an der Riss, FRG) were dissolved in 97% ethanol at a concentration of 5 x 10 2 m ; subsequent dilutions were made with ethanol and drugs used in 1 to 2-/d aliquots. Platelet aggregation was studied by the photometric technique of Born and C ross [1] using a MK IV Born-Michal aggregometer (Pharmacological Research, Cambridge, UK) connected to a two-channel potentiometer (Venture Servoscribe 2, Smiths Industries, Ltd., London, UK), as previously described [9], Separation of platelets from plasma pro teins was obtained by gel filtration, as previously described [12]. Adenosine-5'-diphosphate (ADP: Sigma, Mascia Brunelli, Milan, Italy), collagen (Hormon Chemie, Munich, FRG) and Thrombofax (Ortho Diagnostic, Cilag-Chemie, Milan, Italy) [10] were used as aggregation stimuli. Reptilase clot retraction test (RCR) was performed as previously described [5]. Physical properties of platelet-rich plasma (PRP) clots were studied by thrombelastograplty using a Clotscanner apparatus (Elvi-Logos, Milan, Italy) [6]. Uptake o fu C-serotonin and its release from platelets under the influence of Thrombofax were investigated as previously described [11],
Aggregation by ADP was studied in 8 series of samples (table I). Although ditazole as well as ASA and indomethacin reduced both the slope and the maximal amplitude in comparison to control tracings, none of the differences were statistically significant (Duncan new multiple range lest). In the pre sence of these drugs, aggregation was invariably reversed within 10 min. At the concentration used (10 4 m ), both VK 744 and SH 869 almost com pletely inhibited ADP-induced platelet aggregation. Platelet aggregation induced by ADP was also studied in PRP from a normal volunteer given 500 mg ASA the day before blood collection. The results of this experiment are shown in table II and indicate that the in vitro addition of ditazole, ASA or indomethacin did not modify the degree of platelet aggregation; both VK 744 and SH 869 were, in contrast, strongly inhibitory.
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Results
Ditazole and Human Platelets
129
Aggregation by Thrombofax was studied using final concentrations (f.c.) of ditazole, ASA and indomethacin ranging between 10 4 and 2.10-7 m . Indomethacin completely blocked aggregation at all the concentrations tested, whereas the inhibitory activity of both ditazole and ASA was less constant (table III, IV). As shown in table V ditazole strongly inhibited the slope and the maximal amplitude of aggregation induced by collagen; however, the latent period was not affected. The inhibitory effect of ditazole on collagen-induced platelet aggregation was not influenced in vitro by ASA. The two drugs were added to platelets alone or together (in various combinations, ranging from 10 ‘ to 10 7 m ) : the inhibitory effect of combined drugs was never different from that of the single compound used at the same final concentration. Particularly, when a threshold concentration of ditazole (inhibiting only minimally platelet aggregation) was tested together with various concentra tions of ASA, the inhibitory effect of the latter was unchanged. Effect o f Ditazole on the Aggregation o f ‘ Washed’ Platelets PRP was preincubated with ditazole at concentrations totally inhibiting collagen and/or Thrombofax-induced platelet aggregation. Thereafter PRP was subjected to gel filtration on a Sepharose 2-B column; gel-filtered platelets (GFP) were then challenged with Thrombofax and aggregated normally. When ditazole was added to GFP, Thrombofax-induced aggre gation was again inhibited. GFP were poorly aggregated by collagen, unless one quarter of platelet-poor plasma (PPP) was added to GFP. When normal PPP was added to GFP (the latter obtained from PRP preincubated with ditazole) collagen-induced aggregation was normal. In contrast, when PPP was preincubated for 3 min with ditazole (1.25 x 104 m , f.c.) and subsequently added to GFP, aggregation was inhibited. Aggregation was also inhibited when ditazole was added to GFP before PPP.
Effect o f Ditazole on Thrombelastogram o f PRP Clots Ditazole reduced the maximal amplitude of TEG tracing in a manner which was statistically significant (p < 0 .0 1 ) (table VII). In addition, in 7 out of the 14 samples tested, the maximal amplitude was followed either by
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Effect o f Ditazole on RCR Test Neither ASA, nor indomethacin significantly modified the degree of ADP-stimulated RCR; the latter was, in contrast, significantly reduced by ditazole and almost completely inhibited by VK 744 (table VI).
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Table I. Effect of ditazole, ASA and indomethacin (10* m , f.c.) on ADP (5.10 ®m f.c.) induced human platelet aggregation; means ± SE of 8 experiments
Slope, mm/min Maximal amplitude, mm
Control
Ditazole
ASA
IndoVK 744 SH 869 methacin
149 ± 1 0 86 ± 8
131 ± 1 0 73 ± 8
146 ± 12 75 ± 7
127 ± 1 4 20 ± 1 0 72 ± 8 1 1 ± 7
15 ± 11 10± 9
Table II. Effect of ditazole, ASA, indomethacin, VK 744 or SH 869 (101 m f.c.) on ADPinduced platelet aggregation in plasma from a subject given 500 mg ASA orally 24 h before blood collection; means of two determinations
Slope, mm/min Maximal amplitude, mm
Control
Ditazole
ASA
Indo VK 744 SH 869 methacin
154 65
150 72
154 59
130 55
10 6
10 6
Table III. Comparison of the inhibitory effect of ditazole and ASA on platelet aggregation induced by Thrombofax: altogether, 12 pairs of samples obtained from 5 normal volunteers were compared Pair
Concentration M
Inhibition of aggregation ditazole
ASA
± ± ± ± +
+ ± + + +
1
10*
2
io -4
3 4 5 6 7 8 9
10“4 io -4
106
-
-
10
10-‘
11
io -6
12
10-*
+ ± + + +
+ + +
io -5 10 s io -5 10 s
± ± ± + +
-
±± + +
+ ± + + +
-
+ +
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+ + = Complete inhibition; ± = partial inhibition; - = no inhibition.
Ditazole and Human Platelets
131
Table IV. Minimal concentrations of ditazole required in vitro to inhibit completely Thrombofax-induced platelet aggregation in 5 volunteers Volunteer No.
Concentration,
1
1.3 X IO'5
2
2.5 X 10'6
3
1.0 X 10-*
4
2.0 X io - 7
5
2.0 x 1 0 6
m
Table V. Effect of ditazole on collagen-induced human platelet aggregation in vitro1 Drug final concentration M
8.2 8.2 1.6 8.2
x x x x
10-7 IO6 10 5 106
Latent period ditazole
control
48 41 39 46
50 40 37 43
±4 ± 3 ± 2 ± 8
± ± ± ±
3 3 2 7
Slope % inhibition
Maximal amplitude % inhibition
13.9 27.4 38.1 71.6
2.5 19.2 34.4 60.0
± 5.9 ± 7.9 ± 13.9 ± 5 .1
± ± ± ±
1.7 6.4 12.5 12.7
1 The drug was preincubated with platelets for 3 min at 37 °C before adding collagen. The results of latent period are expressed as absolute values in seconds, whereas the results of slope and maximal amplitude are expressed as percent inhibition as compared to control experiments performed in parallel.
Table VI. Effect of ditazole and other compounds (2 x 101 m f.c.) on ADP reptilase clot retraction; mean ± SE of 10 experiments % clot Retraction Control Ditazole ASA Indomethacin VK 744
70.6 ± 3.8 50.2 ± 4 .6 * 70.2 ± 2.8 72.8 ± 4.0 < 10
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* p < 0.05 (Duncan’s test).
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Fig.l. Thrombelastographic tracings of human PRP clotted by thrombin. Test system: 0.25 ml PRP were preincubated for 3 min at 37 °C with 2 ¡i\ ethanol or test compound, afterwards 0.10 ml 1.20% CaCl2 was added to each sample. Sample A = control; B = ditazole, 10-4 m ; C = ditazole, 2.8 x 10'4 m ; D = ASA, 2.8 x 10'4 m ; E = SH 869, 2.8 x 10 4 m .
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a rapid, partial convergence of the two lines or by several waves of decreasing amplitude and subsequent slow convergence of the 2 lines (fig. 1). Similar TEG patterns have been previously observed in the presence of prosta glandin Ei and some pyrimido-pyrimidine compounds and have been inter preted as postmaximal relaxation-contraction waves dependent on modifi cations of platelet cyclic AMP [7]. Neither ASA nor indomethacin (at equi molar concentrations) exerted any effect on TEG, a finding already re ported [6]. The effect of ditazole on TEG was different from that of SH 869; the latter indeed not only significantly reduced (p < 0 .0 1 ) the maximal amplitude but also prolonged both the reaction time (r) (p < 0.01) and clot formation time (K) (p < 0.05). These results are analogous to those pre viously reported using other pyrimido-pyrimidine compounds [6].
Ditazole and Human Platelets
133
Table VII. In vitro effects of ditazole, ASA, indomethacin and SH 869 (10'4 m f.c.) on TEG parameters*1
Control Ditazole Control ASA Control Indomethacin Control SH 869
r, min
K, min
ma, mm
7.3 8.2 7.1 8.5 7.0 8.2 6.5 12.3
4.6 ± 5.1 ± 4.6 ± 4.1 ± 4.3 ± 5.3 ± 4.5 ± 8.0 ±
56.7 43.7 56.0 58.8 55.7 56.0 59.0 33.8
± 0.4 ± 0.5 ± 0.6 ± 0.9 ± 0.8 ± 0.9 ± 0.3 ± 0 .7 » *
0.3 0.3 0.3 0.4 0.6 1.0 0.3 1.4*
± 1.1 ± 2 .3 * * ± 1.5 ± 1.8 ± 1.4 ± 1.4 ± 1.1 ± 3 .6 * *
* p < 0 .0 5 ; ** p < 0.01. 1 The number of paired experiments with controls was 14, 6, 4 and 6 respectively for the 4 drugs. Data were analyzed by Student’s test (paired comparisons).
Table VIII. Effect of ditazole on l4C-5HT release from human platelets induced by Thrombofax1 Drug
Final concentration,
_
_
Ditazole Ditazole Ditazole ASA Indomethacin VK 744
10-« 2.10-' io -4 io -4 io -4 io -4
m
% release 43.3 ± 16.8 ± 12.1 ±
Ditazole and Platelets I. Effect of Ditazole on Human Platelet Function in vitro
G.
de
G aetano, M .C .T onolli, M .P. Bertoni and M .C . R oncaglioni1 Laboratory for Haemostasis and Thrombosis Research, Istituto di Ricerche Farmacologiche ‘Mario Negri’, Milan
Key Words. Platelet aggregation • Ditazole • Clot retraction • Thrombelastography • Gel-filtered platelets • Serotonin Abstract. Ditazole (4,5-diphenyl-2-bis-(2-hydroxyethyl)-aminoxazol) has been shown to be a strong in vitro inhibitor of human platelet aggregation brought about by release reaction inducers; in contrast, it did not significantly affect primary ADP-induced aggre gation. Ditazole strongly inhibited the release of platelet-bound 14C-serotonin under the influence of Thrombofax, whereas it did not interfere with the transport and storage of serotonin in nonstimulated platelets. The effect of ditazole was not potentiated by acetylsalicylic acid. Ditazole also inhibited ADP-reptilase clot retraction and modified thrombininduced clot formation. The inhibition of platelet aggregation exerted by ditazole in plasma could be removed following gel filtration of platelets on Sepharose 2-B gel. This would indicate that ditazole does not act on platelets by a ‘hit and run’ mechanism.
Introduction Ditazole (4,5-diphenyl-2-bis-(2-hydroxyethyl)-aminoxazol) is a new drug shown by C a p r in o et al. [3] to inhibit human and rabbit platelet aggregation and to prolong bleeding time in mice. Ditazole is also endowed with mod erate anti-inflammatory, analgesic and antipyretic properties [2], The inhibitory effect of this compound on some platelet functions in vitro has been confirmed by other investigators, including our group [4, 12]. The
Received: August 6, 1976; accepted by editor J.V ermylen: November 25, 1976.
Downloaded by: Lund University Libraries 130.235.66.10 - 1/1/2019 5:56:58 PM
1 Mrs. A my C rook, Miss A nna M ancini and Mrs. G raziella Scalvini kindly helped us in preparing this manuscript.
128
de
G aetano/T onolli/B ertoni/R oncaguoni
present study reports on further investigations performed to better charac terize the in vitro effect of ditazole on human platelets.
Materials and Methods Ditazole (Serono, Rome, Italy) and other compounds tested in parallel (acetylsalicylic acid, ASA: Bayer, Milan, Italy; indomethacin, Merck, Sharp & Dohme, Rome, Italy; pyrimido-pyrimidine compounds VK 744 and SH 869, Karl Thomae, Biberach an der Riss, FRG) were dissolved in 97% ethanol at a concentration of 5 x 10 2 m ; subsequent dilutions were made with ethanol and drugs used in 1 to 2-/d aliquots. Platelet aggregation was studied by the photometric technique of Born and C ross [1] using a MK IV Born-Michal aggregometer (Pharmacological Research, Cambridge, UK) connected to a two-channel potentiometer (Venture Servoscribe 2, Smiths Industries, Ltd., London, UK), as previously described [9], Separation of platelets from plasma pro teins was obtained by gel filtration, as previously described [12]. Adenosine-5'-diphosphate (ADP: Sigma, Mascia Brunelli, Milan, Italy), collagen (Hormon Chemie, Munich, FRG) and Thrombofax (Ortho Diagnostic, Cilag-Chemie, Milan, Italy) [10] were used as aggregation stimuli. Reptilase clot retraction test (RCR) was performed as previously described [5]. Physical properties of platelet-rich plasma (PRP) clots were studied by thrombelastograplty using a Clotscanner apparatus (Elvi-Logos, Milan, Italy) [6]. Uptake o fu C-serotonin and its release from platelets under the influence of Thrombofax were investigated as previously described [11],
Aggregation by ADP was studied in 8 series of samples (table I). Although ditazole as well as ASA and indomethacin reduced both the slope and the maximal amplitude in comparison to control tracings, none of the differences were statistically significant (Duncan new multiple range lest). In the pre sence of these drugs, aggregation was invariably reversed within 10 min. At the concentration used (10 4 m ), both VK 744 and SH 869 almost com pletely inhibited ADP-induced platelet aggregation. Platelet aggregation induced by ADP was also studied in PRP from a normal volunteer given 500 mg ASA the day before blood collection. The results of this experiment are shown in table II and indicate that the in vitro addition of ditazole, ASA or indomethacin did not modify the degree of platelet aggregation; both VK 744 and SH 869 were, in contrast, strongly inhibitory.
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Results
Ditazole and Human Platelets
129
Aggregation by Thrombofax was studied using final concentrations (f.c.) of ditazole, ASA and indomethacin ranging between 10 4 and 2.10-7 m . Indomethacin completely blocked aggregation at all the concentrations tested, whereas the inhibitory activity of both ditazole and ASA was less constant (table III, IV). As shown in table V ditazole strongly inhibited the slope and the maximal amplitude of aggregation induced by collagen; however, the latent period was not affected. The inhibitory effect of ditazole on collagen-induced platelet aggregation was not influenced in vitro by ASA. The two drugs were added to platelets alone or together (in various combinations, ranging from 10 ‘ to 10 7 m ) : the inhibitory effect of combined drugs was never different from that of the single compound used at the same final concentration. Particularly, when a threshold concentration of ditazole (inhibiting only minimally platelet aggregation) was tested together with various concentra tions of ASA, the inhibitory effect of the latter was unchanged. Effect o f Ditazole on the Aggregation o f ‘ Washed’ Platelets PRP was preincubated with ditazole at concentrations totally inhibiting collagen and/or Thrombofax-induced platelet aggregation. Thereafter PRP was subjected to gel filtration on a Sepharose 2-B column; gel-filtered platelets (GFP) were then challenged with Thrombofax and aggregated normally. When ditazole was added to GFP, Thrombofax-induced aggre gation was again inhibited. GFP were poorly aggregated by collagen, unless one quarter of platelet-poor plasma (PPP) was added to GFP. When normal PPP was added to GFP (the latter obtained from PRP preincubated with ditazole) collagen-induced aggregation was normal. In contrast, when PPP was preincubated for 3 min with ditazole (1.25 x 104 m , f.c.) and subsequently added to GFP, aggregation was inhibited. Aggregation was also inhibited when ditazole was added to GFP before PPP.
Effect o f Ditazole on Thrombelastogram o f PRP Clots Ditazole reduced the maximal amplitude of TEG tracing in a manner which was statistically significant (p < 0 .0 1 ) (table VII). In addition, in 7 out of the 14 samples tested, the maximal amplitude was followed either by
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Effect o f Ditazole on RCR Test Neither ASA, nor indomethacin significantly modified the degree of ADP-stimulated RCR; the latter was, in contrast, significantly reduced by ditazole and almost completely inhibited by VK 744 (table VI).
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Table I. Effect of ditazole, ASA and indomethacin (10* m , f.c.) on ADP (5.10 ®m f.c.) induced human platelet aggregation; means ± SE of 8 experiments
Slope, mm/min Maximal amplitude, mm
Control
Ditazole
ASA
IndoVK 744 SH 869 methacin
149 ± 1 0 86 ± 8
131 ± 1 0 73 ± 8
146 ± 12 75 ± 7
127 ± 1 4 20 ± 1 0 72 ± 8 1 1 ± 7
15 ± 11 10± 9
Table II. Effect of ditazole, ASA, indomethacin, VK 744 or SH 869 (101 m f.c.) on ADPinduced platelet aggregation in plasma from a subject given 500 mg ASA orally 24 h before blood collection; means of two determinations
Slope, mm/min Maximal amplitude, mm
Control
Ditazole
ASA
Indo VK 744 SH 869 methacin
154 65
150 72
154 59
130 55
10 6
10 6
Table III. Comparison of the inhibitory effect of ditazole and ASA on platelet aggregation induced by Thrombofax: altogether, 12 pairs of samples obtained from 5 normal volunteers were compared Pair
Concentration M
Inhibition of aggregation ditazole
ASA
± ± ± ± +
+ ± + + +
1
10*
2
io -4
3 4 5 6 7 8 9
10“4 io -4
106
-
-
10
10-‘
11
io -6
12
10-*
+ ± + + +
+ + +
io -5 10 s io -5 10 s
± ± ± + +
-
±± + +
+ ± + + +
-
+ +
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+ + = Complete inhibition; ± = partial inhibition; - = no inhibition.
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Table IV. Minimal concentrations of ditazole required in vitro to inhibit completely Thrombofax-induced platelet aggregation in 5 volunteers Volunteer No.
Concentration,
1
1.3 X IO'5
2
2.5 X 10'6
3
1.0 X 10-*
4
2.0 X io - 7
5
2.0 x 1 0 6
m
Table V. Effect of ditazole on collagen-induced human platelet aggregation in vitro1 Drug final concentration M
8.2 8.2 1.6 8.2
x x x x
10-7 IO6 10 5 106
Latent period ditazole
control
48 41 39 46
50 40 37 43
±4 ± 3 ± 2 ± 8
± ± ± ±
3 3 2 7
Slope % inhibition
Maximal amplitude % inhibition
13.9 27.4 38.1 71.6
2.5 19.2 34.4 60.0
± 5.9 ± 7.9 ± 13.9 ± 5 .1
± ± ± ±
1.7 6.4 12.5 12.7
1 The drug was preincubated with platelets for 3 min at 37 °C before adding collagen. The results of latent period are expressed as absolute values in seconds, whereas the results of slope and maximal amplitude are expressed as percent inhibition as compared to control experiments performed in parallel.
Table VI. Effect of ditazole and other compounds (2 x 101 m f.c.) on ADP reptilase clot retraction; mean ± SE of 10 experiments % clot Retraction Control Ditazole ASA Indomethacin VK 744
70.6 ± 3.8 50.2 ± 4 .6 * 70.2 ± 2.8 72.8 ± 4.0 < 10
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* p < 0.05 (Duncan’s test).
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Fig.l. Thrombelastographic tracings of human PRP clotted by thrombin. Test system: 0.25 ml PRP were preincubated for 3 min at 37 °C with 2 ¡i\ ethanol or test compound, afterwards 0.10 ml 1.20% CaCl2 was added to each sample. Sample A = control; B = ditazole, 10-4 m ; C = ditazole, 2.8 x 10'4 m ; D = ASA, 2.8 x 10'4 m ; E = SH 869, 2.8 x 10 4 m .
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a rapid, partial convergence of the two lines or by several waves of decreasing amplitude and subsequent slow convergence of the 2 lines (fig. 1). Similar TEG patterns have been previously observed in the presence of prosta glandin Ei and some pyrimido-pyrimidine compounds and have been inter preted as postmaximal relaxation-contraction waves dependent on modifi cations of platelet cyclic AMP [7]. Neither ASA nor indomethacin (at equi molar concentrations) exerted any effect on TEG, a finding already re ported [6]. The effect of ditazole on TEG was different from that of SH 869; the latter indeed not only significantly reduced (p < 0 .0 1 ) the maximal amplitude but also prolonged both the reaction time (r) (p < 0.01) and clot formation time (K) (p < 0.05). These results are analogous to those pre viously reported using other pyrimido-pyrimidine compounds [6].
Ditazole and Human Platelets
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Table VII. In vitro effects of ditazole, ASA, indomethacin and SH 869 (10'4 m f.c.) on TEG parameters*1
Control Ditazole Control ASA Control Indomethacin Control SH 869
r, min
K, min
ma, mm
7.3 8.2 7.1 8.5 7.0 8.2 6.5 12.3
4.6 ± 5.1 ± 4.6 ± 4.1 ± 4.3 ± 5.3 ± 4.5 ± 8.0 ±
56.7 43.7 56.0 58.8 55.7 56.0 59.0 33.8
± 0.4 ± 0.5 ± 0.6 ± 0.9 ± 0.8 ± 0.9 ± 0.3 ± 0 .7 » *
0.3 0.3 0.3 0.4 0.6 1.0 0.3 1.4*
± 1.1 ± 2 .3 * * ± 1.5 ± 1.8 ± 1.4 ± 1.4 ± 1.1 ± 3 .6 * *
* p < 0 .0 5 ; ** p < 0.01. 1 The number of paired experiments with controls was 14, 6, 4 and 6 respectively for the 4 drugs. Data were analyzed by Student’s test (paired comparisons).
Table VIII. Effect of ditazole on l4C-5HT release from human platelets induced by Thrombofax1 Drug
Final concentration,
_
_
Ditazole Ditazole Ditazole ASA Indomethacin VK 744
10-« 2.10-' io -4 io -4 io -4 io -4
m
% release 43.3 ± 16.8 ± 12.1 ±
