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Effect of gliclazide on platelet-activating factor-induced platelet aggregation in patients with non-insulin-dependent diabetes mellitus
Effect of Gliclazide on Platelet-Activating Factor-Induced Platelet Aggregation in Patients With Non-Insulin-Dependent Diabetes Mellitus C. Phenekos, A. Siafaka-Kapadai,
P
LATELET-ACTIVATING factor (PAF) is a phospholipid secreted by platelets, leukocytes, and endothelial cells, and possesses a wide spectrum of biological activities. It is a potent mediator of inflammation and allergic reactions, and it induces bronchoconstriction and bronchial hyperresponsiveness, lowers blood pressure, and impairs cardiac contractility and coronary blood flow. It acts on the kidney, reducing the renal blood flow and glomerular filtration rate (GFR), and it is also involved in modulating the immune response by affecting lymphocyte function.‘-3 Some of its best-documented actions are related to the blood cells and vessels. It increases vascular permeability. and, in very low concentrations, induces platelet aggregation and secretion, hence, its name: platelet-activating factor.4 The effect of PAF on platelet aggregation and vascular permeability, both of which predispose to the development of diabetic complications, led us to evaluate the effect of gliclazide* on PAF-induced platelet asegation. We recognized both the well-documented increased sensitivity of platelets from diabetic subjects to aggregation’-’ and the reported antiaggregating properties of the sulphonylurea derivative, gliclazide.‘,” MATERIALS For
AND METHODS
the platelet aggregation studies, we used platelet-rich plasma
(PRP) from seven healthy and eight non-insulin-dependent diabetes mellitus (NIDDM) patients, men aged 45 to 65 years. The patients were treated by diet only and were reasonably controlled. Their glycosylated hemoglobin ranged between 7% and 10.3% (normal, 4% to 8%). None ofthem presented with clinically evident complications oftheir disease, and all had normal hepatic and renal function. None were on any medication known to affect platelet function. Blood was obtained from fasting subjects by venipuncture in the morning, and was collected in ACD (citric acid, trisodium citrate, and dextrose)-containing tubes. It was immediately centrifuged for 10 minutes at 100 X g, and the supernatant PRP was transferred, whereas the remaining blood was centrifuged again for 20 minutes at 1000 X g to obtain platelet poor plasma. This was subsequently used to dilute the PRP to achieve a final concentration of 300,000 platelets/mm’. The adjusted PRP was kept in plastic tubes covered with parafilm at room temperature, and the aggregation studies were performed within 50 minutes of blood sampling. Platelet aggregation was measured using a properly calibrated aggregometer (Chronolog, Haverstown, PA). Five hundred microliters of PRP was added in the aggregometer cuvette under constant stirring
M. Trapali, E. Botitsi, and M. Mavris (1.200 rpm) at 37°C. PAF was added at a final concentration of 2.5 X 10m7mol/L. This concentration was known to induce submaximal reversible platelet aggregation in platelets from healthy subjects. The effect of gliclazide on PAF-induced platelet aggregation was studied by preincubating the PRP for I minute in the aggregometer cuvette with different concentrations of the drug. ranging from IO-’ to IO-” mol/L, achieved by diluting it with DMSO. The PAF-induced aggregation in NIDDM and healthy subjects was assessed by estimating the amplitude of the primary aggregation wave. The gliclazide-effected inhibition of PAF-induced platelet aggregation was calculated by estimating the percent change in the aggregation wave amplitude achieved following preincubation of the plasma sample with the drug. Statistical analysis of the results was performed by using one-way ANOVA. For the calculation of differences between groups, the Wilcoxon signed-rank test was used. The gliclazide concentration inducing 50% inhibition ofaggregation was calculated by applying simple regression analysis. All the results are expressed as mean values + SEM percent inhibition of aggregation relative to aggregation induced by PAF. RESULTS
The inhibition of PAF-induced platelet aggregation effected by four different concentrations of gliclazide ( 10m3to 10e5) is shown in Fig 1. This inhibition is statistically significant (P < .OOOl), and it is dose-related, with an ECSoof 6 X 10m4 mol/L of gliclazide for the whole group of controls. The effect of six different concentrations of gliclazide ( 10m3 to 10e6 mol/L) on PAF-induced platelet aggregation in the group of diabetic individuals is shown in Fig 2. The inhibition is also significant, with P < .OOOl and an ECSo of 8 X 10m4 for the whole group. No statistically significant difference was detected in the gliclazide-induced inhibition ofaggregation between patients and controls for the same concentrations of gliclazide tested. Finally, by using regression analysis, the dose-dependent effect of gliclazide on PAF-induced platelet aggregation in X Inhlbltlon Of
aggregatton 60
1
* Developed and produced in France by LES LABORATOIRES SERVIER under the registered trademark DIAMICRON”. From the Department of Endocrinology, Red Cross Hospital, Athens, Greece; and the Department of Chemistry, University qfilthens, Athens, Greece. Address reprint requests to C. Phenekos. MD, Hellenic Red Cross Hospital 1, Erythrou Statou, I IS 26 Athens, Greece. Copyright 0 I992 by W.B. Saunders Company 0026-0495/92/4105-1008$0~.00/0
30
Fig 1.
Inhibition of PAF-induced aggregation in the control group.
Met&d&m,
Vol41, No 5,
Suppl 7 (May), 1992: pp 30-32
GLICLAZIDE EFFECT ON PAF-INDUCED PLATELET AGGREGATION
the group of diabetics is demonstrated. P = .006) (Fig 3).
(R’ = 8.71; r = .93;
DISCUSSION
The hemobiological abnormalities caused by the diabetic syndrome and their relationship to the development of chronic complications has been the object of extensive research recently.“.” The abnormal platelet function in NIDDM patients is well documented and is functionally expressed by increased adhesiveness and aggregation in vitro and in vivo.13 PAF not only induces platelet aggregation, but it is also synthesized and secreted by the endothelial cells of the blood vessels and the platelets; therefore, its role in the pathogenesis of microangiopathy and macroangiopathy is of great interest. Extensive studies have shown that PAF induces platelet aggregation by binding to specific receptors on the platelet
1
InhIbItion “,
Gllclazlde
Fig 2.
Inhibition
of PAF-induced
aggregation
in the diabetic
group.
Fig 3. Dose-dependent effect of gliclazide platelet aggregation in the diabetic group.
on PAF-induced
membrane that probably belong to the guanosine triphosphate (GTP)-coupled group.14 It induces phosphatidyl inositol biphosphate hydrolysis by phospholipase-C activation.‘5 inhibition of adenyl cyclase, and stimulation of arachidonate production through activation of phospholipase A2.16 All of these metabolic changes undoubtedly contribute to platelet aggregation and secretion. The inhibitory effect of gliclazide on PAF-induced platelet aggregation, as was shown in this study, is probably due to the reported properties of this drug on platelet metabolism. It is known that gliclazide stimulates adenyl cyclase. It also inhibits phospholipase A2 activity, thereby reducing the production of arachidonates.‘7.‘8 Finally, it was recently shown in human studies that it reduces the synthesis of thromboxane A2 and lipid peroxides.” There is no doubt that further research is needed to establish the inhibitory effects of gliclazide on PAF-induced platelet aggregation in vitro and in vivo, and to define the intracellular events related to signal transduction that lead to platelet aggregation, as well as to establish the mechanisms by which this result of platelet activation may be attenuated in conditions in which it is abnormal. such as in the diabetic syndrome.
REFERENCES
1. Hanahan DJ. Kumar R: Platelet activating factor. Chemical and biochemical characteristics. Lipid Res 26: l-28, 1987 2. Kroegel C: The potential pathophysiological role of platelet activating factor in human diseases. Klin Wochenschr 66:373-378, 1988 3. Lazanas M, Demopoulos CA, Toumis S, et al: PAF of biological fluids in disease. IV. Levels in blood in allergic reactions induced by drugs. Arch Dermatol Res 280: 124- 126, 1988 4. Braquet P, Touqui L, Shen TY. et al: Perspectives in platelet activating factor research. Pharmacol Rev 39:97- 145, 1987 5. Bern MM: Platelet function in diabetes mellitus. Diabetes 27: 342-352, 1978 6. Dallinger KJC. Jennings PE, Toop MJ, et al: Platelet aggregation and coagulation factors in insulin dependent diabetics with and without microangiopathy. Diabetic Med 4:44-48, 1978 7. Kobbah AM, Ewald U, Tuvemo T: Platelet aggregability during the first two years of type 1 (insulin dependent) diabetes mellitus in children. Diabetologia 32:729-735, 1989 8. Mustard JF, Pacham MA: Platelets and diabetes mellitus. Lancet 297: I345- 1347, 1977
9. Shim& M. Tsuboi T, Fujitani B. et al: Pharmacological studies on gliclazide (2). Effect ofgliclazide on platelet aggregation, adhesion and blood coagulation. Pharmacometrics 12:295-299. 1976 10. Ponari 0, Cirardi E, Megha S. et al: Anti-platelet effects of long term treatment with ghclazide in diabetic patients. Thromb Res 16:191-196. 1979
I I. Davi G, Catalan0 1. Averna M. et al: Thromboxane biosynthesis and platelet function in type II diabetes mellitus. N Engl J Med 322: 1769- 1774, 1990 12. Livingstone C. McLellan AR. McGrecor MA. et al: Altered G-protein expression of adenylate cyclase activity in platelets of noninsulin-dependent diabetic (NIDDM) male subjects. Biochim Biophys Acta 1096:127-133, 1991 13. Chirkov YY, Tyshchuk IA. Severina IS: Guanylate cyclase in human platelets with different aggregability. Experientia 46:687-699. 1990 14. Hwang SB. Lam MH, Pong SS: Ionic and GTP regulation of binding of platelet activating factor to receptors and platelet activating
32
PHENEKOS ET AL
factor induced activation of GTPase in rabbit platelet membranes. J Biol Chem 261:532-538, 1986
0,~
~1
nr.
r
~1~l’
c.n
no
.I_
-_’
-_-
of phospholipase C in platelets by platelet activating factor and thrombin causes hydrolysis of a common pool of phosphatidylinositol4,5 biphosphate. Biochim Biophys Acta 929: 134-141, 1987 I~
I
16. Brass LF, Woolkalis MJ, Manning DR: Interactions in platelets between G proteins and the agonists that stimulate phospholipase C and inhibit adenyl cyclase. J Biol Chem 263:5348-5353, 1988
17. Lagard M. Dechavanne M, Thouverez JP, et al: Effects of gliclazide, a new antidiabetic agent, on the platelet release reaction. Role of adenyl cyclase. Thromb Res 6:345-355. 1975 18. Tsuboi T, Fujitani B. Maeda J. et al: Effect of ghclazide on prostaglandin and thromboxane synthesis in guinea pig platelets. Thromb Res 21:103-l IO. 1981 19. Florkowski CM, Richardson MR, Guen LC, et al: Effect of gliclazide on thromboxane B2, parameters of haemostasis. fluorescent IgG and lipid peroxides in non-insulin-dependent diabetes mellitus. Diabetes Res 9:87-90. 1988
P
LATELET-ACTIVATING factor (PAF) is a phospholipid secreted by platelets, leukocytes, and endothelial cells, and possesses a wide spectrum of biological activities. It is a potent mediator of inflammation and allergic reactions, and it induces bronchoconstriction and bronchial hyperresponsiveness, lowers blood pressure, and impairs cardiac contractility and coronary blood flow. It acts on the kidney, reducing the renal blood flow and glomerular filtration rate (GFR), and it is also involved in modulating the immune response by affecting lymphocyte function.‘-3 Some of its best-documented actions are related to the blood cells and vessels. It increases vascular permeability. and, in very low concentrations, induces platelet aggregation and secretion, hence, its name: platelet-activating factor.4 The effect of PAF on platelet aggregation and vascular permeability, both of which predispose to the development of diabetic complications, led us to evaluate the effect of gliclazide* on PAF-induced platelet asegation. We recognized both the well-documented increased sensitivity of platelets from diabetic subjects to aggregation’-’ and the reported antiaggregating properties of the sulphonylurea derivative, gliclazide.‘,” MATERIALS For
AND METHODS
the platelet aggregation studies, we used platelet-rich plasma
(PRP) from seven healthy and eight non-insulin-dependent diabetes mellitus (NIDDM) patients, men aged 45 to 65 years. The patients were treated by diet only and were reasonably controlled. Their glycosylated hemoglobin ranged between 7% and 10.3% (normal, 4% to 8%). None ofthem presented with clinically evident complications oftheir disease, and all had normal hepatic and renal function. None were on any medication known to affect platelet function. Blood was obtained from fasting subjects by venipuncture in the morning, and was collected in ACD (citric acid, trisodium citrate, and dextrose)-containing tubes. It was immediately centrifuged for 10 minutes at 100 X g, and the supernatant PRP was transferred, whereas the remaining blood was centrifuged again for 20 minutes at 1000 X g to obtain platelet poor plasma. This was subsequently used to dilute the PRP to achieve a final concentration of 300,000 platelets/mm’. The adjusted PRP was kept in plastic tubes covered with parafilm at room temperature, and the aggregation studies were performed within 50 minutes of blood sampling. Platelet aggregation was measured using a properly calibrated aggregometer (Chronolog, Haverstown, PA). Five hundred microliters of PRP was added in the aggregometer cuvette under constant stirring
M. Trapali, E. Botitsi, and M. Mavris (1.200 rpm) at 37°C. PAF was added at a final concentration of 2.5 X 10m7mol/L. This concentration was known to induce submaximal reversible platelet aggregation in platelets from healthy subjects. The effect of gliclazide on PAF-induced platelet aggregation was studied by preincubating the PRP for I minute in the aggregometer cuvette with different concentrations of the drug. ranging from IO-’ to IO-” mol/L, achieved by diluting it with DMSO. The PAF-induced aggregation in NIDDM and healthy subjects was assessed by estimating the amplitude of the primary aggregation wave. The gliclazide-effected inhibition of PAF-induced platelet aggregation was calculated by estimating the percent change in the aggregation wave amplitude achieved following preincubation of the plasma sample with the drug. Statistical analysis of the results was performed by using one-way ANOVA. For the calculation of differences between groups, the Wilcoxon signed-rank test was used. The gliclazide concentration inducing 50% inhibition ofaggregation was calculated by applying simple regression analysis. All the results are expressed as mean values + SEM percent inhibition of aggregation relative to aggregation induced by PAF. RESULTS
The inhibition of PAF-induced platelet aggregation effected by four different concentrations of gliclazide ( 10m3to 10e5) is shown in Fig 1. This inhibition is statistically significant (P < .OOOl), and it is dose-related, with an ECSoof 6 X 10m4 mol/L of gliclazide for the whole group of controls. The effect of six different concentrations of gliclazide ( 10m3 to 10e6 mol/L) on PAF-induced platelet aggregation in the group of diabetic individuals is shown in Fig 2. The inhibition is also significant, with P < .OOOl and an ECSo of 8 X 10m4 for the whole group. No statistically significant difference was detected in the gliclazide-induced inhibition ofaggregation between patients and controls for the same concentrations of gliclazide tested. Finally, by using regression analysis, the dose-dependent effect of gliclazide on PAF-induced platelet aggregation in X Inhlbltlon Of
aggregatton 60
1
* Developed and produced in France by LES LABORATOIRES SERVIER under the registered trademark DIAMICRON”. From the Department of Endocrinology, Red Cross Hospital, Athens, Greece; and the Department of Chemistry, University qfilthens, Athens, Greece. Address reprint requests to C. Phenekos. MD, Hellenic Red Cross Hospital 1, Erythrou Statou, I IS 26 Athens, Greece. Copyright 0 I992 by W.B. Saunders Company 0026-0495/92/4105-1008$0~.00/0
30
Fig 1.
Inhibition of PAF-induced aggregation in the control group.
Met&d&m,
Vol41, No 5,
Suppl 7 (May), 1992: pp 30-32
GLICLAZIDE EFFECT ON PAF-INDUCED PLATELET AGGREGATION
the group of diabetics is demonstrated. P = .006) (Fig 3).
(R’ = 8.71; r = .93;
DISCUSSION
The hemobiological abnormalities caused by the diabetic syndrome and their relationship to the development of chronic complications has been the object of extensive research recently.“.” The abnormal platelet function in NIDDM patients is well documented and is functionally expressed by increased adhesiveness and aggregation in vitro and in vivo.13 PAF not only induces platelet aggregation, but it is also synthesized and secreted by the endothelial cells of the blood vessels and the platelets; therefore, its role in the pathogenesis of microangiopathy and macroangiopathy is of great interest. Extensive studies have shown that PAF induces platelet aggregation by binding to specific receptors on the platelet
1
InhIbItion “,
Gllclazlde
Fig 2.
Inhibition
of PAF-induced
aggregation
in the diabetic
group.
Fig 3. Dose-dependent effect of gliclazide platelet aggregation in the diabetic group.
on PAF-induced
membrane that probably belong to the guanosine triphosphate (GTP)-coupled group.14 It induces phosphatidyl inositol biphosphate hydrolysis by phospholipase-C activation.‘5 inhibition of adenyl cyclase, and stimulation of arachidonate production through activation of phospholipase A2.16 All of these metabolic changes undoubtedly contribute to platelet aggregation and secretion. The inhibitory effect of gliclazide on PAF-induced platelet aggregation, as was shown in this study, is probably due to the reported properties of this drug on platelet metabolism. It is known that gliclazide stimulates adenyl cyclase. It also inhibits phospholipase A2 activity, thereby reducing the production of arachidonates.‘7.‘8 Finally, it was recently shown in human studies that it reduces the synthesis of thromboxane A2 and lipid peroxides.” There is no doubt that further research is needed to establish the inhibitory effects of gliclazide on PAF-induced platelet aggregation in vitro and in vivo, and to define the intracellular events related to signal transduction that lead to platelet aggregation, as well as to establish the mechanisms by which this result of platelet activation may be attenuated in conditions in which it is abnormal. such as in the diabetic syndrome.
REFERENCES
1. Hanahan DJ. Kumar R: Platelet activating factor. Chemical and biochemical characteristics. Lipid Res 26: l-28, 1987 2. Kroegel C: The potential pathophysiological role of platelet activating factor in human diseases. Klin Wochenschr 66:373-378, 1988 3. Lazanas M, Demopoulos CA, Toumis S, et al: PAF of biological fluids in disease. IV. Levels in blood in allergic reactions induced by drugs. Arch Dermatol Res 280: 124- 126, 1988 4. Braquet P, Touqui L, Shen TY. et al: Perspectives in platelet activating factor research. Pharmacol Rev 39:97- 145, 1987 5. Bern MM: Platelet function in diabetes mellitus. Diabetes 27: 342-352, 1978 6. Dallinger KJC. Jennings PE, Toop MJ, et al: Platelet aggregation and coagulation factors in insulin dependent diabetics with and without microangiopathy. Diabetic Med 4:44-48, 1978 7. Kobbah AM, Ewald U, Tuvemo T: Platelet aggregability during the first two years of type 1 (insulin dependent) diabetes mellitus in children. Diabetologia 32:729-735, 1989 8. Mustard JF, Pacham MA: Platelets and diabetes mellitus. Lancet 297: I345- 1347, 1977
9. Shim& M. Tsuboi T, Fujitani B. et al: Pharmacological studies on gliclazide (2). Effect ofgliclazide on platelet aggregation, adhesion and blood coagulation. Pharmacometrics 12:295-299. 1976 10. Ponari 0, Cirardi E, Megha S. et al: Anti-platelet effects of long term treatment with ghclazide in diabetic patients. Thromb Res 16:191-196. 1979
I I. Davi G, Catalan0 1. Averna M. et al: Thromboxane biosynthesis and platelet function in type II diabetes mellitus. N Engl J Med 322: 1769- 1774, 1990 12. Livingstone C. McLellan AR. McGrecor MA. et al: Altered G-protein expression of adenylate cyclase activity in platelets of noninsulin-dependent diabetic (NIDDM) male subjects. Biochim Biophys Acta 1096:127-133, 1991 13. Chirkov YY, Tyshchuk IA. Severina IS: Guanylate cyclase in human platelets with different aggregability. Experientia 46:687-699. 1990 14. Hwang SB. Lam MH, Pong SS: Ionic and GTP regulation of binding of platelet activating factor to receptors and platelet activating
32
PHENEKOS ET AL
factor induced activation of GTPase in rabbit platelet membranes. J Biol Chem 261:532-538, 1986
0,~
~1
nr.
r
~1~l’
c.n
no
.I_
-_’
-_-
of phospholipase C in platelets by platelet activating factor and thrombin causes hydrolysis of a common pool of phosphatidylinositol4,5 biphosphate. Biochim Biophys Acta 929: 134-141, 1987 I~
I
16. Brass LF, Woolkalis MJ, Manning DR: Interactions in platelets between G proteins and the agonists that stimulate phospholipase C and inhibit adenyl cyclase. J Biol Chem 263:5348-5353, 1988
17. Lagard M. Dechavanne M, Thouverez JP, et al: Effects of gliclazide, a new antidiabetic agent, on the platelet release reaction. Role of adenyl cyclase. Thromb Res 6:345-355. 1975 18. Tsuboi T, Fujitani B. Maeda J. et al: Effect of ghclazide on prostaglandin and thromboxane synthesis in guinea pig platelets. Thromb Res 21:103-l IO. 1981 19. Florkowski CM, Richardson MR, Guen LC, et al: Effect of gliclazide on thromboxane B2, parameters of haemostasis. fluorescent IgG and lipid peroxides in non-insulin-dependent diabetes mellitus. Diabetes Res 9:87-90. 1988