THROMBOSIS RESEARCH 59; 489-495,1990 0049-3848190 $3.00 + .OOPrinted in the USA. Copyright (c) 1990 Pergamon Press pk. All rights reserved.
J. May, W. Loesche*and S. Heptinstall Department of Medicine, Ugiversity Hospital, Queen's Medical Centre, NottinghamNG7 2UEL U.K.,and Instituteof PathologicalBiochemistry,Medical Academy, DDR-5010 Erfurt,GDR
(Received 13.11.1989; accepted in revised form 16.51990 by Editor S. Moncada)
ABsTRAcr
Glucose (lo-50mM) added to samples of titratedwhole blood increased the spontaneous platelet aggregation (SPA) that was observed upon stirringthe samples,in a dose-dependentbut time-independentmanner. EUIA preventedand also reversedthe SPA that occurred in the presence of glucose. Galactose, fructose and 2-deoxyglucose,but not sorbitol and sucrose, had effects on SPA similar to those of glucose. The effect of glucose on SPA in whole blood could not be specifically inhibited by tetramethylene glutaric acid (an inhibitor of aldose reductase).SPA was not affectedby pentoxifylline,but was markedly reduced by chlorpromazine. We previously found that glucose, galactose, fructose and 2ileoxyglucose, but not sorbitoland sucrose, render red blood cells more fragile, and the results obtained in the present study provide further evidence that glucose as well as other reducing sugars potentiate SPA in whole blood by liberating ADP or other aggregatingmaterial from red blood cells.
INrRDJXJc!rIQN Diabetesmellitus is frequentlycomplicatedby thromboembolicevents and it is believed that blood platelets contributeto diabetic vascular disease [l-31. We have previously shown that glucose added to samples of wholeblood (WB) potentiates spontaneous platelet aggregation (SPA) 141, and evidence was obtained that this increased SPA is caused by liberation of adenosine diphosphate(ADP)from red blood cells @EC): The effect of glucose on SPA in WB was diminishedby apyrase (an enzyme that removes ADP from blood plasma) ard glucose was found to increasethe mechanicaland osmotic fragilityof RBC [4,51. In the sttiy presentedhere we determinedthe time-dependenceof the effectof glucose on SPA in samples of WB and the effects of EDNA (an inhibitor Key words:platelet aggregation, redblood cells, glucose, reducing sugars, diabetesmellitus 489
490
GLUCOSE INCREASES SPA IN WHOLE BLOOD
Vol59, No. 3
of platelet aggregation),tetramethyleneglutaric acid (TMG; an inhibitorof the polyolpathway [61),pentoxifylline (which increases BBC deformability [73) and chlorpromazine(which stabilizesBBC membranes [81). Furthermorethe effect of glucose on SPA was compared with those of other reducing sugars and with those of sugars that do not have reducinggroups.
MATERIAL AND MEYlwom
Blood was obtainedfrom healthy volunteersby clean venepunctureand collected intoplastic tubes that contained l/10 volume of 0.11 mM trisodium citrate dihydrate as anticoagulant. Glucose or other sugars were added to samples (1 ml)of WBas 300 mM solutions (to avoid changes in osmolarity) to obtain final concentrationsranging from 10 to 50 mM. Saline was used in controlsand to make up the total volume of the incubates to 1.2 ml. The incubates were then kept for various length of time (up to lhour) at 37OC in small plastic tubes under an athmosphere of 5% CO2 in air. In some experiments TMG (Sigma Chemicap), chlorpromazine (LargactilR; May & Baker) or pentoxifylline (Trental; Boechst)were includedin the incubatesof WB. SPA was assessed after stirring the WB samples for 8 min at 1000 rpm, by counting the number of single platelets using an Ultra-F10 100 Whole Blood Platelet Counter [9). The extent of platelet aggregationis expressedas the percentplatelet loss that occurredconsequentto stirring the samples.
RESULTS When samples of WB were incubatedwith glucose (10 - 50 mM) for 1 hour there was a significantand dose-dependentincrease in SPA (Fig.la). Potentiation of SPA was evident immediately after adding glucose (Fig-lb) and remained significantly increased when SPAwas measuredat different times uptoone hour after adding glucose (HO.02 at all time points). 40 7
40]b)
a)
0
10
20
30
Glucose (mM)
40
50
Control
0
102030405060
Minutes
&l: Effect of glucose on SPA in WB. A: Samples were incubated before stirrzg for 60 min with glucose at various concentrations. Significant differences compared to controls (0 mM glucose) are indicated: * p
J. May, W. Loesche*and S. Heptinstall Department of Medicine, Ugiversity Hospital, Queen's Medical Centre, NottinghamNG7 2UEL U.K.,and Instituteof PathologicalBiochemistry,Medical Academy, DDR-5010 Erfurt,GDR
(Received 13.11.1989; accepted in revised form 16.51990 by Editor S. Moncada)
ABsTRAcr
Glucose (lo-50mM) added to samples of titratedwhole blood increased the spontaneous platelet aggregation (SPA) that was observed upon stirringthe samples,in a dose-dependentbut time-independentmanner. EUIA preventedand also reversedthe SPA that occurred in the presence of glucose. Galactose, fructose and 2-deoxyglucose,but not sorbitol and sucrose, had effects on SPA similar to those of glucose. The effect of glucose on SPA in whole blood could not be specifically inhibited by tetramethylene glutaric acid (an inhibitor of aldose reductase).SPA was not affectedby pentoxifylline,but was markedly reduced by chlorpromazine. We previously found that glucose, galactose, fructose and 2ileoxyglucose, but not sorbitoland sucrose, render red blood cells more fragile, and the results obtained in the present study provide further evidence that glucose as well as other reducing sugars potentiate SPA in whole blood by liberating ADP or other aggregatingmaterial from red blood cells.
INrRDJXJc!rIQN Diabetesmellitus is frequentlycomplicatedby thromboembolicevents and it is believed that blood platelets contributeto diabetic vascular disease [l-31. We have previously shown that glucose added to samples of wholeblood (WB) potentiates spontaneous platelet aggregation (SPA) 141, and evidence was obtained that this increased SPA is caused by liberation of adenosine diphosphate(ADP)from red blood cells @EC): The effect of glucose on SPA in WB was diminishedby apyrase (an enzyme that removes ADP from blood plasma) ard glucose was found to increasethe mechanicaland osmotic fragilityof RBC [4,51. In the sttiy presentedhere we determinedthe time-dependenceof the effectof glucose on SPA in samples of WB and the effects of EDNA (an inhibitor Key words:platelet aggregation, redblood cells, glucose, reducing sugars, diabetesmellitus 489
490
GLUCOSE INCREASES SPA IN WHOLE BLOOD
Vol59, No. 3
of platelet aggregation),tetramethyleneglutaric acid (TMG; an inhibitorof the polyolpathway [61),pentoxifylline (which increases BBC deformability [73) and chlorpromazine(which stabilizesBBC membranes [81). Furthermorethe effect of glucose on SPA was compared with those of other reducing sugars and with those of sugars that do not have reducinggroups.
MATERIAL AND MEYlwom
Blood was obtainedfrom healthy volunteersby clean venepunctureand collected intoplastic tubes that contained l/10 volume of 0.11 mM trisodium citrate dihydrate as anticoagulant. Glucose or other sugars were added to samples (1 ml)of WBas 300 mM solutions (to avoid changes in osmolarity) to obtain final concentrationsranging from 10 to 50 mM. Saline was used in controlsand to make up the total volume of the incubates to 1.2 ml. The incubates were then kept for various length of time (up to lhour) at 37OC in small plastic tubes under an athmosphere of 5% CO2 in air. In some experiments TMG (Sigma Chemicap), chlorpromazine (LargactilR; May & Baker) or pentoxifylline (Trental; Boechst)were includedin the incubatesof WB. SPA was assessed after stirring the WB samples for 8 min at 1000 rpm, by counting the number of single platelets using an Ultra-F10 100 Whole Blood Platelet Counter [9). The extent of platelet aggregationis expressedas the percentplatelet loss that occurredconsequentto stirring the samples.
RESULTS When samples of WB were incubatedwith glucose (10 - 50 mM) for 1 hour there was a significantand dose-dependentincrease in SPA (Fig.la). Potentiation of SPA was evident immediately after adding glucose (Fig-lb) and remained significantly increased when SPAwas measuredat different times uptoone hour after adding glucose (HO.02 at all time points). 40 7
40]b)
a)
0
10
20
30
Glucose (mM)
40
50
Control
0
102030405060
Minutes
&l: Effect of glucose on SPA in WB. A: Samples were incubated before stirrzg for 60 min with glucose at various concentrations. Significant differences compared to controls (0 mM glucose) are indicated: * p
