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Glucose increases spontaneous platelet aggregation in whole blood
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
491
GLUCOSE INCREASES SPA IN WHOLE BLOOD
Vol59, No. 3
40 -
: ._
SallEDTA
30-
z z! &
20-
P 10 is
OI
a
6
10
12
16
14
16
Minutes Fig. 2: Effect of EDTA on SPA in WB that had been stirred for 8 min in absence (circles)or presence (squares)of 50 mM glucose. ELYIA(4 mM, open symbols)or saline (closedsymbols)was then added and stirringwas continued for a further8 min period.n=6, means%em. In a previous investigationwe showed that EDNA (4mM) prevents SPA in WB [91. In the present investigationwe determined whether it is able to reverse the SPA that occurs either in the absenceor presenceof 50mM glucose.It is clear (Fig. 2) that EDTA reversesthe aggregationin both cases. We have shown elsewhere [51 that glucose and other reducingsugars,galactose, fructose and 2-deoxyglucose, render BBC more fragile. In contrast, the nonreducing sugars, sorbitoland sucrose,had no effect on BBC fragility. There** 40
T
1
c 0
30
F b
20
2 s
10
._ ii
q q
l
Control Sugar
0 Glu
Gal
Fru
DOG
Sor
sue
Fig. 3: Effect of various sugars on SPA in WB. The samples were incubated before stirring for 60 min with either sugar at a concentration of 50 mM. Glu = glucose, Gal = galactose, Fru = fructose, DOG = deoxyglucose, SOr = sorbitol, Sue = sucrose.n=6, means+sem. Significantdifferencescompared to controls are indicated:* p(O.02; *Tp
492
GLUCOSE INCREASES SPA IN WHOLE BLOOD
30
1
Control
Vol59, No. 3
Gslsctose
Glucose
E ‘g
20
F 68 p
10
8 o............... 0
0
1310
1310
0
1310
TMG (mM) Fig. 4: Effects of TMG on SPA in WB. Before stirring the samples were inch% for 60 min in absenceor presenceof 50 mM glucose or galactoseand various concentrationsof TMG. n=6, means+sem. fore, we examined the effects of these sugars onSPAin samples of WB. It is shown in Fig. 3 that galactose,fructoseand 2-deoxyglucose(10 - 50 mM) had a potentiatiq effecton SPA that was similar to that observedwith glucose,but that no potentiationwas observedwith sorbitolor sucrose. TMG which is known to inhibit the conversion of glucose and galactose to sorbitolby the enzyme aldose reductaseat concentrationsas low as 100 uM [61 inhibited SPA in WB in a dose-dependent manner in both the absence and presence of glucose or galactose.Bowever significantinhibition(p
Glucose
Control v)
WI
a 8
20 10
0
100
0
100
Chlorpromazine (PM) Fig. 5: Effect of chlorpromazineon SPA in WB. Samples were incubatedbefore stirring for 30 min in absence or presence of 50 mM glucose and/or 100 PM chlorpromazine.n=6, means+sem. -
Vol59, No. 3
GLUCOSE INCREASES SPA IN WHOLE BLOOD
493
Pentoxifylline has been shown to increase FEZ deformability 171. In our experiments additionof pentoxifylline (100 uM) to samples of WB that were incubatedfor 30 min before stirringdid not alter significantlythe SPA that was observed in both the absence or presence of 10 - 50 mM glucose (not shown). The effects of chlorpromazine on SPA in samples of WB are shown in Fig. 5. Chlorpromazineis a membrane stabilizingagent [El whi& also interactswith free radicalspreventingoxidativeinjury to cells [lo].At a concentrationof 100 TM, chlorpromazinecaused a marked irihibition (p(O.02)of SPA in both the absence and presence of glucose.
DISCUSSION It has previously been shown that addition of glucose or other reducing sugars, such as galactose, 2-deoxyglucose and fructose, to samples of WB rerrders PBC more fragilein a concentration-dependent but time-independent manner [4,51. An increased RBC fragility may influence the platelet aggregaticnthat is observed in samples of WB upcm stirring, by encouraging liberation of ADP or other aggregating material from RBC. Indeed, SPA in samples of WB, but not in platelet-richplasma, was found to be increasedby glucose and this effect of glucose could be prevented,at least partially,by the ADP-removingenzyme apyrase [41. The involvement of RBC in the enhancement of SPA in WB brought about by glucose finds further supportbythe results obtained in the present study. Similar to its effect on RBC stability, glucose increased SPA in WB in a concentration-dependent but time-independent manner. The lack of timedependence on both RBC stability [5] and SPA indicates that metabolic processes may not be involved. This hypothesis is in agreement with the observationthat 2-deoxyglucose,an inhibitorof glucose metabolism, has an effect on SPA as well as on PBC fragility [5] that is similar to that of glucose.TMG that inhibitsthe conversionof glucose and galactoseto sorbitol via aldose reductase 161 inhibited SPA in both the absence and presence of glucose or galactose. However, the concentration of TMG needed to obtain a significantinhibitionof SPA (3 mM) was more than lOO-foldhigher than that needed to inhibit sorbitol accumulationin RX (61. We have shown elsewhere [51 that TMG in concentrations ashigh as 10 mMdid not significantly alter RBC fragility.Thereforewe assume that the effect of TMG on SPA in WB samples is a non-specificone and is not linked to an inhibitianof aldose reductase. The fact that galactose, fructose and 2_deoxyglucose,but not sorbitoland sucrose,increasesSPA in WB and render RBC more fragile in a similar manner to glucose provides further evidence for a direct link between increasedSPA ard decreased PBC stability. The increasedSPA in WB, measured as loss of single platelets,brought about by glucose could bs preventedby EXYTA.FurthermoreElXA was shown to reverse SPA that had occurred in the absenceas well as in the presenceof glucose,as would be espected for an aggregationprocess E9l. According to the suggestion that glucose may render RBC more fragile and encourageADP liberationfrom them, cne should expect that compoundsknown to increaseRIX membrane stabilitysuch as chlorpromazine[El or to increaseREJC
494
GLUCOSE INCREASES SPA IN WHOLE BLOOD
Vol59, No. 3
deformabilitysuch as pentoxifylline171 would interferewith the effect of glucose on SPA in WB. Indeed, chlorpromazine markedly reduced SPA in the absence as well as the in presenceof glucose,whereas pentoxifyllineproved to be ineffectivein alteringsignificantlythe glucose-induced increase in SPA. Interestingly pentoxifylline does not prevent the glucose-induced increasein RBC membrane fragility(unpublishedobservation). The mechanism by which glucose and other reducing sugars render RBC more fragile and thereby encourage SPA in WB is not yet clear. It has been speculated that an interaction of the aldehyde or keto groups of reducing sugars with membrane constituentsmay increase RBC membrane fragility 15I. Such interactioncould be a fast and reversiblebinding of the sugars to amino group of proteins (and/orlipids?)as is known to occur as the initial step in non-enzymaticprotein glycation [llI. Enhanced platelet aggregation in the presence of RBC and glucose may be relevant to clinical conditions in which high blood levels of glucose are obtained.For example,such a phenomenoncould contributeto the high risk for thromboemboliccomplicationsin diabetesmellitus.
REFERENCES
1. WINOCOUR, P.D.,HALUSKA, P.V.and COLWELL, J.A.:Platelet involvement in diabetes mellitus. in: The Platelets: Physiology and Pharmaco1ogy.G.L. Iongenecker(ed.).Academic Press, Orlando 1985, pp. 341-366. 2. KUBISZ, P., ARABI, A., HOLAN, J. and CRONBERG, S.: Investigations on platelet functionin diabetesmellitus.Haemostasis-14, 347-353,1984. 3. COLWELL, J.A., WINOCOUR, P.D. and HALUSKA, P.V.: Do platelets have anything to do with diabetic microvascular disease? Diabetes -32 (sup@. 2), 14-19, 1983. 4. LOESCHE, W., BEVAN, J., MICHEL, E., BURCHARDT, A. and HEPTINSTALL, S.: Effects of glucose on platelet aggregation in whole blood. -Diab. Nutr. Metab. 3, 209-214, 1988. -5. ICESCHE,w., MAY, J., HEPTINSTALL,S. and TILL, U.: High concentrationsof glucose impair red blood cell membrane integrity.St&a Biophysics134, 87-90, 1990. 6. MALONE, J.I.,KNOX, G., BENFORD, S. and TEDESCO, T.A.:Red cell sorbitol: an indicatorof diabeticcontrol.Diabetes29, 861-864, 1980. 7. SEIFFGE, D. and KIESEWE'ITER,H.: Effect of pentoxifylline on single red cell deformability.Klin. Wochenschr.59, 1271-1272,1981. 8. BORN, G.V.R and WEHMEYER, A.: Inhibitionof platelet thrombus formation by chlorpromazineacting to diminish haemodynamicallyinducedhaemolysis. Nature 282, 212-213,1979. -9. FOX, S.C., BURGESS-WILSON, M., HEPTINSTALL, S. and MITCHELL, J.R.A.: Platelet aggregationinwhole blood determined using the Ultra Flo 100 PlateletCounter.Thromb. Haemostas.48, 327-329, 1982.
Vol59, No. 3
GLUCOSE INCREASES SPA IN WHOLE BLOOD
495
10. JANERO, D.R, and BIXXUWT, B.: Preventionof oxidative injury to cardiac phospholipid by membrane-active 'stabilizingagents'.--Res. Comm. Chem. Pathol. Pharmacol.63, 163-173, 1989. 11. COHEN, M.P.: Diabetes and Protein Glycosylation. Springer-Verlag, New York, Heidelberg 1986, pp. 5-16.
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
491
GLUCOSE INCREASES SPA IN WHOLE BLOOD
Vol59, No. 3
40 -
: ._
SallEDTA
30-
z z! &
20-
P 10 is
OI
a
6
10
12
16
14
16
Minutes Fig. 2: Effect of EDTA on SPA in WB that had been stirred for 8 min in absence (circles)or presence (squares)of 50 mM glucose. ELYIA(4 mM, open symbols)or saline (closedsymbols)was then added and stirringwas continued for a further8 min period.n=6, means%em. In a previous investigationwe showed that EDNA (4mM) prevents SPA in WB [91. In the present investigationwe determined whether it is able to reverse the SPA that occurs either in the absenceor presenceof 50mM glucose.It is clear (Fig. 2) that EDTA reversesthe aggregationin both cases. We have shown elsewhere [51 that glucose and other reducingsugars,galactose, fructose and 2-deoxyglucose, render BBC more fragile. In contrast, the nonreducing sugars, sorbitoland sucrose,had no effect on BBC fragility. There** 40
T
1
c 0
30
F b
20
2 s
10
._ ii
q q
l
Control Sugar
0 Glu
Gal
Fru
DOG
Sor
sue
Fig. 3: Effect of various sugars on SPA in WB. The samples were incubated before stirring for 60 min with either sugar at a concentration of 50 mM. Glu = glucose, Gal = galactose, Fru = fructose, DOG = deoxyglucose, SOr = sorbitol, Sue = sucrose.n=6, means+sem. Significantdifferencescompared to controls are indicated:* p(O.02; *Tp
492
GLUCOSE INCREASES SPA IN WHOLE BLOOD
30
1
Control
Vol59, No. 3
Gslsctose
Glucose
E ‘g
20
F 68 p
10
8 o............... 0
0
1310
1310
0
1310
TMG (mM) Fig. 4: Effects of TMG on SPA in WB. Before stirring the samples were inch% for 60 min in absenceor presenceof 50 mM glucose or galactoseand various concentrationsof TMG. n=6, means+sem. fore, we examined the effects of these sugars onSPAin samples of WB. It is shown in Fig. 3 that galactose,fructoseand 2-deoxyglucose(10 - 50 mM) had a potentiatiq effecton SPA that was similar to that observedwith glucose,but that no potentiationwas observedwith sorbitolor sucrose. TMG which is known to inhibit the conversion of glucose and galactose to sorbitolby the enzyme aldose reductaseat concentrationsas low as 100 uM [61 inhibited SPA in WB in a dose-dependent manner in both the absence and presence of glucose or galactose.Bowever significantinhibition(p
Glucose
Control v)
WI
a 8
20 10
0
100
0
100
Chlorpromazine (PM) Fig. 5: Effect of chlorpromazineon SPA in WB. Samples were incubatedbefore stirring for 30 min in absence or presence of 50 mM glucose and/or 100 PM chlorpromazine.n=6, means+sem. -
Vol59, No. 3
GLUCOSE INCREASES SPA IN WHOLE BLOOD
493
Pentoxifylline has been shown to increase FEZ deformability 171. In our experiments additionof pentoxifylline (100 uM) to samples of WB that were incubatedfor 30 min before stirringdid not alter significantlythe SPA that was observed in both the absence or presence of 10 - 50 mM glucose (not shown). The effects of chlorpromazine on SPA in samples of WB are shown in Fig. 5. Chlorpromazineis a membrane stabilizingagent [El whi& also interactswith free radicalspreventingoxidativeinjury to cells [lo].At a concentrationof 100 TM, chlorpromazinecaused a marked irihibition (p(O.02)of SPA in both the absence and presence of glucose.
DISCUSSION It has previously been shown that addition of glucose or other reducing sugars, such as galactose, 2-deoxyglucose and fructose, to samples of WB rerrders PBC more fragilein a concentration-dependent but time-independent manner [4,51. An increased RBC fragility may influence the platelet aggregaticnthat is observed in samples of WB upcm stirring, by encouraging liberation of ADP or other aggregating material from RBC. Indeed, SPA in samples of WB, but not in platelet-richplasma, was found to be increasedby glucose and this effect of glucose could be prevented,at least partially,by the ADP-removingenzyme apyrase [41. The involvement of RBC in the enhancement of SPA in WB brought about by glucose finds further supportbythe results obtained in the present study. Similar to its effect on RBC stability, glucose increased SPA in WB in a concentration-dependent but time-independent manner. The lack of timedependence on both RBC stability [5] and SPA indicates that metabolic processes may not be involved. This hypothesis is in agreement with the observationthat 2-deoxyglucose,an inhibitorof glucose metabolism, has an effect on SPA as well as on PBC fragility [5] that is similar to that of glucose.TMG that inhibitsthe conversionof glucose and galactoseto sorbitol via aldose reductase 161 inhibited SPA in both the absence and presence of glucose or galactose. However, the concentration of TMG needed to obtain a significantinhibitionof SPA (3 mM) was more than lOO-foldhigher than that needed to inhibit sorbitol accumulationin RX (61. We have shown elsewhere [51 that TMG in concentrations ashigh as 10 mMdid not significantly alter RBC fragility.Thereforewe assume that the effect of TMG on SPA in WB samples is a non-specificone and is not linked to an inhibitianof aldose reductase. The fact that galactose, fructose and 2_deoxyglucose,but not sorbitoland sucrose,increasesSPA in WB and render RBC more fragile in a similar manner to glucose provides further evidence for a direct link between increasedSPA ard decreased PBC stability. The increasedSPA in WB, measured as loss of single platelets,brought about by glucose could bs preventedby EXYTA.FurthermoreElXA was shown to reverse SPA that had occurred in the absenceas well as in the presenceof glucose,as would be espected for an aggregationprocess E9l. According to the suggestion that glucose may render RBC more fragile and encourageADP liberationfrom them, cne should expect that compoundsknown to increaseRIX membrane stabilitysuch as chlorpromazine[El or to increaseREJC
494
GLUCOSE INCREASES SPA IN WHOLE BLOOD
Vol59, No. 3
deformabilitysuch as pentoxifylline171 would interferewith the effect of glucose on SPA in WB. Indeed, chlorpromazine markedly reduced SPA in the absence as well as the in presenceof glucose,whereas pentoxifyllineproved to be ineffectivein alteringsignificantlythe glucose-induced increase in SPA. Interestingly pentoxifylline does not prevent the glucose-induced increasein RBC membrane fragility(unpublishedobservation). The mechanism by which glucose and other reducing sugars render RBC more fragile and thereby encourage SPA in WB is not yet clear. It has been speculated that an interaction of the aldehyde or keto groups of reducing sugars with membrane constituentsmay increase RBC membrane fragility 15I. Such interactioncould be a fast and reversiblebinding of the sugars to amino group of proteins (and/orlipids?)as is known to occur as the initial step in non-enzymaticprotein glycation [llI. Enhanced platelet aggregation in the presence of RBC and glucose may be relevant to clinical conditions in which high blood levels of glucose are obtained.For example,such a phenomenoncould contributeto the high risk for thromboemboliccomplicationsin diabetesmellitus.
REFERENCES
1. WINOCOUR, P.D.,HALUSKA, P.V.and COLWELL, J.A.:Platelet involvement in diabetes mellitus. in: The Platelets: Physiology and Pharmaco1ogy.G.L. Iongenecker(ed.).Academic Press, Orlando 1985, pp. 341-366. 2. KUBISZ, P., ARABI, A., HOLAN, J. and CRONBERG, S.: Investigations on platelet functionin diabetesmellitus.Haemostasis-14, 347-353,1984. 3. COLWELL, J.A., WINOCOUR, P.D. and HALUSKA, P.V.: Do platelets have anything to do with diabetic microvascular disease? Diabetes -32 (sup@. 2), 14-19, 1983. 4. LOESCHE, W., BEVAN, J., MICHEL, E., BURCHARDT, A. and HEPTINSTALL, S.: Effects of glucose on platelet aggregation in whole blood. -Diab. Nutr. Metab. 3, 209-214, 1988. -5. ICESCHE,w., MAY, J., HEPTINSTALL,S. and TILL, U.: High concentrationsof glucose impair red blood cell membrane integrity.St&a Biophysics134, 87-90, 1990. 6. MALONE, J.I.,KNOX, G., BENFORD, S. and TEDESCO, T.A.:Red cell sorbitol: an indicatorof diabeticcontrol.Diabetes29, 861-864, 1980. 7. SEIFFGE, D. and KIESEWE'ITER,H.: Effect of pentoxifylline on single red cell deformability.Klin. Wochenschr.59, 1271-1272,1981. 8. BORN, G.V.R and WEHMEYER, A.: Inhibitionof platelet thrombus formation by chlorpromazineacting to diminish haemodynamicallyinducedhaemolysis. Nature 282, 212-213,1979. -9. FOX, S.C., BURGESS-WILSON, M., HEPTINSTALL, S. and MITCHELL, J.R.A.: Platelet aggregationinwhole blood determined using the Ultra Flo 100 PlateletCounter.Thromb. Haemostas.48, 327-329, 1982.
Vol59, No. 3
GLUCOSE INCREASES SPA IN WHOLE BLOOD
495
10. JANERO, D.R, and BIXXUWT, B.: Preventionof oxidative injury to cardiac phospholipid by membrane-active 'stabilizingagents'.--Res. Comm. Chem. Pathol. Pharmacol.63, 163-173, 1989. 11. COHEN, M.P.: Diabetes and Protein Glycosylation. Springer-Verlag, New York, Heidelberg 1986, pp. 5-16.