THROMBOSIS RESEARCH 66; 239-246,1992 0049-3848/92 $5.00 + .OOPrinted in the USA. Copyright (c) 1992 Pergamon Press Ltd. All rights reserved.
HYPERSENSITIVITY OF DIABETIC HUMAN PLATELETS TO PLATELET ACTIVATING FACTOR
Shivendra D. Shukla, Anjan Paul and David M. Klachkot Departments of Pharmacology and Medicinet University of Missouri School of Medicine Columbia, Missouri 65212 U.S.A. (Received 20.10.1991; accepted in revised form 18.2.1992 by Editor I. Danishefsky)
ABSTRACT
Platelet activating factor (PAF) stimulated aggregation and [“‘Plphosphatidic acid (PA) production was compared in normal and diabetic human subjects in platelet rich plasma. The concentration of PAF for half maximal (50%) aggregation of normal and diabetic platelets was 50 nM and 8 nM, respectively. PAF stimulated [32P]-I’A production (a metabolite of phospholipase C pathway) was also greater in the platelets from diabetic subjects. This [32P]-PA production was inhibited by the PAF receptor antagonists SRI-63441 and SRI-63675. When the levels of glycosylated hemoglobin (HbAlc) were compared with the PAF stimulated [“*PI-PA production a significant relationship was observed. These studies have demonstrated for the first time that diabetic human platelets show hypersensitivity to PAF in both aggregation and [321’l-PA production compared to normal subjects. This may be a result of some modification in phospholipid turnover mechanism and is receptor mediated. Further, the relationship of the degree of aggregation and [3’Pl-PA production to the level of HbAlc suggest that the insulin deficiency may contribute to these effects.
INTRODUCTION Patients with diabetes mellitus develop vascular complications and neuropathy. The macrovascular disease leads to an increased incidence of coronary artery dis#ease and stroke (1). Platelets from diabetic subjects show increased sensitivity to aggregation induced by many agonists e.g., ADP, epinephrine or collagen (1). These platelets have Key words: Platelets, diabetes, platelet activating factor 239
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elevated levels of arachidonic acid and prostaglandin E (2,3,4) and platelets also synthesize more thromboxane from the arachidonic acid in response to ADP, collagen or thrombin. Platelet activating factor (PAF), an ether phospholipid plays an important role in allergy, inflammation, asthma, and in other cardiovascular processes (5). PAF stimulates phosphoinositide specific phospholipase C (PLC) which in turn produces inositol trisphosphate (II’,) and diglyceride (DG) (6). DG activates protein kinase C (PKC) which increases the phosphorylation of proteins. An increased level of II’, within the cell stimulates Ca++ release from intracellular stores (7,8). There is evidence of enhanced degradation of PAF in serum from diabetic patients (9). In platelets PAF causes rapid turnover of phosphoinositides in a receptor dependent manner which then leads to platelet aggregation and serotonin secretion (10,ll). PAF receptor responses in diabetic platelets and its relationship to platelets hypersensitivity is poorly understood. We have therefore investigated this issue and addressed its relevance to PAF receptor coupled activation of PLC. MATERIALS
AND METHODS
Platelet activating factor, PAF (I-O-hexadecyl-2-acetyl-sn-glyceryl-3phosphocholine) was purchased from Bachem (Torrance, CA). Carrier-free 32P was obtained from ICN Radiochemicals (Irvine, CA). PAF receptor antagonists SRI-63675 and SRI-63441 were kindly provided by Dr. D.A. Handly of Sandoz Research Institute, East Hanover, NJ. All other chemicals and solvents used were of the highest analytical grade available. Preparation of platelet rich plasma Blood from normal and diabetic volunteers was collected in sterile vacutainer tubes containing 0.5 ml of 0.105 molar (3.2%) buffered citrate solution (12.35 mg, Na, citrate, .2H20 and 2.21 mg citric acid .lH,O) (Becton Dickinson tube #6415) at the Cosmopolitan International Diabetes Center, UMC Hospital and Clinics. No subjects were taking acetylsalicylic acid or antiplatelet drugs. In this project, the goal was to examine if diabetic platelets, in general, show any difference in their PAF responses compared to normal subjects. In particular, the relationship between the level of glycosylated hemoglobin in diabetic patients and the PAF receptor function was studied. Blood was drawn from both type I and type II diabetic human subjects of both male and female sexes. The age of patients ranged between 29-61 years. The blood was centrifuged at 200 x g for 8 min. at 24°C. The platelet rich plasma (PRP) was used to conduct the experiments and the platelets counts ranged from 0.2 to 0.3 x lo9 per ml. The tubes were kept capped to prevent pH changes. Platelet ag;arepation Aggregation patterns of PRP from normal and diabetic individuals were monitored using PAF at various concentrations in Chronolog Aggregometer Model 330, (Havertown, PA). 32P-labelling;, lipid extraction and separation of phospholipids PRP from normal and diabetic subjects were labelled with 32P (50 @i/ml) for 90 min. at 37°C. One ml aliquots (triplicate) were challenged with different
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concentrations of PAF for 30 sets. and the reaction was stopped by adding 4 ml of 1% ice-cold formalin (11). The suspension was centrifuged at 1500 x g for 10 min. The supernatant was discarded. The pellets were resuspended in 0.9 ml 1% formalin (ice-cold), 3.75 ml C-M-12NHCl (200-400-l-6, v/v) was added, mixed well and allowed to stand for 30 min. at room temperature. This was followed by the addition of 1.25 ml chloroform and 1.2 ml distilled water. The samples were mixed, centrifuged and the lower phase was collected and dried under N,. The lipid samples were applied to silica gel G plates for TLC and run in a solvent system of chloroform, pyridine and formic Phospholipid spots were visualized by spraying with acid (50:30:5, by volume). I-toluidino-2-naphthelene sulfonic acid (lo), scraped into the radioactive vials and counted with 10 ml of scintillation cocktail. Glycosylated hemoglobin (HbAlc) was measured on the Diamat HPLC system (Bio-Rad Laboratories, Hercules, PA). The normal range for HbAlc is 4.0-6.0%.
,_ IO
-Y
-7
-8
log [PAFI
__I
-6
M
Fig;. 1. Effect of PAF on aRgTeaation of platelets from normal and diabetic piatients. Platelet rich plasma (PRP) was isolated from freshly drawn human blood. Aggregation Values of platelets in PRP was monitored using PAF at different concentrations. represent percent aggregation relative to 100% aggregation obtained using 1 x 10m7M PAF. Bars represent + S.D. Cl, Normal (n=lO); 0, Diabetic (n=lO)
Fip. 2. Time course of [32P]-PA increase bv PAF. [“2Pl-labelled PRP was treated with PAF (1 x 10e7 M) for different times. The reaction was terminated and [32P]-PA level analyzed as described in Methods. Values represent percent over control (without PAF) incubations. The results are expressed as mean f S.D. (5 subjects).
242
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0
__*---
___--___--8
100 50- jj!
0:s 0
I
I
I
I
I
I
I
I
I
I
10 20 30 40 50 60 70 80 90 100J PAF (nM)
Fig. 3. PAF stimulated 13’Pl-PA production in normal and diabetic human platelets. r2P]-labelled platelets in PRP from normal or diabetic subjects were treated with different concentrations of PAF for 30 s and their r2Pl-PA levels were analyzed as described in Methods. Results from 5-8 diabetic or normal subjects are presented in the scattergram. The solid line indicates mean values for the groups. Using two tailed El, Normal t test, p values at 10 nM and 100 nM were O-0205 and 09156. (mean value ?? ); 0, Diabetic (mean value 0). The dotted and solid lines are drawn through the mean values. 300
250
?? -
0 4
I
I
I
I
I
I
I
I
I
5
6
7
6
9
10
11
12
13
14
HbAlc(%)
Fia. 4. Relationshiu between levels of alvcosvlated hemoglobin and PAF stimulated rPl-PA uroduction in diabetic platelets. Diabetic PRP was labelled with 32P (see text) and challenged with PAF (1 x lo-’ M) for 30 s. Increases in the 32P]-PA were determined in these platelets (see Methods) and plotted against the glycosylated hemoglobin (HbAlc) levels. Each point represents one subject. Values for [32P]-PA levels were obtained using 10e7M PAF. In order to indicate the variations among subjects all data points are presented. Slope is significantly different from zero; p=Og337. Correlation coefficient (r) = 06139.
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RESULTS AND DISCUSSION PAF induced aggregation in normal and diabetic human ulatelets One of the major limitations in the study of platelets from diabetic human subjects is the availability of large quantities of platelets. Further, human platelets respond well to PAF in PRP but the response is gradually weakened when platelets are washed (see references in 11). To overcome these limitations we have therefore used PRl? in the present study. Different concentrations of PAF were used to examine the aggregation of normal and diabetic human platelets in PRP. The results are shown in Fig. 1 and demanstrate a greater platelet aggregation in the diabetic subjects. For example, at a PAF concentration of 50 nM, the aggregation was 50% and 90% in normal and diabetic platelets, respectively. For half maximal (50%) aggregation, the concentrations of PAF needed for normal and diabetic platelets were about 50 nM and 8 nM, respectively. This demonstrated hypersensitivity of diabetic platelets to PAF (Fig. 1). PAF stimulated PA production in normal and diabetic human subiects It has been shown that PAF causes phosphoinositide turnover producing IP3 and DG. The DG is phosphorylated to PA. A measurement of PA therefore reflects PAF receptor coupled phosphoinositide (PPI) turnover (6). PA can also be directly produced by phospholipase D (PLD) pathways (13) but in human platelets such a pathway for PAF is negligible (unpublished). Using [32P]-labelled human PRP it was observed that PAF caused a maximum increase in [321’]-PA in 30 s and declined thereafter (Fig. 2). This timecourse profile was the same for both normal and diabetic human platelets. Treatment of PRP with the receptor antagonists SRI-63441 and SRI 63675 inhibited the PAF stimulated [32P]-PA production by 87 f 3% and 91 + 4%, respectively. It is relevant to note that these antagonists have been shown to block other receptor responses, e.g. II’, production (14). With the above experimental protocol, production of [32P]-PA was examined using two concentrations of PAF in normal and diabetic human platelets. The results are shown in Fig. 3. In order to standardize the pattern among different samples, the results are presented as percent increase in r*P]-PA over the control samples (i.e., without PAF). As can be seen the normal platelets exhibited a lower level of PAF stimulated [32P]-PA Data points from different subjects are production as compared to diabetic samples. given to reflect upon the inherent variability in such experiments. Overall the pattern is consistently indicative of an increased production of [3’P]-PA in diabetic subjects. The poorer the control of diabetes mellitus and the higher the average blood glucose concentration, the greater the proportion of hemoglobin that is glycosylated. Therefore the relationship between HbAlc level and PAF stimulated [321’l-l’Alevels was examined. Results are presented in Fig. 4. The scattergram clearly points a tendency toward an increased [“*PI-PA as the level of HbAlc increased. Of the 12 subjects shown in Fig. 4, the data for 5 of these subjects are also presented in Fig. 3. The use of PRP in these experiments has several advantages but a few limitations too. Advantages have been stated earlier. Among the limitations are the presence of hydrolytic enzymes in plasma which can cleave PAF to 1ysoPAF. In a recent study it was shown that plasma of diabetic patients contains increased levels of acetylhydrolase (9). However, in the presence of EDTA, which inhibits Ca’+ dependent plasma enzymes, hypersensitivity of platelets to PAF, as monitored by [“‘PIPA production, was still
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observed. It may be mentioned that in the presence of EDTA, PAF stimulates PA production but not the platelet aggregation (11). If there is an increase in plasma acetylhydrolase then diabetic PRP will inactivate PAF more efficiently; hence a reduction in response (in aggregation and [32P]-PA) will be expected. This was not observed in the present study. In contrast, an increase in aggregation and [32Pl-PA production was noted. It was suggested in one report that increased aggregation observed in platelets from the group with diabetes in response to PAF results in part from their higher production of thromboxane A, and release of 5-hydroxy-tryptamine (15). The present study indicates that PAF receptor coupled PLC activation is also affected in diabetes. In contrast to our observations the [32P]-PA formation was shown to be decreased in diabetic human platelets stimulated with thrombin (16). However, in another report thrombin induced phosphoinositide hydrolysis, intracellular Ca” mobilization and P20 phosphorylation was significantly increased in human platelets from non-insulin dependent diabetes mellitus with enhanced platelet aggregation rates (17). Further, an increase in phospholipase activity in platelets from diabetic subjects was observed (18). These reports (17, 18) corroborate our studies with PAF. We have established that diabetic human platelets show hypersensitivity to PAF in both aggregation and [32P]-PA production compared to normal subjects. The fact that receptor antagonists blocked this PA production indicated that this response is PAF receptor related. Thus platelets of diabetic patients exhibit some modification in PAF receptor associated PPI turnover mechanism. The nature of this modification is not known. It could be at the receptor level, at the level of transduction elements or at some other step (19). Furthermore, the increase in the content of HbAlc showed significant correlationship to [32Pl-PA production. The relationship of the degree of aggregation and [3’P]-PA production to the level of glycosylated hemoglobin suggest that the insulin deficiency may contribute to these effects. ACKNOWLEDGEMENTS We are grateful to Ms. Kristin Nelson for her expert typing of the manuscript. This work was supported by National Institutes of Diabetes and Digestive and Kidney Diseases Grant (DK 35170) and by a NIH Research Career Development Award (DK 01782) to Shivendra D. Shukla.
REFERENCES 1. MUSTARD, J.F. AND PACKHAM, M.A. Platelets and Diabetes Mellitus. New Engl. J. Med. 311, 665-666 (1984). 2. MORITA, I., TAKAHASHI, R., ITO, H., ORIMO, H. AND MUROTA, S. Increased Arachidonic Acid Content in Platelet Phospholipids from Diabetic Patients. Prost@mfins Leukotrietzes Med. II, 33-41 (1983). 3. HALUSHKA, A.V., LURIE, D. AND COLWELL, J.A. Increased Synthesis of Prostaglandin-E Like Material by Platelets for Patients with Diabetes Mellitus. New Engl. J. Med. 297, 1306-1310 (1977).
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4. HALUSKKA, P.V., CURTIS ROGERS, R., LOADHOLT, C.V. AND COLWELL, J.A. Increased Platelet Thromboxane Synthesis in Diabetes Mellitus. I. Lab. Clin. Med. 97, 87-96 (1981). 5. BRAQUET, P.L., TOUQUI, L., SHEN, T.Y., VARGAFTIG, Research. Pharmacological Rev. 39, 97-145 (1987). 6. SHUKLA, S.D. Inositol Phospholipid Lipids 26, 1028-1033 (1991).
Turnover
B.B.
Perspectives
in PAF Transmembrane
in PAF
Signalling.
7. BERRIDGE, M.J. AND IRVINE, R.F. Inositol Triphosphate, a Novel Second Messenger in Cellular Signal Transduction. Nature 312, 315-321 (1984). 8. ABDEL-LATIF, A.A. Ca” Mobilizing Receptors, Phosphoinositides Pharmacol. Rev. 38, 246-272 (1986). of Second Messengers.
and the Generation
9. HOFMANN, B., RUHLING, K., SPANGENBERG, I’. AND OSTERMANN, G. Enhanced Degradation of Platelet Activating Factor in Serum from Diabetic Patients. Hfremostasis 19, 180-184 (1989). 10. SHUKLA, S.D. AND HANAHAN, D.J. AGEPC (Platelet Activating Factor) Induced Stimulation of Rabbit Platelets: Effects on Phosphatidylinositol, Di-, and TriPhosphoinositides and Phosphatidic Acid. Biochem. Biophys. Res. Commun. 106, 697-703 (1982). 11. SHUKLA, S.D. PAF Stimulated Formation of Inositol Triphosphate in Platelets and its Regulation by Various Agents including Ca”, Indomethacin, CA3988 and Forskolin. Arch. Biochem. Biophys. 240, 674-681 (1985). 12. SHUKLA, S.D., MORRISON, W.J. AND KLACHKO, D.M. Response to Platelet Activating Factor in Human Platelets Stored and Aged in Plasma: Decrease in Aggregation, Phosphoinositide Turnover and Receptor Affinity. Transfusion 29,528-533 (1989). 13. SHUKLA, S.D. AND HALENDA, S.P. Phospholipase D in Cell Signalling Relationship to Phospholipase C. Life Sci. 48, 851-866 (1991). 14. MORRISON, W.J. AND SHUKLA, SD. Antagonism Receptor Binding and Stimulated Phosphoinositide-Specific Platelets. 1. Pharmncol. Exp. Ther. 250, 831-835 (1989).
and its
of Platelet Activating Factor Phospholipase C in Rabbit
15. GRECO, N.G., ARNOLD, J.H., O’DORISIO, T.M., CATALAND, S. AND PANGANMALA, R.V. Action of Platelet Activating Factor on Type 1 Diabetic Human Platelets. I. Lab. Clin. Med. 105, 410-416 (1985). 16. BASTYR III, E.J., KADROFSKE, Decreased Platelet Phosphoinositide IDDM. Diabetes 38, 1097-1102 (1989).
M.M., DERSHIMER, R.C. AND VINIK, A.I. Turnover and Enhanced Platelet Activation in
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17. ISHII, H., UMEDA, F., HASHIMOTO, T. AND NAWATA, H. Changes in Phosphoinositide Turnover, Ca2’Mobilization and Protein Phosphorylation in Platelets from NIDDM Patients. Diabetes 39, 1561-1568 (1990).
18. TAKEDA, H., MAEDA, H., FUKUSHIMA, H., NAKAMURA, N. AND UZAWA, H. Increased Platelet Phospholipase Activity in Diabetic Subjects. Thromb. Res. 24, 131-141 (1981). 19. SHUKLA, S.D. Platelet Activating Factor Receptor and Signal Transduction Mechanisms. FASEB J. (in press, 1992).
HYPERSENSITIVITY OF DIABETIC HUMAN PLATELETS TO PLATELET ACTIVATING FACTOR
Shivendra D. Shukla, Anjan Paul and David M. Klachkot Departments of Pharmacology and Medicinet University of Missouri School of Medicine Columbia, Missouri 65212 U.S.A. (Received 20.10.1991; accepted in revised form 18.2.1992 by Editor I. Danishefsky)
ABSTRACT
Platelet activating factor (PAF) stimulated aggregation and [“‘Plphosphatidic acid (PA) production was compared in normal and diabetic human subjects in platelet rich plasma. The concentration of PAF for half maximal (50%) aggregation of normal and diabetic platelets was 50 nM and 8 nM, respectively. PAF stimulated [32P]-I’A production (a metabolite of phospholipase C pathway) was also greater in the platelets from diabetic subjects. This [32P]-PA production was inhibited by the PAF receptor antagonists SRI-63441 and SRI-63675. When the levels of glycosylated hemoglobin (HbAlc) were compared with the PAF stimulated [“*PI-PA production a significant relationship was observed. These studies have demonstrated for the first time that diabetic human platelets show hypersensitivity to PAF in both aggregation and [321’l-PA production compared to normal subjects. This may be a result of some modification in phospholipid turnover mechanism and is receptor mediated. Further, the relationship of the degree of aggregation and [3’Pl-PA production to the level of HbAlc suggest that the insulin deficiency may contribute to these effects.
INTRODUCTION Patients with diabetes mellitus develop vascular complications and neuropathy. The macrovascular disease leads to an increased incidence of coronary artery dis#ease and stroke (1). Platelets from diabetic subjects show increased sensitivity to aggregation induced by many agonists e.g., ADP, epinephrine or collagen (1). These platelets have Key words: Platelets, diabetes, platelet activating factor 239
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elevated levels of arachidonic acid and prostaglandin E (2,3,4) and platelets also synthesize more thromboxane from the arachidonic acid in response to ADP, collagen or thrombin. Platelet activating factor (PAF), an ether phospholipid plays an important role in allergy, inflammation, asthma, and in other cardiovascular processes (5). PAF stimulates phosphoinositide specific phospholipase C (PLC) which in turn produces inositol trisphosphate (II’,) and diglyceride (DG) (6). DG activates protein kinase C (PKC) which increases the phosphorylation of proteins. An increased level of II’, within the cell stimulates Ca++ release from intracellular stores (7,8). There is evidence of enhanced degradation of PAF in serum from diabetic patients (9). In platelets PAF causes rapid turnover of phosphoinositides in a receptor dependent manner which then leads to platelet aggregation and serotonin secretion (10,ll). PAF receptor responses in diabetic platelets and its relationship to platelets hypersensitivity is poorly understood. We have therefore investigated this issue and addressed its relevance to PAF receptor coupled activation of PLC. MATERIALS
AND METHODS
Platelet activating factor, PAF (I-O-hexadecyl-2-acetyl-sn-glyceryl-3phosphocholine) was purchased from Bachem (Torrance, CA). Carrier-free 32P was obtained from ICN Radiochemicals (Irvine, CA). PAF receptor antagonists SRI-63675 and SRI-63441 were kindly provided by Dr. D.A. Handly of Sandoz Research Institute, East Hanover, NJ. All other chemicals and solvents used were of the highest analytical grade available. Preparation of platelet rich plasma Blood from normal and diabetic volunteers was collected in sterile vacutainer tubes containing 0.5 ml of 0.105 molar (3.2%) buffered citrate solution (12.35 mg, Na, citrate, .2H20 and 2.21 mg citric acid .lH,O) (Becton Dickinson tube #6415) at the Cosmopolitan International Diabetes Center, UMC Hospital and Clinics. No subjects were taking acetylsalicylic acid or antiplatelet drugs. In this project, the goal was to examine if diabetic platelets, in general, show any difference in their PAF responses compared to normal subjects. In particular, the relationship between the level of glycosylated hemoglobin in diabetic patients and the PAF receptor function was studied. Blood was drawn from both type I and type II diabetic human subjects of both male and female sexes. The age of patients ranged between 29-61 years. The blood was centrifuged at 200 x g for 8 min. at 24°C. The platelet rich plasma (PRP) was used to conduct the experiments and the platelets counts ranged from 0.2 to 0.3 x lo9 per ml. The tubes were kept capped to prevent pH changes. Platelet ag;arepation Aggregation patterns of PRP from normal and diabetic individuals were monitored using PAF at various concentrations in Chronolog Aggregometer Model 330, (Havertown, PA). 32P-labelling;, lipid extraction and separation of phospholipids PRP from normal and diabetic subjects were labelled with 32P (50 @i/ml) for 90 min. at 37°C. One ml aliquots (triplicate) were challenged with different
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concentrations of PAF for 30 sets. and the reaction was stopped by adding 4 ml of 1% ice-cold formalin (11). The suspension was centrifuged at 1500 x g for 10 min. The supernatant was discarded. The pellets were resuspended in 0.9 ml 1% formalin (ice-cold), 3.75 ml C-M-12NHCl (200-400-l-6, v/v) was added, mixed well and allowed to stand for 30 min. at room temperature. This was followed by the addition of 1.25 ml chloroform and 1.2 ml distilled water. The samples were mixed, centrifuged and the lower phase was collected and dried under N,. The lipid samples were applied to silica gel G plates for TLC and run in a solvent system of chloroform, pyridine and formic Phospholipid spots were visualized by spraying with acid (50:30:5, by volume). I-toluidino-2-naphthelene sulfonic acid (lo), scraped into the radioactive vials and counted with 10 ml of scintillation cocktail. Glycosylated hemoglobin (HbAlc) was measured on the Diamat HPLC system (Bio-Rad Laboratories, Hercules, PA). The normal range for HbAlc is 4.0-6.0%.
,_ IO
-Y
-7
-8
log [PAFI
__I
-6
M
Fig;. 1. Effect of PAF on aRgTeaation of platelets from normal and diabetic piatients. Platelet rich plasma (PRP) was isolated from freshly drawn human blood. Aggregation Values of platelets in PRP was monitored using PAF at different concentrations. represent percent aggregation relative to 100% aggregation obtained using 1 x 10m7M PAF. Bars represent + S.D. Cl, Normal (n=lO); 0, Diabetic (n=lO)
Fip. 2. Time course of [32P]-PA increase bv PAF. [“2Pl-labelled PRP was treated with PAF (1 x 10e7 M) for different times. The reaction was terminated and [32P]-PA level analyzed as described in Methods. Values represent percent over control (without PAF) incubations. The results are expressed as mean f S.D. (5 subjects).
242
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0
__*---
___--___--8
100 50- jj!
0:s 0
I
I
I
I
I
I
I
I
I
I
10 20 30 40 50 60 70 80 90 100J PAF (nM)
Fig. 3. PAF stimulated 13’Pl-PA production in normal and diabetic human platelets. r2P]-labelled platelets in PRP from normal or diabetic subjects were treated with different concentrations of PAF for 30 s and their r2Pl-PA levels were analyzed as described in Methods. Results from 5-8 diabetic or normal subjects are presented in the scattergram. The solid line indicates mean values for the groups. Using two tailed El, Normal t test, p values at 10 nM and 100 nM were O-0205 and 09156. (mean value ?? ); 0, Diabetic (mean value 0). The dotted and solid lines are drawn through the mean values. 300
250
?? -
0 4
I
I
I
I
I
I
I
I
I
5
6
7
6
9
10
11
12
13
14
HbAlc(%)
Fia. 4. Relationshiu between levels of alvcosvlated hemoglobin and PAF stimulated rPl-PA uroduction in diabetic platelets. Diabetic PRP was labelled with 32P (see text) and challenged with PAF (1 x lo-’ M) for 30 s. Increases in the 32P]-PA were determined in these platelets (see Methods) and plotted against the glycosylated hemoglobin (HbAlc) levels. Each point represents one subject. Values for [32P]-PA levels were obtained using 10e7M PAF. In order to indicate the variations among subjects all data points are presented. Slope is significantly different from zero; p=Og337. Correlation coefficient (r) = 06139.
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RESULTS AND DISCUSSION PAF induced aggregation in normal and diabetic human ulatelets One of the major limitations in the study of platelets from diabetic human subjects is the availability of large quantities of platelets. Further, human platelets respond well to PAF in PRP but the response is gradually weakened when platelets are washed (see references in 11). To overcome these limitations we have therefore used PRl? in the present study. Different concentrations of PAF were used to examine the aggregation of normal and diabetic human platelets in PRP. The results are shown in Fig. 1 and demanstrate a greater platelet aggregation in the diabetic subjects. For example, at a PAF concentration of 50 nM, the aggregation was 50% and 90% in normal and diabetic platelets, respectively. For half maximal (50%) aggregation, the concentrations of PAF needed for normal and diabetic platelets were about 50 nM and 8 nM, respectively. This demonstrated hypersensitivity of diabetic platelets to PAF (Fig. 1). PAF stimulated PA production in normal and diabetic human subiects It has been shown that PAF causes phosphoinositide turnover producing IP3 and DG. The DG is phosphorylated to PA. A measurement of PA therefore reflects PAF receptor coupled phosphoinositide (PPI) turnover (6). PA can also be directly produced by phospholipase D (PLD) pathways (13) but in human platelets such a pathway for PAF is negligible (unpublished). Using [32P]-labelled human PRP it was observed that PAF caused a maximum increase in [321’]-PA in 30 s and declined thereafter (Fig. 2). This timecourse profile was the same for both normal and diabetic human platelets. Treatment of PRP with the receptor antagonists SRI-63441 and SRI 63675 inhibited the PAF stimulated [32P]-PA production by 87 f 3% and 91 + 4%, respectively. It is relevant to note that these antagonists have been shown to block other receptor responses, e.g. II’, production (14). With the above experimental protocol, production of [32P]-PA was examined using two concentrations of PAF in normal and diabetic human platelets. The results are shown in Fig. 3. In order to standardize the pattern among different samples, the results are presented as percent increase in r*P]-PA over the control samples (i.e., without PAF). As can be seen the normal platelets exhibited a lower level of PAF stimulated [32P]-PA Data points from different subjects are production as compared to diabetic samples. given to reflect upon the inherent variability in such experiments. Overall the pattern is consistently indicative of an increased production of [3’P]-PA in diabetic subjects. The poorer the control of diabetes mellitus and the higher the average blood glucose concentration, the greater the proportion of hemoglobin that is glycosylated. Therefore the relationship between HbAlc level and PAF stimulated [321’l-l’Alevels was examined. Results are presented in Fig. 4. The scattergram clearly points a tendency toward an increased [“*PI-PA as the level of HbAlc increased. Of the 12 subjects shown in Fig. 4, the data for 5 of these subjects are also presented in Fig. 3. The use of PRP in these experiments has several advantages but a few limitations too. Advantages have been stated earlier. Among the limitations are the presence of hydrolytic enzymes in plasma which can cleave PAF to 1ysoPAF. In a recent study it was shown that plasma of diabetic patients contains increased levels of acetylhydrolase (9). However, in the presence of EDTA, which inhibits Ca’+ dependent plasma enzymes, hypersensitivity of platelets to PAF, as monitored by [“‘PIPA production, was still
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observed. It may be mentioned that in the presence of EDTA, PAF stimulates PA production but not the platelet aggregation (11). If there is an increase in plasma acetylhydrolase then diabetic PRP will inactivate PAF more efficiently; hence a reduction in response (in aggregation and [32P]-PA) will be expected. This was not observed in the present study. In contrast, an increase in aggregation and [32Pl-PA production was noted. It was suggested in one report that increased aggregation observed in platelets from the group with diabetes in response to PAF results in part from their higher production of thromboxane A, and release of 5-hydroxy-tryptamine (15). The present study indicates that PAF receptor coupled PLC activation is also affected in diabetes. In contrast to our observations the [32P]-PA formation was shown to be decreased in diabetic human platelets stimulated with thrombin (16). However, in another report thrombin induced phosphoinositide hydrolysis, intracellular Ca” mobilization and P20 phosphorylation was significantly increased in human platelets from non-insulin dependent diabetes mellitus with enhanced platelet aggregation rates (17). Further, an increase in phospholipase activity in platelets from diabetic subjects was observed (18). These reports (17, 18) corroborate our studies with PAF. We have established that diabetic human platelets show hypersensitivity to PAF in both aggregation and [32P]-PA production compared to normal subjects. The fact that receptor antagonists blocked this PA production indicated that this response is PAF receptor related. Thus platelets of diabetic patients exhibit some modification in PAF receptor associated PPI turnover mechanism. The nature of this modification is not known. It could be at the receptor level, at the level of transduction elements or at some other step (19). Furthermore, the increase in the content of HbAlc showed significant correlationship to [32Pl-PA production. The relationship of the degree of aggregation and [3’P]-PA production to the level of glycosylated hemoglobin suggest that the insulin deficiency may contribute to these effects. ACKNOWLEDGEMENTS We are grateful to Ms. Kristin Nelson for her expert typing of the manuscript. This work was supported by National Institutes of Diabetes and Digestive and Kidney Diseases Grant (DK 35170) and by a NIH Research Career Development Award (DK 01782) to Shivendra D. Shukla.
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4. HALUSKKA, P.V., CURTIS ROGERS, R., LOADHOLT, C.V. AND COLWELL, J.A. Increased Platelet Thromboxane Synthesis in Diabetes Mellitus. I. Lab. Clin. Med. 97, 87-96 (1981). 5. BRAQUET, P.L., TOUQUI, L., SHEN, T.Y., VARGAFTIG, Research. Pharmacological Rev. 39, 97-145 (1987). 6. SHUKLA, S.D. Inositol Phospholipid Lipids 26, 1028-1033 (1991).
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17. ISHII, H., UMEDA, F., HASHIMOTO, T. AND NAWATA, H. Changes in Phosphoinositide Turnover, Ca2’Mobilization and Protein Phosphorylation in Platelets from NIDDM Patients. Diabetes 39, 1561-1568 (1990).
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