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Refractoriness of diabetic platelets to inhibitory prostaglandins
Prostaglandins and Medicine 7:
341-347,
1981
REFRACTORINESS OF DIABETIC PLATELETS TO INHIBITORY PROSTAGLANDINS M. Lagarde, P. Berciaud, M. Burtin & M. Dechavanne INSERM U63, Institut Pasteur, Laboratoire d'Hemobiologie Faculte Alexis Carrel 69372 Lyon Cedex 2, France (reprint requests to ML) ABSTRACT Inhibition of collagen-induced platelet aggregation by either endothelial extracts, prostacyclin, prostaglandin El or prostaglandin D2 was investigated. The inhibition was less efficient with diabetic platelets than with platelets from normal donors. The refractoriness of diabetic platelets to inhibitory prostaglandins was observed both with platelet-rich plasma and platelets isolated from their plasma. Moreover, levels of cyclic AMP in resting platelets and after stimulation by either PGEl or PGD2 were lower in diabetic platelets than in normal platelets. It is concluded that the weaker response of diabetic platelets to inhibitory prostaglandins could be related to their content in cyclic AMP. INTRODUCTION Platelets in patients with diabetes mellitus have been shown to be hypersensitive to aggregating agents by several groups (See the review of Bern, I). One of these groups has recently reported an increased production of a prostaglandin E-like material in diabetic-rich plasma (2) and a refractoriness of diabetic platelets to imidazole (3), a thrombosane synthetase inhibitor (4). Another group has reported an increase of thromboxane B2 formation by diabetic platelet-rich plasma (5). In a recent paper, using diabetic platelets isolated from their plasma, we described a normal synthesis of thromboxane BP from exogenous arachidonic acid but an increased synthesis under thrombin stimulation (6). In the same paper, we also described an increase of the half-life of thromboxane A2 in diabetic plasma. In this study, we investigated the response of collagen-induced aggregation to endothelial cell extract and to PGI2, PGE1, PGD2, all pros-
341
taglandins known to inhibit platelet aggregation (7-9). We also measured the level of platelet cyclic AMP under resting conditions and after stimulation by either PGE 1 or PGD2 since it is well known that these prostaglandins inhibit platelets by increasing the level of cyclic AMP (10). METHODS
Subjects. Diabetic patients (17 to 64 years old, average 29.3) had various durations of diabetes (0.25 to 32 years, average 14.2). The group was consisted of 12 men and 16 women. 4 of them had no complications : 16 had only a retinopathy ; 8 had other associated complications ; neuropathy (4 cases), peripheral vascular disease (2 cases), neuropathy + peripheral vascular disease (2 cases). Every patient had not taken any drug for two weeks, except for insulin. Platelets from diabetics and normal donors of the same age and sex were simultaneously investigated. Reagents. Collagen was obtained from Horm, Munich. PGI2 (sodium salt), PGEl and PGD2 were generous gifts from Dr. J.E. Pike of Upjohn, Kalamazoo. Endothelial cell extract provided a prostacyclin synthetase activity. Briefly, endothelial layers were separated from the vein of human umbilical cord and put into cold distilled water in order to induce an osmotic shock. Then, the mixture was filtered through millipore papers (0 = 0.45 pm). The filtrate was lyophilised and stored at -70°C until utilisation. The powder was resuspended in a Tyrode solution just before use and added in the aggregometer cuvette to inhibit platelet aggregation. This inhibition was counteracted by either 15hydroperoxyeicosatetraenoic acid or tranylcypromine indicating that the inhibition of platelet aggregation was due to prostacyclin synthetase activity (II). Platelet studies. Platelet aggregation was performed according to the method of Born (12), either on titrated platelet-rich plasma or platelets isolated from their plasma as previously described (13). Inhibitors were preincubated for 2 min. at 37°C before addition of collagen. The same concentrations of collagen and inhibitors were used for each control diabetic pair. The collagen concentrations were 1.25 or 2.5 I-g/ml.The range of prosta landin concentrations was 1.4 x IO-sM 5.6 x IO-'M for PGI,, 17 x IO-8M - 34 x IO-'M for PGEl and 11 x IO-'M22 x IO-'M for PGD2. Platelet cyclic AMP was measured as described (14) with a radioimmunoassay kit provided by the Institut Pasteur. PGE 2 x 10m6M and PGD, 1 x 10s6M were incubated at 37°C for 2 min. behore platelet lysis. Statistics. Student'spaired test or plain student'stest were used. RESULTS
Inhibition of platelet aggregation. We investigated the inhibition of collagen-induced platelet aggregation by an endothelial cell extract and three different inhibitory prostaglandins (PGI,, PGE, and PGD,).
342
Table I shows the results expressed as percentage inhibition of platelet aggregation. This percentage was significantly decreased in diabetic platelet-rich plasma.
I
I
I Endothelial extract (n = 26)
Controls
I
Diabetics
I
40.8
55.8
Paired test
15.0 + 36.3
p < 0.05
1
I 4
PGL, (n =L28)
53.6
PGE, (n = 17)
66.4 I
t
I
PGD, (n =L13)
27.3
]
37.5 I
67.4
I
40.4
I
I
26.3 + 27.7
p < 0.001
28.8 2 42.8
p < 0.02
27.0 zk 42.0
p < 0.05
I
T!SLE I. Effect of endathelial extract and inhibitory prostaglandins on aggregation of platelet-rich plasma induced by collagen. Results represent the mean percentage inhibition. This difference was found to a similar extent with platelets isolated from their plasma, thus indicating its relation to platelets (Table II).
Controls
Diabetics
63.0
48.7
14.3 f 16.6
p < 0.01
PG12 (n = 19)
61.5
47.1
14.4 +_22.6
p < 0.02
PGE, (n = 11)
67.9
29.8
38.0 f 28.2
p < 0.01
PGD, (n = 12)
71.8
44.2
27.5 + 25.7
p co.01
Endothelial extract (n = 15)
Paired test
TABLE II. Effect of endothelial extract and inhibitory prostaglandins on aggregation of platelets isolated from their plasma induced by collagen. Results represent the mean percentage inhibition. Platelet cyclic AMP. Measuring levels of platelet cyclic AMP under resting conditions and after stimulation by either PGEl 2 x 10 M or PGD2 10B6M, we found a lower level of cyclic AMP in diabetic platelets both under basal conditions and after prostaglandin stimulation (Table III).
343
r
Paired test
Controls
Diabetics
None (n = 19)
83.3
72.1
11.2 * 21.9
p < 0.05
PGE, (n = 19)
l&2.5
131.3
51.2 klO4.8
p < 0.05
PGD, (n = 16)
222.2
169.7
52.5 ?r 93.3
p < 0.05
TABLE III. Levels of platelet cyclic AMP under resting conditions (none) and after 2 min. stimulation by either PGEl 2 x 10m6 M or PGD, 1 x 10-6M. Results in pmoles/lOg platelets.
In contrast, the ratio of stimulated to control CAMP levels with either PGE, or PGD2 was not different in diabetic platelets as compared to normal platelets (Table IV). C El/c
DB1/D
cD*/c:
DD,/D
2.19 ?r 1.09
1.82 + 0.49
2.88 + 1.29
2.57 + 1.11
(n = 16) N.S.
(n = 19) N.S.
TABLE IV. Ratios of cyclic AMP stimulation by either PGE, or PGD, and C : levels of cyclic TGdiabetic and control platelets. C, C AMP controls in resting platelets, afte!'stimuli:ions by PGEr and PGD, respectively. In the same way for D, DE, and DD, in diabetics.
DISCUSSION Besides an increase of thromboxane A2 formation in diabetic platelets (5,6) a decrease of prostacyclin synthetase activity in vascular walls of diabetic rats and humans (15.16) was observed. We investigated the platelet aggregation response to prostacyclin (PGI.,_) and also to other prostaglandins, known to inhibit through the increase of platelet cyclic AMP (10.17). Our results show a refractoriness of collagen-induced aggregation to inhibition by three different prostaglandins as well as prostacyclin synthetase. This refractoriness was observed to the same extent in either the presence or the absence of plasma. This fact indicates that the refractoriness is related to platelets and not to plasma. This point is important to specify since we have previously found a different aggregation response of diabetic platelets to arachidonate in the presence of plasma (6). The refractoriness was observed to the
344
same extent with PGI,, PGE, or PGD, which are known to have different membrane sites on human platelets (18,19). This suggests an abnormality of the mechanism of inhibition rather than of the receptor sites. On the other hand, we believe that the inhibition of collagen-induced platelet aggregation by prostacyclin synthetase is relevant because it indicates the possibility of producing prostacyclin from platelet arachidonate or/and prostaglandin endoperoxides under collagen stimulation. This possibility has been recently described under thrombin stimulation (20). In addition, we have found a low level of platelet cyclic AMP in diabetics under resting conditions as well as after stimulation by either PGE, or PGD,. In contrast, the ratio of cyclic AMP increase by either PGEl or PGD, was not significantly different in diabetic platelets as compared to controls. Thus, the refractoriness of diabetic platelets to inhibitory prostaglandins could be related to the lower level of cyclic AMP rather than to a deficient response of adenylate cyclase. In our previous study (6) we have described that diabetic platelets exhibit an increase of phospholipase activity induced by thrombin. Since it is well known that cyclic AMP inhibits platelet phospholipase activity (21,22), we suggest that the increase of this activity that we observed is directly related to the low cyclic AMP level. Lastly, preliminary results showed a decrease of the basal level of platelet PGE, in diabetics (results not shown). This fact and those mentioned above lead us to the following hypothesis. Hepatic A-6 desaturase, able to convert linoleic acid (9,12-octadecadienoic acid) to gammalinolenic acid (6,9,12-octadecatrienoicacid), has a reduced activity in different conditions including the lack of insulin (23-25). So, it is conceivable to imagine a decrease of gamma linolenic acid formation in diabetes mellitus and subsequently a decrease of dihomogammalinolenic acid (8,11,14-eicosatrienoicacid) in tissues. Thus dihomogammalinolenic acid, the precursor of PGE,, could be diminished in diabetic platelets, consequently leading to the diminution of PGE, and cyclic AMP. ACKNOWLEDGMENTS This work was supported by a grant from INSERM (ASR N'5). We thank Miss B. Toor who reviewed the english manuscript. REFERENCES 1. Bern MM. Platelet functions in diabetes mellitus. Diabetes 27: 342350, 1978. 2. Halushka PV, Lurie D & Colwell JA. Increased synthesis of prostaglandin E-like material by platelets from patients with diabetes mellitus. New Engl. Med. 297: 1306-1310, 1977. 3. Colwell JA, Nair RMG, Halushka PV, Rogers C, Whetsell A & Sage1 J. Platelet adhesion and aggregation in diabetes mellitus. Metabolism 28: 394-400, 1979.
345
4. Moncada S, Bunting S, Mullane K, Thorogood P, Vane JR, Raz A & Needleman P. Imidazole : a selective inhibitor of thromboxane synthetase. Prostaglandins 13: 611-618, 1977. 5. Butkus A, Skrinska VA & Schumacher OP. Thromboxane production and platelet aggregation in diabetic subjects with clinical complications. Thrombos. Res. 19: 211-223, 1980. 6. Lagarde M, Burtin M, Berciaud P, Blanc M, Velardo B & Dechavanne M. Increase of platelet thromboxane Az formation and of its plasmatic half-life in diabetes mellitus. Thrombos. Res. 19: 823-830, 1980. 7. Moncada S, Gryglewski R, Bunting S & Vane SR. An enzyme isolated from arteries transforms prostaglandin endoperoxides to an unstable substance that inhibits platelet aggregation. Nature 263: 663-665, 1976. 8. Kloeze J. Relationship between chemical structure and plateletaggregation activity of prostaglandins. Biochim. Biophys. Acta 187: 285-292, 1969. 9. Smith JB, Silver MI, Ingerman C & Kocsis JJ. Prostaglandin D, inhibits the aggregation of human platelets. Thrombos. Res. 5: 291199, 1974. IO. Mills DCB & Macfarlane DE. Prostaglandins and platelet adenylate cyclase. p. 219-233 in Prostaglandins in Hematology (MJ Silver, JB Smith & JJ Kocsis eds) Spectrum Publications, New York, 1977. 11. Moncada S, Gryglewski R, Bunting S & Vane JR. A lipid peroxide inhibits the enzyme in blood vessel microsomes that generates from prostaglandin endoperoxides the substance (prostaglandin X) which prevents platelet aggregation. Prostaglandins 12: 715-737, 1976. 12. Born GVR. Aggregation of blood platelets by adenosine di-phosphate and its reversal. Nature 194: 927-929, 1962. 13. Lagarde M, Bryon PA, Guichardant M & Dechavanne M. A simple and efficient method for platelet isolation from their plasma. Thrombos. Res. 17: 581-588, 1980. 14. Lagarde M, Bryon PA, Vargaftig BB & Dechavanne M. Impairment of platelet thromboxane A, generation and of the platelet release reaction in two patients with congenital deficiency of platelet cyclo-oxygenase. Brit. J. Haematol. 38: 251-266, 1978. 15. Johnson M, Harrison HE, Raftery AT & Elder JB. Vascular prostacyclin may be reduced in diabetes in man. Lancet i: 325-326, 1979. 16. Silberbauer K, Schernthaner G, Sinzinger H, Piza-Katzer H & Winter M. Decreased vascular prostacyclin in juvenile-onset diabetes. New Engl. Med. 300: 366-367, 1979. 17. Gorman RR, Bunting S & Miller OV. Modulation of human platelet adenylate cyclase by prostacyclin (PGX). Prostaglandins 13: 377-388, 1977, 346
18. Miller OG & Gorman RR. Evidence for distinct prostaglandin I2 and D2 receptors in human platelets. J. Pharmacol. Exp. Ther. 210: 134-140, 1979. 19. Schafer AI, Cooper B, O'Hara & Handin RI. Identification of platelet receptors for prostaglaudin I2 and Dz. J. Biol. Chem. 254: 2914-2917, 1979. 20. Marcus AJ, Weksler BB, Jaffe EA & Broekman MJ. Synthesis of prostacyclin from platelet-derived endoperoxides by cultured human endothelial cells. J. Clin. Invest. 66: 979-986, 1980. 21. Lapetina EG, Schmitges CJ, Chandrabose K & Cuatrecasas P. Cyclic AMP and prostaglandin inhibit membrane phospholipase activity in platelets. Biochem. Biophys. Res. Commun. 76: 828-835, 1977. 22. Minkes M, Stanford N, Chy My, Roth GJ, Raz A, Needleman P & Majerus PW. Cyclic AMP inhibits the availability of arachidonate to prostaglandin synthetase in human platelet suspensions. J. Clin. Invest. 59: 449-454, 1977. 23. Mercuri 0, Peluffo RO & Brenner RR. Depression of microsomal desaturation of linoleic to gamma-linolenic acid in the alloxan diabetic rat. Biochim. Biophys. Acta 116: 409-411, 1966. 24. Poisson JP, Lemarchal P, Blond JP, Lecerf J & Mendy F. Influence du diabete alloxanique sur la conversion des acides linoleyque et gamma-linolenique en acide arachidonique chez le rat in vivo. Diabete et Metabolisme 4: 39-45, 1978. 25. Faas FH & Carter WJ. Altered fatty acid desaturation and microsoma1 fatty acid composition in the streptozotocin diabetic rat. Lipids 15: 953-961, 1980.
347
341-347,
1981
REFRACTORINESS OF DIABETIC PLATELETS TO INHIBITORY PROSTAGLANDINS M. Lagarde, P. Berciaud, M. Burtin & M. Dechavanne INSERM U63, Institut Pasteur, Laboratoire d'Hemobiologie Faculte Alexis Carrel 69372 Lyon Cedex 2, France (reprint requests to ML) ABSTRACT Inhibition of collagen-induced platelet aggregation by either endothelial extracts, prostacyclin, prostaglandin El or prostaglandin D2 was investigated. The inhibition was less efficient with diabetic platelets than with platelets from normal donors. The refractoriness of diabetic platelets to inhibitory prostaglandins was observed both with platelet-rich plasma and platelets isolated from their plasma. Moreover, levels of cyclic AMP in resting platelets and after stimulation by either PGEl or PGD2 were lower in diabetic platelets than in normal platelets. It is concluded that the weaker response of diabetic platelets to inhibitory prostaglandins could be related to their content in cyclic AMP. INTRODUCTION Platelets in patients with diabetes mellitus have been shown to be hypersensitive to aggregating agents by several groups (See the review of Bern, I). One of these groups has recently reported an increased production of a prostaglandin E-like material in diabetic-rich plasma (2) and a refractoriness of diabetic platelets to imidazole (3), a thrombosane synthetase inhibitor (4). Another group has reported an increase of thromboxane B2 formation by diabetic platelet-rich plasma (5). In a recent paper, using diabetic platelets isolated from their plasma, we described a normal synthesis of thromboxane BP from exogenous arachidonic acid but an increased synthesis under thrombin stimulation (6). In the same paper, we also described an increase of the half-life of thromboxane A2 in diabetic plasma. In this study, we investigated the response of collagen-induced aggregation to endothelial cell extract and to PGI2, PGE1, PGD2, all pros-
341
taglandins known to inhibit platelet aggregation (7-9). We also measured the level of platelet cyclic AMP under resting conditions and after stimulation by either PGE 1 or PGD2 since it is well known that these prostaglandins inhibit platelets by increasing the level of cyclic AMP (10). METHODS
Subjects. Diabetic patients (17 to 64 years old, average 29.3) had various durations of diabetes (0.25 to 32 years, average 14.2). The group was consisted of 12 men and 16 women. 4 of them had no complications : 16 had only a retinopathy ; 8 had other associated complications ; neuropathy (4 cases), peripheral vascular disease (2 cases), neuropathy + peripheral vascular disease (2 cases). Every patient had not taken any drug for two weeks, except for insulin. Platelets from diabetics and normal donors of the same age and sex were simultaneously investigated. Reagents. Collagen was obtained from Horm, Munich. PGI2 (sodium salt), PGEl and PGD2 were generous gifts from Dr. J.E. Pike of Upjohn, Kalamazoo. Endothelial cell extract provided a prostacyclin synthetase activity. Briefly, endothelial layers were separated from the vein of human umbilical cord and put into cold distilled water in order to induce an osmotic shock. Then, the mixture was filtered through millipore papers (0 = 0.45 pm). The filtrate was lyophilised and stored at -70°C until utilisation. The powder was resuspended in a Tyrode solution just before use and added in the aggregometer cuvette to inhibit platelet aggregation. This inhibition was counteracted by either 15hydroperoxyeicosatetraenoic acid or tranylcypromine indicating that the inhibition of platelet aggregation was due to prostacyclin synthetase activity (II). Platelet studies. Platelet aggregation was performed according to the method of Born (12), either on titrated platelet-rich plasma or platelets isolated from their plasma as previously described (13). Inhibitors were preincubated for 2 min. at 37°C before addition of collagen. The same concentrations of collagen and inhibitors were used for each control diabetic pair. The collagen concentrations were 1.25 or 2.5 I-g/ml.The range of prosta landin concentrations was 1.4 x IO-sM 5.6 x IO-'M for PGI,, 17 x IO-8M - 34 x IO-'M for PGEl and 11 x IO-'M22 x IO-'M for PGD2. Platelet cyclic AMP was measured as described (14) with a radioimmunoassay kit provided by the Institut Pasteur. PGE 2 x 10m6M and PGD, 1 x 10s6M were incubated at 37°C for 2 min. behore platelet lysis. Statistics. Student'spaired test or plain student'stest were used. RESULTS
Inhibition of platelet aggregation. We investigated the inhibition of collagen-induced platelet aggregation by an endothelial cell extract and three different inhibitory prostaglandins (PGI,, PGE, and PGD,).
342
Table I shows the results expressed as percentage inhibition of platelet aggregation. This percentage was significantly decreased in diabetic platelet-rich plasma.
I
I
I Endothelial extract (n = 26)
Controls
I
Diabetics
I
40.8
55.8
Paired test
15.0 + 36.3
p < 0.05
1
I 4
PGL, (n =L28)
53.6
PGE, (n = 17)
66.4 I
t
I
PGD, (n =L13)
27.3
]
37.5 I
67.4
I
40.4
I
I
26.3 + 27.7
p < 0.001
28.8 2 42.8
p < 0.02
27.0 zk 42.0
p < 0.05
I
T!SLE I. Effect of endathelial extract and inhibitory prostaglandins on aggregation of platelet-rich plasma induced by collagen. Results represent the mean percentage inhibition. This difference was found to a similar extent with platelets isolated from their plasma, thus indicating its relation to platelets (Table II).
Controls
Diabetics
63.0
48.7
14.3 f 16.6
p < 0.01
PG12 (n = 19)
61.5
47.1
14.4 +_22.6
p < 0.02
PGE, (n = 11)
67.9
29.8
38.0 f 28.2
p < 0.01
PGD, (n = 12)
71.8
44.2
27.5 + 25.7
p co.01
Endothelial extract (n = 15)
Paired test
TABLE II. Effect of endothelial extract and inhibitory prostaglandins on aggregation of platelets isolated from their plasma induced by collagen. Results represent the mean percentage inhibition. Platelet cyclic AMP. Measuring levels of platelet cyclic AMP under resting conditions and after stimulation by either PGEl 2 x 10 M or PGD2 10B6M, we found a lower level of cyclic AMP in diabetic platelets both under basal conditions and after prostaglandin stimulation (Table III).
343
r
Paired test
Controls
Diabetics
None (n = 19)
83.3
72.1
11.2 * 21.9
p < 0.05
PGE, (n = 19)
l&2.5
131.3
51.2 klO4.8
p < 0.05
PGD, (n = 16)
222.2
169.7
52.5 ?r 93.3
p < 0.05
TABLE III. Levels of platelet cyclic AMP under resting conditions (none) and after 2 min. stimulation by either PGEl 2 x 10m6 M or PGD, 1 x 10-6M. Results in pmoles/lOg platelets.
In contrast, the ratio of stimulated to control CAMP levels with either PGE, or PGD2 was not different in diabetic platelets as compared to normal platelets (Table IV). C El/c
DB1/D
cD*/c:
DD,/D
2.19 ?r 1.09
1.82 + 0.49
2.88 + 1.29
2.57 + 1.11
(n = 16) N.S.
(n = 19) N.S.
TABLE IV. Ratios of cyclic AMP stimulation by either PGE, or PGD, and C : levels of cyclic TGdiabetic and control platelets. C, C AMP controls in resting platelets, afte!'stimuli:ions by PGEr and PGD, respectively. In the same way for D, DE, and DD, in diabetics.
DISCUSSION Besides an increase of thromboxane A2 formation in diabetic platelets (5,6) a decrease of prostacyclin synthetase activity in vascular walls of diabetic rats and humans (15.16) was observed. We investigated the platelet aggregation response to prostacyclin (PGI.,_) and also to other prostaglandins, known to inhibit through the increase of platelet cyclic AMP (10.17). Our results show a refractoriness of collagen-induced aggregation to inhibition by three different prostaglandins as well as prostacyclin synthetase. This refractoriness was observed to the same extent in either the presence or the absence of plasma. This fact indicates that the refractoriness is related to platelets and not to plasma. This point is important to specify since we have previously found a different aggregation response of diabetic platelets to arachidonate in the presence of plasma (6). The refractoriness was observed to the
344
same extent with PGI,, PGE, or PGD, which are known to have different membrane sites on human platelets (18,19). This suggests an abnormality of the mechanism of inhibition rather than of the receptor sites. On the other hand, we believe that the inhibition of collagen-induced platelet aggregation by prostacyclin synthetase is relevant because it indicates the possibility of producing prostacyclin from platelet arachidonate or/and prostaglandin endoperoxides under collagen stimulation. This possibility has been recently described under thrombin stimulation (20). In addition, we have found a low level of platelet cyclic AMP in diabetics under resting conditions as well as after stimulation by either PGE, or PGD,. In contrast, the ratio of cyclic AMP increase by either PGEl or PGD, was not significantly different in diabetic platelets as compared to controls. Thus, the refractoriness of diabetic platelets to inhibitory prostaglandins could be related to the lower level of cyclic AMP rather than to a deficient response of adenylate cyclase. In our previous study (6) we have described that diabetic platelets exhibit an increase of phospholipase activity induced by thrombin. Since it is well known that cyclic AMP inhibits platelet phospholipase activity (21,22), we suggest that the increase of this activity that we observed is directly related to the low cyclic AMP level. Lastly, preliminary results showed a decrease of the basal level of platelet PGE, in diabetics (results not shown). This fact and those mentioned above lead us to the following hypothesis. Hepatic A-6 desaturase, able to convert linoleic acid (9,12-octadecadienoic acid) to gammalinolenic acid (6,9,12-octadecatrienoicacid), has a reduced activity in different conditions including the lack of insulin (23-25). So, it is conceivable to imagine a decrease of gamma linolenic acid formation in diabetes mellitus and subsequently a decrease of dihomogammalinolenic acid (8,11,14-eicosatrienoicacid) in tissues. Thus dihomogammalinolenic acid, the precursor of PGE,, could be diminished in diabetic platelets, consequently leading to the diminution of PGE, and cyclic AMP. ACKNOWLEDGMENTS This work was supported by a grant from INSERM (ASR N'5). We thank Miss B. Toor who reviewed the english manuscript. REFERENCES 1. Bern MM. Platelet functions in diabetes mellitus. Diabetes 27: 342350, 1978. 2. Halushka PV, Lurie D & Colwell JA. Increased synthesis of prostaglandin E-like material by platelets from patients with diabetes mellitus. New Engl. Med. 297: 1306-1310, 1977. 3. Colwell JA, Nair RMG, Halushka PV, Rogers C, Whetsell A & Sage1 J. Platelet adhesion and aggregation in diabetes mellitus. Metabolism 28: 394-400, 1979.
345
4. Moncada S, Bunting S, Mullane K, Thorogood P, Vane JR, Raz A & Needleman P. Imidazole : a selective inhibitor of thromboxane synthetase. Prostaglandins 13: 611-618, 1977. 5. Butkus A, Skrinska VA & Schumacher OP. Thromboxane production and platelet aggregation in diabetic subjects with clinical complications. Thrombos. Res. 19: 211-223, 1980. 6. Lagarde M, Burtin M, Berciaud P, Blanc M, Velardo B & Dechavanne M. Increase of platelet thromboxane Az formation and of its plasmatic half-life in diabetes mellitus. Thrombos. Res. 19: 823-830, 1980. 7. Moncada S, Gryglewski R, Bunting S & Vane SR. An enzyme isolated from arteries transforms prostaglandin endoperoxides to an unstable substance that inhibits platelet aggregation. Nature 263: 663-665, 1976. 8. Kloeze J. Relationship between chemical structure and plateletaggregation activity of prostaglandins. Biochim. Biophys. Acta 187: 285-292, 1969. 9. Smith JB, Silver MI, Ingerman C & Kocsis JJ. Prostaglandin D, inhibits the aggregation of human platelets. Thrombos. Res. 5: 291199, 1974. IO. Mills DCB & Macfarlane DE. Prostaglandins and platelet adenylate cyclase. p. 219-233 in Prostaglandins in Hematology (MJ Silver, JB Smith & JJ Kocsis eds) Spectrum Publications, New York, 1977. 11. Moncada S, Gryglewski R, Bunting S & Vane JR. A lipid peroxide inhibits the enzyme in blood vessel microsomes that generates from prostaglandin endoperoxides the substance (prostaglandin X) which prevents platelet aggregation. Prostaglandins 12: 715-737, 1976. 12. Born GVR. Aggregation of blood platelets by adenosine di-phosphate and its reversal. Nature 194: 927-929, 1962. 13. Lagarde M, Bryon PA, Guichardant M & Dechavanne M. A simple and efficient method for platelet isolation from their plasma. Thrombos. Res. 17: 581-588, 1980. 14. Lagarde M, Bryon PA, Vargaftig BB & Dechavanne M. Impairment of platelet thromboxane A, generation and of the platelet release reaction in two patients with congenital deficiency of platelet cyclo-oxygenase. Brit. J. Haematol. 38: 251-266, 1978. 15. Johnson M, Harrison HE, Raftery AT & Elder JB. Vascular prostacyclin may be reduced in diabetes in man. Lancet i: 325-326, 1979. 16. Silberbauer K, Schernthaner G, Sinzinger H, Piza-Katzer H & Winter M. Decreased vascular prostacyclin in juvenile-onset diabetes. New Engl. Med. 300: 366-367, 1979. 17. Gorman RR, Bunting S & Miller OV. Modulation of human platelet adenylate cyclase by prostacyclin (PGX). Prostaglandins 13: 377-388, 1977, 346
18. Miller OG & Gorman RR. Evidence for distinct prostaglandin I2 and D2 receptors in human platelets. J. Pharmacol. Exp. Ther. 210: 134-140, 1979. 19. Schafer AI, Cooper B, O'Hara & Handin RI. Identification of platelet receptors for prostaglaudin I2 and Dz. J. Biol. Chem. 254: 2914-2917, 1979. 20. Marcus AJ, Weksler BB, Jaffe EA & Broekman MJ. Synthesis of prostacyclin from platelet-derived endoperoxides by cultured human endothelial cells. J. Clin. Invest. 66: 979-986, 1980. 21. Lapetina EG, Schmitges CJ, Chandrabose K & Cuatrecasas P. Cyclic AMP and prostaglandin inhibit membrane phospholipase activity in platelets. Biochem. Biophys. Res. Commun. 76: 828-835, 1977. 22. Minkes M, Stanford N, Chy My, Roth GJ, Raz A, Needleman P & Majerus PW. Cyclic AMP inhibits the availability of arachidonate to prostaglandin synthetase in human platelet suspensions. J. Clin. Invest. 59: 449-454, 1977. 23. Mercuri 0, Peluffo RO & Brenner RR. Depression of microsomal desaturation of linoleic to gamma-linolenic acid in the alloxan diabetic rat. Biochim. Biophys. Acta 116: 409-411, 1966. 24. Poisson JP, Lemarchal P, Blond JP, Lecerf J & Mendy F. Influence du diabete alloxanique sur la conversion des acides linoleyque et gamma-linolenique en acide arachidonique chez le rat in vivo. Diabete et Metabolisme 4: 39-45, 1978. 25. Faas FH & Carter WJ. Altered fatty acid desaturation and microsoma1 fatty acid composition in the streptozotocin diabetic rat. Lipids 15: 953-961, 1980.
347