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Biological effects of 15-hydroperoxythromboxane A2 on platelets and aorta
Prostaglandinsand Medicine 4: 297-302, 1980
BIOLOGICAL
EFFECTS
OF 15-HYDROPEROXYTHROMBOXANE
PLATELETS
Department Sweden
A2 ON
AN'D AORTA
Sven HammarstrZjm of Chemistry, Karolinska Institutet,
S-104 01 Stockholm,
ABSTRACT
The conversion of PGG2 to 15-hydroperoxythromboxane A2 by purified thromboxane synthase from human platelets was recently reported (HammarstrZjm, S. (1980) J. Biol. Chem.255, 518). The hydroperoxythromboxane is approximately 25 times more potent than PGG2 in causing aggregation of human platelet rich plasma and 30 times more potent than PGG2 as an inducer of contractions of rabbit aorta.
INTRODUCTION
An enzyme from human platelets (1) and bovine lung (2) converts the prostaglandin (PG) endoperoxide PGH2 into thromboxane A2(1,2)and PGH3 (3) into thromboxane A3. PGHl is not however, efficiently transformed to thromboxane Al (4) and a A5 double bond in the substrate seems to be essential for the conversion (5). Thromboxane A has profound biological effects on platelets and smooth muscles (6f . Recently, the conversion of PGG2 to 15-hydroperoxythromboxane A2 (Fig. 1) @as demonstrated using purified platelet enzyme (7). The present report describes some biological effects of the hydroperoxythromboxane.
MATERIALS
AND
METHODS
PGG2 and PGH2 were prepared from [l-14C] arachidonic acid as described (7). Platelet thromboxane s-ynthase was solubilized from microsomes with Triton X-100 and purified by DEAE cellulose chromatography (1).
297
b-0-H PGG2
FH+
OH
6:: _ -
15-hydroperoxy-
15-hydroperox y Fig. 1
-TX62
Conversions synthase.
Generation
HPHT
TXA2
0 MDA
of prostaglandin
and determination
G2 by platelet thromboxane
of thromboxanes
Enzyme solution (0.2 M eluate (11, 0.45 mg protein in 0.18 ml) and 20 ~1 1 M Tris buffer H 7.4 were preincubated at 37OC for 1 min. [l-l4C] PGG2 or [I-14CrPGH2 (10 nmol) was added. After 10 set at 370C aliquots were removed for bioassay on PRP or rabbit aorta strips. Simultaneously, 100 1-11 of the incubation mixture was mixed with 5 vol of ethanol containing SnC12 (5 mg/ml). After 2 min, 0.5 ml water, 10 ug thromboxane B2 and 10 ug PGF2, were added. The mixture was acidified to pH 3 and extracted with diethyl ether. The washed extracts were methylated with diazomethane and analyzed by TLC (solvent, diethyl ether/methanol, 49:l v/v, = solvent A). The remainder of the incubation mixture was mixed with 80 vol of methanol, left at 20°C for 1 hr, evaporated to dryness, dissolved in 0.1 ml 0.1 M Tris buffer, pH 7.4 and treated with ethanolic SnCl2 as described above. TLC analysis was performed with ethyl acetate/2,2,4 trimethylpentane/water, 75:75:100 v/v/v (organic phase = solvent B). Zones containing PGF2c, thromboxane B2 (solvent A), and O-methyl thromboxane B2 (solvent B) were removed and counted in a Packard Tri-Carb liquid scintillation counter. 298
Platelet
aggregation
Blood from healthy donors was collected with 0.13 volumes of 0.1 M trisodium citrate and centrifuged at 200 x g for 15 min. The platelet rich plasma (PRP) thus obtained was mixed with 0.25 M CaC12 (30 ul/ml PRP) and preincubated with indomethacin (10B5M, 2 min, 37OC). Aggregation was monitored with a Payton model 300 B aggregometer.
Superfused
rabbit aorta strip
Thoracic aorta from male rabbits (ca 2 kg) was cut spirally into 3 mm wide and 3 cm long strips. The lower end was attached to a glass rod with a platinum needle and the upper end to a Grass FT.03C transducer, connected to a Grass 79C polygraph. The strips (stretched by 0.5 ponds) were superfused (8) with 2 ml/min KrebsHenseleit bicarbonate medium (9) supplemented with 0.2% glucose and gassed with 6.5% CO2 in 02. Compounds were dissolved in 0.5 ml Krebs medium and applied to strips during 15 sec. Before starting an experiment, dose-response curves for PGG2 and PGH2 were deterhyomined. At the indicated concentrations, mepyramine maleate seine bromide, phenoxybenzamine hydrochloride (each at 10-lo g/l), methyser ide bimaleate (2 x lo-10 g/l) and propranol hydrochloride (2 x lo- 3 g/l) did not influence the responses obtained.
RESULTS
Effects of 15-hydroperoxythromboxane
A2 on platelets
PRP supplemented with CaC12 and preincubated with indomethacin was used for these experiments. Fig. 2 shows the effects of 350 ng/ml PGH2 and 1 ug/ml PGG2 on light transmission through PRP.Thm oxane A2 and 15-hydroperoxythromboxane A2 were generated by short (10 set) incubations of PGH2 and PGG2 with purified thromboxane synthase. Aliquots were added to PRP and the remainders of the incubation mixtures were used for analyses of prostaglandin endoperoxide and (hydroperoxy) thromboxane A concentrations: PGG2 and PGH2 were converted to PGF2c by SnC12 reduction; 15-hydroperoxythromboxane A2 and thromboxane A2 were trapped as O-methyl derivatives and O-methyl 15-hydroperoxythromboxane B2 was reduced by SnC12 (7). After TLC, the amounts of radioactive PGF2, and O-methyl thromboxane B2 were determined. Fig. 2 shows the effects of 20 ng/ml thromboxane A2 (plus 22 ng/ml PGH2) and 24 ng/ml 15-hydroperoxythromboxane A2 (plus 56 ng/ml of PGG2) on the turbidity of human PRP. In both cases the concentrations of unconverted endoperoxides were too low to induce aggregation. Comparisons of dose-response curves for PGG2 and 15-hydroperoxythromboxane A2 showed that the thromboxane was approximately 25 times more potent than the endoperoxide in causing platelet aggregation. 299
long TXA2 (11ng PGH2 I
L
0
s 20.E 40: 'i 60: c 80+ loo-
'1 12ng HPTXA2 (28ngPGG2)
Fig. 2
Platelet aggregation induced by 175 ng PGH2, 10 ng thromboxane A2, 500 ng PGG2, and 12 ng 15-hydroperoxythromboxane A2 in recalcified, indomethacin treated human PRP (0.5 ml).
Effects of 15-hydroperoxythromboxane
A2 on rabbit aorta strips
Superfused rabbit aorta strips were used. Fig. 3 shows the effect of 400 ng/ml PGH2 and 890 ng/ml PGG2 (A and C, respectively) Thromboxane A2 (75 ng/ml plus 23 ng/ml PGH2) and 15-hydroperoxythromboxane A2 (180 ng/ml plus 115 ng/ml PGG2) gave the responses shown by tracings B and D. From dose-response curves, 15-hydroperoxythromboxane A2 was approximately 30 times more potent than PGG2 as an inducer of rabbit aorta contractions.
DISCUSSION PGH is presumably the ordinary substrate for thromboxane synthase Tcf. 10). Under pathological conditions PGG2 may accumulate (11) and 15-hydroperoxythromboxane A2 formation occur. 300
0.5g L
5min
Fig. 3
Contractile responses of superfused rabbit aorta strips B: 75 ng/ml thromboxane A2 (plus to A: 400 ng/ml PGH2; C: 890 ng/ml PGG2 and D: 180 ng/ml 23 ng/ml PHG2); 15-hydroperoxythromboxane A2 (plus 115 ng/ml PGG2).
Thromboxane A2 is a powerful inducer of platelet aggregation and rabbit aorta contractions (6). The results presented here show that 15-hydroperoxythromboxane A2 has similar properties. Furthermore, its potency is similar to that of thromboxane A2.
ACKNOWLEDGEMENTS The excellent technical assistance of Mrs Saga Elwe is gratefully acknowledged. This work was supported by a grant from the Swedish Medical Research Council (03X-217).
REFERENCES 1. Hammarstrom, S. and Falardeau, P. Resolution of prostaglandin endoperoxide synthase and thromboxane synthase of human platelets. Proc. Natl. Acad. Sci. USA 74:3691, 1977. 2. Wlodawer, P. and Hammarstrom, S. Thromboxane synthase from bovine 1ung:solubilization and partial purification. Biochem. Biophys. Res. Commun 80:525, 1978. 3. Needleman, P., Raz, A., Minkes, M.S., Ferrendelli, J.A. and Sprecher, H. Triene prostaglandins: Prostacyclin and thromboxane biosynthesis and unique biological properties. Proc. Nat1 Acad. Sci. USA 76:944, 1979. 301
4.
Diczfalusy, U., Falardeau, P. and HammarstrBm, S. Conversion of prostaglandin endoperoxides to Cl7 hydroxy acids catalyzed by human platelet thromboxane synthase. FEBS Lett 84:271, 1977,
5. Dicafalusy, U. and HammarstrBm, S. A structural requirement for the conversion of prostaglandin endoperoxides to thromboxanes. FEBS Lett. 105:291, 1979. 6. Samuelsson, B., Goldyne, M., Granstrom, E., Hamberg, M., Hammarstrem, S. and Malmsten, C. Prostaglandins and thromboxanes. Ann. Rev. Biochem. 47:997, 1978 7. Hammarstrijm, S. Enzymatic synthesis of 15-hydroperoxythromboxane A2 and 12-hydroperoxy-5,8,10-heptadecatrienoic acid. J. Biol. Chem. 255:518, 1980. 8. Piper, P. and Vane, J.R. Release of additional factors in anaphylaxis and its antagonism by anti-inflammatory drugs. Nature 223:29, 1969. 9. Krebs, H.A. and Henseleit, K. Untersuchungen iiber die Harnstoffbildung im Tierkgrper. Hoppe Seyler's 2. Physiol. Chem. 210:33, 1932. 10. Miyamoto, T., Ogino, N., Yamamoto, S. and Hayaishi, 0. Purification of prostaglandin endoperoxide synthetase from bovine vesicular gland microsomes. J. Biol. Chem. 251:2629, 1976. 11. Kuehl, F.A., Humes, J.L., Egan, R.W., Ham, E.A., Beveridge, G.C and van Arman, C.G. Role of prostaglandin endoperoxide PGG2 in inflammatory processes. Nature 265:170, 1977.
302
BIOLOGICAL
EFFECTS
OF 15-HYDROPEROXYTHROMBOXANE
PLATELETS
Department Sweden
A2 ON
AN'D AORTA
Sven HammarstrZjm of Chemistry, Karolinska Institutet,
S-104 01 Stockholm,
ABSTRACT
The conversion of PGG2 to 15-hydroperoxythromboxane A2 by purified thromboxane synthase from human platelets was recently reported (HammarstrZjm, S. (1980) J. Biol. Chem.255, 518). The hydroperoxythromboxane is approximately 25 times more potent than PGG2 in causing aggregation of human platelet rich plasma and 30 times more potent than PGG2 as an inducer of contractions of rabbit aorta.
INTRODUCTION
An enzyme from human platelets (1) and bovine lung (2) converts the prostaglandin (PG) endoperoxide PGH2 into thromboxane A2(1,2)and PGH3 (3) into thromboxane A3. PGHl is not however, efficiently transformed to thromboxane Al (4) and a A5 double bond in the substrate seems to be essential for the conversion (5). Thromboxane A has profound biological effects on platelets and smooth muscles (6f . Recently, the conversion of PGG2 to 15-hydroperoxythromboxane A2 (Fig. 1) @as demonstrated using purified platelet enzyme (7). The present report describes some biological effects of the hydroperoxythromboxane.
MATERIALS
AND
METHODS
PGG2 and PGH2 were prepared from [l-14C] arachidonic acid as described (7). Platelet thromboxane s-ynthase was solubilized from microsomes with Triton X-100 and purified by DEAE cellulose chromatography (1).
297
b-0-H PGG2
FH+
OH
6:: _ -
15-hydroperoxy-
15-hydroperox y Fig. 1
-TX62
Conversions synthase.
Generation
HPHT
TXA2
0 MDA
of prostaglandin
and determination
G2 by platelet thromboxane
of thromboxanes
Enzyme solution (0.2 M eluate (11, 0.45 mg protein in 0.18 ml) and 20 ~1 1 M Tris buffer H 7.4 were preincubated at 37OC for 1 min. [l-l4C] PGG2 or [I-14CrPGH2 (10 nmol) was added. After 10 set at 370C aliquots were removed for bioassay on PRP or rabbit aorta strips. Simultaneously, 100 1-11 of the incubation mixture was mixed with 5 vol of ethanol containing SnC12 (5 mg/ml). After 2 min, 0.5 ml water, 10 ug thromboxane B2 and 10 ug PGF2, were added. The mixture was acidified to pH 3 and extracted with diethyl ether. The washed extracts were methylated with diazomethane and analyzed by TLC (solvent, diethyl ether/methanol, 49:l v/v, = solvent A). The remainder of the incubation mixture was mixed with 80 vol of methanol, left at 20°C for 1 hr, evaporated to dryness, dissolved in 0.1 ml 0.1 M Tris buffer, pH 7.4 and treated with ethanolic SnCl2 as described above. TLC analysis was performed with ethyl acetate/2,2,4 trimethylpentane/water, 75:75:100 v/v/v (organic phase = solvent B). Zones containing PGF2c, thromboxane B2 (solvent A), and O-methyl thromboxane B2 (solvent B) were removed and counted in a Packard Tri-Carb liquid scintillation counter. 298
Platelet
aggregation
Blood from healthy donors was collected with 0.13 volumes of 0.1 M trisodium citrate and centrifuged at 200 x g for 15 min. The platelet rich plasma (PRP) thus obtained was mixed with 0.25 M CaC12 (30 ul/ml PRP) and preincubated with indomethacin (10B5M, 2 min, 37OC). Aggregation was monitored with a Payton model 300 B aggregometer.
Superfused
rabbit aorta strip
Thoracic aorta from male rabbits (ca 2 kg) was cut spirally into 3 mm wide and 3 cm long strips. The lower end was attached to a glass rod with a platinum needle and the upper end to a Grass FT.03C transducer, connected to a Grass 79C polygraph. The strips (stretched by 0.5 ponds) were superfused (8) with 2 ml/min KrebsHenseleit bicarbonate medium (9) supplemented with 0.2% glucose and gassed with 6.5% CO2 in 02. Compounds were dissolved in 0.5 ml Krebs medium and applied to strips during 15 sec. Before starting an experiment, dose-response curves for PGG2 and PGH2 were deterhyomined. At the indicated concentrations, mepyramine maleate seine bromide, phenoxybenzamine hydrochloride (each at 10-lo g/l), methyser ide bimaleate (2 x lo-10 g/l) and propranol hydrochloride (2 x lo- 3 g/l) did not influence the responses obtained.
RESULTS
Effects of 15-hydroperoxythromboxane
A2 on platelets
PRP supplemented with CaC12 and preincubated with indomethacin was used for these experiments. Fig. 2 shows the effects of 350 ng/ml PGH2 and 1 ug/ml PGG2 on light transmission through PRP.Thm oxane A2 and 15-hydroperoxythromboxane A2 were generated by short (10 set) incubations of PGH2 and PGG2 with purified thromboxane synthase. Aliquots were added to PRP and the remainders of the incubation mixtures were used for analyses of prostaglandin endoperoxide and (hydroperoxy) thromboxane A concentrations: PGG2 and PGH2 were converted to PGF2c by SnC12 reduction; 15-hydroperoxythromboxane A2 and thromboxane A2 were trapped as O-methyl derivatives and O-methyl 15-hydroperoxythromboxane B2 was reduced by SnC12 (7). After TLC, the amounts of radioactive PGF2, and O-methyl thromboxane B2 were determined. Fig. 2 shows the effects of 20 ng/ml thromboxane A2 (plus 22 ng/ml PGH2) and 24 ng/ml 15-hydroperoxythromboxane A2 (plus 56 ng/ml of PGG2) on the turbidity of human PRP. In both cases the concentrations of unconverted endoperoxides were too low to induce aggregation. Comparisons of dose-response curves for PGG2 and 15-hydroperoxythromboxane A2 showed that the thromboxane was approximately 25 times more potent than the endoperoxide in causing platelet aggregation. 299
long TXA2 (11ng PGH2 I
L
0
s 20.E 40: 'i 60: c 80+ loo-
'1 12ng HPTXA2 (28ngPGG2)
Fig. 2
Platelet aggregation induced by 175 ng PGH2, 10 ng thromboxane A2, 500 ng PGG2, and 12 ng 15-hydroperoxythromboxane A2 in recalcified, indomethacin treated human PRP (0.5 ml).
Effects of 15-hydroperoxythromboxane
A2 on rabbit aorta strips
Superfused rabbit aorta strips were used. Fig. 3 shows the effect of 400 ng/ml PGH2 and 890 ng/ml PGG2 (A and C, respectively) Thromboxane A2 (75 ng/ml plus 23 ng/ml PGH2) and 15-hydroperoxythromboxane A2 (180 ng/ml plus 115 ng/ml PGG2) gave the responses shown by tracings B and D. From dose-response curves, 15-hydroperoxythromboxane A2 was approximately 30 times more potent than PGG2 as an inducer of rabbit aorta contractions.
DISCUSSION PGH is presumably the ordinary substrate for thromboxane synthase Tcf. 10). Under pathological conditions PGG2 may accumulate (11) and 15-hydroperoxythromboxane A2 formation occur. 300
0.5g L
5min
Fig. 3
Contractile responses of superfused rabbit aorta strips B: 75 ng/ml thromboxane A2 (plus to A: 400 ng/ml PGH2; C: 890 ng/ml PGG2 and D: 180 ng/ml 23 ng/ml PHG2); 15-hydroperoxythromboxane A2 (plus 115 ng/ml PGG2).
Thromboxane A2 is a powerful inducer of platelet aggregation and rabbit aorta contractions (6). The results presented here show that 15-hydroperoxythromboxane A2 has similar properties. Furthermore, its potency is similar to that of thromboxane A2.
ACKNOWLEDGEMENTS The excellent technical assistance of Mrs Saga Elwe is gratefully acknowledged. This work was supported by a grant from the Swedish Medical Research Council (03X-217).
REFERENCES 1. Hammarstrom, S. and Falardeau, P. Resolution of prostaglandin endoperoxide synthase and thromboxane synthase of human platelets. Proc. Natl. Acad. Sci. USA 74:3691, 1977. 2. Wlodawer, P. and Hammarstrom, S. Thromboxane synthase from bovine 1ung:solubilization and partial purification. Biochem. Biophys. Res. Commun 80:525, 1978. 3. Needleman, P., Raz, A., Minkes, M.S., Ferrendelli, J.A. and Sprecher, H. Triene prostaglandins: Prostacyclin and thromboxane biosynthesis and unique biological properties. Proc. Nat1 Acad. Sci. USA 76:944, 1979. 301
4.
Diczfalusy, U., Falardeau, P. and HammarstrBm, S. Conversion of prostaglandin endoperoxides to Cl7 hydroxy acids catalyzed by human platelet thromboxane synthase. FEBS Lett 84:271, 1977,
5. Dicafalusy, U. and HammarstrBm, S. A structural requirement for the conversion of prostaglandin endoperoxides to thromboxanes. FEBS Lett. 105:291, 1979. 6. Samuelsson, B., Goldyne, M., Granstrom, E., Hamberg, M., Hammarstrem, S. and Malmsten, C. Prostaglandins and thromboxanes. Ann. Rev. Biochem. 47:997, 1978 7. Hammarstrijm, S. Enzymatic synthesis of 15-hydroperoxythromboxane A2 and 12-hydroperoxy-5,8,10-heptadecatrienoic acid. J. Biol. Chem. 255:518, 1980. 8. Piper, P. and Vane, J.R. Release of additional factors in anaphylaxis and its antagonism by anti-inflammatory drugs. Nature 223:29, 1969. 9. Krebs, H.A. and Henseleit, K. Untersuchungen iiber die Harnstoffbildung im Tierkgrper. Hoppe Seyler's 2. Physiol. Chem. 210:33, 1932. 10. Miyamoto, T., Ogino, N., Yamamoto, S. and Hayaishi, 0. Purification of prostaglandin endoperoxide synthetase from bovine vesicular gland microsomes. J. Biol. Chem. 251:2629, 1976. 11. Kuehl, F.A., Humes, J.L., Egan, R.W., Ham, E.A., Beveridge, G.C and van Arman, C.G. Role of prostaglandin endoperoxide PGG2 in inflammatory processes. Nature 265:170, 1977.
302