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Arachidonic acid induced platelet aggregation and thromboxane formation is inhibited by OKY-1581
Prostaglandins Leukotrienes and Medicine 12:
299 - 303, 1983
ARACHIDONICACID INDUCEDPLATELETAUGREGATIONAND THROMBOXANE FORMATIONIS INHIBITEDBY OKY-1581 Pekka Uotila,Martti Matintaloand Marja-LiisaDahl Departmentof Physiology,Universityof Turku SF-20520Turku, Finland (Reprintrequeststo PU) ABSTRACT Addition of arachidonic acid (AA) to human platelet rich plasma caused a reversibleplatelet aggregation and a concomitant formation of thromboxane B2 (TXB >. Both AA-induced platelet aggregationand TXB formation were in%Iibited by OKY-1581 and the amounts of POE 2P formed were correspondingly increased. The formation oT ZetZQF was smaller and was increasedonly slightlyby OKY-1581. ADP-inducedoplatelet aggregation did not result in the formation of TXB and it was inhibited only slightly by OKY-1581. When ADP was added to platelet rich plasma six minutes after the addition of AA, the ADP-inducedaggregationwas greatly decreasedin the presence and absence of OKY-1581. The present study indicates that the AA-induced platelet aggregation is inhibited by OKY-1581 and that OKY-1581 is a specificinhibitorof thromboxane synthesis in platelet rich human plasma. INTRODUCTION Arachidonic acid (AA)is metabolized in humanplateletsby cycle-oxygenese to thromboxane A (TXA 1, a potent aggregatory compound, which is rapidly converted2to a stable metabolitethromboxaneB (TXB > (I 2). The specificinhibitionof thromboxane formation would 2De fa;?vourlble in the treatment of many cardiovascular diseases. Pyridineand its derivatives,e.g. OKY-1581 (Kisseiand Ono Pharmaceutical Co, Japan), have been reported to be specificinhibitorsof thromboxanesynthetase (3). We have investigated the effects of OKY-1581 in vitro on the AA- and ADP-inducedplateletaggregationand on the concomitantforxsttion of TXB2 and some other cycle-oxygenase productsin human plasma. METHODS Venous blood was taken from seven healthy male volunteers aged 21-32 who had not taken aspirin-like drugs during the precedingtwo weeks. Platelet rich plasma was separated and platelet aggregation was measuredspectrophotometrically as describedearlier(4). samples consisting of 0.9 ml of platelet rich plasma (about3 x ?10 platelets) and 0.3 ml. of 0.9 % NaCl were incubatedin a spectrophotometer
at 37 'C and were continuouslymixed by an electrically rotatedpolyethylene stirrer. OKY-1581 (sodium (El-3-[4-(+pyridylmethyl)phenyl]-2-methylpropenoate; Kissei Pharmaceutical Co. Ltd. and Ono Pharmaceutical Co. Ltd.,Japan)was addedin 25 pl of 0.9 $ NaClto give finalconcentrations of 1 nM, 100 nM, 10 pM or 1 mM. In control experiments the same volume of 0.9 % NaCl was added. Five minutes after the addition of OKY-1581 aggregation was induced with arachidonic acid (AA, 5.5 mM, sodium salt, in 100 ~1 0.9 % NaCl, Sigma, St. Louis, MO, U.S.A.) and six minutes later ADP (15 PM, in 50 pl of 0.9 $ NaCl, Sigma) was added to the same aggregationsamples to investigate the possible effect of arachidonate metabolites on ADP-induced aggregation. To investigate the effect of OKY-1581 on the ADP-induced aggregation other aggregation samples were incubatedwith or without 1 mM OKY-1581 and the aggregationwas inducedwith 15 uM ADP without previousaddition of AA. About seven minutes after the addition of ADP the samples were taken from the aggregometerand acetylsalicylicacid (1 mM) was added to prevent further metabolism of AA. The samples were then stored at -20 'C until the amounts of TXB2, PGE2, PGF and 6-keto-PGF were measured by radioimmunoassay. The aggregation z samples we% dilutedwith ethanol to appropriatesolutionsfor differentradioimmunoassays. The antiserum for TXB was from Upjohn (Kalamazoo,Michigan, U.S.A.) and those for PGE PG$ and 6-keto-PGF from Seragen Inc. (Boston, Mass, U.S.A.). ?&lab& ed TXB was frbamCayman Chemical (Denver, Colorado, y.S.A.1, PGE2 and 6-$eto-PGFla from Upjohn and H-TXB w s purcha ed from New England Nuclear PGF from Sigma. 9H-PGF2a and 6-keto-3H-PGF1a (Bogton, Mass, U.S.A.) aifd2 H-PGE from Amersham (Buckinghamshire, Englan8' 1. RESULTS Arachidonicacid (5.5 mM) caused a reversible aggregation of human platelets and a concomitant formation of thromboxane. ADP (15 uM) alone induced an irreversible aggregation which was greater than the aggregation induced with AA. The formationof TXB2 was, however, very small in ADt-induced aggregation, as the amount of TXB2 was 0.12+0.04 pmol/lg platelets (mean+SEM, n=6) in ADP experimentsand 17.5+6.2 pmol/lO platelets (n=7) in AA-inducedcontrol eXperiaentS. At 150 nM OKY-1581 caused a partial inhibition of both AA-induced platelet aggregation and the concomitant formation of TXB (Figures 1 and 2). Higher concentrations(IO pM and 1 mM) completely % nhibited thromboxane formation, although the AA-induced aggregation was not completely suppressed. When the formation of TXB was decreased by OKY-1581 that of PGE2 and PGF was correspond1ngly increased (Figure 2). The formed amount of %Gketo-PGF was small and it was increased only slightly by OKY-1581. The co&ined amountsof PGE2, PGF d 6-keto-PGF in AA-induced controlaggregationswas 2.720.6 pmo$a fi0 plateletsand% 1 nM, 100 nM, 10 UM p 1 x&f OKY-1581 eXpeP~ts 3.920.9, 5.020.5,5.521.1and 4.820.4 pmol/lO platelets,respectively. When the aggregationwas induced with ADP without previous addition of AA, OKY-1581 at 1 mM had only a slight inhibitory effect on the aggregation(FigureI).
300
m
AA-ImucED
0
ADP-IMDUCED
OKY-1581(M) FIGURE 1. The effect of OKY-1581on the AA- and ADP-inducedaggregation of human platelets. Platelet aggregation was induced with 5.5 mM AA or 15 PM ADP as described in methods. Results (mean+SEM, n=T) were comparedto the controlby Student's t-test for paired-data: s** = 2P < 0.001, ?? = 2P < 0.05.
I
0
TXB2
CONTROL
PGE2
PGF2cY
6-keto-PCW,,
FIGURE 2. The effect of OKY-1581 on the formation of immunoreactive TXB2, PGE PGF and 6-keto-PGF during AA-induced aggregation of human pf' atelet& The means and'!!EMsfrom seven experiments are given. Compared to the controlby Student'st-test for paired data: ?? = 2P < 0.05, + 2P < 0.1. ??
301
When ADP was added to platelet rich plasma six minutes after the addition of AA, the ADP-induced aggregation was greatly decreased as it was only II28 $ (n=7) of the aggregationinducedwith ADP only. In the presenceof OKY-1581ADP did not induce any clear aggregation in six cases when added to platelet rich plasma after AA. In the remaining one case ADP did induce plateletaggregationafter arachidonate additionbut the aggregationwas decreasedwhen increasingconcentrations of OKY-1581were added. DISCUSSION The present study indicates that OKY-1581 selectively inhibits the formation of aggregatory TXA in human platelets in vitro and that this inhibitionresults in the decreasedaggregabilityof platelets with arachidonic acid. The effect of OKY-1581 on the ADP-induced platelet aggregation was, however, small. Thus the results of the present study are rather similar to those reportedearlier i3,5,6). When the formation of TXB was inhibitedby OKY-1581,the formation of other cycle-oxygenase produgts was correspondinglyincreased (Figure 2). As the formation of TXB was almost completely inhibited by both IO uM and 1 ?M? OKY-1581 a%d the combined amount of PGE $' PGFfo and 6-keto-PGF in 1 mM OKY-1581 experiments was not signi icant y smaller than th&?in 10 uM experiments, this indicates that OKY-1581 does not significantlyinhibit the activityof plateletCyClO-OxygenaSS even at the rather high concentrationof 1 mM in human plasma. Although the formation of TXB2 was almost totally inhibited by OKY-1581,the AA-inducedaggregationwas not. The degree of inhibitionof AA-induced aggregationwas, however,similar to that caused by 1 mM acetylsalicylic acid (7). When ADP is added to platelet rich plasma after arachidonate, the ADP-induced aggregation is greatly decreased and this decrease can also be seen after aspirin ingestion (cf. 4). This decrease is obviouslydue to the metabolitesof AA formed via the 12-lipoxygenase pathway in human plateletas 12-HPETEhas been reportedto haveinhibitory effects on platelets functions (8,9). The present study indicates that a similar inhibition of ADP-induced platelet aggregationafter the additionof AA can be seen also in the presenceof OKY-1581. ACKNOWLEDGEMENTS The excellenttechnicalassistanceof MS Tarja Laiho is gratefully acknowledged. This study was supportedby a grant from YrjB Jahnsson Foundation(Finland). OKY-1581was a generous gift from Dr. A. Ujiie (KisseiPharmaceuticalCo. Ltd., Japan). REFERENCES 1. Hamberg M, Samuelsson B. Prostaglandin endoperoxides. Novel transformations of arachidonic acid in human platelets. Proc Nat1 Acad Sci USA 71:3400, 1974.
302
2.
Hamberg H, Svensson J, Samuelsson B- Throaboxanes: of’ biologiaally active oawmlde derived fruo promglmdin Proc Flat1 Acad Sci USA 72:2994, 1975.
A new group eMopemdde8.
3. Miymoto T, Taniguchi K, Tanouchi T, Hirata F. Selectiveinhibitor of thromboxane synthetase: Pyridine and its derivatives. Page 443 in Advances in Prostaglandin and ThromboxaneResearch,Vol. 6. (B Samuelsson,PW Ramwell, R Paoletti eds) Raven Press, New York, 1980. 4. Dahl M-L, Puustinen T, Uotila P. The effect of arachidonicacid on the aggregabilityof human plateletrich plasma. Prostaglandins Leukotrienesand Medicine8:133, 1982. 5. Hiraku S, Wakitani K, Katsube N, Kawasaki A, Tsuboshima M, Naito J, Ujiee A, Komatsu H, Iizuka K. Pharmacologicalstudies on OKY-1581, a selectivethromboxanesynthetaseinhibitor. Page 373 in Abstract Book of the Fifth International Conference on Prostaglandins, Florenze,Italy, 1982. 6. Ito T, Ogawa K, Sakai K, Watanabe J, Satake T, Seiji H, Jun N. Effectsof a selectiveinhibitorof thromboxanesynthetase(OKY-1581) in man. Page 374 in Abstract Book of the Fifth International Conferenceon Prostaglandins,Florenze,Italy, 1982. 7. Dahl M-L, Puustinen T, Uotila P. Sodium salicylate interferes with the inhibitory effects of aspirin and indomethacin on human platelets. Submittedfor publication. 8. Siegel MI, McConnell RT, Abrahams SL, Porter NA, Cuatrecasas P. Regulationof arachidonate metabolism via liv and cyclo-cacglganase by 12-HPETE,the product of human platelet lipoxygenase. Biochem Biophys Res Counnun 89:1273, 1979. 9. Aharony D, Smith JB, Silver MJ. Regulationof arachidonate-induced plateletaggregationby the lipoxygenaseproduct, 12-hydroperoxyeicosatetraenoicacid. Biochim Biophys Acta 718:193, 1982.
303
299 - 303, 1983
ARACHIDONICACID INDUCEDPLATELETAUGREGATIONAND THROMBOXANE FORMATIONIS INHIBITEDBY OKY-1581 Pekka Uotila,Martti Matintaloand Marja-LiisaDahl Departmentof Physiology,Universityof Turku SF-20520Turku, Finland (Reprintrequeststo PU) ABSTRACT Addition of arachidonic acid (AA) to human platelet rich plasma caused a reversibleplatelet aggregation and a concomitant formation of thromboxane B2 (TXB >. Both AA-induced platelet aggregationand TXB formation were in%Iibited by OKY-1581 and the amounts of POE 2P formed were correspondingly increased. The formation oT ZetZQF was smaller and was increasedonly slightlyby OKY-1581. ADP-inducedoplatelet aggregation did not result in the formation of TXB and it was inhibited only slightly by OKY-1581. When ADP was added to platelet rich plasma six minutes after the addition of AA, the ADP-inducedaggregationwas greatly decreasedin the presence and absence of OKY-1581. The present study indicates that the AA-induced platelet aggregation is inhibited by OKY-1581 and that OKY-1581 is a specificinhibitorof thromboxane synthesis in platelet rich human plasma. INTRODUCTION Arachidonic acid (AA)is metabolized in humanplateletsby cycle-oxygenese to thromboxane A (TXA 1, a potent aggregatory compound, which is rapidly converted2to a stable metabolitethromboxaneB (TXB > (I 2). The specificinhibitionof thromboxane formation would 2De fa;?vourlble in the treatment of many cardiovascular diseases. Pyridineand its derivatives,e.g. OKY-1581 (Kisseiand Ono Pharmaceutical Co, Japan), have been reported to be specificinhibitorsof thromboxanesynthetase (3). We have investigated the effects of OKY-1581 in vitro on the AA- and ADP-inducedplateletaggregationand on the concomitantforxsttion of TXB2 and some other cycle-oxygenase productsin human plasma. METHODS Venous blood was taken from seven healthy male volunteers aged 21-32 who had not taken aspirin-like drugs during the precedingtwo weeks. Platelet rich plasma was separated and platelet aggregation was measuredspectrophotometrically as describedearlier(4). samples consisting of 0.9 ml of platelet rich plasma (about3 x ?10 platelets) and 0.3 ml. of 0.9 % NaCl were incubatedin a spectrophotometer
at 37 'C and were continuouslymixed by an electrically rotatedpolyethylene stirrer. OKY-1581 (sodium (El-3-[4-(+pyridylmethyl)phenyl]-2-methylpropenoate; Kissei Pharmaceutical Co. Ltd. and Ono Pharmaceutical Co. Ltd.,Japan)was addedin 25 pl of 0.9 $ NaClto give finalconcentrations of 1 nM, 100 nM, 10 pM or 1 mM. In control experiments the same volume of 0.9 % NaCl was added. Five minutes after the addition of OKY-1581 aggregation was induced with arachidonic acid (AA, 5.5 mM, sodium salt, in 100 ~1 0.9 % NaCl, Sigma, St. Louis, MO, U.S.A.) and six minutes later ADP (15 PM, in 50 pl of 0.9 $ NaCl, Sigma) was added to the same aggregationsamples to investigate the possible effect of arachidonate metabolites on ADP-induced aggregation. To investigate the effect of OKY-1581 on the ADP-induced aggregation other aggregation samples were incubatedwith or without 1 mM OKY-1581 and the aggregationwas inducedwith 15 uM ADP without previousaddition of AA. About seven minutes after the addition of ADP the samples were taken from the aggregometerand acetylsalicylicacid (1 mM) was added to prevent further metabolism of AA. The samples were then stored at -20 'C until the amounts of TXB2, PGE2, PGF and 6-keto-PGF were measured by radioimmunoassay. The aggregation z samples we% dilutedwith ethanol to appropriatesolutionsfor differentradioimmunoassays. The antiserum for TXB was from Upjohn (Kalamazoo,Michigan, U.S.A.) and those for PGE PG$ and 6-keto-PGF from Seragen Inc. (Boston, Mass, U.S.A.). ?&lab& ed TXB was frbamCayman Chemical (Denver, Colorado, y.S.A.1, PGE2 and 6-$eto-PGFla from Upjohn and H-TXB w s purcha ed from New England Nuclear PGF from Sigma. 9H-PGF2a and 6-keto-3H-PGF1a (Bogton, Mass, U.S.A.) aifd2 H-PGE from Amersham (Buckinghamshire, Englan8' 1. RESULTS Arachidonicacid (5.5 mM) caused a reversible aggregation of human platelets and a concomitant formation of thromboxane. ADP (15 uM) alone induced an irreversible aggregation which was greater than the aggregation induced with AA. The formationof TXB2 was, however, very small in ADt-induced aggregation, as the amount of TXB2 was 0.12+0.04 pmol/lg platelets (mean+SEM, n=6) in ADP experimentsand 17.5+6.2 pmol/lO platelets (n=7) in AA-inducedcontrol eXperiaentS. At 150 nM OKY-1581 caused a partial inhibition of both AA-induced platelet aggregation and the concomitant formation of TXB (Figures 1 and 2). Higher concentrations(IO pM and 1 mM) completely % nhibited thromboxane formation, although the AA-induced aggregation was not completely suppressed. When the formation of TXB was decreased by OKY-1581 that of PGE2 and PGF was correspond1ngly increased (Figure 2). The formed amount of %Gketo-PGF was small and it was increased only slightly by OKY-1581. The co&ined amountsof PGE2, PGF d 6-keto-PGF in AA-induced controlaggregationswas 2.720.6 pmo$a fi0 plateletsand% 1 nM, 100 nM, 10 UM p 1 x&f OKY-1581 eXpeP~ts 3.920.9, 5.020.5,5.521.1and 4.820.4 pmol/lO platelets,respectively. When the aggregationwas induced with ADP without previous addition of AA, OKY-1581 at 1 mM had only a slight inhibitory effect on the aggregation(FigureI).
300
m
AA-ImucED
0
ADP-IMDUCED
OKY-1581(M) FIGURE 1. The effect of OKY-1581on the AA- and ADP-inducedaggregation of human platelets. Platelet aggregation was induced with 5.5 mM AA or 15 PM ADP as described in methods. Results (mean+SEM, n=T) were comparedto the controlby Student's t-test for paired-data: s** = 2P < 0.001, ?? = 2P < 0.05.
I
0
TXB2
CONTROL
PGE2
PGF2cY
6-keto-PCW,,
FIGURE 2. The effect of OKY-1581 on the formation of immunoreactive TXB2, PGE PGF and 6-keto-PGF during AA-induced aggregation of human pf' atelet& The means and'!!EMsfrom seven experiments are given. Compared to the controlby Student'st-test for paired data: ?? = 2P < 0.05, + 2P < 0.1. ??
301
When ADP was added to platelet rich plasma six minutes after the addition of AA, the ADP-induced aggregation was greatly decreased as it was only II28 $ (n=7) of the aggregationinducedwith ADP only. In the presenceof OKY-1581ADP did not induce any clear aggregation in six cases when added to platelet rich plasma after AA. In the remaining one case ADP did induce plateletaggregationafter arachidonate additionbut the aggregationwas decreasedwhen increasingconcentrations of OKY-1581were added. DISCUSSION The present study indicates that OKY-1581 selectively inhibits the formation of aggregatory TXA in human platelets in vitro and that this inhibitionresults in the decreasedaggregabilityof platelets with arachidonic acid. The effect of OKY-1581 on the ADP-induced platelet aggregation was, however, small. Thus the results of the present study are rather similar to those reportedearlier i3,5,6). When the formation of TXB was inhibitedby OKY-1581,the formation of other cycle-oxygenase produgts was correspondinglyincreased (Figure 2). As the formation of TXB was almost completely inhibited by both IO uM and 1 ?M? OKY-1581 a%d the combined amount of PGE $' PGFfo and 6-keto-PGF in 1 mM OKY-1581 experiments was not signi icant y smaller than th&?in 10 uM experiments, this indicates that OKY-1581 does not significantlyinhibit the activityof plateletCyClO-OxygenaSS even at the rather high concentrationof 1 mM in human plasma. Although the formation of TXB2 was almost totally inhibited by OKY-1581,the AA-inducedaggregationwas not. The degree of inhibitionof AA-induced aggregationwas, however,similar to that caused by 1 mM acetylsalicylic acid (7). When ADP is added to platelet rich plasma after arachidonate, the ADP-induced aggregation is greatly decreased and this decrease can also be seen after aspirin ingestion (cf. 4). This decrease is obviouslydue to the metabolitesof AA formed via the 12-lipoxygenase pathway in human plateletas 12-HPETEhas been reportedto haveinhibitory effects on platelets functions (8,9). The present study indicates that a similar inhibition of ADP-induced platelet aggregationafter the additionof AA can be seen also in the presenceof OKY-1581. ACKNOWLEDGEMENTS The excellenttechnicalassistanceof MS Tarja Laiho is gratefully acknowledged. This study was supportedby a grant from YrjB Jahnsson Foundation(Finland). OKY-1581was a generous gift from Dr. A. Ujiie (KisseiPharmaceuticalCo. Ltd., Japan). REFERENCES 1. Hamberg M, Samuelsson B. Prostaglandin endoperoxides. Novel transformations of arachidonic acid in human platelets. Proc Nat1 Acad Sci USA 71:3400, 1974.
302
2.
Hamberg H, Svensson J, Samuelsson B- Throaboxanes: of’ biologiaally active oawmlde derived fruo promglmdin Proc Flat1 Acad Sci USA 72:2994, 1975.
A new group eMopemdde8.
3. Miymoto T, Taniguchi K, Tanouchi T, Hirata F. Selectiveinhibitor of thromboxane synthetase: Pyridine and its derivatives. Page 443 in Advances in Prostaglandin and ThromboxaneResearch,Vol. 6. (B Samuelsson,PW Ramwell, R Paoletti eds) Raven Press, New York, 1980. 4. Dahl M-L, Puustinen T, Uotila P. The effect of arachidonicacid on the aggregabilityof human plateletrich plasma. Prostaglandins Leukotrienesand Medicine8:133, 1982. 5. Hiraku S, Wakitani K, Katsube N, Kawasaki A, Tsuboshima M, Naito J, Ujiee A, Komatsu H, Iizuka K. Pharmacologicalstudies on OKY-1581, a selectivethromboxanesynthetaseinhibitor. Page 373 in Abstract Book of the Fifth International Conference on Prostaglandins, Florenze,Italy, 1982. 6. Ito T, Ogawa K, Sakai K, Watanabe J, Satake T, Seiji H, Jun N. Effectsof a selectiveinhibitorof thromboxanesynthetase(OKY-1581) in man. Page 374 in Abstract Book of the Fifth International Conferenceon Prostaglandins,Florenze,Italy, 1982. 7. Dahl M-L, Puustinen T, Uotila P. Sodium salicylate interferes with the inhibitory effects of aspirin and indomethacin on human platelets. Submittedfor publication. 8. Siegel MI, McConnell RT, Abrahams SL, Porter NA, Cuatrecasas P. Regulationof arachidonate metabolism via liv and cyclo-cacglganase by 12-HPETE,the product of human platelet lipoxygenase. Biochem Biophys Res Counnun 89:1273, 1979. 9. Aharony D, Smith JB, Silver MJ. Regulationof arachidonate-induced plateletaggregationby the lipoxygenaseproduct, 12-hydroperoxyeicosatetraenoicacid. Biochim Biophys Acta 718:193, 1982.
303