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Inhibition of platelet aggregation and reduced formation of thromboxane and lipoxygenase products in platelets by oil of cloves
Prostaglandins, Leukotrienes and Medicine Longman Group UK Ltd 1987
0
(1987)
29,
I l-18
INHIBITION OF PLATELET AGGREGATION AND REDUCED FORMATION OF THROMBOXANE AND LIPOXYGENASE PRODUCTS IN PLATELETS BY OIL OF CLOVES K.C. Srivastava and Ulla Justesen, Department of Environmental Medicine, Institute of Community Health, Odense University, J.B. Winsl0ws Vej 19, DK-5000 Odense, Denmark. ABSTRACT Oil of cloves (OC) was found to be a potent inhibitor of platelet aggregation induced by arachidonic acid (AA), collagen and epinephrine; in this respect it was most effective against AA-induced aggregation. Inhibition of aggregation by OC seems to be mediated through a reduced formation of thromboxane as indicated by the following experimental evidence. (i) OC inhibited TxB2 formation in intact as well as lysed platelet preparations from added arachidonate, and (ii) it inhibited the formation of TxB2 from AA-labelled platelets after activation with CaZf-ionophore A23187. The formation of lipoxygenase derived products was dependent on the concentration of OC used; at its lower concentration their amounts increased but this was found to be reversed at higher concentrations. At all concentrations thromboxane was decreased with a concomitant increase in mused AA. INTRODUCTION Since our observation with onion, garlic and ginger - the three spices used most frequently in Asia and other parts of the world - on their effects on human platelet aggregation and inhibition of fatty acid oxygenases (l-4),, we have felt that a general screening of various spices frequently consumed should be undertaken. Here we report. our preliminary results on the in vitro effects of oil of cloves (OC) (Hindustani: laung; Danish: nelliker) on platelet aggregation and on endogenous formation of thromboxane and lipoxygenase products in platelets. Cloves are used as a spice and are included frequently in spice mixtures (called Garam Masala in India) used to give flavour and taste to vegetable- and meat dishes. MATERIALS AND METHODS Arachidonic acid (I-14C) (specific activity 59.6 mCi/mmol) was purchased from the Radiochemical Centre, Amersham, England. Calcium ionophore A23187 was obtained from Sigma Chemical Co. Thromboxane B2 (TxB2) was obtained gratis from ON0 Pharmaceutical Co., Ltd., Osaka (Japan). TLC plates were prepared in
11
our laboratory. All solvents were of analytical reagent grade except that in the extraction of AA-metabolites technical grade eth& was used. Cloves (18 g) were powdered in a mixer/grinder and extracted in 150 ml ether overnight at 4oC. The ethereal extract was filtered in a desiccator and the filtrate The extract was filtered and ether treated with anhydrous sodium sulphate. distilled by bubbling nitrogen at 450C; the yield of the oily material was ca. 10%. A portion (450 mg) of the extracted material was dissolved in 5 ml alcohol (96%). Appropriate dilutions were made from this solution; 5 ul of the solution were used in all experiments - incubation as well as aggregation. Controls were treated with 5 ul alcohol. Blood was obtained from healthy donors who had not taken aspirin or any such drug known to affect platelet function for at least 7-10 days prior to giving blood. For aggregation experiments platelet-rich plasma (PRP) and platelet-poor plasma They were used in aggregation as were prepared by differential centrifugation. described earlier with minor changes (2). After mixing the alcoholic solution of OC for 30 set in the aggregometer, the contents were allowed to stay at room temperature for ca. 5 min before producing aggregation. Preparation of washed platelet suspension, incubation with labelled AA and extraction of AA metabolites were carried out as described elsewhere (1). Thromboxane B2 was resolved from other products of AA by TLC using the solvent system ethyl acetate-isooctaneacetic acid-water (110:50:20:10, v/v, of which upper organic phase was used after mixing for 5 min) (I). For the separation of lipoxygenase products from AA and its metabolites solvent II (n-hexane-ether-acetic acid, 80:20:1, v/v) was used. Hydroxy derivatives of AA (HHT and HETE) were localized in a certain area of the plate (between the application point containing TxB2, ptostaglandins and phospholipids, and AA, Rf value 0.32). The entire area between the application point and AA was scraped off and its radioactivity was measured. The amount of HETE was determined by subtracting TxB2 counts (TxB2 = HHT) from the total counts due to HHT and HETE (5). We had to resort to this method because of the lack of reference standards (HHT and HETE). Labelling of platelets with arachidonate was achieved by incubating them (as PRP) with low concentrations (0.10 uM) of AA. At low concentrations, AA is not metabolized by the platelet enzymes - cyclooxygenase and lipoxygenase; AA is mainly incorporated into platelet phospholipids, and of the amount of AA finding access to the platelets, more than 95% is incorporated into phospholipids (6). were used in two different experiments. (i) The effect of Labelled platelets OC was examined on the incorporation of labelled AA in platelet phospholipids. For this purpose platelets (PRP were first treated with 5 ul alcoholic solution of the oil (112 ug/ml PRP) (experimental) or with 5 ul ethanol (control) by incubating for 5 min at 37OC followed by incubation with labelled AA at 37OC for 2 h. The contents were centrifuged at 1500 x g, supernatant decanted and the platelets resuspended in I ml Ringer-citrate-dextrose (RCD). Finally the suspension was extracted in 8 ml chloroform-methanol (2:l) overnight at 4oC. (ii) The effect of OC was examined on platelet phospholipase activity and on the formation of TxB2 and lipoxygenase products from endogenous AA. The method used by us is a modification of that described by Bills et al. (6). In our method labelled platelets were washed with, and resuspended in RCD. Details of the procedure describing mixing of platelets with the test material, incubation, activation of labelled platelets with calcium ionophore A23187 (5 uM), extraction of phospholipids and AA metabolites have been described recently by us (3). For the TLC separation of TxB2 from phospholipids and other AA metabolites solvent I was used. Lipoxygenase ptoducts were separated and quantified as described above. Statistics Statistical significance was evaluated by Student’s t-test for paired data.
12
RESULTS Influence of OC on the metabolism of exogenous arachidonate in platelets. Washed platelet suspensions were treated with various concentrations of OC, control platelets were treated with 5 ul alcohol prior to treatment with arachidonate (2). Arachidonic acid metabolites were extracted in ether after acidification and resolved by TLC. It was found that OC reduced thromboxane formation at the three concentrations used; it reduced the lipoxygenase products at higher concentrations but at the lowest concentration the lipoxygenase products were increased. At all concentrations of OC more unused AA was recovered (Fig. 1).
Figure 1. Autoradiogram of the TLC plate showing the effect of OC on the formation of arachidonic acid metabolites by human platelets. C = control platelets; I, II and III = experimental platelets treated respectively with 11.25 ug, 45 ug and 450 ug OC/200 ul platelet suspension. Arachidonic acid (6.5 uM). a. Separation of TxB2 from other products by applicating 50 ul of 200 ul extract and using solvent ethyl acetate-isooctane-acetic acid-water (110:50:20:100, v/v). b. Separation of HETE, HHT and AA from TxB2 and prostaglandins (PCs) which remained at the application point together with the phospholipids (Rf = 0.00) by applicating 25 ul of 200 ul extract using solvent n-hexane-ether-acetic acid (80:20:1, v/v). Control values (n = 2) for TxB2 = 183 picomoles and lipoxygenase products = 811 picomoles/108 platelets. Values (% of control) for OC-treated platelets TxB2: I = 13.2%, II = 5.2%, III = 0.6%. Lipoxygenase products: I = 146%, II = 56%, III = 1.5%. Influence of OC on the platelet phospholipase activity and on the endogenous formation of thromboxane and lipoxygenase products in platelets. The experimental set up used here required the use of AA-labelled platelets which were challenged
13
by Ca2+-ionophore A23187 after treatment with 5 ul OC solution (experimental platelets) or with 5 ul alcohol (control platelets). On activation platelets liberated AA which was metabolized into various products of which TxB2 and lipoxygenase products were determined. By this method the effect of clove oil was examined at three dose levels - 450 ug, 45 ug and 11.25 q/500 ul platelet suspension. At 450 ug/500 ul dose there was observed no change in the phospholipase activity from control platelets; the amounts of TxB2 and lipoxygenase products decreased significantly with a concomitant increase in the AA counts (Fig. 2). At 45 ug and 11.25 ug/500 ~1 dose levels TxB2 was significantly decreased; lipoxygenase products were sli htly (by 15%) reduced at 45 ug/500 dose level without any effect at 11.25 ugB500 ul level (Fig. 3). CPM x lo-’ 8 1
I
PL
I3 ControlIn=41
II
APPL
TxB?
HETE
AA
Figure 2. Effect of OC on the formation of arachidonic acid metabolites from endogenous phospholipid (14C)-arachidonic acid. Washed platelets previously treated with (I4Qarachidonic acid to permit incorporation into platelet phospholipids were exposed to calcium ionophore A23187 (5 uM). In a typical experiment the effect of OC (450 ug/500 ul) added 10 min prior to platelet stimulation is shown. The values (CPM, Mean + SD) are l/6 of the total amount produced. Control and experimental determinations were done in duplicate. * p < 0.05 ** p < 0.01 PL = phospholipids and TxB2 = thromboxane B2 (separated by solvent I). APPL = application point; HETE and AA (separated by solvent II). Influence of OC on the incorporation of labelled arachidonate into platelet hos holi ids. Incorporation of AA into platelets was studied by incubating AA 0.12 uM with PRP (4 ml) previously treated with 5 ul alcohol (control) or with 5 ul Y+-of alcoholic solution of OC (112 ug/ml PRP). At this concentration OC potentiated incorporation of AA into platelets (p < 0.05) (control 1797 + 502, OC-treated 1981 t 519, values are in CPM, Mean + SD obtained with ca. 1 x 109 platelets). Aggregation experiments. OC inhibited aggregation induced by arachidonate, collagen and epinephrine; this effect was dose-related. It was found to be most effective in AA-induced aggregation; even 11.25 ug OC/ml PRP were sufficient to abolish AA-induced platelet aggregation (Fig. 4). In five blood samples half of this dose (5.5 ug/ml PRP) produced total inhibition of aggregation. Collagen-induced aggregation was completely inhibited by 45 ug OC/mI PRP (3 blood samples). In two other blood samples abolition of collagen-induced aggregation was achieved 14
IZZI Control
CPMxlO-'
t?3 OC-treated
6
CPM 80-
I
LIPOXYGENASE PRODUCTS
J :.: :, :.:
4-
TxBz
2-
:;I ::: ::: :b: L
OI
II
Figure 3. Effect of OC on the formation of thromboxane B2 and hydroxy acid (HETE) in Ca2+-ionophore A23187-treated platelets, whose phospholipids were prelabelled with (14C)-arachidonic acid. I = OC 45 ug/500 ul (n = B), II = OC 1I .25 ug/500 ul (n = 7). *p < 0.05 The values (CPM, Mean + SD) are l/6 of the total amount produced by 0.5 x lo9 platelets. Arachldonatel0.5mMl
Arach~donotelO.SmtlI
Figure 4. Representative tracings showing the inhibitory effect of OC on arachidonate-induced aggregation with two separate blood samples (A and B). A. 1 and 6 control tracings. Tracings 2-5 were obtained by treating respectively with 22.5 ug, 11.25 ug, 5.5 ug and 45 ug OC/ml PRP. 8. 1 control tracing. Tracings 2-5 were obtained by treating respectively with 90 ug, 45 ug, 22.5 ug and 11.25 ug OC/ml PRP. Aggregation was performed in the sequence of numbers appearing on the tracings.
15
with 90 ug/ml. With lower doses (22.5-45 ug/ml) the first phase of aggregation was reversed suggesting disaggregation effects (Fig. 5). In epinephrine-induced aggregation OC (11.25-90 ug/ml) inhibited the second phase of aggregation; the first phase was reversed as in the case of collagen-induced aggregation (Fig. 6). C0llogsn Ilopglml,
Collagen IlOyglmli
Figure 5. Representative tracings showing the effect of OC on collagen-induced aggregation with two separate blood samples (A and B). A. 1 and 4 control tracings. Tracings 2 and 3 were obtained by treating respectively with 45 ug and 90 ug OC/ml PRP. B. 1 control tracing. Tracings 2-6 were obtained by treating respectively with 22.5 ug, 90 ug, 11.25 ug, 45 ug and 450 ug OC/ml PRP. Aggregation was performed in the sequence of numbers on the tracings. Epinephrine IlO@II
Epinephrine
(lOpt41
7
Figure 6. Representative tracings showing the inhibitory effect of OC on epinephrine-induced platelet aggregation. A and B were obtained with the same blood sample. 1 and 7 control tracings. Tracings 2-6 were obtained by treating respectively with 45 ug, 90 ug, 450 ug, 22.5 ug and 11.25 ug OC/ml PRP. Aggregation was performed in the sequence of numbers on the tracings. 16
2 q
5 uM)
796 + 274**
1207 f 274
Phospholipidsa
pointb
902 + 277* *
1297 + 305
Application
28 + 13**
423
TxB2c
acid from platelet
accounted
productsc§
297 + 72**
724
Lipoxygenase
phospholipids
by the
28 t 25*
826
AAc@
for mainly
The values (CPM, Mean + SD) are l/6 of the total amount produced by 0.5 x IO9 platelets. a counts are due to phospholipids. Thromboxane B2, prostanglandins (PCs) and other hydroxy metabolites of arachidonic acid (AA) were resolved completely from the phospholipids using solvent system I. b Counts are due to phospholipids, TxB2, PCs (Rf = 0.00). HHT and HETE (HPETE) and AA were resolved by using solvent II. c Counts are shown after substracting the background counts. 9 Includes counts mainly due to lipoxygenase products (HETE, HPETE). $0 Excess of the liberated AA from platelets phospholipids. * p < 0.05 ** p < 0.01
Experimental U&3187-treated,
Control (n__lgcoholtreated)
Exp. condition
Table 1. Calcium ionophore A23187 induced loss of (L4C) arachidonic appearance of oxygenated products of arachidonic acid
DISCUSSION The results of the present study suggest that OC contains component(s) that How this is achieved, we have tried to explain by inhibit platelet aggregation. examining the formation of cyclooxygenaseand lipoxygenase products from AA by human platelets. It was found that thromboxane was reduced. This was demonstrated by TxB2 formation from exogenous AA in washed (intact) and lysed platelet preparations; the effect was dose-dependent. At low-dose levels an increased formation of lipoxygenase products was observed suggesting a mechanism that could involve a redirection of AA from cyclooxygenase pathway to lipoxygenase pathway and/or potentiation of lipoxygenase enzyme. Use of AAlabelled platelets and their activation by Ca2+ -ionophore A23187 has been found to be suitable for the determination of metabolites produced from endogenous AApool (Table 1). Formation of these AA metabolites from AA-labelled platelets activated by Ca2+ -ionophore shows that reduced formation of TxB2 was due to the effect of OC on cyclooxygenase/Tx-synthetase enzyme(s) as no effect on the phospholipase activity was observed at the three concentrations used. At the lower two concentrations lipoxygenase products were not reduced. In conclusion, it may be said that inhibition of AA-, collagenand epinephrine-induced aggregation by OC may, in part, be due to inhibition of cyclooxygenase/Tx-synthetase enzymes. This may find support from its inability to inhibit A23187 and ADP-induced aggregation. ACKNOWLEDGEMENTS We wish to thank the personnel of the Blood Bank, Odense University Hospital for collection of blood samples. Ruth B. Alexandersen provided technical assistance. Mrs. Inge Bggelund typed the manuscript. REFERENCES 1. Srivastava, K.C. Effect of aqueous platelet aggregation and metabolism system: in vitro study. Prostaglandins 1984. 2.
Srivastava, K.C. Aqueous extracts of onion, garlic aggregation and alter arachidonic acid metabolism. 335-346, 1984.
3. Srivastava, aggregation. 4.
extracts of onion, garlic and ginger on of arachidonic acid in the blood vascular Leukotrienes and Medicine 13: 227-235.
K.C. Evidence Prostaglandins
and ginger inhibit platelet Biomed. Biochim. Acta 43:
for the mechanism by which garlig inhibits platelet Leukotrienes and Medicine 22: 313-321, 1986.
Vanderhoek, J.Y., Makheja, A.N., Bailey, J.M. Inbibition of fatty acid oxygenases by onion and garlic oils. Evidence for the mechanism by which these oils inhibit platelet aggregation. Biochemical Pharmacology 29: 3169-3173, 1980.
5. Srivastava, K.C., Tiwari, K.P. A simple procedure for the thin-layer chromatographic separation and determination of prostaglandins and other metabolites formed from 14C-arachidonic acid in human blood platelets. Fresenius Zeitschrift fiir Analytische Chemie 304: 412-416, 1980. 6.
Bills, T.K., Smith, J.B., Silver, M.J. Metabolism of (14C) arachidonic human platelets. Biochimica et Biophysics Acta 424: 303-314, 1976.
18
acid
by
0
(1987)
29,
I l-18
INHIBITION OF PLATELET AGGREGATION AND REDUCED FORMATION OF THROMBOXANE AND LIPOXYGENASE PRODUCTS IN PLATELETS BY OIL OF CLOVES K.C. Srivastava and Ulla Justesen, Department of Environmental Medicine, Institute of Community Health, Odense University, J.B. Winsl0ws Vej 19, DK-5000 Odense, Denmark. ABSTRACT Oil of cloves (OC) was found to be a potent inhibitor of platelet aggregation induced by arachidonic acid (AA), collagen and epinephrine; in this respect it was most effective against AA-induced aggregation. Inhibition of aggregation by OC seems to be mediated through a reduced formation of thromboxane as indicated by the following experimental evidence. (i) OC inhibited TxB2 formation in intact as well as lysed platelet preparations from added arachidonate, and (ii) it inhibited the formation of TxB2 from AA-labelled platelets after activation with CaZf-ionophore A23187. The formation of lipoxygenase derived products was dependent on the concentration of OC used; at its lower concentration their amounts increased but this was found to be reversed at higher concentrations. At all concentrations thromboxane was decreased with a concomitant increase in mused AA. INTRODUCTION Since our observation with onion, garlic and ginger - the three spices used most frequently in Asia and other parts of the world - on their effects on human platelet aggregation and inhibition of fatty acid oxygenases (l-4),, we have felt that a general screening of various spices frequently consumed should be undertaken. Here we report. our preliminary results on the in vitro effects of oil of cloves (OC) (Hindustani: laung; Danish: nelliker) on platelet aggregation and on endogenous formation of thromboxane and lipoxygenase products in platelets. Cloves are used as a spice and are included frequently in spice mixtures (called Garam Masala in India) used to give flavour and taste to vegetable- and meat dishes. MATERIALS AND METHODS Arachidonic acid (I-14C) (specific activity 59.6 mCi/mmol) was purchased from the Radiochemical Centre, Amersham, England. Calcium ionophore A23187 was obtained from Sigma Chemical Co. Thromboxane B2 (TxB2) was obtained gratis from ON0 Pharmaceutical Co., Ltd., Osaka (Japan). TLC plates were prepared in
11
our laboratory. All solvents were of analytical reagent grade except that in the extraction of AA-metabolites technical grade eth& was used. Cloves (18 g) were powdered in a mixer/grinder and extracted in 150 ml ether overnight at 4oC. The ethereal extract was filtered in a desiccator and the filtrate The extract was filtered and ether treated with anhydrous sodium sulphate. distilled by bubbling nitrogen at 450C; the yield of the oily material was ca. 10%. A portion (450 mg) of the extracted material was dissolved in 5 ml alcohol (96%). Appropriate dilutions were made from this solution; 5 ul of the solution were used in all experiments - incubation as well as aggregation. Controls were treated with 5 ul alcohol. Blood was obtained from healthy donors who had not taken aspirin or any such drug known to affect platelet function for at least 7-10 days prior to giving blood. For aggregation experiments platelet-rich plasma (PRP) and platelet-poor plasma They were used in aggregation as were prepared by differential centrifugation. described earlier with minor changes (2). After mixing the alcoholic solution of OC for 30 set in the aggregometer, the contents were allowed to stay at room temperature for ca. 5 min before producing aggregation. Preparation of washed platelet suspension, incubation with labelled AA and extraction of AA metabolites were carried out as described elsewhere (1). Thromboxane B2 was resolved from other products of AA by TLC using the solvent system ethyl acetate-isooctaneacetic acid-water (110:50:20:10, v/v, of which upper organic phase was used after mixing for 5 min) (I). For the separation of lipoxygenase products from AA and its metabolites solvent II (n-hexane-ether-acetic acid, 80:20:1, v/v) was used. Hydroxy derivatives of AA (HHT and HETE) were localized in a certain area of the plate (between the application point containing TxB2, ptostaglandins and phospholipids, and AA, Rf value 0.32). The entire area between the application point and AA was scraped off and its radioactivity was measured. The amount of HETE was determined by subtracting TxB2 counts (TxB2 = HHT) from the total counts due to HHT and HETE (5). We had to resort to this method because of the lack of reference standards (HHT and HETE). Labelling of platelets with arachidonate was achieved by incubating them (as PRP) with low concentrations (0.10 uM) of AA. At low concentrations, AA is not metabolized by the platelet enzymes - cyclooxygenase and lipoxygenase; AA is mainly incorporated into platelet phospholipids, and of the amount of AA finding access to the platelets, more than 95% is incorporated into phospholipids (6). were used in two different experiments. (i) The effect of Labelled platelets OC was examined on the incorporation of labelled AA in platelet phospholipids. For this purpose platelets (PRP were first treated with 5 ul alcoholic solution of the oil (112 ug/ml PRP) (experimental) or with 5 ul ethanol (control) by incubating for 5 min at 37OC followed by incubation with labelled AA at 37OC for 2 h. The contents were centrifuged at 1500 x g, supernatant decanted and the platelets resuspended in I ml Ringer-citrate-dextrose (RCD). Finally the suspension was extracted in 8 ml chloroform-methanol (2:l) overnight at 4oC. (ii) The effect of OC was examined on platelet phospholipase activity and on the formation of TxB2 and lipoxygenase products from endogenous AA. The method used by us is a modification of that described by Bills et al. (6). In our method labelled platelets were washed with, and resuspended in RCD. Details of the procedure describing mixing of platelets with the test material, incubation, activation of labelled platelets with calcium ionophore A23187 (5 uM), extraction of phospholipids and AA metabolites have been described recently by us (3). For the TLC separation of TxB2 from phospholipids and other AA metabolites solvent I was used. Lipoxygenase ptoducts were separated and quantified as described above. Statistics Statistical significance was evaluated by Student’s t-test for paired data.
12
RESULTS Influence of OC on the metabolism of exogenous arachidonate in platelets. Washed platelet suspensions were treated with various concentrations of OC, control platelets were treated with 5 ul alcohol prior to treatment with arachidonate (2). Arachidonic acid metabolites were extracted in ether after acidification and resolved by TLC. It was found that OC reduced thromboxane formation at the three concentrations used; it reduced the lipoxygenase products at higher concentrations but at the lowest concentration the lipoxygenase products were increased. At all concentrations of OC more unused AA was recovered (Fig. 1).
Figure 1. Autoradiogram of the TLC plate showing the effect of OC on the formation of arachidonic acid metabolites by human platelets. C = control platelets; I, II and III = experimental platelets treated respectively with 11.25 ug, 45 ug and 450 ug OC/200 ul platelet suspension. Arachidonic acid (6.5 uM). a. Separation of TxB2 from other products by applicating 50 ul of 200 ul extract and using solvent ethyl acetate-isooctane-acetic acid-water (110:50:20:100, v/v). b. Separation of HETE, HHT and AA from TxB2 and prostaglandins (PCs) which remained at the application point together with the phospholipids (Rf = 0.00) by applicating 25 ul of 200 ul extract using solvent n-hexane-ether-acetic acid (80:20:1, v/v). Control values (n = 2) for TxB2 = 183 picomoles and lipoxygenase products = 811 picomoles/108 platelets. Values (% of control) for OC-treated platelets TxB2: I = 13.2%, II = 5.2%, III = 0.6%. Lipoxygenase products: I = 146%, II = 56%, III = 1.5%. Influence of OC on the platelet phospholipase activity and on the endogenous formation of thromboxane and lipoxygenase products in platelets. The experimental set up used here required the use of AA-labelled platelets which were challenged
13
by Ca2+-ionophore A23187 after treatment with 5 ul OC solution (experimental platelets) or with 5 ul alcohol (control platelets). On activation platelets liberated AA which was metabolized into various products of which TxB2 and lipoxygenase products were determined. By this method the effect of clove oil was examined at three dose levels - 450 ug, 45 ug and 11.25 q/500 ul platelet suspension. At 450 ug/500 ul dose there was observed no change in the phospholipase activity from control platelets; the amounts of TxB2 and lipoxygenase products decreased significantly with a concomitant increase in the AA counts (Fig. 2). At 45 ug and 11.25 ug/500 ~1 dose levels TxB2 was significantly decreased; lipoxygenase products were sli htly (by 15%) reduced at 45 ug/500 dose level without any effect at 11.25 ugB500 ul level (Fig. 3). CPM x lo-’ 8 1
I
PL
I3 ControlIn=41
II
APPL
TxB?
HETE
AA
Figure 2. Effect of OC on the formation of arachidonic acid metabolites from endogenous phospholipid (14C)-arachidonic acid. Washed platelets previously treated with (I4Qarachidonic acid to permit incorporation into platelet phospholipids were exposed to calcium ionophore A23187 (5 uM). In a typical experiment the effect of OC (450 ug/500 ul) added 10 min prior to platelet stimulation is shown. The values (CPM, Mean + SD) are l/6 of the total amount produced. Control and experimental determinations were done in duplicate. * p < 0.05 ** p < 0.01 PL = phospholipids and TxB2 = thromboxane B2 (separated by solvent I). APPL = application point; HETE and AA (separated by solvent II). Influence of OC on the incorporation of labelled arachidonate into platelet hos holi ids. Incorporation of AA into platelets was studied by incubating AA 0.12 uM with PRP (4 ml) previously treated with 5 ul alcohol (control) or with 5 ul Y+-of alcoholic solution of OC (112 ug/ml PRP). At this concentration OC potentiated incorporation of AA into platelets (p < 0.05) (control 1797 + 502, OC-treated 1981 t 519, values are in CPM, Mean + SD obtained with ca. 1 x 109 platelets). Aggregation experiments. OC inhibited aggregation induced by arachidonate, collagen and epinephrine; this effect was dose-related. It was found to be most effective in AA-induced aggregation; even 11.25 ug OC/ml PRP were sufficient to abolish AA-induced platelet aggregation (Fig. 4). In five blood samples half of this dose (5.5 ug/ml PRP) produced total inhibition of aggregation. Collagen-induced aggregation was completely inhibited by 45 ug OC/mI PRP (3 blood samples). In two other blood samples abolition of collagen-induced aggregation was achieved 14
IZZI Control
CPMxlO-'
t?3 OC-treated
6
CPM 80-
I
LIPOXYGENASE PRODUCTS
J :.: :, :.:
4-
TxBz
2-
:;I ::: ::: :b: L
OI
II
Figure 3. Effect of OC on the formation of thromboxane B2 and hydroxy acid (HETE) in Ca2+-ionophore A23187-treated platelets, whose phospholipids were prelabelled with (14C)-arachidonic acid. I = OC 45 ug/500 ul (n = B), II = OC 1I .25 ug/500 ul (n = 7). *p < 0.05 The values (CPM, Mean + SD) are l/6 of the total amount produced by 0.5 x lo9 platelets. Arachldonatel0.5mMl
Arach~donotelO.SmtlI
Figure 4. Representative tracings showing the inhibitory effect of OC on arachidonate-induced aggregation with two separate blood samples (A and B). A. 1 and 6 control tracings. Tracings 2-5 were obtained by treating respectively with 22.5 ug, 11.25 ug, 5.5 ug and 45 ug OC/ml PRP. 8. 1 control tracing. Tracings 2-5 were obtained by treating respectively with 90 ug, 45 ug, 22.5 ug and 11.25 ug OC/ml PRP. Aggregation was performed in the sequence of numbers appearing on the tracings.
15
with 90 ug/ml. With lower doses (22.5-45 ug/ml) the first phase of aggregation was reversed suggesting disaggregation effects (Fig. 5). In epinephrine-induced aggregation OC (11.25-90 ug/ml) inhibited the second phase of aggregation; the first phase was reversed as in the case of collagen-induced aggregation (Fig. 6). C0llogsn Ilopglml,
Collagen IlOyglmli
Figure 5. Representative tracings showing the effect of OC on collagen-induced aggregation with two separate blood samples (A and B). A. 1 and 4 control tracings. Tracings 2 and 3 were obtained by treating respectively with 45 ug and 90 ug OC/ml PRP. B. 1 control tracing. Tracings 2-6 were obtained by treating respectively with 22.5 ug, 90 ug, 11.25 ug, 45 ug and 450 ug OC/ml PRP. Aggregation was performed in the sequence of numbers on the tracings. Epinephrine IlO@II
Epinephrine
(lOpt41
7
Figure 6. Representative tracings showing the inhibitory effect of OC on epinephrine-induced platelet aggregation. A and B were obtained with the same blood sample. 1 and 7 control tracings. Tracings 2-6 were obtained by treating respectively with 45 ug, 90 ug, 450 ug, 22.5 ug and 11.25 ug OC/ml PRP. Aggregation was performed in the sequence of numbers on the tracings. 16
2 q
5 uM)
796 + 274**
1207 f 274
Phospholipidsa
pointb
902 + 277* *
1297 + 305
Application
28 + 13**
423
TxB2c
acid from platelet
accounted
productsc§
297 + 72**
724
Lipoxygenase
phospholipids
by the
28 t 25*
826
AAc@
for mainly
The values (CPM, Mean + SD) are l/6 of the total amount produced by 0.5 x IO9 platelets. a counts are due to phospholipids. Thromboxane B2, prostanglandins (PCs) and other hydroxy metabolites of arachidonic acid (AA) were resolved completely from the phospholipids using solvent system I. b Counts are due to phospholipids, TxB2, PCs (Rf = 0.00). HHT and HETE (HPETE) and AA were resolved by using solvent II. c Counts are shown after substracting the background counts. 9 Includes counts mainly due to lipoxygenase products (HETE, HPETE). $0 Excess of the liberated AA from platelets phospholipids. * p < 0.05 ** p < 0.01
Experimental U&3187-treated,
Control (n__lgcoholtreated)
Exp. condition
Table 1. Calcium ionophore A23187 induced loss of (L4C) arachidonic appearance of oxygenated products of arachidonic acid
DISCUSSION The results of the present study suggest that OC contains component(s) that How this is achieved, we have tried to explain by inhibit platelet aggregation. examining the formation of cyclooxygenaseand lipoxygenase products from AA by human platelets. It was found that thromboxane was reduced. This was demonstrated by TxB2 formation from exogenous AA in washed (intact) and lysed platelet preparations; the effect was dose-dependent. At low-dose levels an increased formation of lipoxygenase products was observed suggesting a mechanism that could involve a redirection of AA from cyclooxygenase pathway to lipoxygenase pathway and/or potentiation of lipoxygenase enzyme. Use of AAlabelled platelets and their activation by Ca2+ -ionophore A23187 has been found to be suitable for the determination of metabolites produced from endogenous AApool (Table 1). Formation of these AA metabolites from AA-labelled platelets activated by Ca2+ -ionophore shows that reduced formation of TxB2 was due to the effect of OC on cyclooxygenase/Tx-synthetase enzyme(s) as no effect on the phospholipase activity was observed at the three concentrations used. At the lower two concentrations lipoxygenase products were not reduced. In conclusion, it may be said that inhibition of AA-, collagenand epinephrine-induced aggregation by OC may, in part, be due to inhibition of cyclooxygenase/Tx-synthetase enzymes. This may find support from its inability to inhibit A23187 and ADP-induced aggregation. ACKNOWLEDGEMENTS We wish to thank the personnel of the Blood Bank, Odense University Hospital for collection of blood samples. Ruth B. Alexandersen provided technical assistance. Mrs. Inge Bggelund typed the manuscript. REFERENCES 1. Srivastava, K.C. Effect of aqueous platelet aggregation and metabolism system: in vitro study. Prostaglandins 1984. 2.
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