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A new 1,4-dihydropyridine derivative (PCA-4230) prevents PIP2 hydrolysis and protein phosphorylation in PAF-stimulated rabbit platelets
345
et al., 1988). The evidence for existence of an additional mice plasma factor (AF) acting synergistically with know proaggregatory stimuli is being reported in the present communication. Male albino mice (20-25 g) of swiss strain were used in the study. They were injected with 100 pl of a mixture containing 30 pg collagen and 10 pg adrenaline in saLine into the tail vein. Blood was collected in sodium euate (0.129 M, 8: 2 v/v) by cardiac punctttre under ether anesthesia from treated and umreated animals. Plasma was obtained by high speed centrifugation. Platelet aggregation was monitored in normal rhesus monkey platelet rich plasma on Payton single channel aggregometer following Born’s turbidimetric technique (1962). Collagen and adrenaline administration resulted in death/paralysis of hind limbs of 100% of the animals. Incubation of monkey PRP with 100 gl of thrombotic mice plasma (TP) increased the ADP (2 X 10m5 M) induced aggregation by 40.0 f 7% (n = 40) in comparison to control. Pre-incubation of monkey PRP with TP also potentiated the aggregatory response of arachidonic acid (5 X lob6 M), calcium ionophore A23187 (2 pg) and collagen (5 pg) in a similar manner. However, addition of TP (upto 200 ~1) to monkey PRP did not initiate the aggregatory response. Indomethacin (3 x lo-” M), nordiiydroguaertic acid (3 x lo-’ M), BW755C (5 x 10V4 M), phentolamine (4 x 10m5 M), cimetidine (4 x 10m4 M) and ketanserin (2 x lO-4 M) could not b!ock the response of AF. The active component was not present in ether or chloroform: methanol (2 : 1) extract. In addition, no change in malonaldehyde levels and synthesis of prostacyclin was observed in presence of AF. Mice plasma was incubated with creatine phosphate (8 x 10V4 M) and creatine phosphokinase (80 units) for 1 hr at 37’C. Increase in aggregatory response due to AF remained unaltered. Preincubation of TP with heparin (150 pg), phospholipase A, (40 units) and collagenase (100 pg) had no effect on the aggregatory response. These results suggest that the AF is not arachidonic acid, cyclooxygenase product, lipa,xygenase product, adenosine diphosphate, platelet activating factor, platelet factor, collagen or biogenic amines. Fractionation of the plasma indicated the presence of AF in acetone precipitate. Activity was destroyed by pronase (O.OSW),charcoal treatment and had molecular weight less than 10 Kda but more than 3.5 Kda. Existence of such a factor which is heat resistant, is of low molecular weight and is proaggregatory in nature and which requires calcium ions for the expression of its activity, has not been reported earlier.
eferences Guameli, L., IMolinari, A., Cassci, E.. Pace], C., Cerletti, C. and G. deGaetano, 1988, J. Pharmacol. Methods 20, 161. Born, G.V.R., 1962. Nature 194, 927.
I
P.mo.i61
new 1,
ine derivative phosphorylation in
ysis
irn
Cat.alhn *, R.E., Martinez * *, A.M., Aragonts * *, M.D., Garde *, E., Lombardia * * *, M., Encabo * * *, P. and Wem&ndez * * *, F. y Biologic MJechr II, KM, Madrid * Dcpto. de BiologiaMolecular,CBM-UAM, Madrid * * Repto. de Bioquimicry and * * * Depto. de Investigackh, LaborotoriosAlter, Madrid Spain
Stimulation of platelets with physiological agents such as thrombin, collagen and platelet activating factor induces rapid changes in membrane phospholipids via the activation of phospholipase C, leading to the sequential formation of 1,3 diacyiglycerol which may play a direct role in mediating platelet activation by increasing the protein kinase C activity. Recently, a new potent antithrombotic drug has been synthetized by Laboratorios Alter (Madrid. Spain) (Sunkel et al., 1988). The compound, 2(-1,1,3-trioxo-2,3-dihydro-l,l-benzisot~~zo~-~-yl)ethyl 2,6-diiethyl-5-(ethoxycarbonyl)-4 methyl-l&lihydrophyridine-carboxylate (code name: PCA-4230) was the most efective inhibitor of collagen-induced platelet aggregation. Some preliminary data on the mode of action of PCA-4230 has been reported (Catalan et al,, 1989a). Thus, the effects of extracellular calcium on protein phosphorylation stimulated by collagen in rabbit platelets have been investigated. The drug inhibited the phosphorylation of 40 kDa and 20 kDa protein
tIy of the extra&h&u calcium. The inhibitory effect was dose-dependent but not time-dependent and was t when the drug was added before or simultaneously with collagen. In order to study further the mechanism of action of the drug we describe the results obtained on the PCA-4230 effects on protein tion and on phosphoinositide metabolism in platelets by using platelet activating factor as agonist. gterials, platelet preparation, platelet agregation and platelet protein phosphorylation have been reported previously et al., 1989b); lipid extraction and phosphoinositide separation were carried out as previously described et al., 1989a); diacylglycerol separation and phosphorous determination were carried out as described in the literature. We have demonstrated thath PCA-4230 is able to prevent both phosphatidylinositol bisphosphate hydrolysis and 1,2diacylglycerol generation evoked by platelet activating factor but not by thrombin in washed rabbit platelets. Furthermore the treatment with PCA-4230 also prevents the phosphorylation of both 20 kDa and 40 kDa proteins caused by platelet activating factor. When the platelets were stimulated with thrombin the drug was not able to prevent the protein phosphorylation. On the other hand the inhibitory action of PCA-4230 is also observed when the drug is added simultaneously with the agonist but if the treatment is made after exposure to platelet activating factor the drug does not exert its action. In addition, PCA-4230 is able to prevent the platelet activating factor evoked responses in the presence of extracellular calcium as well as in a calcium free medium (with EGTA), a fact that suggest that the drug effect does not depend on calcium entry. To fully characterize the precise mechanism of this new dihydropyridine derivative, further studies are required. Experiments are being conducted in our laboratory to explore these mechanisms further.
Sunk& C.E. Casa-Juana, M.F., Ciiero, F.J., P&go, J.G. and Ortega, P., (1988), J. Med. Chem. 31,1886. titalkn, RE, (1989a) Internal Report, Laboratorios Alter, Madrid, Spain. CataS, RE, Martinez, A.M., Aragonk, M.D., Eucabo, P. and HemBndez, F., (1989b), B&hem. Int. 19,919. jP.mo.1621
d blocker on human Wang, Z. and Wang, L. Department of Phamacology,Instituteof Basic Medical Sciences, Chinese Aca&my of Medical Sciences/Peking Union Medical College, 5 Dong Dan San Tkw, Dong Dan, Beijing 100730, People’s Reptddic of China
Ilexonin A is an effective compound isolated from Ilex pubescens hook. et Am. It has been primarily used in clink experiments have shown that it inhibits thrombos&, platelet adhesion, and platelet aggregation and 5-HT release induced by collagen or ADP in vitro. It also inhibits r&t platelet aggregation in vivo. In the present experiments, the effects of Ilexonin A on the level of cytoplasmic free calcium concentration ([Ca”],) and CAMP in platelets were observed. These effects were compared with those of Verapamil-a known calcium blocker. The results iudicated that the percentage of platelet aggregation induced by ionophore A23187 (3 pM) was 85.9 f 9.0. The addition of Ilexonin A (208 PM) or Verapamil (400 PM) to PRP resulted in inhibition of platelet aggregation by the same inducer, the aggregation percentages were 21.7 f 5.5 and 31.3 f 10.2 respectively. However the inhibitory effects of both drugs were significantly antagonized by adding 1 mM CaCI, to the medium. It suggests that the effect of Ilexonin A on platelet function is possibly related to calcium. Experiments were then designed to investigate the effects of both drugs on thrombin-induced calcium fluxes of platelets in which the fluorescent probe, quin-2, was used to measure [Ca*+]i. It was found that, in the presence of 1 mM external calcium, thrombm raised [Ca*+]i around 10 fold a few seconds to peak near 1 CM. In the absence of external calcium there was a much smaller increase in /Ca*+], of similar pattern. These findings suggest that thrombin increases [Ca*+]i partly be discharge of internal caltium, but mainly by stimulated influx. Ilexonin A (17, 87,124 and 261 @I) and Verapamil (32, 64, 96 and 128 PM) m-n;lrkedly iuhibited the thrombin-induced calcium influx in a dose-dependent pattern. The I&were 76.8 PM and 67.5 PM respectively, but both had no effect on thrombin-induced calcium release from dense tubular system. M treat cerebral embolism. coronary heart disease, cardiac failure tilr --A vasculitis etc. Pharmacological
et al., 1988). The evidence for existence of an additional mice plasma factor (AF) acting synergistically with know proaggregatory stimuli is being reported in the present communication. Male albino mice (20-25 g) of swiss strain were used in the study. They were injected with 100 pl of a mixture containing 30 pg collagen and 10 pg adrenaline in saLine into the tail vein. Blood was collected in sodium euate (0.129 M, 8: 2 v/v) by cardiac punctttre under ether anesthesia from treated and umreated animals. Plasma was obtained by high speed centrifugation. Platelet aggregation was monitored in normal rhesus monkey platelet rich plasma on Payton single channel aggregometer following Born’s turbidimetric technique (1962). Collagen and adrenaline administration resulted in death/paralysis of hind limbs of 100% of the animals. Incubation of monkey PRP with 100 gl of thrombotic mice plasma (TP) increased the ADP (2 X 10m5 M) induced aggregation by 40.0 f 7% (n = 40) in comparison to control. Pre-incubation of monkey PRP with TP also potentiated the aggregatory response of arachidonic acid (5 X lob6 M), calcium ionophore A23187 (2 pg) and collagen (5 pg) in a similar manner. However, addition of TP (upto 200 ~1) to monkey PRP did not initiate the aggregatory response. Indomethacin (3 x lo-” M), nordiiydroguaertic acid (3 x lo-’ M), BW755C (5 x 10V4 M), phentolamine (4 x 10m5 M), cimetidine (4 x 10m4 M) and ketanserin (2 x lO-4 M) could not b!ock the response of AF. The active component was not present in ether or chloroform: methanol (2 : 1) extract. In addition, no change in malonaldehyde levels and synthesis of prostacyclin was observed in presence of AF. Mice plasma was incubated with creatine phosphate (8 x 10V4 M) and creatine phosphokinase (80 units) for 1 hr at 37’C. Increase in aggregatory response due to AF remained unaltered. Preincubation of TP with heparin (150 pg), phospholipase A, (40 units) and collagenase (100 pg) had no effect on the aggregatory response. These results suggest that the AF is not arachidonic acid, cyclooxygenase product, lipa,xygenase product, adenosine diphosphate, platelet activating factor, platelet factor, collagen or biogenic amines. Fractionation of the plasma indicated the presence of AF in acetone precipitate. Activity was destroyed by pronase (O.OSW),charcoal treatment and had molecular weight less than 10 Kda but more than 3.5 Kda. Existence of such a factor which is heat resistant, is of low molecular weight and is proaggregatory in nature and which requires calcium ions for the expression of its activity, has not been reported earlier.
eferences Guameli, L., IMolinari, A., Cassci, E.. Pace], C., Cerletti, C. and G. deGaetano, 1988, J. Pharmacol. Methods 20, 161. Born, G.V.R., 1962. Nature 194, 927.
I
P.mo.i61
new 1,
ine derivative phosphorylation in
ysis
irn
Cat.alhn *, R.E., Martinez * *, A.M., Aragonts * *, M.D., Garde *, E., Lombardia * * *, M., Encabo * * *, P. and Wem&ndez * * *, F. y Biologic MJechr II, KM, Madrid * Dcpto. de BiologiaMolecular,CBM-UAM, Madrid * * Repto. de Bioquimicry and * * * Depto. de Investigackh, LaborotoriosAlter, Madrid Spain
Stimulation of platelets with physiological agents such as thrombin, collagen and platelet activating factor induces rapid changes in membrane phospholipids via the activation of phospholipase C, leading to the sequential formation of 1,3 diacyiglycerol which may play a direct role in mediating platelet activation by increasing the protein kinase C activity. Recently, a new potent antithrombotic drug has been synthetized by Laboratorios Alter (Madrid. Spain) (Sunkel et al., 1988). The compound, 2(-1,1,3-trioxo-2,3-dihydro-l,l-benzisot~~zo~-~-yl)ethyl 2,6-diiethyl-5-(ethoxycarbonyl)-4 methyl-l&lihydrophyridine-carboxylate (code name: PCA-4230) was the most efective inhibitor of collagen-induced platelet aggregation. Some preliminary data on the mode of action of PCA-4230 has been reported (Catalan et al,, 1989a). Thus, the effects of extracellular calcium on protein phosphorylation stimulated by collagen in rabbit platelets have been investigated. The drug inhibited the phosphorylation of 40 kDa and 20 kDa protein
tIy of the extra&h&u calcium. The inhibitory effect was dose-dependent but not time-dependent and was t when the drug was added before or simultaneously with collagen. In order to study further the mechanism of action of the drug we describe the results obtained on the PCA-4230 effects on protein tion and on phosphoinositide metabolism in platelets by using platelet activating factor as agonist. gterials, platelet preparation, platelet agregation and platelet protein phosphorylation have been reported previously et al., 1989b); lipid extraction and phosphoinositide separation were carried out as previously described et al., 1989a); diacylglycerol separation and phosphorous determination were carried out as described in the literature. We have demonstrated thath PCA-4230 is able to prevent both phosphatidylinositol bisphosphate hydrolysis and 1,2diacylglycerol generation evoked by platelet activating factor but not by thrombin in washed rabbit platelets. Furthermore the treatment with PCA-4230 also prevents the phosphorylation of both 20 kDa and 40 kDa proteins caused by platelet activating factor. When the platelets were stimulated with thrombin the drug was not able to prevent the protein phosphorylation. On the other hand the inhibitory action of PCA-4230 is also observed when the drug is added simultaneously with the agonist but if the treatment is made after exposure to platelet activating factor the drug does not exert its action. In addition, PCA-4230 is able to prevent the platelet activating factor evoked responses in the presence of extracellular calcium as well as in a calcium free medium (with EGTA), a fact that suggest that the drug effect does not depend on calcium entry. To fully characterize the precise mechanism of this new dihydropyridine derivative, further studies are required. Experiments are being conducted in our laboratory to explore these mechanisms further.
Sunk& C.E. Casa-Juana, M.F., Ciiero, F.J., P&go, J.G. and Ortega, P., (1988), J. Med. Chem. 31,1886. titalkn, RE, (1989a) Internal Report, Laboratorios Alter, Madrid, Spain. CataS, RE, Martinez, A.M., Aragonk, M.D., Eucabo, P. and HemBndez, F., (1989b), B&hem. Int. 19,919. jP.mo.1621
d blocker on human Wang, Z. and Wang, L. Department of Phamacology,Instituteof Basic Medical Sciences, Chinese Aca&my of Medical Sciences/Peking Union Medical College, 5 Dong Dan San Tkw, Dong Dan, Beijing 100730, People’s Reptddic of China
Ilexonin A is an effective compound isolated from Ilex pubescens hook. et Am. It has been primarily used in clink experiments have shown that it inhibits thrombos&, platelet adhesion, and platelet aggregation and 5-HT release induced by collagen or ADP in vitro. It also inhibits r&t platelet aggregation in vivo. In the present experiments, the effects of Ilexonin A on the level of cytoplasmic free calcium concentration ([Ca”],) and CAMP in platelets were observed. These effects were compared with those of Verapamil-a known calcium blocker. The results iudicated that the percentage of platelet aggregation induced by ionophore A23187 (3 pM) was 85.9 f 9.0. The addition of Ilexonin A (208 PM) or Verapamil (400 PM) to PRP resulted in inhibition of platelet aggregation by the same inducer, the aggregation percentages were 21.7 f 5.5 and 31.3 f 10.2 respectively. However the inhibitory effects of both drugs were significantly antagonized by adding 1 mM CaCI, to the medium. It suggests that the effect of Ilexonin A on platelet function is possibly related to calcium. Experiments were then designed to investigate the effects of both drugs on thrombin-induced calcium fluxes of platelets in which the fluorescent probe, quin-2, was used to measure [Ca*+]i. It was found that, in the presence of 1 mM external calcium, thrombm raised [Ca*+]i around 10 fold a few seconds to peak near 1 CM. In the absence of external calcium there was a much smaller increase in /Ca*+], of similar pattern. These findings suggest that thrombin increases [Ca*+]i partly be discharge of internal caltium, but mainly by stimulated influx. Ilexonin A (17, 87,124 and 261 @I) and Verapamil (32, 64, 96 and 128 PM) m-n;lrkedly iuhibited the thrombin-induced calcium influx in a dose-dependent pattern. The I&were 76.8 PM and 67.5 PM respectively, but both had no effect on thrombin-induced calcium release from dense tubular system. M treat cerebral embolism. coronary heart disease, cardiac failure tilr --A vasculitis etc. Pharmacological