Karl J. Romstedt

Antagonism of prostaglandin-mediated responses in platelets and vascular smooth muscle by 13-azaprostanoic acid analogs: Evidence for selective blockade of thromboxane A2 responses

Studies were undertaken to examine the pharmacological properties and stereochemical requirements of a limited series of prostanoic acid analogs for inhibition of arachidonic acid (AA) and/or endoperoxide (U46619)-mediated responses in human platelets and rat aorta. To assess the role of stereochemistry, a set of trans- and cis-isomers of 13-azaprostanoic acid (APA) and 11a-homo-13-azaprostanoic acid (HAPA) were prepared. Each prostanoic acid analog blocked AA- or U46619-induced aggregatory and secretory responses in platelets, and U46619-mediated contractions of rat aorta in a concentration-dependent manner (0.1 to 100 microM). The azaprostanoic acid analogs blocked responses to both inducers of platelet activation with IC50 values ranging from 3.4 to 27.5 microM. Trans-APA was about 2- to 3-fold more active as an antagonist of serotonin release induced by AA or U46619 than the remaining analogs. The rank order of inhibitory potency (IC50; microM) for these analogs against U46619-induced serotonin release in human platelets was trans-APA (3.4) greater than cis-APA (8.9) = cis-HAPA (8.7) = trans-HAPA (9.1). Concentrations of the prostanoic acid analogs required to block these responses to AA and U46619 were similar, and the highest concentration used (100 microM) did not modify AA-induced malondialdehyde production in human platelet preparations. In contrast, the isomers of APA and HAPA were equally active as antagonists of U46619-induced contractions of rat vascular tissue, possessing KB values varying from 7.1 to 13.2 microM. Each azaprostanoic acid analog shifted the concentration-response curve of U46619 in rat aorta to the right, indicating a competitive-type inhibition. In addition, the azoprostanoic acid analog (U51605) was a more potent competitive antagonist of U46619 in this preparation and possessed an average pKB value of 6.18. In summary, the results show that (1) expansion of the five-membered ring of APA to the six-membered ring analogs (HAPA) led to a retention of potent inhibitory activity against U46619 in human platelets and rat vascular smooth muscle, (2) the antiaggregatory and antisecretory actions of the azaprostanoic acid analogs were mediated by a blockade of the responses to AA and U46619, and not by an inhibition of AA metabolism, (3) the blocking activity for the APA isomers was stereoselective (trans greater than cis) whereas the isomers of HAPA were equally effective as inhibitors of platelet function; and (4) these azaprostanoic acid analogs act as selective endoperoxide (U46619)/thromboxane A2 antagonists in these two tissues.

Characterization of the inhibition of U46619-mediated human platelet activation by the trimetoquinol isomers: Evidence for endoperoxide/thromboxane A2 receptor blockade

Sites of inhibition for the trimetoquinol (TMQ) isomers on 15S-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid (U46619)-, 12-O-tetradecanoylphorbol 13-acetate (TPA)- and A23187-induced human platelet activation were investigated. Experiments using washed human platelets were designed to characterize relationships among functional (aggregation, secretion) and biochemical (protein phosphorylation, metabolism of inositol phospholipids and radioligand displacement analysis) processes of platelet activation by U46619 and the specificity of inhibition by the TMQ isomers. Thromboxane A2 receptor stimulation by U46619 in human platelets resulted in a time- and concentration-dependent breakdown of inositol phospholipids [phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-monophosphate (PIP), and phosphatidylinositol (PI)], phosphatidic acid (PA) accumulation, phosphorylation of 20 and 45 kD proteins, aggregation and serotonin secretion. The TMQ isomers stereoselectively inhibited all U46619-mediated platelet activation processes. R(+)-TMQ was 40- and 22-fold more potent than S(-)-TMQ as an inhibitor of U46619-induced platelet aggregation and serotonin secretion respectively. In addition, R(+)-TMQ blocked U46619-induced 20 kD protein phosphorylation, 45 kD protein phosphorylation, PIP2, PIP and PI breakdown, and PA accumulation with a potency which was 8-, 13-, 45-, 37-, 33- and 33-fold greater than the S(-)-isomer respectively. In contrast to S(-)-TMQ, R(+)-TMQ produced a concentration-dependent inhibition of specific [3H]U46619 binding to endoperoxide/thromboxane A2 receptor sites in washed platelets. In other experiments, S(-)-TMQ was more potent than R(+)-TMQ as an inhibitor of TPA- and A23187-induced platelet aggregation and serotonin secretion, and of TPA-induced phosphorylation of 45 and 20 kD proteins. The inhibitory potencies of S(-)-TMQ against TPA- or A23187-induced responses were similar to those needed for antagonism of U46619-mediated platelet activation. In contrast, much higher concentrations of R(+)-TMQ were required for blockade of TPA or A23187 versus U46619-mediated responses in human platelets. Taken collectively, the data show that the TMQ isomers interfered with the endoperoxide/thromboxane A2 receptor-mediated phospholipase C-signal cascade of inositol phospholipid hydrolysis, calcium mobilization, and protein phosphorylation leading to platelet aggregation and secretion. R(+)-TMQ acted as a pharmacologically selective and highly stereospecific [R(+)-TMQ much greater than S(-)-TMQ] antagonist of endoperoxide/thromboxane A2 receptor sites in platelets.(ABSTRACT TRUNCATED AT 400 WORDS)

Benzodiazepines inhibit human platelet activation: comparison of the mechanism of antiplatelet actions of flurazepam and diazepam

These studies were undertaken to examine the effects and the mechanism of action of flurazepam and diazepam on human platelet activation. One minute preincubation with flurazepam (3-300 microM) or diazepam (3-300 microM) inhibited platelet aggregation, serotonin secretion and prostaglandin synthesis induced by ADP (1-5 microM), epinephrine (1-5 microM), and arachidonic acid (600-1000 microM). However, 357% higher concentration of diazepam (265 microM) as compared to flurazepam (58 microM), was required to inhibit arachidonic acid induced production of malondialdehyde (MDA) by 50%. In addition, flurazepam and not diazepam inhibited the release of arachidonic acid from platelet phospholipids in a concentration dependent manner. In other experiments flurazepam but not diazepam also blocked aggregation and secretion induced by U46619 (2 microM), a stable analog of prostaglandin H2. Platelet aggregation and serotonin secretion induced by collagen (40-300 micrograms/ml) was inhibited by flurazepam with an IC-50 of 153 microM and 136 microM respectively, whereas higher than 300 microM diazepam was required to inhibit collagen-induced aggregation and secretion by 50%. Flurazepam and diazepam both exhibited their most potent antiplatelet effects against phospholipase C-induced aggregation which is mediated by prostaglandin-independent mechanisms. Only 15 microM and 11 microM flurazepam and 31 microM and 27 microM diazepam were needed to inhibit PLC-induced aggregation and secretion of serotonin by 50% respectively. Effects of these benzodiazepines on platelet cyclic AMP and cyclic GMP were also examined. Neither flurazepam nor diazepam caused any significant change in cyclic AMP or cyclic GMP levels in platelets. These findings suggest that: (a) flurazepam, as compared to diazepam, is 106% - 357% more effective in inhibiting platelet aggregation and serotonin secretion induced by arachidonic acid, collagen and phospholipase C; (b) flurazepam inhibits platelet activation by inhibiting the release of arachidonic acid, its conversion into prostaglandins and by blocking the action of prostaglandins on platelets; (c) diazepam does not inhibit thrombin-induced release of arachidonic acid, conversion of exogenously added arachidonic acid into MDA, or the action of prostaglandins; (d) both flurazepam and diazepam inhibit PLC-mediated activation of platelets; and (e) neither diazepam nor flurazepam achieve their antiplatelet actions by affecting platelet cyclic nucleotide levels.

Synthesis and in vitro platelet aggregation and TP receptor binding studies on bicyclic 5,8-ethanooctahydroisoquinolines and 5,8-ethanotetrahydroisoquinolines.

Eighteen novel bicyclic 1-substituted benzyl octahydro- and tetrahydroisoquinolines were synthesized and evaluated for human thromboxane A(2)/prostaglandin H(2) (TP) receptor affinity and antagonism of TP receptor-mediated platelet aggregation. In both cases, potency depended more on the presence of methoxy groups on the 1-benzyl moiety than on nitrogen substitution or extent of oxidation of the isoquinoline ring system. The most potent of the bicyclic compounds retained the 5,8-ethanooctahydroisoquinoline ring structure of the parent molecule (1) and required the 3,4,5-trimethoxybenzyl substitution pattern found in the well-characterized tetrahydroisoquinoline antiplatelet agent trimetoquinol. Differences in nitrogen substituent SAR were noted between the mono-methoxylated compounds and the 3,4,5-trimethoxybenzyl derivatives.

Human platelet activation by bacterial phospholipase C is mediated by phosphatidylinositol hydrolysis but not generation of phosphatidic acid: Inhibition by a selective inhibitor of phospholipase C

We have shown earlier that phospholipase C (PLC) from Clostridium perfringens causes human platelet aggregation and secretion in a concentration dependent manner. The present study was undertaken to further characterize the specificity of the effects of PLC and to better understand the mechanism of the action of this inducer. A methylene-dioxybenzazepine (MDBA) analog of trimetoquinol was synthesized and tested for antiplatelet activity. MDBA (3-30 microM) inhibited PLC-induced aggregation in a concentration dependent manner. Whereas up to 200 microM MDBA did not inhibit aggregation induced by either thrombin, arachidonic acid, or U46619. Effects of PLC (0.05 U/ml) on hydrolysis of phosphatidylinositol, production of phosphatidic acid and thromboxane B2 (TXB2) synthesis were investigated using [32P]-phosphate and [14C]-arachidonic acid labeled platelets. PLC (0.05 U/ml) caused a time dependent decrease in platelet phosphatidylinositol. Up to 50% of labeled phosphatidylinositol was lost from platelets in five minutes. MDBA (3-30 microM) inhibited PLC-induced loss of phosphatidylinositol in a concentration dependent manner. An increase in phosphatidic acid was also observed in PLC-stimulated platelets. Up to 100 microM MDBA did not inhibit production of phosphatidic acid. PLC-treated platelets did not produce any TXB2. In other experiments possible protease contamination of PLC preparations was tested by incubating PLC (0.03-0.5 U/ml) with [14C]-casein. PLC in concentrations up to ten times higher than the concentrations used in aggregation studies did not cause hydrolysis of [14C]-casein, whereas more than 30% of [14C]-casein was hydrolyzed by trypsin. PLC-induced aggregation was not inhibited by up to 300 microM adenosine or ATP. In other experiments, platelet aggregation by ADP was inhibited by adenosine and ATP in a concentration dependent manner. The addition of calcium (0.5- 2.0 mM) increased aggregation by PLC in a concentration dependent manner. These findings suggest that PLC-induced activation of platelets is: (a) dependent on phosphatidylinositol hydrolysis but not on the production of phosphatidic acid, TXB2 or secretion of ADP; (b) not caused by protease contaminants; (c) calcium dependent; and (d) MDBA inhibits PLC-induced aggregation by blocking phosphatidylinositol hydrolysis.

Halogen-substituted trimetoquinol analogs as thromboxane A2 receptor antagonists in platelets and aorta.

Trimetoquinol (TMQ) is a non-prostanoid compound that blocks prostaglandin H2/thromboxane A2 (TXA2) receptor-mediated responses initiated by a prostaglandin (PG) H2 analog, U46619, in human platelets and rat aorta. Ring fluorine-substituted TMQ analogs selectively antagonized PG-dependent human platelet activation induced by U46619, arachidonic acid, collagen, ADP or epinephrine; and were about 300-fold less potent as inhibitors of PG-independent responses mediated by thrombin or bacterial phospholipase C. For each inducer of the PG-dependent pathway, the rank order of inhibitory potency was identical (TMQ > 8-fluoro-TMQ > 5-fluoro- TMQ). Iodine substitution yielded a similar rank order of antagonism against U46619-induced platelet activation (TMQ > 8-iodo-TMQ > 5-iodo-TMQ), and all TMQ analogs inhibited platelet aggregation in whole blood as well as in platelet-rich plasma. Inhibition of specific [3H]SQ 29,548 binding by TMQ analogs was highly correlated with inhibition of functional responses to U46619. Radioligand binding experiments using TMQ analogs with rat platelets showed no interspecies difference in comparison with human platelets. The rank order of inhibitory potencies for the fluorinated (but not iodinated) TMQ analogs changed in rat thoracic aorta with 8-fluoro-TMQ > TMQ > or = 5-fluoro-TMQ as antagonists of U46619-induced vascular contraction. These findings demonstrate that the primary mechanism of antiplatelet action of TMQ analogs is related to a blockade of TXA2 receptor sites, and ring-halogenated TMQ analogs distinguish between TXA2-mediated functional responses in vascular smooth muscle and platelets.

Structure-activity studies of new imidazolines on adrenoceptors of rat aorta and human platelets

SummaryPotencies of new aromatic substituted fluoro or iodo analogues of catecholimidazolines on functional responses in rat aorta (α1) and platelets (α2) were quantified.(1) When compared either on the basis of EC50 or the dissociation constant (KA), 5-fluorocatecholimidazoline was as potent as the reference α1-adrenoceptor agonist, phenylephrine in the vascular tissue. The maximum contraction of aorta produced by the fluoro analogue was, however, 17% higher than that of phenylephrine. The time required for 1/2 relaxation of the tissue after 5-fluoro hydroxy imidazoline was at least twice as long as that of the phenylephrine. The catechol moiety as well as fluorine substitution at the critical 5-position of the aromatic ring is essential for higher α1 adrenoceptor-mediated potency. (2) As compared to the fluoro analogues, the adrenoceptor-mediated potencies of iodo-analogues were relatively weak on vascular tissue. Naphazoline and its analogues were partial agonists on vascular tissue with dissociation constants which ranged from 110 to 2600 nmol/l. (3) Imidazole analogues were generally less potent agonist than the imidazolines by one order of magnitude. (4) The vascular effects of all agonists were competitively blocked by prazosin with KB values which ranged from 0.04 to 0.48 nmol/l. Since the variation in KB values were within normal limits, the action of new imidazolines on rat aorta appears to be mediated mainly by the activation of the α1-adrenoceptor. Prazosin 10 nmol/l abolished the vascular response of some partial agonists. This indicates a slightly different mode of interaction of agonists with the transduction process. (5) Carbon 4-substituted imidazolines produced little or no α1 adrenoceptor-mediated intrinsic activity, but competitive receptor blocking potency was comparable to that of phentolamine. (6) Medetomidine was a partial agonist on the rat aorta with a KA of 260 nmol/l. When investigated as a blocker, the KB of medetomidine against phenylephrine was approximately 5600 nmol/l. The variation in the latter value was high. (7) In acetylsalicylic acid-treated human platelets, the α2-adrenoceptor-mediated aggregatory effect of all fluoro analogues was weak. lodo or naphazoline analogues did not initiate platelet aggregation but blocked the aggregation induced by epinephrine. The affinity of naphazoline for the α2-adrenoceptor was 1100 nmol/l. The IC50 of medetomidine for platelet anti-aggregatory effect was 3300 nmol/l, which compares favorably with other imidazoline type of blockes of platelet aggregation. (8) Sympathomimetic vasoconstrictor actions and platelet aggregation effects of these compounds can be dissociated. Some vasoconstrictors were antiaggregatory. The structure-activity relationships of the two receptor systems, namely rat aorta (α1) and platelets (α2), are discussed.

Human platelet activation by bacterial phospholipase C: Mechanism of inhibition by flurazepam

We have shown earlier that phospholipase C (PLC) from Clostridium perfringens causes platelet activation possibly by inducing turnover of phosphoinositides and phosphorylation of a 47,000 Dalton protein (P47). Moreover, only 15 microM and 11 microM flurazepam inhibits PLC-induced platelet aggregation and serotonin secretion by 50% respectively. This study was conducted to better understand the mechanism of platelet activation by PLC and its inhibition by flurazepam. Incubation of (14C)-arachidonic acid labelled platelets with PLC produced diacylglycerol in a time- and concentration-dependent manner. Flurazepam did not inhibit diacylglycerol production by PLC. Paranitrophenolphosphorylcholine and prostaglandin E1 inhibited diacylglycerol production by 75% and 20% respectively. In a platelet-free system PLC hydrolyzed 14C-choline-phosphatidylcholine (14C-PC) in a time- and calcium ions-dependent manner. Flurazepam had no effect on PLC-induced hydrolysis of 14C-PC. Platelet cytosolic fraction (PCF), containing phosphatidylinositol-specific PLC (PI-PLC), hydrolyzed (3H-inositol)-phosphatidylinositol (3H-PI) in a platelet-free system. Flurazepam did not inhibit hydrolysis of 3H-PI by PCF. Phospholipase C caused phosphorylation of P47 in 32P-labelled platelets. Flurazepam did not block phosphorylation of P47 in the first three minutes and had very little inhibitory effect by five minutes. However, flurazepam completely blocked phosphorylation of P47 by seven minutes. Platelet aggregation induced by ionomycin, a calcium ionophore, was completely inhibited by 100 microM flurazepam whereas platelet aggregation induced by 12-O-Tetradecanoylphorbol-13-acetate (TPA), which mimics the action of diacylglycerol, was partially inhibited by 300 microM flurazepam. These findings suggest that PLC induced platelet activation depends, at least in part, on diacylglycerol production and phosphorylation of P47. These data also suggest that flurazepam does not inhibit PLC-induced platelet activation by inhibiting: (a) the production of diacylglycerol from phosphatidylcholine; and (b) the action of PI-PLC on phosphatidylinositol. The ability of flurazepam to inhibit ionomycin-induced platelet aggregation indicates that flurazepam is able to block platelet activation by inhibiting the increase in free cytosolic calcium ions in platelets or by inhibiting a step subsequent to the rise in intraplatelet calcium ions.

Pharmacological properties of novel bicyclic isoquinoline analogs in isolated guinea pig atria, trachea and in human platelets: Relationship to trimetoquinol

1. Antiplatelet and beta-adrenoceptor activities of a set of secondary and tertiary N-methyl substituted amine analogs of trimetoquinol (TMQ, I and II, respectively) and 5,8-ethano-l-(p-methoxybenzyl)-1,2,3,4,5,6,7,8-octahydroisoquin oline (bicyclic isoquinoline compounds III and IV, respectively) were examined. 2. Compounds III and IV induced relaxations of guinea pig trachea which were blocked by propranolol whereas neither compound acted as an agonist nor antagonist of beta-adrenoceptors (chronotropy) in guinea pig atria. TMQ analogs (I and II) were agonists in both beta-adrenoceptor systems. 3. When tested in human platelets, compounds III and IV, like the TMQ analogs, blocked several inducers of the prostaglandin-dependent and -independent pathways, and the alpha 2-adrenoceptor-mediated pathway of platelet activation. 4. The bicyclic isoquinoline analogs (III and IV) possessed more selective beta 2-adrenoceptor stimulatory activity and equal or greater inhibitory activity against inducers of the prostaglandin-independent pathways of platelet function than the corresponding TMQ analogs (I and II). 5. These chemically novel lipophilic bicyclic compounds provide a new lead to the development of agents useful for the treatment of asthma and thrombotic disorders.

Beclobrinic acid ― a new hypolipidemic agent ― inhibits in vitro human platelet activation by blocking prostaglandin synthesis

Effects and the mechanism of the antiplatelet actions of beclobrinic acid, free acid form of a new hypolipidemic agent beclobrate [(+)-2-[d-(P-chlorophenyl)p-tolyl)oxy)-2-methyl-butyrate), were examined using human platelets. Platelet-rich plasma (PRP) which has been prelabeled with (14C)-serotonin was incubated with beclobrinic acid (BBA) for one minute before the addition of various agonists. BBA (0.1-1.5 mM) inhibited platelet aggregation and serotonin secretion induced by ADP, epinephrine, arachidonic acid and collagen in a concentration dependent manner. BBA also inhibited arachidonic acid-induced production of malondialdehyde (MDA), a byproduct of prostaglandins, in a concentration dependent manner. However, up to 1.0 mM BBA did not inhibit platelet aggregation induced by U46619, a stable analog of prostaglandin H2. In other experiments BBA also blocked thrombin-induced release of (3H)-arachidonic acid from platelet phospholipids. These findings suggest that: (a) BBA inhibits platelet aggregation and serotonin secretion by inhibiting prostaglandin synthesis at two steps. First by interfering in the release of arachidonic acid from platelet phospholipids and second by inhibiting its conversion into prostaglandins; and (b) BBA does not inhibit the action of prostaglandins on human platelets.