On the multiplicity of platelet prostaglandin receptors. I. Evaluation of competitive antagonism by aggregometry

Methods for the evaluation of competitive interactions at receptors associated with platelet activation and inhibition using aggregometry of human PRP have been developed. The evidence supports the suggestion that PGE1 and PGI2 share a common receptor for inhibition of platelet reactivity, but only a portion (if any) of the aggregation stimulation associated with PGE2 is the result of PGE2 binding (without efficacy) to this receptor. PGE2 (at .3-20 microM) is an effective antagonist of PGE1, PGI2, and PGD2 producing a shift of about one order of magnitude in the IC50-values obtained from complete aggregation inhibition dose response curves. The antagonism of PGD2 inhibition is particularly notable, 80 nM PGE2 levels are detectable. This and other actions of PGE2 indicate another platelet receptor for PGE2. PGE1 acts at both the PGE2 and PGI2 receptor. Other substances showing PGI2-like actions only at high doses (1-30 microM), display additive responses with PGI2 indicative of decreased affinity for the I2/E1 receptor and the absence of PGE2-like aggregation stimulation activity. PGI2 methyl ester has intrinsic inhibitory action not associated with in situ ester hydrolysis. The methyl ester is dissaggregatory showing particular specificity for inhibition of release and second wave aggregation.     

On the multiplicity of platelet prostaglandin receptors. II. The use of N-0164 for distinguishing the loci of action for PGI2, PGD2, PGE2 and hydantoin analogs

The omega-chain variant analogs of prostacyclin (PGI2) and PGD2 in which n-amyl side-chain has been replaced by a cyclohexyl group have been prepared and their cardiovascular activities have been compared to those of BW-245C(Fig. 1) a potent anti-aggregatory vasodilator bearing a cyclohexyl-terminated side-chain on a hydantoin skeleton. The cyclohexyl group has little effect on PGI2, but converts PGD2 to a long lasting hypotensive agent and increases the platelet anti-aggregatory potency of PGD2 by a factor of 8. The prostaglandin antagonist N-0164 selectively blocks the anti-aggregatory actions of PGD2, cyclohexyl-PGD2, and BW-245C; with essentially no effect on PGI2, cyclohexyl-PGI2 and PGE2 at comparably effective doses. The latter observation is contrary to an earlier report by MacIntyre, but supports the view that the anti-aggregatory effect of high doses of PGE2 (EC50=50 microM) is mediated by the PGI2 receptor. The hydantoin acts at the platelet PGD2 receptor.     

On the multiplicity of platelet prostaglandin receptors: II. The use of N-0164 for distinguishing the loct of action for PGI2, PGD2, PGE2 and hydantoin analogs

The ω-chain variant analogs of prostacyclin (PGI₂) and PGD₂ in which the n-amyl side-chain has been replaced by a cyclohexyl group have been prepared and their cardiovascular activities have been compared to those of BW-245C(Fig. 1)(1) a potent anti-aggregatory vasodilator bearing a cyclohexyl-terminated side-chain on a hydantoin skeleton. The cyclohexyl group has little effect on PGI₂, but converts PGD₂ to a long lasting hypotensive agent and increases the platelet anti-aggregatory potency of PGD₂ by a factor of 8. The prostaglandin antagonist N-0164 selectively blocks the anti-aggregatory actions of PGD₂, cyclohexyl-PGD₂, and BW-245C; with essentially no effect on PGI₂, cyclohexyl-PGI₂ and PGE₂ at comparably effective doses. The latter observation is contrary to an earlier report by MacIntyre (2,3), but supports the view that the anti-aggregatory effect of high doses of PGE₂ ($\text{EC}{}_{50}\text{=50μ}\text{M}$) is mediated by the PGI₂ receptor (4). The hydantoin acts at the platelet PGD₂ receptor.     

Human platelet aggregation induced by prostaglandin endodisulfide.

The effect on human platelet functions of 9,11-dithio analogues of prostaglandin endoperoxide was investigated. Methyl (5Z, 9alpha, 11alpha, 13E, 15S)-9,11-epidithio-15-hydroxyprosta-5,13-dienoate induced platelet aggregation, while the 9beta,11beta-epimer was inactive. The platelet aggregation caused by the 9alpha,11alpha-dithio analogue was associated with serotonin release from platelets, and was inhibited by methyl ester of prostaglandin I2 (prostacyclin) but not by indomethacin.     

Amino acid residues conferring ligand binding properties of prostaglandin I and prostaglandin D receptors. Identification by site-directed mutagenesis

Using chimeras of the mouse prostaglandin (PG) I receptor (mIP) and the mouse PGD receptor (mDP), we previously revealed that the cyclopentane ring recognition by these receptors is specified by a region from the first to third transmembrane domain of each receptor; recognition by this region of mIP is broad, accommodating the D, E, and I types of cyclopentane rings, whereas that of mDP binds the D type of PGs alone (Kobayashi, T., Kiriyama, M., Hirata, T., Hirata, M., Ushikubi, F., and Narumiya, S. (1997) J. Biol. Chem. 272, 15154-15160). In the present study, we performed a more detailed chimera analysis, and narrowed the domain for the ring recognition to a region from the first transmembrane domain to the first extracellular loop. One chimera with the replacement of the second transmembrane domain and the first extracellular loop of mDP with that of mIP bound only iloprost. The amino acid substitutions in this chimera suggest that Ser(50) in the first transmembrane domain of mIP confers the broad ligand recognition of mIP and that Lys(75) and Leu(83) in the second transmembrane domain of mDP confer the high affinity to PGD(2) and the strict specificity of ligand binding of mDP, respectively.     

On the molecular structure of some prostaglandin receptors

Hypotheses are presented of the detailed molecular structure of two prostaglandin receptors both concerned in tumor-promotion processes. These structures have been derived by the comparison of the molecular structure of agents active at the site with (i) a simple theoretical protein structure and (ii) the known x-ray structure of phospholipase A₂. The first model receptor is stimulatory to the tumor-promotion process and may be located on the control system for ornithine decarboxylase. The binding of PG here is cooperative with the binding of Ca++. Naturally-occurring agonists at this receptor may include members of the cathartic class of drugs such as colocynth, chrysarobin, etc. Naturally-occurring antagonists at this site may include a number of anti-tumor compounds such as datiscoside. The second model receptor (PGE₁) is inhibitory to the tumor-promotion process and is located at a specific allosteric site on the x-ray-determined structure of phospholipase A₂. This site overlaps for one for lysolecithin (excitatory), for which tumor-promoting phorbol esters such as TPA are agonists and some anti-tumor drugs such as maytansine may be antagonists.     

Cholinergic antagonism by beta-blockers. I. Spectrum of interactions

Complete final steady state selective antagonism by beta-blockers of different conventional classes versus acetylcholine effects on isolated frog rectus abdominis, guinea pig ileum and spontaneous beating auricles had been measured. The results do not support common beta-blockers groupings, nor binary conventional subdivision of "nicotinic" and "muscarinic" cholinergic receptors, confirming our previous findings.     

SQ-27986 inhibition of platelet aggregation is mediated through activation of platelet prostaglandin D2 receptors

SQ-27986, a oxabicycloheptane derivative, potently inhibits ADP-, collagen- and arachidonic acid-induced platelet aggregation in human platelet-rich plasma. Human platelet aggregation induced by ADP is inhibited by SQ-27986 (EC50 = 22nM), and the inhibitory action of SQ-27986 can be prevented with N-0164, a PGD2 antagonist. By comparison, ADP-induced rat platelet aggregation is unaffected by SQ-27986 (IC50 greater than 80 microM). Washed human platelets treated with SQ-27986 exhibit elevated cAMP levels and activated cAMP-dependent protein kinase. Elevation of platelet cAMP levels (greater than 4 fold basal) and activation of the cAMP-dependent protein kinase (greater than 4 fold) are observed with SQ-27986 concentrations above 100 nM. The SQ-27986-induced elevation of cAMP can be prevented by N-0164. Lysed platelets treated with SQ-27986 showed stimulated adenylate cyclase activity. SQ-27986 competes with [3H]prostaglandin D2 binding to isolated platelet membranes (EC50 for SQ-27986 is 20 nM, which was more potent than cold PGD2 itself). Radiolabeled Iloprost binding is virtually unaffected by SQ-27986 (EC50 greater than 100 microM), indicating that SQ-27986 does not interact with platelet prostacyclin receptors. These studies indicate that SQ-27986 inhibits platelet aggregation by activating platelet adenylate cyclase via stimulation of platelet PGD2 receptors.     

DuP 753, the selective angiotensin II receptor blocker, is a competitive antagonist to human platelet thromboxane A2/prostaglandin H2 (TP) receptors.

DuP 753 is a potent, selective angiotensin II type 1 (AT1) receptor antagonist. The possibility was investigated that DuP 753 may crossreact with thromboxane A2/prostaglandin H2 (TP) receptors. DuP 753 inhibited the specific binding of the TP receptor antagonist [3H]SQ 29,548 (5 nM) in human platelets with kd/slope factor values of 9.6 +/- 1.4 microM/1.1 +/- 0.02. The AT2-selective angiotensin receptor ligand, PD 123,177 was a very weak inhibitor of specific [3H]SQ 29,548 binding in platelets (Kd/slope factor:200 microM/0.86). [3H]SQ 29,548 saturation binding in the absence and presence of DuP 753 resulted in an increase in equilibrium affinity constant (Kd: 9.3, 22, 33 nM, respectively) without a concentration-dependent reduction in binding site maxima (Bmax: 3597, 4597, 3109 fmol/mg protein, respectively). Platelet aggregation induced by the TP receptor agonist U 46,619 was concentration-dependently inhibited by DuP 753 (IC50 = 46 microM). These data indicate for the first time that DuP 753 is a weak but competitive antagonist at human platelet TP receptors.     

On the interrelationship of prostaglandin endoperoxide G2 and cyclic nucleotides in platelet function.

The prostaglandin endoperoxide G2 caused rapid aggregation and relase of ADP and [14C]serotonin in human platelets. Since the presence of the ADP phosphorylating system creatine phosphate/creatine phosphokinase markedly inhibited the aggregation caused by the endoperoxide, this effect seemed to be mediated mainly by ADP, which is instantaneously released by the endoperoxide. The prostaglandin G2 counteracted the increasing effect of prostaglandin E1 on the adenosine 3':5'-monophosphate (cAMP) levels in platelet-rich plasma. This effect of prostaglandin G2 was only observed when ADP was released by the endoperoxide. This finding indicates that the effect of prostaglandin G2 on the cAMP levels in platelet-rich plasma is principally mediated by ADP. The rapid release of ADP by prostaglandin G2 and the time courses for the effects of the endoperoxide and ADP on the level of cAMP give further evidence for this hypothesis. ADP also caused primary aggregation in the presence of indomethacin, and prostaglandin synthesis inhibitors did not influence the decreasing effect of ADP on the cAMP levels. N2,O2-Dibutyrylguanosine 3':5'-monophosphate did not influence the aggregation and release-reaction caused by ADP and no changes of the cGMP levels were observed after addition of prostaglandin G2.     

Amplification of prostaglandin I2 production by thapsigargin.

Rat liver cells (the C-9 cell line) are synergistically stimulated to produce prostaglandin I2 (PGI2) when incubated in the presence of thapsigargin and several recombinant human growth factors or tumor promoters, including basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), transforming growth factor-alpha (TGF-alpha), 12-O-tetradecanoylphorbol-13-acetate (TPA), teleocidin and aplysiatoxin, but not acidic fibroblast growth factor (aFGF). The production of PGI2 by exogenous arachidonic acid in the presence of thapsigargin is not amplified. The effects of varying levels of bFGF on PGI2 production in the presence of thapsigargin are biphasic: at low levels (0.05 nM) bFGF's effect is synergistic; at high levels (24 nM) it is not.     

Reduction in rat mesentery mast cell staining after degranulation by protamine. A competitive antagonism.

Degranulation of rat mesentery mast cells by increasing concentrations of protamine causes a parallel decrease in the numbers of mast cells stained with toluidine blue or with berberine sulfate. No decrease in mast cell numbers occurs when degranulation is inhibited. Since protamine does not enter into non stimulated mast cells, these results suggest that this reduction in mast cell numbers is caused by the binding of protamine to the anionic sites of heparin of exocytosed granules thereby preventing their staining. There seems to be a competitive antagonism between protamine and toluidine blue at the anionic sites of heparin for increasing concentrations of toluidine blue progressively reverse the reduction in mast cell numbers.     

Agonist regulation of the human platelet prostaglandin D2 receptor.

The effect of exposing platelets to prostaglandin D₂ (PGD₂) on hormone binding was studied. Incubation of platelets with PGD₂ for 2 hr resulted in a decrease in [³H]PGD₂ binding that was dose dependent. Inhibition of binding was 14% after incubation with 10^?8M PGD₂, 19% after incubation with 10^?7M PGD₂, and 40% after exposure to 10^?6M PGD₂. This decreased binding (desensitization) was specific for [³H]PGD₂ as binding to platelets by [³H]PGE₁ and the α-adrenergic antagonist [³H] dihydroergocryptine (DHEC) was comparable to control platelets. Saturation of [³]PGD₂ binding to desensitization platelets was at 27 fmole ligand/10⁸ platelets compared to 43 fmoles/10⁸ platelets in control platelets. Half-maximal saturation occured at 20 nM PGD₂ both for desensitized and control platelets, suggesting that decreased binding sites rather than altered affinity between ligand and receptor accounted for these results. These platelets had a partial increase in [³H]PGD₂ binding a few hours after plasma was washed free of PGD₂ with complete resensitization after 24 hr. Since prostaglandins such as PGI₂, PGD₂, and PGE₁ are potent inhibitors of platelet aggregation, decreased binding of platelets to these hormones after prostaglandin exposure may provide a mechanism for altered responsiveness of platelets to aggregating stimuli.     

Structure-function relationships in the activation of platelet thrombin receptors by receptor-derived peptides.

According to present models, thrombin activates platelets by cleaving its receptors after Arg41, creating a new N terminus which acts as a tethered ligand. In support of this model, a peptide (SFLLRNPNDKYEPF or TRP42/55) corresponding to residues 42-55 has been shown to activate the receptor. In the present studies, the structural basis for thrombin receptor activation was examined using fragments of this peptide, as well as variants of the peptide with selected amino acid substitutions. The results show that the features of SFLLRNPNDKYEPF required to mimic the effects of thrombin reside within the first 6 residues, SFLLRN. A hexapeptide comprised of these residues was approximately 5 times more potent than the parent peptide in assays of platelet aggregation and, in addition, caused tyrosine phosphorylation, inhibition of cAMP formation, and an increase in cytosolic Ca2+. Omission of either the Ser residue or the Arg and Asn residues greatly diminished peptide activity, as did the substitution of Ala for Phe or Arg. Substitution of Ala for Ser or the initial Leu, on the other hand, had little adverse effect. The inactive peptides SALLRN and NPNDKYEPF had no effect on platelet activation initiated by SFLLRN, but FLLRN inhibited platelet aggregation in response to both SFLLRN and thrombin. These results suggest that within SFLLRN the Phe and Arg residues are particularly important and that Phe must be preceded by another amino acid, the identity of which is not tightly constrained. This observation and comparisons with the homologous domains of proteins whose tertiary structure is known were used to predict the conformation of the SFLLR sequence. The model which emerged suggests that the SFLLR domain may be part of an extended beta structure in the intact receptor and that cleavage by thrombin causes it to contract and assume a modified helical configuration. In this predicted conformation the side chains of Phe and Arg point in the same direction, potentially into a pocket formed by the remainder of the receptor.     

Binding to prostaglandin (PG) receptors and activation of adenyl cyclase by 20-isopropylidene-PGS in rabbit platelet membranes

20-Isopropylidene-PGE1 (Isop-PGE1) was about 10 times more potent than PGE1 in inhibition of thrombin-induced aggregation of rabbit washed platelets. Likewise, 20-isopropylidene-17(R)-methyl-carbacyclin (CS-570), a stable PGI2 analogue, was more potent than carbacyclin in the anti-aggregatory activity. In order to define the platelet-prostaglandin interactions, a binding assay was done using platelet membranes with [3H]-PGE1 as a radioligand. Isop-PGE1 (IC50 = 0.18 microM) bound to the PG receptors more potently than PGE1 (IC50 = 2.1 microM). CS-570 (IC50 = 0.39 microM) was more potent than carbacyclin (IC50 = 1.9 microM). These indicate that introduction of an isopropylidene group to the carbon 20 of PGs increases the binding ability to the receptors. These PGE1 and PGI2 analogues activated platelet membrane adenyl cyclase and increased intracellular cAMP levels with the same potency series obtained in the binding experiments. All these results suggest that the binding to the receptors by these PGs is coupled to the activation of adenyl cyclase, followed by the increase in cAMP levels in platelets and the inhibition of platelet aggregation. Thus, the increased anti-aggregatory activity of 20-isop-PGs may be explained by their increased affinity for the PG receptors and stimulation of adenyl cyclase. 15-Epimeric-20-isopropylidene-PGE1 (15-Epi-isop-PGE1), which has an unnatural configuration of the 15-hydroxyl group, was much less potent than isop-PGE1 in the binding experiment and the other three investigations. This indicates that the configuration of the 15-hydroxyl group is important for the binding to the PG receptors and the consequent activities in platelets.     

Isoelectric and mass characterization of human platelet thromboxane A2/prostaglandin H2 receptors

To further characterize the human thromboxane A2 (TXA2)/prostaglandin H2 (PGH2) receptor, preparative isoelectric focusing (IEF) was performed on solubilized platelet membranes. TXA2/PGH2 receptors, assayed by specific binding of the TXA2/PGH2 antagonist [125I]PTA-OH, were electrofocused at pH 5.6. Scatchard analysis of IEF fraction pH 5.6 revealed a 180-fold concentration of TXA2/PGH2 receptors (Bmax = 3650 +/- 228 pM/mg focused, 19 +/- 4 pM/mg unfocused) with no change in binding affinity (Kd = 47 +/- 7 nM focused, 36 +/- 14 nM unfocused). SDS-polyacrylamide gel electrophoresis of photoaffinity-labelled electrofocused receptors revealed concentration of specifically labelled proteins having molecular masses of 49,000 and 27,000 Daltons. These results suggest that the human platelet TXA2/PGH2 receptor has a pI of 5.6, molecular mass of 49,000 Daltons, and may exist as a dimer. Preparative IEF should prove useful in the eventual purification of this receptor.     

Successful therapy of the prostaglandin defect "Wien-Hietzing" (lack of platelet high-affinity binding sites for prostaglandin I2)

Some years ago we detected a lack of platelet high-affinity PGI2 binding sites in a 10 year-old girl who presented to the outpatient unit with clinical symptoms and signs of acute popliteal artery occlusion. We named this new defect in the prostaglandin system "Wien-Hietzing". Acute surgery was successful. On the basis of earlier findings that aspirin is able to sensitize platelets to the action of PGI2 and produce beneficial changes in platelet sensitivity, we decided to treat this girl with a daily dosage of 20 mg aspirin orally. Repeated control examinations during the total follow-up period of about 6 years revealed normalized platelet sensitivity and normalized receptor behaviour. The girl is symptom-free to date. It is concluded that this prostaglandin defect may be successfully treated with long-term, low-dose aspirin administration.     

Modulation of platelet cyclic nucleotide content by PGE1 and the prostaglandin endoperoxide PGG2.

The effects of prostaglandin E1 and prostaglandin G2, the prostaglandin endoperoxide, on platelet cyclic nucleotide concentrations were measured in platelet rich plasma (PRP), and in washed intact platelets. PGE1 was found to be a potent stimulator of platelet cAMP levels in both PRP and washed cells, and to inhibit aggregation in both systems. PGE1 did not change platelet cGMP levels in either PRP or washed cells. PGG2 which is a potent inducer of platelet aggregation, did not affect either the basal cAMP or the basal cGMP concentration. However, PGG2 was found to antagonize the increases in cAMP content in response to PGE1 in both PRP and washed platelets. The addition to our system of a cyclic nucleotide phosphodiesterase inhbitor, theophylline, did not change our findings. It is suggested that PGG2 may induce platelet aggregation by inhibiting PGE1-stimulated cAMP accumulation.