Activation of nuclear receptors by prostaglandins

Deletion of membrane receptors for prostaglandins has revealed their importance in diverse biological systems. Some evidence has accrued to support the contention that they may also ligate nuclear receptors, particularly peroxisomal proliferator activator receptors (PPARs). This is most pronounced in the case of 15-deoxy PGJ₂, a cyclopentanone derivative of PGJ₂ as a ligand for PPARγ. However, while this compound can ligate the PPAR, the quantities formed in vivo suggest that this is an unlikely endogenous ligand. Furthermore, biosynthesis is unaltered in murine atherosclerosis and other inflammatory and metabolic disorders where activation of this PPAR has been implicated. The suggestion that prostaglandins serve as endogenous ligands for nuclear receptors is presently configured on the use of synthetic compounds and immunoreactive quantitation of dubious validity. The application of quantitatively precise and sensitive physicochemical methodology will enhance experiments designed to address this hypothesis.     

Protective effects of a specific platelet activating factor (PAF) antagonist, WEB 2086, in traumatic shock

We have investigated the role of platelet activating factor (PAF) in the pathogenesis of a murine model of traumatic shock using WEB 2086, a specific antagonist of PAF. WEB 2086 (0.5 mg/kg) significantly reversed the decrease in mean arterial blood pressure (MABP) induced by PAF (0.3 micrograms/kg) in anesthetized rats. Anesthetized rats were subjected to Noble-Collip drum trauma. Traumatized rats treated with WEB 2086 (0.5 mg/kg bolus followed by infusion at 0.5 mg/kg/hr) maintained a higher MABP than those receiving only the vehicle (0.9% NaCl). Improvement in MABP paralleled a significant increase in overall survival time (p less than 0.01) in rats receiving WEB 2086 (0.5 mg/kg). WEB 2086 also significantly attenuated the plasma accumulation of the lysosomal hydrolase, cathepsin D and of free amino-nitrogen compounds, compared to shocked rats receiving only the vehicle. Furthermore, the production of the cardiotoxic peptide, myocardial depressant factor (MDF) was also blunted by WEB 2086. These results suggest that PAF may be an important mediator in the pathogenesis of traumatic shock in rats. Furthermore, PAF receptor antagonists may be useful as therapeutic agents when given early in the course of ischemic and shock states.     

Activation of platelet prostaglandin biosynthesis pathway during neoplastic cell-induced platelet aggregation

In a previous study we found a correlation between metastatic potential and platelet aggregating activity in sublines of a benzopyrene-induced murine fibrosarcoma (mFS6); the purpose of the present work was to elucidate the role of thromboxane biosynthesis by platelets and/or by neoplastic cells in the activation of platelets in this system. The cells of the more malignant subline induced higher aggregation and TxB₂ production than those of the non metastasizing one. The supernatants of aggregating cell suspensions contained very few TxB₂; furthermore, preincubation of platelets with ASA or Apyrase resulted in inhibition of aggregation and TxB₂ production, while preincubation of the cells was ineffective; these results suggest the platelet origin of the measured TxB₂ and indicate that platelet-derived ADP plays an important role in their activation, while the production of ADP by the cells does not seem to be relevant in this model. The involvement of platelet prostaglandin biosynthesis pathway in neoplastic cell induced platelet activation could play an important role in the development of platelet-dependent tumour metastasis.     

Deaggregation of human platelets in vitro by an RGD analog antagonist of platelet glycoprotein IIb/IIIa receptors

Binding of fibrinogen to platelet glycoprotein (GP) IIb/IIIa receptors is essential for normal platelet aggregation. We investigated whether inhibition of GP IIb/IIIa receptors with arginine-glycine-aspartate-O-methyltyrosine amide (RGDY), an analog of the receptor recognition sequence found in fibrinogen, is associated with platelet deaggregation. Platelets from five healthy human subjects that were maximally aggregated by addition of adenosine diphosphate to platelet-rich plasma were deaggregated in a dose-dependent manner by subsequent addition of RGDY (86.5 ± 6.2%, 68.2 ± 4.3%, 44.9 ± 6.4%, and 31.6 ± 4.1% for RGDY concentrations of 400, 200, 133, and 67 μM, respectively vs. 7.8 ± 2.9% for saline control samples, p < 0.0001). The extent of deaggregation was decreased as the time of addition of RGDY (400 μM) after maximal aggregation increased (85.6 ± 6.7%, 58.5 ± 12.6%, 47.1 ± 2.7%, and 37.1 ± 4.2% for 0, 1, 3, and 5 minute intervals, respectively, vs. 8.3 ± 3.1% in control samples, N = 4, p < 0.0001) consistent with the occurrence of irreversible aggregation. Thus, RGDY can rapidly and extensively deaggregate human platelets under certain conditions which may enhance its antithrombotic efficacy.     

Sheep platelets as a model for human platelets: evidence for specific PAF (platelet activating factor) receptors

In a number of animals, the platelet response to Platelet Activating Factor (PAF) has been shown to differ considerably from that in humans. However, aggregation, release and particularly shape change were quite similar for human and sheep platelets. In this study, aggregation and shape change analysis were used to measure the response of sheep platelets to various synthetic analogs of PAF. Response is greatly reduced with no alkyl group in position 2 of PAF and decreases progressively as the number of carbons of the alkyl gets larger than three. A reduction of activity is also seen as the ether linkage at position 1 of PAF is replaced by an ester linkage. These changes are indicative of a specific membrane receptor for PAF in sheep platelets and confirm the usefulness of sheep platelets as a model for PAF-platelet interaction in humans.     

Molecular requirements in the recognition of low-density lipoproteins (LDL) by specific platelet membrane receptors

We have demonstrated that platelet low-density lipoprotein (LDL) receptors differ from classic LDL receptors of nucleated cells. Although positively charged Arg and Lys residues of apoprotein B-100 are known to play a key role in LDL recognition by classic LDL receptors, there are no conclusive data on platelet LDL receptors. This study investigated the molecular requirements of LDL particle recognition by platelet LDL receptors. The involvement of lipid and protein fractions was determined by displacement studies of the binding of 125I-LDL to platelets and fibroblasts (used as a classical LDL receptor model). The role of the protein moiety was evaluated by chemically modifying positively charged apoB residues (Lys, Arg, and Tyr) via copper-induced oxidation, cyclohexanedione, and tetranitromethane, respectively. The involvement of the lipid fraction was determined by ligand binding assays using 125I-LDL particles that had previously been delipidated and subjected to apoB solubilization. The degree of particle modification was analyzed by agarose/acrylamide gel electrophoresis and anion exchange chromatography. Modifying the amino acid residues increased particle electronegativity in the following order of potency: CHD-LDL>TNM-LDL>ox-LDL>native LDL. The results obtained by displacement studies in fibroblasts suggested that the gain in the LDL negative charge was the most important factor in the loss of receptor affinity. The chemical models of protein modification used in our study greatly affected LDL binding to the classical fibroblast receptor. In contrast, there was very slight difference in the displacement capacity on platelet 125I-LDL binding, which suggests that the protein fraction does not play a major role in the interaction of LDL with its platelet receptor. On the other hand, whereas modifying the lipid moiety did not alter the ability of solubilized 125I-apoB to interact with the classical fibroblast LDL receptor, platelet LDL receptors were unable to recognize these particles. In conclusion, our results confirm that the protein fraction plays a key role in the fibroblast LDL-receptor recognition process, whereas the lipid fraction appears to have a more relevant role in platelet LDL-receptor recognition.     

Binding of aggregated immunoglobulins to the human platelet Fc receptors: a mechanism of platelet to platelet bridging

Aggregated immunoglobulins react with human platelets by occupying the Fc receptors present on their surface, inducing aggregation and the release reaction. We studied the effect of heat aggregated gammaglobulins (HAGG) on ADP-induced aggregation of platelets. We used the minimum concentration of ADP required to induce a reversible aggregation of platelets without any substantial amount of serotonin (14C-5HT) release. EDTA (5 mM) added at the peak of platelet aggregation resulted in rapid deaggregation of these platelets. However, incubation of platelets with HAGG at a dose that did not by itself induce any aggregation or release reaction, followed by ADP addition resulted in an irreversible platelet aggregation of greater magnitude accompanied by a substantial release of 14C-5HT. The addition of EDTA at the peak of platelet aggregation failed to deaggregate these platelets. To determine whether the augmented aggregation response and the inhibition of deaggregation was due to HAGG or a consequence of platelet release products, we used thrombin-degranulated platelets. The augmented aggregation response and the inhibition of deaggregation due to HAGG and ADP could be demonstrated using these platelets. To confirm that the binding of HAGG to the platelet Fc receptors was responsible for these observations, we incubated platelets with an excess of Fc fragments of IgG prior to the addition of HAGG and ADP. This abolished the aggregation response observed previously. From this study we conclude that interplatelet bridging by HAGG renders the platelets hyperaggregable and appears to be a mechanism involved in maintaining platelet aggregates.     

Interaction of stable prostaglandin endoperoxide analogs U46619 and U44069 with human platelet membranes: coupling of receptors with high-affinity GTPase and adenylate cyclase

It has been demonstrated using a membrane preparation of human platelets that stable analogs of PGH2, U46619 and U44069, control the activity of adenylate cyclase and a high-affinity hormone-sensitive GTPase. At 10(-8)-10(-6) M, the analogs inhibit the basal activity of adenylate cyclase by 20-25%. With a further rise in U46619 and U44069 concentrations up to 10(-5)-10(-4) M, the inhibition is abolished and adenylate cyclase activity is stimulated in a dose-dependent fashion. In the presence of PGE1, only inhibitory action of U46619 was observed at all the concentrations tested. The inhibitory action of the analogs on adenylate cyclase correlates with the activation of the high-affinity hormone-sensitive GTPase. It is concluded that U46619 and U44069 inhibit human platelet adenylate cyclase via specific receptors coupled to the GTP-binding inhibitory protein.     

Anti-thrombotic activity of RG13965, a novel platelet fibrinogen receptor antagonist

RG13965, a pseudotetrapeptide analogue of Arg-Gly-Asp (RGD), inhibited collagen-induced dog, monkey, human, hamster, mouse, and pig platelet aggregation in vitro with IC₅₀ values of 3.7, 4.6, 6.3, 126, 136 and 1600 μM, respectively. RG13965 (3, 10, and 30 mg/kg, i.v.) decreased the incidence of collagen/epinephrine-induced thrombosis in mice from 90% in untreated animals to 63, 37, and 0%, respectively. In hamsters, RG13965 (10 and 30 mg/kg, i.v.) prolonged the time required for formation of a hemostatic plug in severed mesenteric arteries by 1.6- and 3.6-fold, respectively. In a canine model of repetitive platelet thrombus formation in the coronary artery, RG13965 (0.1, 0.3, and 1 mg/kg, i.v.) reversibly inhibited cyclic flow reductions (CFRs) and inhibited ADP-induced ex vivo platelet aggregation by 29, 57, and 77%, respectively. RG13965 (1 mg/kg) completely inhibited CFRs for at least 40 min. Platelet count was not altered at any dose and template bleeding time was prolonged modestly (1.8-fold) at only the highest dose. RG13965 dose-dependently and reversibly inhibited thrombus formation at doses which did not completely inhibit ex vivo platelet aggregation and only modestly prolonged template bleeding time.     

Prevention of aspirin inhibition of platelet release reaction by the fatty acid precursor of platelet prostaglandins

Preincubating human platelet-rich citrated plasma with eicosatetraenoic acid (E-tetra), the immediate precursor of PGE₂ and F_2α, prevents the inhibitory effect of aspirin on the release reaction induced by epinephrine, ADP, thrombin or collagen. The methyl ester of E-tetra as well as a saline solution of arachidonyl C1 had the same effect as the free acid. In striking contrast to E-tetra, E-tri or its methyl ester, E-penta, stearic, oleic, linoleic and γ-linolenic acid failed to prevent the inhibitory effect of aspirin. Although E-tetra alone induces the release reaction, this phenomenon does not explain the action of E-tetra in blocking effects of aspirin. Thus, we tentatively postulate that E-tetra's unique protection of the platelet against the untoward effect of aspirin, may be attributable to the role it plays in platelet prostaglandin synthesis.     

Interaction of a vasopressin antagonist with vasopressin receptors in the septum of the rat brain

The ability of d(CH2)5-Tyr(Me)-arginine-8-vasopressin, an antagonist of peripheral pressoric (V1-type) vasopressin receptors, to label vasopressin binding sites in the septum of the rat brain was evaluated. Using crude membrane preparations from the septum, 3H-arginine-8-vasopressin (AVP) specifically labels a single class of binding sites with a Kd of 2.9 nM and maximum binding site concentration of 19.8 fmole/mg protein. 3H-Antag also labels a single class of membrane sites but with higher affinity (Kd = 0.47 nM) and lower capacity (10.1 fmole/mg protein) than 3H-AVP. The rank order of potency of various competitor peptides for 3H-AVP and 3H-Antag binding was similar. Oxytocin was 100-1,000 fold less potent than AVP in competing for binding with both ligands. 3H-AVP and 3H-Antag showed similar labeling patterns when incubated with septal tissue slices. Unlabeled Antag also effectively antagonized vasopressin-stimulated phosphatidylinositol hydrolysis in septal tissue slices.     

Formation of endogenous glutamatergic receptors antagonist kynurenic acid--differences between cortical and spinal cord slices

Rat spinal cord slices produced kynurenic acid (KYNA) upon exposure to L-kynurenine. Aminooxyacetic acid, non-selective aminotransferase inhibitor, and L-glutamate, but neither N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-metyloisoxazolo-4-propionate (AMPA), nor kainate, diminished synthesis of KYNA. L-Glutamate action was less potent in spinal than in cortical slices. Metabotropic agonists, L-(+)-2-amino-4-phosphonobutyrate (L-AP4) and (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD), used in concentrations inhibiting cortical KYNA synthesis, were ineffective in spinal cord. Spinal KYNA production seems less susceptible to inhibitory modulation.     

Clinical correlates of platelet prostaglandin receptor subsensitivity in schizophrenia

A diminished cAMP response to prostaglandin E1 (PGE1) in platelets from schizophrenic patients has been demonstrated previously. The authors report that among 35 actively psychotic male schizophrenic patients, the platelet cAMP response to PGE1 was negatively correlated with global symptom severity and with several indexes of positive symptom severity but not with negative symptom severity. If this subsensitivity of platelet PGE receptors extends to brain PGE receptors, schizophrenic patients may have an impairment in the ability of endogenous PGEs to inhibit dopaminergic transmission. Such impairment could have a permissive effect on the production of psychotic symptoms during exacerbations in schizophrenic patients.     

Fibrinogen interaction with human platelets: Effect of other coagulation factors,prostaglandins and platelet inhibitors

Fibrinogen (fg) binding to platelets is a critical step in the formation of the hemostatic plug. The interaction requires platelet stimulation and the induction of receptor sites on the platelet membrane. In this paper we report on the effect of other clotting factors on ¹²⁵I-labeled fg binding to gel filtered human platelets. The action of exogenously added or endogenously synthesized prostaglandins and the effect of antiplatelet drugs were also investigated. Prothrombin and active factor X enhance ADP-induced platelet-fg binding whereas active factor VIII and active factor IX, separately or combined, are without effect. Human prothrombin complex (PC) factor concentrates (II-VII-IX-X) cause significant enhancement of platelets-fg binding; this effect is most likely due to activated factors and/or traces of thrombin present in the preparation. In the concentration used, these clotting factors and the PC factor concentrates failed to aggregate platelets in platelet rich plasma. Acetylsalicylic acid, carbenicillin and the calcium channel blocking agents verapamil and nifedipine showed variable degrees of inhibition of ADP-induced platelet-fg binding. Chlorpromazine and propranolol were without effect. Estrogen and progesterone had some enhancing effect on binding. These results suggest that, when the hemostatic mechanism is initiated, thromboxane A₂ synthesis and activated prothrombin complex factors significantly enhance fg binding to platelets, a key step in hemostasis. Inhibitors of aggregation do not necessarily impede platelet fibrinogen interaction.     

Plasma levels of platelet and vascular prostaglandin derivatives in children with sickle cell anaemia

Platelet and vascular endothelial prostaglandin derivatives were measured by radioimmunoassay in the plasma of 39 children with homozygous sickle cell anaemia and 27 control subjects. The levels of both thromboxane B2 and 6-keto-PGF1alpha, the stable end-products of platelet and vascular prostaglandin metabolism, were significantly (p less than 0.001) greater in sickle cell anaemia plasma than in the plasma of the controls. There were no differences in levels of either 6-keto-PGF1alpha or thromboxane B2 between steady state and vaso-occlusive crisis in the sickle cell patients. Injury to the endothelial lining of blood vessels may occur in sickle cell anaemia as a consequence of contact with the abnormal erythrocytes.     

Stimulation of human platelet adenylate cyclase by prostaglandin D2

Prostaglandin D₂ (PGD₂) stimulates the formation of cyclic AMP in human platelets measured as the incorporation of radio-activity from previously labelled intracellular nucleotides. In this action it is similar to, and more powerful than PGE₁. Both inhibition of platelet aggregation and stimulation of cyclic AMP accumulation by PGD₂ and by PGE₁ are potentiated by an inhibitor of platelet phosphodiesterase. A number of minor differences in the response of platelets to PGD₂ and PGE₁ suggest the existence of at least two prostaglandin receptors influencing a single adenylate cyclase.