Platelet Adenylate Cyclase Activity Research Articles

Diminished Platelet Adenylate Cyclase Activation by Prostaglandin D2 in Acute Thrombosis

AbstractProstaglandin D2 (P6D2) produced by platelets can inhibit aggregation via activation of platelet adenylate cyclase. PGD2 activation of adenylate cyclase in platelet membrane fractions was studied in 20 consecutive patients hospitalized with acute deep-vein thrombosis and/or pulmonary embolism. In nine patients, PGD2-stimulated enzyme activity was decreased at all concentrations of PGD2 studied. This altered enzyme sensitivity was specific for PGD2 as basal enzyme activity, and prostaglandin E1, prostaglandin I2, and sodium fluoride stimulated adenylate cyclase was normal. The effect of PGD2 on platelet aggregation and 14C-serotonin release was also studied in one patient where a fourfold higher concentration of PGD2 was required to inhibit collagen-induced 14C-serotonin release. Binding studies using [3H]PGD2 as a radioligand indicated that this patient’s platelets bound 10 fmole PGD2/108 platelets compared to 30 fmole/108 platelets in a normal control. Five patients had follow-up studies between 2 and 7 mo after their acute thrombotic event, and PGD2-stimulated adenylate cyclase activity returned towards normal in four. Since PGD2 is synthesized in platelets at concentrations sufficient to inhibit aggregation and activate adenylate cyclase, diminished platelet sensitivity to this prostaglandin could result in “hyperactivity” and contribute to the thrombosis observed in these patients.

Diminished platelet adenylate cyclase activation by prostaglandin D2 in acute thrombosis

Prostaglandin D2 (PGD2) produced by platelets can inhibit aggregation via activation of platelet adenylate cyclase. PGD2 activation of platelet cyclase in platelet membrane fractions was studied in 20 consecutive patients hospitalized with acute deep-vein thrombosis and/or pulmonary embolism. In nine patients, PGD2-stimulated enzyme activity was decreased at all concentrations of PGD2 studied. This altered enzyme sensitivity was specific for PGD2 as basal enzyme activity, and prostaglandin E1, prostaglandin I2, and sodium fluoride stimulated adenylate cyclase was normal. The effect of PGD2 on platelet aggregation and 14C-serotonin release was also studied in one patient where a four-fold higher concentration of PGD2 was required to inhibit collagen-induced 14C-serotonin release. Binding studies using [3H]PGD2 as a radioligand indicated that this patient's platelets bound 10 fmole PGD2/10(8) platelets compared to 30 fmole/10(8) platelets in a normal control. Five patients had follow-up studies between 2 and 7 mo after their acute thrombotic event, and PGD2-stimulated adenylate cyclase activity returned towards normal in four. Since PGD2 is synthesized in platelets at concentrations sufficient to inhibit aggregation and activate adenylate cyclase, diminished platelet sensitivity to this prostaglandin could result in “hyperactivity” and contribute to the thrombosis observed in these patients.

Triene prostaglandins: Prostacyclin and thromboxane biosynthesis and unique biological properties

Platelets enzymatically convert prostaglandin H(3) (PGH(3)) into thromboxane A(3). Both PGH(2) and thromboxane A(2) aggregate human platelet-rich plasma. In contrast, PGH(3) and thromboxane A(3) do not. PGH(3) and thromboxane A(3) increase platelet cyclic AMP in platelet-rich plasma and thereby: (i) inhibit aggregation by other agonists, (ii) block the ADP-induced release reaction, and (iii) suppress platelet phospholipase-A(2) activity or events leading to its activation. PGI(3) (Delta(17)-prostacyclin; synthesized from PGH(3) by blood vessel enzyme) and PGI(2) (prostacyclin) exert similar effects. Both compounds are potent coronary relaxants that also inhibit aggregation in human platelet-rich plasma and increase platelet adenylate cyclase activity. Radioactive eicosapentaenoate and arachidonate are readily and comparably acylated into platelet phospholipids. In addition, stimulation of prelabeled platelets with thrombin releases comparable amounts of eicosapentaenoate and arachidonate, respectively. Although eicosapentaenoic acid is a relatively poor substrate for platelet cyclooxygenase, it appears to have a high binding affinity and thereby inhibits arachidonic acid conversion by platelet cyclooxygenase and lipoxygenase. It is therefore possible that the triene prostaglandins are potential antithrombotic agents because their precursor fatty acids, as well as their transformation products, PGH(3), thromboxane A(3), and PGI(3), are capable of interfering with aggregation of platelets in platelet-rich plasma.

The effects of dextran sulfate, heparin and PGE 1 on adenylate cyclase activity and aggregation of human platelets

Prostaglandin E 1 (PGE 1) sensitive adenylate cyclase [EC.4.6.1.1 ATP-pyrophosphate lyase (cyclizing)] in human platelet lysates is inhibited by dextran sulfate. Inhibition is dose-dependent at the range of 1–10 μg/ml. Maximal inhibition obtained was 83% at 30 μg/ml. At a similar range of concentrations dextran sulfate was found to antagonize the anti-aggregating effect of PGE 1 on adenosine S' diphosphate (ADP) induced human platelet aggregation. Heparin, like dextran sulfate, effectively antagonizes the anti-aggregating effect of PGE 1 in human platelets. It is possible that the aggregating effects of both heparin and dextran sulfate on human platelets may be mediated via inhibition of platelet PGE 1-sensitive adenylate cyclase.

Platelet Resistance to Prostaglandin D2 in Patients With Myeloproliferative Disorders

We investigated the effect of prostaglandin D2 (PGD2) on platelet adenylate cyclase activity in 23 patients with myeloproliferative disorders, including eight patients with polycythemia vera, seven with myeloid metaplasia, four with chronic myelogenous leukemia, and four with essential thrombocythemia. In 20 of these patients there was less activation of platelet adenylate cyclase at all concentrations of PGD2 studied when compared to normal controls. In 5 of these patients we also studied the effect of PGD, on platelet aggregation and the release of 14C-serotonin. Each of these patients required ten fold higher than normal concentrations of PGD2 to inhibit collagen-induced 14C-serotonin release. This diminished response was specific for PGD2; the stimulation of adenylate cyclase by PGE1 and PGI2 was normal in these patients’ platelets, as was the inhibition of 14C-serotonin release by PGE1. Aspirin therapy in 5 patients and anticoagulation with sodium warfarin in 1 patient did not correct this platelet abnormality. Five subjects with reactive thrombocytosis who were studied had a normal platelet adenylate cyclase response to PGD2. Since sufficient PGD2 is synthesized by platelets to inhibit aggregation, the resistance of these platelets to physiologic concentrations of PGD2 could contribute to the high incidence of thrombosis in patients with myeloproliferative disorders.

Characterization of alpha- and beta-adrenergic receptors linked to human platelet adenylate cyclase.

Adrenaline and noradrenaline cause aggregation of human platelets through α-adrenergic receptors, whereas isoprenaline through β-adrenergic receptors can inhibit aggregation. Either type of adrenergic receptors is coupled to platelet adenylate cyclase. Stimulation and inhibition of adenylate cyclase by β-and α-adrenergic stimulants, respectively, had been demonstrated in human platelet lysates. These effects were characterized with regard to the effectiveness of various agonists and antagonists. Reduction of platelet adenylate cyclase activity was observed only with L-adrenaline and L-noradrenaline. This inhibitory effect, which was increased in the presence of a β-adrenergic blocking agent, was half-maximal at about 1 to 2×10−6 M adrenaline, and maximal inhibition (by 50–60%) was observed at about 3×10−5M. Various other catecholamine and imidazoline derivatives that act as α-adrenergic agonists in other cell types neither induced aggregation nor affected the enzyme activity. Adrenaline-induced inhibition of platelet adenylate cyclase was prevented by α-adrenergic blocking agents. These compounds inhibited the effects of adrenaline on aggregation and on adenylate cyclase with similar efficacies. Dihydrogenated ergot alkaloids were more effective than phentolamine and yohimbine; phenoxybenzamine, tolazoline and azapetine were least effective. Adrenaline-induced inhibition of platelet adenylate cyclase was reversed by phentolamine without apparent lag phase. In the presence of α-adrenergic blocking agents, adrenaline was capable of increasing adenylate cyclase activity between 20 and 50%. Only adrenaline and isoprenaline stimulated adenylate cyclase activity; other compounds that stimulate β-adrenergic receptors in other cell types, including β-adrenergic stimulants, had no effect on the activity of the platelet enzyme. The stimulatory effect of adrenaline was prevented by various β-adrenergic blocking agents including pindolol and propranolol. Preferentially β-adrenergic receptor blocking agents such as practolol and atenolol were without effect. These findings indicate that the spectrum of compounds capable of exhibiting intrinsic activity through α- and β-adrenergic receptors of human platelets is very narrow and that either type of platelet adrenergic receptors appears to differ from those found in other cell types.

Prostacyclin increases cyclic AMP levels and adenylate cyclase activity in platelets

PROSTACYCLIN (PGX) (ref. 1) is the name proposed for the metabolic product of a recently discovered enzymic transformation of the prostaglandin endoperoxides PGG2 and PGH2 (refs 2–4). Prostacyclin synthetase, the enzyme which catalyses this conversion, is particularly active in the microsomal fraction of blood vessel walls3,4 although there is evidence that it is also present in the rat stomach fundus3. The prostaglandin endoperoxides, PGG2 and PGH2, have been shown to contract arterial smooth muscle and to cause platelet aggregation5,6, whereas, in contrast, prostacyclin relaxes arterial strips and prevents platelets aggregation3. A biochemical interaction has therefore been postulated to exist between platelets and the blood vessels wall, such that endoperoxides derived from platelets are converted by the vessel wall into prostacylin, which in turn actively prevents the deposition of platelets on the vascular endothelium2,4. In general, agents which inhibit platelet aggregation, such as PGE1, increase cyclic AMP levels, whereas agents causing aggregation lower platelet cyclic AMP levels7. The ability of prostacyclin to rapidly reverse or prevent platelet aggregation suggested that its action on the platelet might, like that of PGE1, be mediated by an increase in cyclic AMP levels. This hypothesis is supported by the experiments detailed here which strongly suggest that prostacyclin generated by incubation of arachidonic acid, platelets and aortic tissue, causes a rapid and pronounced accumulation of cyclic AMP by intact platelets, as well as stimulation of adenylates cyclase in isolated platelet membranes.

Cyclic 3?,5?-adenosine monophosphate level and adenylate cyclase activity in human blood platelets during storage in ACD solution

The level of cyclic 3',5'-adenosine monophosphate (cAMP) in human platelets and the activity of platelet adenylate cyclase in response to prostaglandin E1 stimulation do not change during two days storage at room temperature in ACD solution. However, the level of cyclic AMP is lower in platelets stored in ACD solution than in platelets from blood anticoagulated by ethylenediamine tetra-acetic acid.

Effect of Chilling on Platelet Cyclic Adenosine 3:5‐Monophosphate and Adenylate Cyclase Activity

Exposure of platelets to 1 C led to a transient increase in cyclic AMP levels (determined either by a protein binding method or by radioimmunoassay) within five to ten minutes reaching a maximum 10 to 15 minutes after chilling was begun and returning subsequently to baseline values. Addition of EDTA to the platelet suspension medium prevented this increase. Rewarming at 37 C produced a sudden reduction in platelet cyclic AMP. To determine whether the cold-induced increase in cyclic AMP was due to a transient stimulation of platelet adenylate cyclase or a rapid inhibition of phosphodiesterase, these enzymes were assayed in ruptured platelet suspensions. Platelet adenylate cyclase activity was found to possess certain characteristics similar to those of the enzyme derived from other sources but there was a marked potentiation of fluoride-stimulated adenylate cyclase activity by 0.001 M EDTA. This effect was limited to low EDTA concentrations. Exposure of platelets to 1 C for up to 60 minutes did not increase adenylate cyclase activity but lowered it substantially compared with controls kept at room temperature. Phosphodiesterase activity at 1 C was depressed sooner and to a greater extent than was adenylate cyclase. The transient rise in cyclic AMP levels in chilled platelets appears to be due to a disproportionate reduction of cyclic nucleotide phosphodiesterase activity.

Interaction of a chick skin collagen fragment (alpha1-CB5) with human platelets. Biochemical studies during the aggregation and release reaction.

The denatured alpha1(I) chain and the cyanogen bromide peptide, alpha1(I)-CB5, of chick skin collagen cause the release of serotonin and leakage of lactic dehydrogenase from human platelets in a manner similar to the release reaction mediated by adenosine diphosphate and native collagen. These peptides also cause a decrease in the level of adenosine 3':5'-monophosphate (cAMP) in platelets. Adenylate cyclase activity of platelets is partially inhibited by these peptides as well as by native collagen, ADP, and epinephrine, but cAMP phosphodiesterase activity is unaltered by these substances. In contrast, the level of platelet guanosine 3':5'-monophosphate (cGMP) is increased by the collagen peptides as well as the other aggregating agents. The increase is associated with increased guanylate cyclase, but normal cGMP phosphodiesterase activities of platelets. Optical rotatory and viscometric measurements of the alpha1 chains and alpha1-CB5 of chick skin in 0.01 M phosphate/0.15 M sodium chloride, pH 7.4, at various temperatures as a function of time indicate that no detectable renaturation occurs at 37 degrees for at least 30 min of observation. Molecular sieve chromatography of alpha1-CB5 in the phosphate buffer at 37 degrees shows that its elution position is identical to that performed under denaturing conditions (at 45 degrees) with no evidence of higher molecular weight aggregates, and the alpha1-CB5 glycopeptide fraction eluting from the column at the position of its monomer retains the platelet aggregating activity. Additionally, electron microscopic examination of the platelet-rich plasma that had been reacted with these peptides fail to show any ordered collagen structures. These data indicate that the denatured alpha1 chain and alpha1-CB5 glycopeptide of chick skin collagen mediate platelet aggregation through the "physiologic" release reaction in a manner similar to that induced by other aggregating agents such as ADP, epinephrine, or native collagen, and support the conclusion that the aggregating activity of the alpha1 chain and alpha1-CB5 is not likely to be due to the formation of polymerized products.

Effects of gliclazide, a new antidiabetic agent, on the platelet release reaction, role of adenylate cyclase

Gliclazide, a sulphonylurea derivative possessing an “in vivo” hypoglycaemic action, inhibits “in vitro” platelet release induced by thrombin or collagen. It does not alter 14CO 2 production from [6- 14C] glucose, but opposes its increase by thrombin. It is an activator of adenylate cyclase increasing the level of [ 3H] cyclic AMP formed within the intact platelets from [ 3H] adenine or [ 3H] adenosine. Finally, it inhibits the effects of thrombin and collagen on platelet adenylate cyclase activity.

Adrenergic receptor function in depression

319 Adrenergic receptor function in depression* A. FRAZER, G. PANDBY, Y.-C. WANG, M. HESS and J. MENDELS Affective Diseases Research Unit, Veterans Administration Hospital and Departments of Psychiatry and Pharmacology, University of Pennsylvania, Philadelphia, Pennsylvania, U.S.A. The adenylate cyclase-adenosine 3’,5’-monophosphate (cyclic AMP) system probably plays an important role in brain function. Consequently, investigators have attempted to examine the activity of this system in patients with affective disorders, by measurement of the urinary excretion of cyclic AMP or its concentration in lumbar fluid. We have employed a different strategy to explore the possible role of this system in affective illness, and propose to summarize the results of three related experiments: (1). The level of act& of adenylate cyclase in intact platelets from eleven depressed male patients (Hamilton score of 24 & 2; x f SEM) was compared with its activity measured in platelets obtained from eight male normal control subjects. Prostaglandin E1 (PGE,) was added in vitro to stimulate enzyme activity and the inhibitory effect of norepinephrine (NE) on this stimulation was observed. There was no sianificant difference in the basal uercent conversion of (3H) nucleotides to (3H) cvclic AMP between the depre the dose-dependent reduction of PGE,stimulated enzyme activity caused by NE was also the same in both groups of subjects. (2) We examined the effect of therapeutic concentrations of lithium (Li) in vitro on adenylate cyclase activity in intact platelets. Li produced a dose-dependent inhibition of the stimulatory effect of PGE, (2 x 1O-6 M) on (3H) cvclic AMP net svnthesis without alter& the basal net svnthesis of the labelled nucleotide. As little as 1 mM Li signitlcantli reduced the stimulato;y effect of PG’E;. This effect was specific to Li as other monovalent cations (sodium, potassium, or rubidium) did not alter PGE,-stimulated enzyme activity. (3) We investigated the effects of a combination of imipramine and Tri-idothyronine (T$ on brain adenylate cyclase, in order to determine whether this might explain the reported therapeutic advantage of combining imipramine with T8 in the treatment of depression. Male rats were injected with either Ts (15 pg), or imipramine (20 mg/kg) or T8 plus imipramine, for 5 consecutive days. Cerebral cortex slices obtained from these animals were incubated with (%I)-adenine and the production of radioactive cyclic AMP in the slices was measured. Results obtained were as follows : (a) none of the treatments altered basal net synthesis of ($H) cyclic AMP; (b) imipramine treatment significantly reduced the stimulatory effect of NE on (%I) cyclic AMP net synthesis; (c) T, treatment did not alter the stimulation produced by NE in this system; (d) the combination of T3 plus imipramine reduced the stimulatory effect of NE. However, the degree of inhibition was less than that observed with imipramine alone. The relevance of the results from these three sets of experiments for the postulated deficit in adrenergic receptors in depression will be discussed together with the signiticance of the interaction of lithium with hormone-stimulated adenylate cyclase. Neuroendocrine strategies in psychobiological research EDWARD J. SACHAR Bronx Municipal Hospital, Bronx, New York, U.S.A. Exciting research of the past 5 yr has established that the hypothalamic neuroendocrine cells which secrete the releasing factors for the anterior pituitary hormones are themselves regulated by monoaminergic neurones. For example, proiactin secretion is regulated by Prolactin Inhibiting Factor (PIF), which in turn * Supported in part by Research Funds from the Veterans Administration.

Effect of lithium on prostaglandin E 1 − stimulated adenylate cyclase activity of human platelets

The effect of lithium (Li) on the stimulation of adenylate cyclase by prostaglandin E 1(PGE 1) was examined. In platelet sonicates, Li, as well as sodium (Na), potassium (K), and rubidium (Rb) significantly reduced the PGE 1-induced stimulation of adenylate cyclase in a dose-dependent manner. The inhibition due to Rb was significantly less than that produced by the other cations; at a high concentration, 64 mM, Li was a more potent inhibitor than the other cations. In intact platelets, only Li reduced the PGE 1-enhanced accumulation of [ 3H]cyclic AMP, K and Rb being ineffective. As little as 1 mM Li significantly reduced the stimulatory effect of PGE 1 on [ 3H]cyclic AMP production in this sytem. The inhibition produced by Li was not blocked by phentolamine, whereas phentolamine did block the inhibition due to norepinephrine (NE). Magnesium enhanced the stimulatory effect of PGE 1 on the production of labeled cyclic AMP in intact platelets and antagonized the inhibition produced by Li on this process. These data show that Li antagonizes PGE 1 − induced stimulation of platelet adenylate cyclase at a site distinct from that at which NE acts.

Activation and inhibition of blood platelet adenylate cyclase by adenosine or by 2-chloroadenosine

The effects of adenosine and of 2-chloroadenosine on the activity of platelet adenylate cyclase were measured using a particulate fraction from washed human platelets that had been lysed by freezing and thawing. At 25 μM these nucleosides increased activity to about 150–160% of the control value. Higher concentrations had less effect and adenosine was inhibitory above 100 μM. The activation was observed in the presence of papaverine or cyclic AMP but not aminophylline. In the presence of 1 μM PGE 1, which increased adenylate cyclase activity more than twelvefold, both nucleosides were inhibitory above 10 μM. The results suggest that the activation and inhibition of platelet adenylate cyclase by adenosine or 2-chloroadenosine are independent processes mediated by different components of a receptor-adenylate cyclase complex.