Platelet Adenylate Cyclase Activity Research Articles

Trauma Leads to a Rise in Intracellular cAMP and Adenylate Cyclase Activity in Rats

Background: Severe trauma and hemorrhage leads to an acute coagulopathy as exemplified by a decrease in clotting firmness and platelet aggregation. Because platelets contribute 70-80% of clot strength, evaluating the intracellular mechanisms that regulate aggregation in platelets may lead to strategies to mitigate the development of coagulopathy. It is known that platelet aggregation can be inhibited by a rise in intracellular cyclic adenosine monophosphate (cAMP) or cyclic guanosine monophosphate (cGMP). Changes in cAMP/cGMP would necessitate a change in activity of those enzymes that either synthesize (adenylate cyclase/ guanylate cyclase) or breakdown (phosphodiesterases) these 2nd messengers.Objective: Determine if the intracellular levels of cAMP and cGMP in platelets rise in rats subjected to polytrauma and hemorrhage. And if so, if there is a change in cyclase or phosphodiesterase activity.Methods: Polytrauma was induced in isoflurane anesthetized Sprague-Dawley rats (n=10 per group) by damage to the small intestines, right and medial lobes of the liver, the right leg skeletal muscle, and by fracturing the right femur. 40% hemorrhage was then performed immediately after trauma. Rats were euthanized at 4hrs. Platelet intracellular intermediates were measured in whole blood before, and 0.5, 1, 2 and 4hrs after trauma. Blood samples were taken before, and 0.5, 2 and 4 hours after trauma. Platelet rich plasma (PRP) was generated by centrifugation of whole blood at 200g for 10min, no brakes. Cyclic AMP, cGMP, AMP and GMP were extracted from 100ul of PRP after adding 1ml of EtOH, 10mM ammonium formate, with 10ug/ml cGMP-Br as an internal control. IP3 and IP1 (metabolic breakdown product of IP3) were extracted from another 100ul PRP by addition of 50% EtOH, 500mM Formic Acid with 10ug/ml ATP-C13 as an internal control. Samples were centrifuged at 20K g for 10min, and supernatant dried. All Samples were brought up in 200ul of 0.1% formic acid for analysis by LC-MS/MS. Cyclase and phosphodiesterases were isolated from 100ul of rat PRP after sonication to lyse cells, centrifugation to release intracellular constituents, and Spin Column (Zeba, ThermoFisher) removal of small molecular weight constituents. Cyclase activity was measured for 15min after addition of either ATP or GTP and measuring cAMP or cGMP with inhibition of PDE activity (IBMX 1uM). Phosphodiesterase was measured by addition of cAMP or cGMP and measuring AMP or GMP. Data was generated by subtracting measurement from control.Results: Trauma and hemorrhage led to a significant rise in the intracellular cAMP and a fall in cGMP. Adenylate cyclase activity in platelets also significantly increase after trauma. However, trauma had no effect on guanylate cyclase activity. Phosphodiesterase activity was elevated after trauma for both conversion of cAMP and cGMP to AMP and GMP, but neither was significant.Conclusion: Trauma and hemorrhage leads to coagulopathy and platelet dysfunction. The platelet dysfunction is likely due to a rise in the intracellular cAMP, but not to cGMP as it fell precipitously after trauma. The rise in cAMP is likely due to an increase in adenylate cyclase activity induced by trauma. Although there was a rise in PDE activity, it was not significant, but may play a role in blunting the rise and cAMP, and contribute to the fall in cGMP. This study was funded by the US Army MRMC and conducted in compliance with the Animal Welfare Act, the implementing of Animal Welfare Regulations, and the principles of the Guide for the Care and Use of Laboratory Animals. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A novel antithrombotic effect of sulforaphane via activation of platelet adenylate cyclase: ex vivo and in vivo studies

Sulforaphane is a naturally occurring isothiocyanate, which can be found in cruciferous vegetables such as broccoli and cabbage. Sulforaphane was found to have very potent inhibitory effects on tumor growth through regulation of diverse mechanisms. However, no data are available concerning the effects of sulforaphane on platelet activation and its relative issues. Activation of platelets caused by arterial thrombosis is relevant to a variety of cardiovascular diseases. Hence, the aim of this study was to examine the in vivo antithrombotic effects of sulforaphane and its possible mechanisms in platelet activation. Sulforaphane (0.125 and 0.25 mg/kg) was effective in reducing the mortality of ADP-induced acute pulmonary thromboembolism in mice. Other in vivo studies also revealed that sulforaphane (0.25 mg/kg) significantly prolonged platelet plug formation in mice. In addition, sulforaphane (15–75 μM) exhibited more-potent activity of inhibiting platelet aggregation stimulated by collagen. Sulforaphane inhibited platelet activation accompanied by inhibiting relative Ca2+ mobilization; phosphorylation of phospholipase C (PLC)γ2, protein kinase C (PKC), mitogen-activated protein kinases (MAPKs) and Akt; and hydroxyl radical (OH●) formation. Sulforaphane markedly increased cyclic (c)AMP, but not cyclic (c)GMP levels, and stimulated vasodilator-stimulated phosphoprotein (VASP) phosphorylation. SQ22536, an inhibitor of adenylate cyclase, but not ODQ (1H-[1,2,4]Oxadiazolo[4,3-a]quinoxal in-1-one), an inhibitor of guanylate cyclase, obviously reversed the sulforaphane-mediated effects on platelet aggregation; PKC activation, p38 MAPK, Akt and VASP phosphorylation; and OH● formation. Furthermore, a PI3-kinase inhibitor (LY294002) and a p38 MAPK inhibitor (SB203580) both significantly diminished PKC activation and p38 MAPK and Akt phosphorylation; in contrast, a PKC inhibitor (RO318220) did not diminish p38 MAPK or Akt phosphorylation stimulated by collagen. This study demonstrates for the first time that in addition to it originally being considered as an agent for prevention of tumor growth, sulforaphane possesses potent antiplatelet activity which may initially activate adenylate cyclase/cAMP, followed by inhibiting intracellular signals (such as the PI3-kinase/Akt and PLCγ2-PKC-p47 cascades) and ultimately inhibiting platelet activation. Therefore, this novel role of sulforaphane may represent a high therapeutic potential for treatment or prevention of cardiovascular diseases.

Exaggerated platelet reactivity to physiological agonists in war veterans with posttraumatic stress disorder

An association between traumatic stress and cardiovascular disease (CVD) is supported by various epidemiological studies. Platelet activation and binding of activated platelets to leukocytes contributes to the pathophysiology of CVD. Evidence of hyperactive sympathetic nervous system, altered expression of platelet α(2)-adrenoreceptors (α(2)AR), and altered platelet adenylate cyclase activity in patients with posttraumatic stress disorder (PTSD) suggest that platelet reactivity in PTSD may be altered as well. We tested whether platelet reactivity to increasing doses of adenosine-diphosphate (ADP), epinephrine (EPI), or their combination differs between war veterans with PTSD (n=15) and healthy controls (n=12). For this purpose, citrated whole blood was incubated with increasing concentrations of ADP (0.1, 1, 10 μM), EPI alone (10 nM, 100 nM, 1000 nM), or EPI (10 nM, 100 nM, 1000 nM) in combination with 0.1 μM ADP. A subset of samples was also incubated with 10 μM yohimbine (YOH), α(2)AR antagonist, to distinguish receptor-specific effects. Platelet CD62P expression and formation of platelet-leukocyte aggregates (PLA) [platelet-monocyte (P-Mo), -lymphocyte (P-Ly), and -neutrophil (P-Ne) aggregates] were measured using three-color flow cytometry. Platelet reactivity was higher in war veterans with PTSD when compared to controls, as determined by greater CD62P expression and formation of PLA in response to ADP alone or in combination with EPI. Platelet reactivity also correlated with the severity of PTSD symptoms. Preliminary experiments with YOH indicate that stress-associated EPI elevations may contribute to platelet activation through a α(2)AR-dependent mechanism. The enhanced platelet reactivity observed in our study may be the underlying mechanism contributing to the development of CVD in PTSD patients.

Adenylate-cyclase activity in platelets of patients with obsessive-compulsive disorder

Although the main biological hypothesis on the pathophysiology of obsessive-compulsive disorder (OCD) is centered on the serotonin system, indications are available that other neurotransmitters, and even second messengers, particularly the cyclic adenosine monophosphate (cAMP) signaling, may be involved, though effective data are few. Therefore, the aim of the present study was to evaluate and compare the basal and isoprenaline (ISO)-stimulated velocity of adenylate-cyclase (AC) in human platelet membranes of patients with OCD and healthy control subjects. The results showed that the basal and ISO-stimulated AC activity, as well as the dose-response curves of ISO by using agonist concentrations ranging between 0.1 nM and 10 muM, were not different in the two groups. However, OCD patients showed lower EC(50) and higher E(max) values than healthy subjects. These findings suggest the presence of supersensitive beta-adrenergic receptors in platelets of OCD patients.

Biological Markers of Alcohol Consumption in Nondrinkers, Drinkers, and Alcohol‐Dependent Brazilian Patients

The purpose of this study was to compare the sensitivity and specificity of some new and traditional biological markers and indicators of health among Brazilian nondrinkers, drinkers, and alcohol-dependent patients. We evaluated 130 nondrinkers, 167 drinkers, and 183 alcohol-dependent drinkers from Brazil who participated in the WHO/ISBRA Study on State and Trait Markers of Alcohol Use and Dependence. A standardized WHO/ISBRA Interview Schedule provided background information on the subjects' characteristics including reported health problems and alcohol consumption. Blood samples were analyzed for aspartate aminotransferase (AST), carbohydrate deficient transferrin (CDT), gamma-glutamyltransferase (GGT), blood alcohol levels (BAL), and platelet adenylate cyclase activity (basal levels [AC] and levels after stimulation with Gpp(NH)p, cesium fluoride, and forskolin). The alcohol-dependent drinkers presented higher levels of AST, GGT, AC, CDT, and BAL than the nondrinkers and drinkers, whose levels were similar. Sex differences in the sensitivity of CDT and AC were found. The alcohol-dependent women presented a lower prevalence of abnormal values of CDT and Gpp(NH)p-stimulated AC than the alcohol-dependent men, despite the fact that they presented similar alcohol consumption levels. The alcohol-dependent drinkers presented a higher prevalence of clinical disorders than the nondrinkers and drinkers. The drinkers and alcohol-dependent patients presented significantly higher rates of gastritis than the nondrinkers. Sex differences in the sensitivity of CDT and AC suggest that these markers are not as sensitive at detecting excessive alcohol use in women as they are in men. If data from this Brazilian sample are compared with those reported for international samples, relevant differences are detected, which suggests that genetic and cultural differences should be considered in the selection of biological markers of heavy alcohol consumption.

Inhibition of human platelet adenylate cyclase activity by adrenaline, thrombin and collagen: analysis and reinterpretation of experimental data

Mathematical models based on the current understanding of stimulation and inhibition of adenylate cyclase (AC) activity have been developed and used to analyse experimental data [Farndale, Winkler, Martin and Barnes (1992) Biochem. J. 282, 25-32] describing the inhibition of human platelet AC by collagen, thrombin and adrenaline. Here it has been demonstrated that neither affinities of receptors specific for adrenaline or thrombin nor the activity of cAMP phosphodiesterase are affected by collagen. Both collagen and thrombin at high doses act as effective inhibitors of AC activity. Inhibition of AC activity by collagen proceeds via two parallel pathways; the same is true for thrombin at moderate concentrations, and the two ligands act independently. The G-protein-dependence of these pathways is distinct from that mediating inhibition of AC activity by adrenaline, i.e. Gi2. Convergence of the inhibitory pathways takes place at the catalytic subunit of AC.

Inhibition of human platelet adenylate cyclase activity by adrenaline, thrombin and collagen: analysis and reinterpretation of experimental data

Mathematical models based on the current understanding of stimulation and inhibition of adenylate cyclase (AC) activity have been developed and used to analyse experimental data [Farndale, Winkler, Martin and Barnes (1992) Biochem. J. 282, 2532] describing the inhibition of human platelet AC by collagen, thrombin and adrenaline. Here it has been demonstrated that neither affinities of receptors specific for adrenaline or thrombin nor the activity of cAMP phosphodiesterase are affected by collagen. Both collagen and thrombin at high doses act as effective inhibitors of AC activity. Inhibition of AC activity by collagen proceeds via two parallel pathways; the same is true for thrombin at moderate concentrations, and the two ligands act independently. The G-protein-dependence of these pathways is distinct from that mediating inhibition of AC activity by adrenaline, i.e. Gi2. Convergence of the inhibitory pathways takes place at the catalytic subunit of AC.

Genetic markers of alcohol abuse

In this paper, we review the current status of genetic markers for the development of alcohol abuse. Family, twin, half-sibling and adoption studies of alcoholic subjects suggest that the heritability of liability to alcoholism is at least 50%. These findings have fuelled intensive investigation in the fields of neurology, biochemistry, genetics and molecular biology aimed at the identification of markers for the risk of alcoholism. The most promising of these are discussed in detail. Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) polymorphisms, specifically the ADH3 ∗1, ADH2 ∗2, and ALDH2 ∗2 genotypes appear to confer a protective effect against alcoholism, most notably in Oriental subjects. Caucasian alcohol abusers and their first-degree relatives exhibit depressed platelet monoamine oxidase activity, the degree of which is greater in Type II than Type I alcoholids. Electrophysiological characteristics of alcoholics and those at risk for developing alcoholism have also been identified, including the reduced amplitude of the event-related brain potential and, after ethanol ingestion, characteristic EEG α-wave activity. Lower platelet adenylate cyclase activity is seen in alcoholics compared to controls, presumably as a result of over-expression of an inhibitory G-protein. Markers related to other signal transduction pathways of the central nervous system including the serotoninergic, muscarinic and dopaminergic systems are also discussed. In this group of markers, the putative association between the inheritance of the A1 allele of the D2 dopamine receptor and the susceptibility to alcoholism provides the most dramatic illustration of the challenges presently existing in this field of scientific investigation. Current limitations in the definition, diagnosis and classification of alcoholism, the confounding influences of race and gender on association studies, as well as the statistical approach of linkage studies are discussed as they relate to the endeavor to uncover valid genetic markers for the risk of alcoholism.

S-56-2 - Schizophrenia: A neurodevelopmental and progressive disorder

In this paper, we review the current status of genetic markers for the development of alcohol abuse. Family, twin, half-sibling and adoption studies of alcoholic subjects suggest that the heritability of liability to alcoholism is at least 50%. These findings have fuelled intensive investigation in the fields of neurology, biochemistry, genetics and molecular biology aimed at the identification of markers for the risk of alcoholism. The most promising of these are discussed in detail. Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) polymorphisms, specifically the ADH3∗1, ADH2∗2, and ALDH2∗2 genotypes appear to confer a protective effect against alcoholism, most notably in Oriental subjects. Caucasian alcohol abusers and their first-degree relatives exhibit depressed platelet monoamine oxidase activity, the degree of which is greater in Type II than Type I alcoholids. Electrophysiological characteristics of alcoholics and those at risk for developing alcoholism have also been identified, including the reduced amplitude of the event-related brain potential and, after ethanol ingestion, characteristic EEG α-wave activity. Lower platelet adenylate cyclase activity is seen in alcoholics compared to controls, presumably as a result of over-expression of an inhibitory G-protein. Markers related to other signal transduction pathways of the central nervous system including the serotoninergic, muscarinic and dopaminergic systems are also discussed. In this group of markers, the putative association between the inheritance of the A1 allele of the D2 dopamine receptor and the susceptibility to alcoholism provides the most dramatic illustration of the challenges presently existing in this field of scientific investigation. Current limitations in the definition, diagnosis and classification of alcoholism, the confounding influences of race and gender on association studies, as well as the statistical approach of linkage studies are discussed as they relate to the endeavor to uncover valid genetic markers for the risk of alcoholism.

Longitudinal studies of platelet cyclic AMP during healthy pregnancy and pregnancies at risk of pre-eclampsia.

1. Platelet behaviour in vitro in relation to cyclic AMP was studied longitudinally during pregnancy and in the same women when they were not pregnant. Subjects comprised a group of healthy primigravidae and a group of women deemed at risk of pre-eclampsia, on the basis of a previous history of the condition. 2. There was a progressive decline during pregnancy in sensitivity of platelets to inhibition of the arachidonic acid-induced release reaction by agents which act via cyclic AMP. This effect was maximum at 36 weeks' gestation. 3. Basal platelet cyclic AMP levels, and those in the presence of a phosphodiesterase inhibitor, did not change throughout the period of the study. 4. By contrast, platelet cyclic AMP accumulation in response to a variety of adenylate cyclase stimulators was reduced from early pregnancy, throughout the gestational period, compared with post-natally. This effect was noted when platelets were incubated with prostaglandins acting via different surface receptors or with forskolin and was most marked on co-incubation with a phosphodiesterase inhibitor. 5. Compared with healthy women, platelets from women with a previous history of pre-eclampsia tended to accumulate less cyclic AMP in response to adenylate cyclase stimulators. This was the case both during pregnancy and post-natally. Further investigation of adenylate cyclase activity in platelets in relation to pre-eclampsia is required.

Absence of reduced platelet adenylate cyclase activity in Vietnam veterans with PTSD

Many studies have been devoted to an examination of radioligand receptor binding and postreceptor signal transduction in peripheral cells obtained from patients suffering from mood or anxiety disorders. Postreceptor signal transduction has been investigated by the use of agents that interact with the adenylate cyclase system at points distal to the receptor, such as aluminum chloride (AIC13) and sodium fluoride (NaF), which affect the guanyl nucleotidebinding (G) protein, and forskolin, which affects the catalytic (C) unit of the enzyme. Post-traumatic stress disorder (PTSD) is a member of the group of anxiety disorders which also includes generalized anxiety disorder, panic disorder, and obsessive-compulsive disorder. PTSD also shares phenomenological features with depression (Sierles et al 1983). Abnormalities in adenylate cyclase activity have been demonstrated in peripheral cells derived both from panic disorder patients (Charney et al 1989) and depressed patients (Ebstein et a11988; Kanof et al 1988). Recent evidence has indicated possible abnormalities at both the receptor and postreeeptor levels in platelets from PTSD patients. Perry et al (1987) found a reduction in alpha-2-adrenergic receptors in 11 Vietnam veterans with PTSD relative to agematched normal controls. This was due to a relative loss of sites labeled with low affinity by the antagonist 3H-rauwolscine, representing those sites coupled via the inhibitory G protein Gi to adenylate cyclase. The involvement of adrenergic receptors in PTSD has been recently reviewed (Lerer et al 1994). In a series of studies, our laboratory has investigated platelet adenylate cyclase in PTSD with mixed results. A first study (Lerer

2-[3-[2-(4,5-diphenyl-2-oxazolyl) ethyl] phenoxy] acetic acid (BMY 42393): A new, structurally-novel prostacyclin partial agonist: 1) inhibition of platelet aggregation and mechanism of action

BMY 42393, (2-[3-[2-(4,5-diphenyl-2-oxazolyl)ethyl]phenoxy]acetic acid), is a new prostacyclin partial agonist that inhibited ADP, collagen and thrombin-induced platelet aggregation (IC 50 range 0.3 - 2.0 μM). BMY 42393 stimulated platelet adenylate cyclase activity (EC 50 = 25 μM), however, the maximal activation was 75–80 % of that observed with maximal iloprost or PGE 1. Platelets treated with BMY 42393 showed an elevation of cAMP levels and activation of cAMP-dependent protein kinase. BMY 42393 also inhibited thrombin-induced elevation of intracellular free calcium. BMY 42393 competed for radiolabeled iloprost and PGE 1 binding to platelet membranes (IC 50: 170 nM and 130 nM, respectively); however, it had little effect on radiolabeled PGE 2, PGD 2, or SQ 29548 binding. These studies indicate that BMY 42393 is a novel platelet aggregation inhibitor which acts by stimulation of platelet prostacyclin receptors to elevate platelet cAMP levels.

Platelet adenylate cyclase and monoamine oxidase in women with alcoholism or a family history of alcoholism.

Characteristic changes of platelet membrane monoamine oxidase and adenylate cyclase activities have been described in men with alcoholism. We studied the occurrence of these changes in abstinent alcoholic women and in nonalcoholic female control subjects with and without family histories of alcoholism. Blood samples were collected from 23 female alcoholics and 39 nonalcoholic female social drinkers. Platelet membrane assays were performed for monoamine oxidase and adenylate cyclase activities. Alcoholic women had lower basal adenylate cyclase (p < 0.01) and adenylate cyclase activities stimulated by cesium fluoride (p < 0.001), by the guanine nucleotide analog 5'-guanylylimidodiphosphate (p < 0.02), and by prostaglandin E1 (p < 0.01). Female control subjects with family histories of alcoholism also had lower basal adenylate cyclase (p < 0.01) and adenylate cyclase activities enhanced by incubation with cesium fluoride (p < 0.005) and 5'-guanylylimidodiphosphate (p < 0.001). Monoamine oxidase activity levels measured with (p < 0.001) and without ethanol (p < 0.01) were higher for alcoholic women. No significant differences were found between female control subjects with and without family histories of alcoholism for monoamine oxidase in the absence or presence of ethanol. In vitro platelet adenylate cyclase activity may facilitate a diagnosis of alcoholism in women and may be a biologic indicator of vulnerability in the offspring of alcoholics.

The role of Gi and the membrane-fluidizing agent benzyl alcohol in modulating the hysteretic activation of human platelet adenylate cyclase by guanylyl 5'-imidodiphosphate.

The non-hydrolysable GTP analogue guanylyl 5'-imidodiphosphate (p[NH]ppG) elicited a profound increase in the adenylate cyclase activity of human platelets. This occurred after a well-defined lag period of around 6 min, whereupon an enhanced steady-state rate was evident. The duration of the lag period was unchanged over a range of concentrations of p[NH]ppG which gave very different steady-state rates of adenylate cyclase activity. Prior activation of the stimulatory G-protein Gs by cholera-toxin pre-treatment abolished the lag period and elicited a small increase in the steady-state rate. Manipulating function of the inhibitory G-protein Gi also led to profound changes in the lag periods. Thus marked decreases in the lag were seen (approximately 70-81%) when Gi function was ablated through pre-treatment of platelet membranes with pertussis toxin, or by using elevated (25 mM) Mg2+ levels in the assay, or when Mg2+ was replaced by 5 mM Mn2+ in the assay. In contrast with this, potentiation of Gi function led to an increase in the lag period, as seen under conditions of agonist occupancy of inhibitory alpha 2-adrenoceptors (increase approximately 74%) or with the addition of 100 mM NaCl to the assays (increase approximately 44%). The local anaesthetic and membrane-fluidizing agent benzyl alcohol elicited both a profound decrease (around 70% at 80 mM) in the p[NH]ppG-induced lag period and a marked augmentation (around 5-fold) in the steady-state adenylate cyclase activity. When adenylate cyclase assays were done at 35 degrees C instead of 25 degrees C, then the lag period for activation by p[NH]ppG was decreased by around 33% and the steady-state rate increased by around 3-fold. At 35 degrees C, the addition of benzyl alcohol led to the apparent abolition of the lag period for p[NH]ppG activation of adenylate cyclase and amplified the steady-state rate by only around 2.2-fold. It is shown that Gi plays a fundamental role in determining the rate of activation of Gs. The proposal is formulated that such an action may be mediated through the release of beta gamma-subunits. Thus beta gamma-subunit dissociation is proposed as providing the rate-limiting step in Gi activation.

Inhibitory effects of S-(1,2-dicarboxyethyl)glutathione on collagen-induced platelet aggregation; enhancements of cyclic AMP level and adenylate cyclase activity in platelets by S-(1,2-dicarboxyethyl)glutathione.

S-(1,2-Dicarboxyethyl)glutathione (DCE-GS) in addition to being present in the liver, lens, and heart, also inhibited platelet aggregation. To clarify these inhibitory effects, the role of DCE-GS in the release of ATP and serotonin from platelets was studied, as was thromoboxane A2 formation, cyclic AMP level and adenylate cyclase activity in human platelets. The results are as follows: DCE-GS at a concentration of 1.3 mM inhibited ATP and serotonin release from platelets induced by collagen, by 77.4 +/- 4.3 and 78.7 +/- 6.3%, respectively. At 1.5 mM DCE-GS also inhibited the formation of thromboxane B2 by 79.6 +/- 4.1%. Incubation of human platelet rich plasma with 2 mM of DCE-GS for 10 min increased the cyclic AMP level and the activity of adenylate cyclase by 204 +/- 28 and 211 +/- 11.7%, respectively. These results suggest that the inhibitory effect of DCE-GS on the platelet aggregation induced by collagen is due to an increase in the cyclic AMP level in platelets, which in turn may be due to enhancement of the activity of adenylate cyclase.

Involvement of the “tethered-ligand” receptor in thrombin inhibition of platelet adenylate cyclase

Thrombin is thought to activate platelets through multiple signaling pathways. Recently a new thrombin receptor was identified (Vu et al., Cell 64 : 1057–1068, 1991) that recognizes α-thrombin's anion-binding exosite. Thrombin cleaves this receptor generating a new N-terminal (“tethered-ligand”) that activates the receptor. We report here that this receptor is involved in α-thrombin inhibition of platelet adenylate cyclase, a process thought mediated by thrombin's high-affinity pathway. In gel-filtered human platelets, iloprost-stimulated cAMP levels were lowered by α- and ζ-thrombin addition and, to a much lesser extent, by γ-thrombin. The α- and ζ-thrombin mediated decreases in cAMP were prevented by the thrombin anion-binding exosite inhibitor, BMS 180742, implying that binding to thrombin's anion-binding exosite was required. The iloprost-stimulated increase in cAMP was also reversed (in a concentration-dependent fashion) by a peptide mimicking the new N-terminal of the “tethered-ligand” thrombin receptor (SFLLRNPNDKYEPF). In broken cell preparations, platelet adenylate cyclase activity was also inhibited by SFLLRNPNDKYEPF (but not by a similar peptide used as a control, FSLLRNPNDKYEPF). These results support the hypothesis that thrombin inhibition of platelet adenylate cyclase activity is mediated, at least in part, via the “tethered-ligand” receptor. Moreover, this data is consistent with the “tethered-ligand” receptor mediating the high affinity actions of α-thrombin.

Inhibition of platelet aggregation by prostacyclin is attenuated after exercise in patients with angina pectoris

We tested whether alteration of platelet sensitivity to prostacyclin (PGI 2) is involved in the activation of platelets induced by exercise in patients with stable angina. Twenty patients and 20 control subjects underwent treadmill testing. Blood samples were obtained before and immediately after exercise for plasma thromboxane B 2 (TXB 2) and 6-keto-PGF 1α (6kP) assays and platelet aggregation studies. Dose-response curves for platelet aggregation to coliagen were obtained in the presence and absence of 1 nmol/L PGI 2 to quantify the antiaggregation effects of PGI 2. At rest, platelet aggregation by collagen was enhanced in the patients. However, platelets were more sensitive to exogenous PGI 2, apparently associated with lower plasma 6kP levels in the patients. After exercise, plasma TXB 2 levels increased in the patients but not in the control subjects. Plasma 6kP levels remained unchanged and platelet sensitivity to PGI 2 decreased in the patients whereas these values increased in the control subjects. The exercise-induced changes in platelet sensitivity to PGI 2 correlated with those of platelet adenylate cyclase activity in response to 1 nmol/L PGI 2 ( r = 0.787, p < 0.01). Thus impaired sensitivity of platelets to PGI 2, in addition to the reduced response of prostanoid secretion, might be relevant to the platelet activation associated with exercise in patients with stable angina.

Decrease in epinephrine-induced attenuation of platelet adenylate cyclase activity in depressed patients: relation with plasma electrolytes.

We have measured the alpha 2-adrenoceptor-mediated inhibition of platelet membrane adenylate cyclase in depressed patients and control subjects. The results showed a decrease in the forskolin-stimulated adenylate cyclase inhibition of depressed patients compared to the healthy subjects. This suggests a subsensitivity of alpha 2-adrenoceptor in depression. However, this subsensitivity was not correlated to the severity of depression as both severely and moderately depressed patients exhibited the same percent of adenylate cyclase inhibition. The antidepressant drugs treatment induced an increase in the percent of adenylate cyclase inhibition with a trend towards the control values. However, this increase did not equal control value, and moreover both remitted and unremitted patients presented a similar change in their alpha 2-adrenoceptor-mediated adenylate cyclase inhibition. This result raises the question about a simple and direct relation between the clinical status of depression and the power of alpha 2-adrenoceptor-mediated adenylate cyclase inhibition. Plasma magnesium and sodium yielded correlations to this alpha 2-adrenoceptor-mediated adenylate cyclase inhibition suggesting a relation between the platelet adrenergic function and plasma electrolytes.

Altered G-protein expression and adenylate cyclase activity in platelets of non-insulin-dependent diabetic (NIDDM) male subjects

Adenylate cyclase activity and levels of guanine nucleotide regulatory proteins (G-proteins) were compared in platelets from normal and non-insulin-dependent diabetic (NIDDM) male subjects. Whilst no differences were noted in basal and NaF-stimulated cyclase activities the degree of stimulation achieved by both forskolin and prostaglandin, E 1 was lower by some 34 and 52% respectively, in platelet membranes from diabetic subjects compared with those from normal control subjects. Altered α 1-adrenoceptor-mediated inhibition of prostaglandin E 1-stimulated adenylate cyclase activity was evident; it being some 34% lower in platelet membranes from diabetic subjects compared to controls. Analysis of G-protein α-subunits, using specific anti-peptide antisera, wshowed that platelets from all subjects exhibited the G i-2 and G i-3, but not the G i-1 forms of the inhibitory G-protein ‘G i’ and all expressed the 42 kDa species of α-subunits of the stimulatory G-protein G s. Whilst platelets of siabetic subjects had levels of G s whihch were comparable to those found in control subjects their levels of G i-2 and G i-3 were some 49 and 75%, respectively, of those found in platelets from control subjects. Its is suggested that changes in adenylate cyclase functioning and G-protein expression may contribute to altered platelet functioning in non-insulin-dependent diabetic subjects.

Ring-deleted analogs of atrial natriuretic factor inhibit adenylate cyclase/cAMP system. Possible coupling of clearance atrial natriuretic factor receptors to adenylate cyclase/cAMP signal transduction system.

We have recently shown that atrial natriuretic factor (ANF) inhibits adenylate cyclase activity in rat platelets where only one population of ANF receptors (ANF-R2) is present, indicating that ANF-R2 receptors may be coupled to the adenylate cyclase/cAMP system. In the present studies, we have used ring-deleted peptides which have been reported to interact with ANF-R2 receptors also called clearance receptors (C-ANF) without affecting the guanylate cyclase/cGMP system, to examine if these peptides can also inhibit the adenylate cyclase/cAMP system. Ring-deleted analog C-ANF4-23 like ANF99-126 inhibited the adenylate cyclase activity in a concentration-dependent manner in rat aorta, brain striatum, anterior pituitary, and adrenal cortical membranes. The maximal inhibition was about 50-60% with an apparent Ki between 0.1 and 1 nM. In addition, C-ANF4-23 also decreased the cAMP levels in vascular smooth muscle cells in a concentration-dependent manner without affecting the cGMP levels. The maximal decrease observed was about 60% with an apparent Ki of about 1 nM. Furthermore, C-ANF4-23 was also able to inhibit cAMP levels and progesterone secretion stimulated by luteinizing hormone in MA-10 cell line. Other smaller fragments of ANF with ring deletions were also able to inhibit the adenylate cyclase activity as well as cAMP levels. Furthermore, the stimulatory effects of various agonists such as 5'-(N-ethyl)carboxamidoadenosine, dopamine, and forskolin on adenylate cyclase activity and cAMP levels were also significantly inhibited by C-ANF4-23. The inhibitory effect of C-ANF4-23 on adenylate cyclase was dependent on the presence of GTP and was attenuated by pertussis toxin treatment. These results indicate that ANF-R2 receptors or so-called C-ANF receptors are coupled to the adenylate cyclase/cAMP signal transduction system through inhibitory guanine nucleotide regulatory protein.