Developmental Differences in Platelet Inhibition Response to Prostaglandin E1.

BACKGROUND: The mechanisms underlying neonatal platelets hyporesponsiveness are not fully understood. While previous studies have demonstrated developmental impairment of agonist-induced platelet activation, differences in inhibitory signaling pathways have been scarcely investigated. OBJECTIVE: To compare neonatal and adult platelets with regard to inhibition of platelet reactivity by prostaglandin E1 (PGE1). METHODS: Platelet-rich plasma from umbilical cord (CB) or adult blood was incubated with PGE1 (0-1 µM). We assessed aggregation in response to adenosine diphosphate (ADP), collagen, and thrombin receptor activating peptide as well as cyclic adenosine 3'5'-monophosphate (cAMP) levels (ELISA). Gαs, Gαi2, and total- and phospho-protein kinase A (PKA) were evaluated in adult and CB ultrapure and washed platelets, respectively, by immunoblotting. RESULTS: Neonatal (vs. adult) platelets display hypersensitivity to inhibition by PGE1 of platelet aggregation induced by ADP and collagen (PGE1 IC50: 14 and 117 nM for ADP and collagen, respectively, vs. 149 and 491 nM in adults). They also show increased basal and PGE1-induced cAMP levels. Mechanistically, PGE1 acts by binding to the prostanoid receptor IP (prostacyclin receptor), which couples to the Gαs protein-adenylate cyclase axis and increases intracellular levels of cAMP. cAMP activates PKA, which phosphorylates different target inhibitor proteins. Neonatal platelets showed higher basal and PGE1-induced cAMP levels, higher Gαs protein expression, and a trend to increased PKA-dependent protein phosphorylation compared to adult platelets. CONCLUSION: Neonatal platelets have a functionally increased PGE1-cAMP-PKA axis. This finding supports a downregulation of inhibitory when going from neonate to adult contributing to neonatal platelet hyporesponsiveness.

Possible roles for ATP release from RBCs exclude the cAMP-mediated Panx1 pathway.

Red blood cell (RBC)-derived adenosine triphosphate (ATP) has been proposed as an integral component in the regulation of oxygen supply to skeletal muscle. In ex vivo settings RBCs have been shown to release ATP in response to a number of stimuli, including stimulation of adrenergic receptors. Further evidence suggested that ATP release from RBCs was dependent on activation of adenylate cyclase (AC)/cyclic adenosine monophosphate (cAMP)-dependent pathways and involved the pannexin 1 (Panx1) channel. Here we show that RBCs express Panx1 and confirm its absence in Panx1 knockout (-/-) RBCs. However, Panx1-/- mice lack any decrease in exercise performance, challenging the assumptions that Panx1 plays an essential role in increased blood perfusion to exercising skeletal muscle and therefore in ATP release from RBCs. We therefore tested the role of Panx1 in ATP release from RBCs ex vivo in RBC suspensions. We found that stimulation with hypotonic potassium gluconate buffer resulted in a significant increase in ATP in the supernatant, but this was highly correlated with RBC lysis. Next, we treated RBCs with a stable cAMP analog, which did not induce ATP release from wild-type or Panx1-/- mice. Similarly, multiple pharmacological treatments activating AC in RBCs increased intracellular cAMP levels (as measured via mass spectrometry) but did not induce ATP release. The data presented here question the importance of Panx1 for exercise performance and dispute the general assumption that ATP release from RBCs via Panx1 is regulated via cAMP.

cAMP-dependent cell differentiation triggered by activated CRHR1 in hippocampal neuronal cells.

Corticotropin-releasing hormone receptor 1 (CRHR1) activates the atypical soluble adenylyl cyclase (sAC) in addition to transmembrane adenylyl cyclases (tmACs). Both cAMP sources were shown to be required for the phosphorylation of ERK1/2 triggered by activated G protein coupled receptor (GPCR) CRHR1 in neuronal and neuroendocrine contexts. Here, we show that activated CRHR1 promotes growth arrest and neurite elongation in neuronal hippocampal cells (HT22-CRHR1 cells). By characterising CRHR1 signalling mechanisms involved in the neuritogenic effect, we demonstrate that neurite outgrowth in HT22-CRHR1 cells takes place by a sAC-dependent, ERK1/2-independent signalling cascade. Both tmACs and sAC are involved in corticotropin-releasing hormone (CRH)-mediated CREB phosphorylation and c-fos induction, but only sAC-generated cAMP pools are critical for the neuritogenic effect of CRH, further highlighting the engagement of two sources of cAMP downstream of the activation of a GPCR, and reinforcing the notion that restricted cAMP microdomains may regulate independent cellular processes.

Role of P2Y12 Receptor in Thrombosis.

P2Y12 receptor is a 342 amino acid Gi-coupled receptor predominantly expressed on platelets. P2Y12 receptor is physiologically activated by ADP and inhibits adenyl cyclase (AC) to decrease cyclic AMP (cAMP) level, resulting in platelet aggregation. It also activates PI3 kinase (PI3K) pathway leading to fibrinogen receptor activation, and may protect platelets from apoptosis. Abnormalities of P2Y12 receptor include congenital deficiencies or high activity in diseases like diabetes mellitus (DM) and chronic kidney disease (CKD), exposing such patients to a prothrombotic condition. A series of clinical antiplatelet drugs, such as clopidogrel and ticagrelor, are designed as indirect or direct antagonists of P2Y12 receptor to reduce incidence of thrombosis mainly for patients of acute coronary syndrome (ACS) who are at high risk of thrombotic events. Studies on novel dual-/multi-target antiplatelet agents consider P2Y12 receptor as a promising part in combined targets. However, the clinical practical phenomena, such as "clopidogrel resistance" due to gene variations of cytochrome P450 or P2Y12 receptor constitutive activation, call for better antiplatelet agents. Researches also showed inverse agonist of P2Y12 receptor could play a better role over neutral antagonists. Personalized antiplatelet therapy is the most ideal destination for antiplatelet therapy in ACS patients with or without other underlying diseases like DM or CKD, however, there is still a long way to go.

Opposing needling promotes behavior recovery and exerts neuroprotection via the cAMP/PKA/CREB signal transduction pathway in transient MCAO rats.

The aim of the present study was to investigate whether the cyclic adenosine 3',5'­monophosphate (cAMP)/protein kinase A(PKA)/cAMP­responsive element binding protein (CREB) signal transduction pathway triggered by γ­aminobutyric acid class B (GABA(B)) receptor activation is involved in neuroprotection against ischemia and behavioral recovery induced by opposing needling (ON). A total of 80 rats were randomly divided into four groups: A sham operation group, an ischemia group, an ON group and an ON group effectively inhibited by the GABA(B) receptor antagonist, CGP35384 (n=20/group). The behavior of the rats was assessed by their neurological deficit score, whereas the impairment of gait was examined using the CatWalk system. The volume of cerebral infarction was examined upon treatment with 2,3,5­triphenyltetrazolium chloride. The expression levels of CREB, GABA(B1) and GABA(B2) were examined by western blotting and reverse transcription­quantitative polymerase chain reaction, and the activity of adenylyl cyclase (AC), cAMP and PKA in the serum was detected using an enzyme­linked immunosorbent assay. In the present study, in comparison with other groups, the ON group exhibited a reduced score for the neurological deficit, the stride length and swing speed were improved, and the volume of infarction was reduced. However, these effects were reversed upon administration of CGP35384. Additionally, the expression levels of CREB, GABA(B1) and GABA(B2) were increased in the ON group. The levels of AC, cAMP and PKA in the serum were also increased in the ON group, whereas the addition of CGP35384 reversed these effects. The results of the present study demonstrated that ON markedly protected the brain against transient cerebral ischemic injury, and this effect was possibly mediated by the activation of the GABAB/cAMP/PKA/CREB signal transduction pathway. These findings implied that ON may be a potential therapeutic method for treating stroke.

Blood transcriptomic biomarkers in adult primary care patients with major depressive disorder undergoing cognitive behavioral therapy.

An objective, laboratory-based diagnostic tool could increase the diagnostic accuracy of major depressive disorders (MDDs), identify factors that characterize patients and promote individualized therapy. The goal of this study was to assess a blood-based biomarker panel, which showed promise in adolescents with MDD, in adult primary care patients with MDD and age-, gender- and race-matched nondepressed (ND) controls. Patients with MDD received cognitive behavioral therapy (CBT) and clinical assessment using self-reported depression with the Patient Health Questionnaire-9 (PHQ-9). The measures, including blood RNA collection, were obtained before and after 18 weeks of CBT. Blood transcript levels of nine markers of ADCY3, DGKA, FAM46A, IGSF4A/CADM1, KIAA1539, MARCKS, PSME1, RAPH1 and TLR7, differed significantly between participants with MDD (N=32) and ND controls (N=32) at baseline (q< 0.05). Abundance of the DGKA, KIAA1539 and RAPH1 transcripts remained significantly different between subjects with MDD and ND controls even after post-CBT remission (defined as PHQ-9 <5). The ROC area under the curve for these transcripts demonstrated high discriminative ability between MDD and ND participants, regardless of their current clinical status. Before CBT, significant co-expression network of specific transcripts existed in MDD subjects who subsequently remitted in response to CBT, but not in those who remained depressed. Thus, blood levels of different transcript panels may identify the depressed from the nondepressed among primary care patients, during a depressive episode or in remission, or follow and predict response to CBT in depressed individuals.

Glyoxylate lowers metabolic ATP in human platelets without altering adenylate energy charge or aggregation.

Human blood platelets adhere to exposed collagen at the site of vascular injury, initiating a signaling cascade leading to fibrinogen activation, secretion of granules and aggregation, thus producing a stable thrombus. All these steps require metabolic ATP. In this study we have labeled the metabolic pool of ATP with nucleotides, treated platelets with various inhibitors and have monitored their ability to be activated. Incubating platelets with glyoxylate dramatically reduced the ATP level without a change in the adenylate energy charge (AEC). This reduction of ATP did not affect ADP-induced primary or secondary aggregation, whereas glyoxal, methyl glyoxal, or the combination of antimycin plus deoxyglucose reduced both ATP and AEC and inhibited aggregation. The reduction of ATP by glyoxylate was almost quantitatively matched by an increase in hypoxanthine without elevation of ADP. AMP, IMP or inosine, acetoacetate, aspartate, or glutamate had no effect on glyoxylate-induced breakdown of ATP, while pyruvate stopped the ATP reduction fast and efficiently. Glyoxylate also lowered the citrate content. The glyoxylate-induced breakdown of ATP coincided with an increase in fructose-1,6-bisphosphate, indicating that the phosphofructokinase reaction was the main ATP-consuming step. Glyoxylate was a substrate for lactate dehydrogenase although with a Km almost 100 times higher than pyruvate. We suggest that glyoxylate primarily competes with pyruvate in the pyruvate dehydrogenase reaction, thus lowering the citrate concentration, which in turn activates phosphofructokinase. Clearly, lowering of ATP in the cytosol by more than 50% does not affect platelet aggregation provided that the AEC is not reduced.

Platelet adenylyl cyclase activity: a biological marker for major depression and recent drug use.

BACKGROUND: Adenylyl cyclase (AC) is an enzyme that can regulate the physiologic effects of numerous drugs and hormones through the production of cyclic adenosine-3',5'-monophosphate (cAMP). Some studies suggest that certain measures of AC activity are lower among depressed subjects. We examined the relationship between various measures of AC activity and major depression, taking into account potential confounders, such as drug use and gender. METHODS: We assessed the relationship between platelet levels of AC activity and lifetime diagnosis of major depression among 1481 participants (226 subjects with a history of major depression and 1255 control subjects) in an international, cross-sectional study initiated by the World Health Organization and the International Society on Biomedical Research on Alcoholism. RESULTS: After accounting for recent drug use, subjects with a history of major depression had markedly lower mean levels for all measures of platelet AC activity compared with control subjects. The adjusted odds ratios for major depression comparing the bottom to the top quartile of AC activity were 2.69 for basal (95% confidence interval [CI] 1.30-5.56), 3.72 for cesium fluoride-stimulated (95% CI 1.54-8.98), 6.20 for forskolin-stimulated (95% CI 2.04-18.80), and 2.20 for Gpp(NH)p-stimulated (95% CI 1.03-4.70). CONCLUSIONS: Subjects with major depression have lower platelet AC activity levels, and this relationship is dramatically attenuated by various types of drug use.

Platelet aggregation and adenosine diphosphate/adenosine triphosphate receptors: a historical perspective.

The work of many investigators since adenosine diphosphate (ADP) was recognized as a platelet aggregating agent in 1961 has led to an appreciation of the important part that ADP plays in hemostasis and thrombosis. Recently, interest has focused on the platelet receptors for ADP and adenosine triphosphate (ATP). Platelets are unique because they have two P2Y receptors that must act in concert to achieve a normal aggregation response. The P2Y (1) receptor is responsible for mobilizing internal calcium, platelet shape change, and weak aggregation. The P2Y (12) receptor inhibits adenylyl cyclase, but the concentration of cyclic AMP is reduced only if it has been raised from its low basal levels by stimulation of adenylyl cyclase by an aggregation inhibitor such as adenosine or prostaglandin I (2). The abnormal bleeding of the rare patients whose platelets lack P2Y (12) and the beneficial clinical effects of ticlopidine and clopidogrel that block this receptor indicate that P2Y (12), in addition to inhibiting adenylyl cyclase, may have an as yet unidentified role that is needed for its cooperative aggregation effect with P2Y (1). ATP stimulates a rapid influx of calcium into platelets through the P2X (1) receptor, and it may synergize with ADP when these two nucleotides are released from platelets at a site of vessel injury.

The P2 receptors in platelet function.

After vessel wall injury, platelets adhere to the exposed subendothelium, are activated, and release mediators such as thromboxane A (2) (TXA (2)) and nucleotides stored at very high concentration in the so-called dense granules. Among other soluble agents, released nucleotides act in a positive feedback mechanism to cause further platelet activation and amplify platelet responses induced by agents such as thrombin or collagen. Adenine nucleotides act on platelets through three distinct P2 receptors: two are G protein-coupled adenosine diphosphate (ADP) receptors, namely the P2Y (1) and P2Y (12) receptor subtypes; the P2X (1) receptor ligand-gated cation channel is activated by adenosine triphosphate (ATP). The P2Y (1) receptor initiates platelet aggregation but is not sufficient for a full platelet aggregation in response to ADP, whereas the P2Y (12) receptor is responsible for completion of the aggregation to ADP. This receptor, the molecular target of the antithrombotic drug clopidogrel, is responsible for most of the potentiating effects of ADP when platelets are stimulated by agents such as thrombin, collagen, or immune complexes. The P2X (1) receptor is involved in platelet shape change and in activation by collagen under shear conditions. Each of these receptors is coupled to specific signal transduction pathways in response to ADP or ATP and is differentially involved in all of the sequential events involved in platelet function and hemostasis. As such, they represent potential targets for antithrombotic drugs.

A2B adenosine receptor activity is reduced in neutrophils from patients with systemic sclerosis.

We conducted the present study to investigate protein expression and functioning of A2A and A2B adenosine receptors (ARs) in neutrophils of patients affected by systemic sclerosis (SSc). The presence of A2A and A2B ARs was assessed by immunoblotting using specific antibodies. Equilibrium A2A and A2B ARs binding parameters were evaluated by radioligand binding assay. Functional studies were conducted to investigate coupling of the A2B AR to the adenylyl cyclase pathway. This is the first report of the use of Western blot analysis to confirm the presence of A2A and A2B ARs in human neutrophils. No significant changes in A2A AR binding parameters or expression levels were detected between SSc patients and healthy control individuals. A significant decrease (65%) in the maximum density of A2B AR binding sites occurred in SSc neutrophils, whereas no changes in the affinity constant values were found. Moreover, a decrease in A2B AR mediated adenylyl cyclase activity was observed in patients with SSc. Our findings demonstrate the occurrence of selective alterations in A2B AR density and signalling in SSc.

Is cyclic AMP formation desensitized in patients with end-stage renal failure?

1 Cyclic AMP formation has consistently been reported to be desensitized in various tissues including heart of animal models of end-stage renal failure (ESRF). In contrast, reports on desensitization of cAMP formation in ESRF patients remain contradictory. Whether this discrepancy results from a difference between human ESRF and its animal models or from the use of circulating blood cells in the human and various solid tissues in the animal studies, remains unclear. Therefore, we performed three studies with heart and platelets of ESRF patients undergoing haemodialysis or continuous ambulatory peritoneal dialysis and age- and gender-matched controls with normal renal function (n = 11-13 each). 2 In platelets from haemodialysis patients adenylyl cyclase activity in response to receptor-dependent and -independent agonists was reduced by approximately 30%, and this could be explained by an alteration at the level of adenylyl cyclase itself. However, no such desensitization was seen in platelets from peritoneal dialysis patients. 3 In hearts from ESRF patients undergoing haemodialysis, beta-adrenoceptor density and subtype distribution, cAMP formation in response to the beta-adrenoceptor agonist isoprenaline or various receptor-independent stimuli, were very similar to those in control patients but activity of G-protein-coupled receptor kinase was increased by approximately 20%. 4 We conclude that conflicting reports on the desensitization of cAMP formation between ESRF patients and ESRF animal models are not explained by the use of solid tissues in animal studies vs. circulating blood cells in patient studies. Rather desensitization of cAMP formation seems to be a less consistent feature of human ESRF than of its animal models.

[Role of adenylate cyclase system in pathogenesis of various types of occupational bronchial asthma]. / Rol' adenilattsiklaznoi sistemy v patogeneze razlichnykh form professional'noi bronkhial'noi astmy.

Occupational bronchial asthma with its prevalence amounting to 14% is one of the main entities in occupational morbidity structure. Clinical evidence in recent decades demonstrates changed phenotype of occupational bronchial asthma. Changes are increased number of patients suffering from the severe asthma, higher occurrence of occupational bronchial asthma which pathogenesis is more significantly mediated by nonimmune mechanisms. Prevalence of these types of occupational bronchial asthma approaches 9.7-22%.

Phosphorylation of the vasodilator-stimulated phosphoprotein (VASP) by the anti-platelet drug, cilostazol, in platelets.

Vasodilator-stimulated phosphoprotein (VASP) is a regulator of actin dynamics in platelets and a common substrate of both cAMP- and cGMP-dependent protein kinases (PKA and PKG). Elevations of the cAMP and cGMP concentration have been shown to inhibit platelet aggregation. Intracellular levels of cAMP and cGMP are regulated by the synthesizing system of adenylate cyclases, and hydrolysis by cyclic nucleotide phosphodiesterases (PDEs). The present study examined the effect of the anti-platelet drug, cilostazol, which inhibits PDE3 activity, on VASP phosphorylation in platelets. VASP phosphorylation was examined by immunoblotting with an anti-VASP antibody, M4, and an anti-phospho-VASP antibody, 16C2. Cilostazol phosphorylated VASP at both Ser157 and Ser239 in a concentration-dependent manner, but EHNA (PDE2 inhibitor), dipyridamole and zaprinast (PDE5 inhibitors) did not. Forskolin (adenylate cyclase activator) and sodium nitroprusside (SNP, NO donor) resulted in the VASP phosphorylation, with increase in the cAMP and cGMP level, respectively. Cilostazol increased cAMP, but not cGMP levels, in platelets. EHNA, zaprinast and dipyridamole, had no effect on cAMP and cGMP levels. The PKA/PKG inhibitor, H-89, inhibited VASP phosphorylation by cilostazol. These results demonstrated that cilostazol phosphorylates VASP through the PDE3 inhibition, increase of cAMP level, and PKA activation in platelets.

[Evaluation of cell membrane permeability for Ca2+ and adenylate cyclase in sheep peripheral blood cells, exposed to low doses of radiation]. / Otsenka pronitsaemosti plazmaticheskoi membrany dlia Ca2+ i aktivnosti adenilattsiklazy v kletkakh perifericheskoi krovi ovets, obluchennykh v malykh dozakh.

Chronic irradiation of sheep with doses of 2.6 and 12.9 mC.kg was characterized by the modification of the adenylatecyclase activity and Ca2+ permeability of plasma membrane in cells of the peripheric blood, with no changes in the clinical and hematological indicators. The observed effects are assumed to result from structural and dynamic variations in the lipids of membranes.

Esmolol improves left ventricular function via enhanced beta-adrenergic receptor signaling in a canine model of coronary revascularization.

BACKGROUND: Recent American Heart Association guidelines highlight the paucity of data on effectiveness and/or mechanisms underlying use of beta-adrenergic receptor (beta AR) antagonists after acute coronary syndromes in patients subsequently undergoing revascularization. It is important to assess whether beta AR antagonists might protect the heart and improve ventricular function in this scenario. The authors therefore used esmolol (an ultra-short-acting beta AR antagonist) to determine whether beta AR antagonist treatment improves left ventricular function in a canine model of acute reversible coronary ischemia followed by coronary reperfusion during cardiopulmonary bypass (CPB). The authors also tested whether the mechanism includes preserved beta AR signaling. METHODS: Dogs were randomized to either esmolol or saline infusions administered during CPB (n = 29). Pre-CPB and end-CPB transmyocardial left ventricular biopsies were obtained; plasma catecholamine concentrations, myocardial beta AR density, and adenylyl cyclase activity were measured. In addition, left ventricular systolic shortening and postsystolic shortening were determined immediately prior to each biopsy. RESULTS: While beta AR density remained unchanged in each group, isoproterenol-stimulated adenylyl cyclase activity decreased 26 +/- 6% in the control group but increased 38 +/- 10% in the esmolol group (pre-CPB to end-CPB, mean +/- SD, P = 0.0001). Left ventricular systolic shortening improved in both groups after release of coronary (LAD) ligature; however, the esmolol group increased to 72 +/- 23% of pre-CPB values compared to 48 +/- 12% for the control group (P = 0.0008). CONCLUSIONS: These data provide prospective evidence that esmolol administration results in improved myocardial function. Furthermore, the mechanism appears to involve enhanced myocardial beta AR signaling.

Impaired activation of murine platelets lacking G alpha(i2).

The intracellular signaling pathways by which G protein-coupled receptors on the platelet surface initiate aggregation, a critical process for hemostasis and thrombosis, are not well understood. In particular, the contribution of the G(i) pathway has not been directly addressed. We have investigated the activation of platelets from mice in which the gene for the predominant platelet G alpha(i) subtype, G alpha(i2), has been disrupted. In intact platelets from G alpha(i2)-deficient mice, the inhibition of adenylyl cyclase by ADP was found to be partially impaired compared with wild-type platelets. Moreover, both ADP-dependent platelet aggregation and the activation of the integrin alpha IIb beta 3 (GPIIb-IIIa) were strongly reduced in platelets from G alpha(i2)-deficient mice. In addition, G alpha(i2)-deficient platelets displayed impaired activation at low thrombin concentrations. This defect was mimicked by blocking the adenylyl cyclase--coupled platelet ADP receptor (P2Y(12)) on wild-type platelets with a selective antagonist. These observations suggest that G alpha(i2) is involved in the inhibition of platelet adenylyl cyclase in vivo and is a critical component of the signaling pathway for integrin activation by ADP, resulting in platelet aggregation. In addition, thrombin-dependent activation of mouse platelets is mediated, at least in part, by secreted ADP acting on the G alpha(i2)-linked ADP receptor.

Platelet adenylyl cyclase activity as a trait marker of alcohol dependence. WHO/ISBRA Collaborative Study Investigators. International Society for Biomedical Research on Alcoholism.

BACKGROUND: There is compelling evidence that genetic factors play a major role in the development of alcohol dependence. Platelet adenylyl cyclase (AC) activity has been proposed as a biochemical marker for differentiating alcohol-dependent and nondependent subjects, but the sensitivity and specificity of this marker have not been ascertained. The objective of this study was to determine the sensitivity and specificity of platelet AC activity in identifying alcohol-dependent subjects and to ascertain the effect of medical/ psychiatric variables, drinking and smoking history, and age and body weight on AC activity. METHODS: The cross-sectional study was conducted from 1995 to 1998. Participants were 210 Australian White men who were community volunteers and alcohol treatment inpatients in Sydney, Australia. There were 41 nondrinkers, 140 drinkers, and 29 men who were entering alcohol treatment. The main outcome measure was platelet AC activity. Classification variables were plasma ethanol, gamma-glutamyltransferase, aspartate aminotransferase, serum carbohydrate-deficient transferrin (CDT), and urinary 5-hydroxytryptophol/5-hydroxyindoleacetic acid (5-HTOL/5-HIAA) levels, and World Health Organization/International Society for Biomedical Research on Alcoholism Interview Schedule variables, which included alcohol use and dependence criteria. RESULTS: Among subjects who reported abstinence for at least 4 days, both cesium fluoride (CsF)- and forskolin-stimulated platelet AC activities were significantly lower in those with a lifetime history of alcohol dependence compared with those with no such history (p < 0.005 and p < 0.05, respectively). The sensitivity and specificity of CsF-stimulated AC activity to discriminate individuals with a lifetime history of alcohol dependence were 75% and 79%, respectively. Similar values for sensitivity and specificity for CsF-stimulated AC activity were calculated when discriminating current alcohol dependence in the subjects in our sample. Irrespective of the history of alcohol dependence, persons who had consumed alcohol recently (within the last 3-4 days) showed significantly higher mean basal, CsF-stimulated, and forskolin-stimulated AC activity (p < 0.001), as did those who had elevated 5-HTOL/5-HIAA ratios or CDT levels, indicative of recent (heavy) drinking. The "normalization" of platelet AC activity to baseline levels after an individual stops drinking may be related to the generation of new platelets during the abstinence period. Conduct disorder and antisocial personality disorder were not associated with low AC activity, but low forskolin-stimulated AC activity was associated with major depression. CONCLUSIONS: We found that CsF- and forskolin-stimulated platelet AC activity discriminates between subjects with and without alcohol dependence in a population of subjects who had not consumed significant quantities of ethanol recently. Recent alcohol consumption is a confounding variable that can alter the measured levels of AC activity. Forskolin-stimulated platelet AC activity also may be influenced by a history of major depression.

Arg60 Leu mutation in the first cytoplasmic loop of the platelet thromboxane A2 receptor is not essential for mediating inhibitory coupling between the receptor and adenylyl cyclase.

Human platelet thromboxane A2 (TXA2) receptor (TXR) has been reported to functionally couple to the inhibitory GTP-binding protein for adenylyl cyclase (Gi). However, it still remains unclear which portions of the TXR structure are critical determinants in that coupling. We have previously reported several patients with platelet dysfunction, whose platelets showed impaired coupling between TXR and phospholipase C caused by an Arg60 to Leu mutation in the first cytoplasmic loop. To investigate whether this portion is essential for mediating inhibitory coupling between TXR and adenylyl cyclase, we analyzed the inhibition by the TXA2 analog of the PGE1 or forskolin-induced platelet cAMP increase in patients' platelets, and found that the inhibition occurred normally. This suggests that Arg60 in the first cytoplasmic loop of the TXR is not involved in TXR-Gi coupling.