Identification and characterization of α1- and β2-adrenergic receptors in human liver

Adrenergic receptors were identified in healthy human hepatic tissue from thirty-nine subjects undergoing elective abdominal surgery by using the specific alpha 1-antagonist [3H]-prazosin and the beta adrenergic antagonist [3H]-dihydroalprenolol ([3H]-DHA). [3H]-prazosin binding to plasma membranes was rapid, of high affinity, saturable and stereospecific with a maximal binding capacity (Bmax) of 74.1 +/- 5.5 fmol mg-1 of protein. The displacement curve for (-)-norepinephrine was better explained by a one-site binding and after addition of GTP 0.1 mM the curve was not right-shifted, suggesting the majority of alpha receptors in healthy human liver are of the alpha 1 subtype and not linked to a GTP-binding protein. [3H]-DHA binding to liver plasma membranes was also rapid, of high affinity, saturable and stereospecific with a Bmax 96.5 +/- 10.3 fmol mg-1 of protein of receptors. Computer aided analysis of the displacement curve of ICI 118,551, a subtype selective beta 2-antagonist (IC50 = 62 +/- 2 nM), indicated a one-site binding, thus, showing that beta adrenergic receptors are of the beta 2 subtype. The displacement curve of [3H]-DHA for (-)-isoproterenol was right shifted by GTP indicating that beta 2 adrenergic receptors are linked to a GTP-binding protein in human liver. These results indicate that alpha 1- and beta 2-receptors co-exist in human liver but only beta 2-receptors are linked to a GTP-binding protein.     

Increased α2- but similar β-adrenergic receptor activities in subcutaneous gluteal adipocytes from females compared with males

alpha 2 and beta-adrenergic binding and action were studied in subcutaneous adipocytes from the gluteal region in females and males. The beta-selective antagonist [3H]dihydroalprenolol and the alpha 2-selective antagonist [3H]yohimbine were used to identify the beta- and the alpha 2-receptor, respectively. The biological effects mediated by these receptors were determined by measuring the glycerol release induced by isoproterenol (beta-receptor agonist) and by clonidine (alpha 2-receptor agonist). The study consisted of health volunteers (eighteen females and fifteen males). Compared to men the alpha 2-receptor binding was increased by 73% (P less than 0.01) in adipocytes from females. From Scatchard analysis it was determined that the increased binding was due to an increased receptor number (438 v. 262 fmol mg-1 protein, P less than 0.001) with unaltered Kd (1.18 v. 1.35 nmol l-1, P greater than 0.05). This increased alpha 2-receptor binding in female and adipocytes was associated with a significantly increased sensitivity (P less than 0.05) and maximal antilipolytic effect of clonidine (P less than 0.05). The beta-receptor binding was similar in adipocytes from females and males. However, isoproterenol was significantly more lipolytic in female adipocytes (P less than 0.01). Since the combination of theophylline and adenosine deaminase also was significantly more lipolytic in female adipocytes the enhanced effect of isoproterenol then seemed to be due to mechanisms not directly related to the hormone-beta-receptor binding. Finally, the mixed beta- and alpha 2-receptor agonist, adrenaline showed biphasic effects on lipolysis, with stimulatory effect at low concentrations (less than 500 nmol l-1) and pronounced inhibitory effect at higher concentrations (greater than 1 mumol l-1).(ABSTRACT TRUNCATED AT 250 WORDS)     

Peripheral α2-adrenoceptor-mediated sympathoinhibitory effects of mivazerol

Summary— Mivazerol is a new compound that could potentially reduce perioperative cardiovascular morbidity and mortality in patients with or at risk of coronary disease and submitted to surgery. This action of mivazerol depends on a well documented centrally mediated reduction in sympathetic nerve activity, but a direct peripheral decrease in sympathetic neurotransmitter release induced by activation of prejunctional α₂-adrenoceptors located on sympathetic nerve endings could also contribute. To investigate this issue, the effects of mivazerol on the pressor, systemic and regional hemodynamic (pulsed Doppler technique) as well as on the cardiac responses to electrical stimulation of the spinal cord (SCS) were measured in pithed rats in the absence and in the presence of mivazerol. Mivazerol exerted strong sympathoinhibitory effects: SCS-induced increases in blood pressure, total peripheral resistance and heart rate were dose-dependently reduced by mivazerol, but among the regional vascular beds investigated, only the hindlimb vasoconstrictor responses were significantly drug-affected. All these sympathoinhibitory effects of mivazerol were abolished by prior yohimbine administration. Simultaneously, mivazerol did not induce any postjunctional adrenoceptor blockade as it did not affect noradrenaline cardiac and hemodynamic effects. On the contrary, through postjunctional α₂-adrenoceptor stimulation, mivazerol, in this pithed preparation, dose-dependently increased blood pressure, total peripheral and hindlimb vascular resistances, but heart rate was not affected. We conclude that, in the pithed rat, mivazerol exerts strong peripheral sympathoinhibitory effects. The mechanism involved is prejunctional α₂-adrenoceptor activation as i) mivazerol does not display any postsynaptic α-adrenoceptor blocking effect — it even behaves as a postsynaptic α₂-adrenoceptor agonist — and ii) yohimbine abolishes mivazerol's sympathoinhibitory effects. Thus, direct peripheral together with central mechanisms contribute to mivazerol's sympathoinhibitory effects and ultimately to its cardioprotective action.     

Changes in plasma glucose and lactate evoked by α and β2-adrenoceptor stimulation in conscious fasted rabbits

In conscious fasted rabbits, the iv infusion of salbutamol (3 micrograms/kg per min) and clonidine (2 micrograms/kg per min) induced a blood glucose increase amenable to blockade, respectively by ICI 118551 (1 micrograms/kg per min) and idazoxan (20 micrograms/kg per min). Amidephrine (10 micrograms/kg per min) and salbutamol mediated an increase in plasma lactate which was attenuated by prazosin (50 micrograms/kg, sc) and ICI 118551 respectively. Clonidine did not alter basal plasma lactate. The iv infusion of adrenaline (0.3 micrograms/kg per min) evoked an increase in plasma lactate more sensitive to blockade by ICI 118551 than by prazosin. ICI 118551 also shortened the hyperglycaemic response to adrenaline, 3-Mercaptopicolinic acid (25 mg/kg) reduced salbutamol- and adrenaline-mediated hyperglycaemia and increased at the same time the lactate/glucose ratio. Our data show that plasma lactate levels may be regulated by alpha 1- and beta 2-excitatory adrenoceptor stimulation. However, only the increase in blood lactate derived from beta 2-adrenergic stimulation seems to contribute to the overall catecholamine-mediated hyperglycaemia.     

The regulation of integrin-linked kinase in human platelets: evidence for involvement in the regulation of integrin α2β1

BACKGROUND: Activation of the platelet integrin alpha 2 beta 1 is closely regulated due to the high thrombogenicity of its ligand. As a beta 1 interacting kinase, ILK represents a candidate intracellular regulator of alpha 2 beta 1 in human platelets. OBJECTIVES: We investigated the regulation of ILK in human platelets and the role of ILK in regulating alpha 2 beta 1 activation in HEL cells, a megakaryocytic cell line. METHODS: An in-vitro kinase assay was used to determine the effect of platelet agonists on ILK kinase activity together with the contribution of PI3K and PKC on ILK activation. Interaction of ILK with beta 1-integrin subunits was investigated by coimmunoprecipitation and the role of ILK in regulating alpha 2 beta 1 function assessed by overexpression studies in HEL cells. RESULTS: We report that collagen and thrombin modulate ILK kinase activity in human platelets in an aggregation-independent manner. Furthermore, ILK activity is dually regulated by PI3K and PKC in thrombin-stimulated platelets and regulated by PI3K in collagen-stimulated cells. ILK associates with the beta 1-integrin subunits immunoprecipitated from platelet cell lysates, an association which increased upon collagen stimulation. Overexpression of ILK in HEL cells enhanced alpha 2 beta 1-mediated adhesion whereas overexpression of kinase-dead ILK reduced adhesion, indicating a role for this kinase in the positive regulation of alpha 2 beta 1. CONCLUSIONS: Our findings that ILK regulates alpha 2 beta 1 in HEL cells, is activated in platelets and associates with beta 1-integrins, raise the possibility that it may play a key role in adhesion events upon agonist stimulation of platelets.     

α2-Antagonist compounds and lipid mobilization: evidence for a lipid mobilizing effect of oral yohimbine in healthy male volunteers

Investigations were carried out to analyse the interactions of alpha 2-antagonists (yohimbine, idazoxan, SK & F-86,466) with human fat cell alpha 2-adrenoceptors. All the alpha 2-antagonists enhanced the lipolytic potencies of epinephrine with an order of potency: yohimbine greater than idazoxan greater than SK & F-86,466; the same order was also found in 3H-yohimbine competition studies on human fat cell membranes. The most potent agent, yohimbine, was administered orally in humans to define the conditions of appearance and the time-course of a putative lipid-mobilizing action. Oral yohimbine administration (0.2 mg kg-1) elevated plasma glycerol and non-esterified fatty acids in fasting healthy subjects without significant action on heart rate or blood pressure during the time-course of the experiment. The lipid-mobilizing action of yohimbine was reinforced during physical exercise, completely suppressed after a meal and partially blocked by administration of propranolol (0.5 mg kg-1; 60 min before yohimbine). Plasma norepinephrine concentrations were increased (40-50%) after oral yohimbine administration. The rise in plasma catecholamine concentration elicited by yohimbine was not modified by propranolol treatment. The lipid-mobilizing effect of yohimbine could be attributable to: (i) the increase in synaptic norepinephrine with a resultant increment in lipolysis by beta-adrenergic agonism; (ii) a decrease in alpha 2-adrenoceptor stimulation of human fat cell alpha 2-adrenoceptors; (iii) a blockade of presynaptic alpha 2-adrenoceptors. The use of highly selective alpha 2-antagonists will allow investigations into alpha 2-adrenoceptors, which may represent a novel locus for pharmacological intervention in lipid-mobilization strategies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Platelet integrin αIIbβ3: activation mechanisms

Integrin alpha(IIb)beta(3) plays a critical role in platelet aggregation, a central response in hemostasis and thrombosis. This function of alpha(IIb)beta(3) depends upon a transition from a resting to an activated state such that it acquires the capacity to bind soluble ligands. Diverse platelet agonists alter the cytoplasmic domain of alpha(IIb)beta(3) and initiate a conformational change that traverses the transmembrane region and ultimately triggers rearrangements in the extracellular domain to permit ligand binding. The membrane-proximal regions of alpha(IIb) and beta(3) cytoplasmic tails, together with the transmembrane segments of the subunits, contact each other to form a complex which restrains the integrin in the resting state. It is unclasping of this complex that induces integrin activation. This clasping/unclasping process is influenced by multiple cytoplasmic tail binding partners. Among them, talin appears to be a critical trigger of alpha(IIb)beta(3) activation, but other binding partners, which function as activators or suppressors, are likely to act as co-regulators of integrin activation.     

PLASMA CATECHOLAMINES AND ADRENOCEPTORS AFTER CHRONIC SINOAORTIC DENERVATION IN DOGS

Chronic sinoaortic denervation (SAD) performed by section of both carotid and aortic nerves induced a significant and sustained increase in blood pressure and heart rate in conscious dogs; under our experimental conditions, the values of systolic blood pressure and heart rate were never lower than 190 mmHg and 120 beats/min, respectively. The present long-term study (8 mo) investigated the time-course of plasma catecholamine levels and circulating blood cell adrenoceptor (leukocyte beta 2 and platelet alpha 2) number. Catecholamine plasma levels were highly correlated with the variations of leukocyte beta adrenoceptors (measured by [125I]cyanopindolol binding and characterized as a beta 2 receptor in dogs). These two parameters followed a biphasic pattern after SAD: during the first 2 mo, significant increases in noradrenaline and adrenaline levels were associated with a decrease in leukocyte beta 2 adrenoceptor number; from the 4th month, they slowly returned to normal values, although blood pressure remained elevated. By contrast, platelet alpha 2 adrenoceptor number (measured by [3H]yohimbine binding) did not follow this pattern since it remained lower than before SAD during the whole experiment. Finally, the development (but not the maintenance) of SAD-induced hypertension is associated with an increase in sympathetic tone. Only leukocyte beta 2 (and not platelet alpha 2) adrenoceptors are directly regulated by the endogenous levels of catecholamines.     

A mechanism to safeguard platelet adhesion under high shear flow: von Willebrand factor–glycoprotein Ib and integrin α2β1–collagen interactions make complementary, collagen-type-specific contributions to adhesion

BACKGROUND: Blood vessels contain different types of collagen, with types I and III being the major components of vascular collagen. Platelet adhesion under high shear stress has been suggested to depend on the binding of von Willebrand factor (VWF) to collagen. OBJECTIVE: We analyzed the collagen type specificity for the interaction with VWF and high shear stress platelet adhesion. METHODS: VWF binding to different types of immobilized collagen and effects of antibodies against glycoprotein Ib (gpIb) and integrin alpha(2)beta(1) on platelet adhesion to type I and III collagens under high shear were analyzed. RESULTS: VWF showed high-affinity, selective binding to human and bovine type III collagens, but weak or no affinity for types I, II, IV and V under static conditions. Anti-integrin alpha(2)beta(1) markedly inhibited adhesion to type I collagen, but did not affect that to type III collagen. Anti-gpIb antibody significantly inhibited adhesion to type III collagen. Adding both antibodies abrogated the adhesion to either type I or III collagen. CONCLUSIONS: Both the gpIb-VWF interaction and the integrin alpha(2)beta(1)-collagen interaction contribute to platelet adhesion to collagen under high shear stress, and integrin alpha(II)beta(1) makes a greater contribution to adhesion to type I collagen because less VWF is bound to it.     

NORADRENALINE RELEASE IN HUMAN CORPUS CAVERNOSUM AND ITS MODULATION VIA PRESYNAPTIC α2-ADRENOCEPTORS

Strips of human corpus cavernosum were incubated with 3H-noradrenaline and subsequently superfused with physiological salt solution containing desipramine and corticosterone. The electrically (0.66 Hz) evoked tritium overflow was abolished by tetrodotoxin or omission of Ca2+ from the superfusion fluid. The alpha 2-adrenoceptor agonist B-HT 920 (6-allyl-2-amino-5, 6, 7, 8-4H-thiazolo-5,4-d-azepine), inhibited the evoked overflow, whereas the alpha 1-adrenoceptor agonist, methoxamine, was ineffective. The alpha 2-adrenoceptor antagonist, rauwolscine, facilitated the evoked tritium overflow. It is concluded that the stimulation-evoked release of noradrenaline from the sympathetic nerve fibres of the human corpus cavernosum is modulated via presynaptic alpha 2-adrenoceptors.     

Effects of high-altitude chronic hypoxia on platelet α2-receptors in man

During chronic high-altitude (HA) exposure, basal and exercise-induced noradrenaline (NA) increases do not parallel blood pressure (BP) changes observed; unlike β-adrenergic receptors, to our knowledge no data are available on α-receptors. We studied platelet α₂- and leucocyte β-receptors and basal catecholamine levels in 11 trained climbers before and after they had spent a 15-day period at a height of over 4400 m. In six of the climbers we also evaluated catecholamines after maximal bicycle ergometer exercise. After chronic high-altitude exposure, a significant decrease was found in platelet α₂-receptor density and affinity [ B _max from 92.6 ± 6.7 to 54.6 ± 4.2 fmol mg⁻¹ protein ( P  < 0.001) and K _D from 1.271 ± 0.034 to 1.724 ± 0.077 nmol L⁻¹ ( P  < 0.05)], although no changes to β-receptors were observed. No changes were found in basal pre- and post-expedition NA and adrenaline (A), and there was only a slight decrease in post-expedition NA after maximal exercise. Our results suggest that prolonged exposure to hypoxia induces a down-regulation of α₂-receptors, which may be a contributory factor in the regulation of the physiological vascular response to acclimatization.     

α1-adrenoceptor subtypes mediating vasoconstriction in the carotid circulation of anaesthetized pigs: possible avenues for antimigraine drug development

It has recently been shown that the alpha-adrenoceptors mediating vasoconstriction of porcine carotid arteriovenous anastomoses resemble both alpha1- and alpha2-adrenoceptors, but no attempt was made to identify the specific subtypes (alpha1A, alpha1B and alpha1D) involved. Therefore, the present study was designed to elucidate the specific subtype(s) of alpha1-adrenoceptors involved in the above response, using the alpha1-adrenoceptor agonist phenylephrine and alpha1-adrenoceptor antagonists 5-methylurapidil (alpha1A), L-765 314 (alpha1B) and BMY 7378 (alpha1D). Ten-minute intracarotid infusions of phenylephrine (1, 3 and 10 microgkg-1.min-1) induced a dose-dependent decrease in total carotid and arteriovenous anastomotic conductance, accompanied by a small tachycardia. These carotid vascular effects were abolished by L-765 314 (1000 microgkg-1; i.v.), while these responses were only attenuated by 5-methylurapidil (1000 microgkg-1; i.v.), and BMY 7378 (1000 microgkg-1; i.v.). Furthermore, intravenous bolus injections of phenylephrine (3 and 10 microgkg-1) produced a dose-dependent vasopressor response, which was only affected by 1000 microgkg-1 of 5-methylurapidil, while the other antagonists were ineffective. These results, coupled to the binding affinities of the above antagonists at the different alpha1-adrenoceptors, suggest that both alpha1A- and alpha1B-adrenoceptors mediate constriction of carotid arteriovenous anastomoses in anaesthetized pigs. In view of the less ubiquitous nature of alpha1B- compared to alpha1A-adrenoceptors, the development of potent and selective alpha1B-adrenoceptor agonists may prove to be important for the treatment of migraine.     

Association of polymorphisms of platelet membrane integrins αIIbβ3 (HPA-1b/PlA2) and α2β1 (α2807TT) with premature myocardial infarction

Conflicting results of an association of the human platelet antigen 1b (HPA-1b/PlA2), localized on the beta-subunit of the integrin alpha(IIb)beta3, and the alpha(2)807TT genotype of the integrin alpha2beta1 with coronary atherosclerosis and myocardial infarction have been reported. Both platelet receptor polymorphisms were genotyped in 3261 patients who had undergone coronary angiography, including 1175 survivors of a myocardial infarction, 1211 individuals with coronary artery disease but no history of myocardial infarction, and 571 control patients without angiographic coronary artery disease, and in 793 blood donors. In a case-control design, the prevalence of HPA-1b and alpha(2)807TT genotypes did not differ significantly between the patient groups with coronary artery disease or myocardial infarction and patient controls or blood donors. By contrast, using a multivariate case-only design, it was found that the median age of onset of myocardial infarction was 5.2 years earlier (P = 0.006) in carriers of the HPA-1b allele and 6.3 years earlier (P = 0.006) in carriers of the alpha(2)807TT genotype in the 264 survivors of myocardial infarction of recent onset with one- or two-vessel coronary artery disease. A significant interaction with the conventional risk factors hypercholesterolemia, smoking, diabetes, hypertension, and hyperfibrinogenemia was excluded. Human platelet antigen 1b and alpha(2)807TT are associated with premature myocardial infarction but not with coronary artery disease, suggesting a role of distinct integrin genotypes for increased platelet thrombogenicity. This association requires confirmation in follow-up studies.     

Differential activation of β1-, β2- and β3-adrenoceptors by catecholamines in white and brown adipocytes

Summary— This review summarizes the experiments performed by various groups to determine how the activation of the different β-adrenoceptors (β-ARs) is ordinated when they are present in the same fat cell and involved in the same biological event. When expressed after the transfection of their genes in Chinese hamster ovary cell (CHO cells), β₁- and β₂-ARs present a higher affinity for catecholamines than β₃-ARs. In vitro, the lipolytic effect induced by low concentrations of catecholamines in dog and rat white fat cells is due to the selective activation of β₁- and/or β₂-ARs. Higher concentrations only are able to activate β₃-ARs. Similar results have been obtained in rat brown adipocytes. On the other hand, the lipolytic effect of catecholamines in human and primate adipocytes does not involve a β₃-AR component whatever the concentration used. In vivo experiments in the dog have also shown that lipomobilization induced by low doses of isoprenaline only involved β₁- and β₂-AR activation, this effect being blocked by β₁-/ β₂-antagonist pretreatment. However, in the same blockade conditions, perfusion of a 10-fold higher dose of isoprenaline revealed a β₃-AR contribution in the lipomobilizing effect. These data showed that brown and white adipocyte β₃-ARs possess a lower affinity for catecholamines than β₁- and β₂-ARs and are only recruited by high concentrations of the amines. Thus, even if the β₃-AR plays an indisputable role in the white and brown adipose tissue of some species, its physiological status and its expression are still subject to debate in human white fat cells.     

Platelet and brain α2-adrenoceptors and cardiovascular sensitivity to agonists in dogs suffering from endotoxic shock

Summary— We examined the changes in α₂-adrenoceptor binding on platelet and brain membranes of dogs treated with a non-lethal dose of endotoxin (0.1 mg/kg intravenously), and the α₂-adrenoceptor mediated cardiovascular effects during endotoxin shock. At 2 h, 24 h, and 7 days after endotoxin administration, the number of binding sites (B_max) of [³H]yohimbine binding decreased and equilibrium dissociation constants (K_d) increased in platelets, whereas both B_max and K_d decreased in either cerebral cortex or medulla oblongata. After 30 days of endotoxin administration, there were no significant differences in B_max or K_d between the treated and untreated animals in both platelets and brain tissues. Significant positive correlations were observed for B_max values between platelets and brain tissues, although negative correlations for K_d values between platelets and brain were not significant. Significant negative correlations were also observed between plasma catecholamine concentrations and platelet α₂-adrenoceptor number, and between plasma noradrenaline and medulla α₂-adrenoceptor number. Pretreatment with E coli endotoxin diminished cardiovascular effects such as bradycardia, hypotension, and increase in systemic vascular resistance induced by either iv clonidine or xylazine. This suggests that α-adrenoceptor activity is impaired in the central nervous system as well as in the peripheral vascular system during endotoxin shock. Therefore, platelets may in part represent a good model which reflects the α₂-adrenoceptor changes in the central nervous system and peripheral vascular system during and after endotoxin shock.     

Platelet adhesion to dimeric β2-glycoprotein I under conditions of flow is mediated by at least two receptors: glycoprotein Ibα and apolipoprotein E receptor 2''

BACKGROUND: The major antigen implicated in the antiphospholipid syndrome is beta2-glycoprotein I (beta2GPI). Dimerized beta2GPI binds to apolipoprotein E receptor 2' (apoER2') on platelets and increases platelet adhesion to collagen under conditions of flow. AIM: To investigate whether the interaction between dimerized beta2GPI and platelets is sufficiently strong to resist shear stresses. METHODS: We studied the interaction of platelets with immobilized dimerized beta2GPI under conditions of flow, and further analyzed the interaction using surface plasmon resonance and solid phase immunoassays. RESULTS: We found that dimerized beta2GPI supports platelet adhesion and aggregate formation under venous flow conditions. Adhesion of platelets to dimerized beta2GPI was completely inhibited by the addition of soluble forms of both apoER2' and GPIbalpha, and the addition of receptor-associated protein and the removal of GPIbalpha from the platelet surface. GPIbalpha co-precipitated with apoER2', suggesting the presence of complexes between GPIbalpha and apoER2' on platelet membranes. The interaction between GPIbalpha and dimeric beta2GPI was of intermediate affinity (Kd = 180 nM) and Zn2+, but not Ca2+-dependent. Deletion of domain V from dimeric beta2GPI strongly reduced its binding to both GPIbalpha and apoER2'. Antibodies that inhibit the binding of thrombin to GPIbalpha inhibited platelet adhesion to dimeric beta2GPI completely, while antibodies blocking the binding of von Willebrand factor to GPIbalpha had no effect. Dimeric beta2GPI showed reduced binding to low-sulfated GPIbalpha compared to the fully sulfated form. CONCLUSION: We show that platelets adhere to dimeric beta2GPI under both arterial and venous shear stresses. Platelets adhere via two receptors: GPIbalpha and apoER2'. These receptors are present in a complex on the platelet surface.     

Thrombin induces GPIb-IX-mediated fibrin binding to αIIbβ3 in a reconstituted Chinese hamster ovary cell model

BACKGROUND: The interaction of thrombin with platelet glycoprotein (GP) Ib-IX-V has been recently suggested to induce fibrin-dependent platelet aggregation associated with signaling events. The approaches used to avoid the protease-activated receptor (PAR) thrombin receptors in platelets have provided controversial conclusions regarding the precise mechanism and molecules involved in the response. OBJECTIVES: In the present study, we developed a cellular model to investigate the functional consequences following the binding of thrombin to GPIb-IX. METHODS: We used Chinese hamster ovary (CHO) cells stably expressing human alpha(IIb)beta(3) and/or GPIb-IX complexes (CHO-alpha(IIb)beta(3)-IbIX cells) to analyze the effect of thrombin on the binding of polymerizing fibrin by using fluorescein isothiocyanate-fibrinogen as precursor. RESULTS: Thrombin induces, in a dose-dependent manner, the binding of polymerizing fibrin to CHO-alpha(IIb)beta(3)-IbIX cells. This response is not observed in cells expressing only one of the receptors, and it can be blocked by monoclonal antibodies against alpha(IIb)beta(3) and GPIbalpha. We show that the reaction is not due to simple cell trapping by the fibrin clot, and provide data supporting a role of a signaling pathway in which the 14-3-3zeta adaptor and calcium-calmodulin-dependent events are involved. CONCLUSIONS: The present data support a significant role of GPIb-IX and alpha(IIb)beta(3) receptors in an alternative fibrin-mediated pathway of platelet activation induced by thrombin.     

von Willebrand factor mediates platelet spreading through glycoprotein Ib and αIIbβ3 in the presence of botrocetin and ristocetin, respectively

BACKGROUND: von Willebrand factor (VWF) plays a critical role in the process of hemostasis by mediating flow-dependent adhesion and spreading of platelets on exposed extracellular matrix proteins following vascular injury. To accomplish this, VWF binds to two distinct platelet receptors: glycoprotein (GP)Ib-IX-V and integrin alpha(IIb)beta3. OBJECTIVE: To evaluate the ability of GPIb and alpha(IIb)beta3 to mediate platelet adhesion and lamellipodia formation on immobilized VWF in the presence of the biochemical modulators, ristocetin and botrocetin. RESULTS: In the presence of botrocetin and inhibitors of adenosine diphosphate (ADP) and thromboxane A2 (TxA2), VWF is able to support formation of lamellipodia through a GPIb-dependent mechanism that is independent of alpha(IIb)beta3 and PI3-kinase. Lamellipodia formation under these conditions is incomplete. In marked contrast, in the presence of ristocetin, VWF stimulates formation of fully spread lamellipodia through a pathway that is dependent upon alpha(IIb)beta3 and PI3-kinase. Furthermore, alpha(IIb)beta3 also supports platelet spreading on VWF alone, but only in the absence of inhibitors of ADP and TxA2. The localization of filamentous actin and the Arp2/3 complex in platelets on VWF in the presence of botrocetin and ristocetin are distinct, yielding disparate lamellipodium kinetic signatures. Interestingly, botrocetin significantly enhances platelet adhesion to VWF under flow in whole blood in an alpha(IIb)beta3-independent manner, while ristocetin augments washed platelet adhesion and spreading to VWF under flow in an alpha(IIb)beta3-dependent manner. CONCLUSIONS: These observations demonstrate that VWF is able to induce lamellipodia formation through distinct receptors, and has important consequences for investigation of the role of VWF-GPIb interactions in the context of platelet regulation.     

Evidence that epinephrine acts preferentially as an antilipolytic agent in abdominal human subcutaneous fat cells: assessment by analysis of beta and alpha2 adrenoceptors properties

Investigations were carried out to demonstrate the function and the possible advantage of the interplay between beta 1 and alpha 2 adrenoceptor sites in the regulation of human subcutaneous fat-cell lipolysis. alpha 2 and beta adrenoceptor binding studies were conducted with antagonist radioligands and revealed that alpha 2-adrenoceptors ([3H]yohimbine and [3H]rauwolscine binding sites) are more numerous than beta 1-adrenoceptors ([3H]dihydroalprenolol and [3H]CGP-12177 binding sites) in human fat-cell membranes. Physiological agonists epinephrine and norepinephrine competed with [3H]-ligand sites with a higher affinity for alpha 2 sites than for beta 1 sites. Epinephrine exhibited a higher affinity than norepinephrine for the alpha 2 sites; the two amines had the same affinity for beta 1 sites. In lipolysis studies conducted in the absence of adenosine deaminase the beta lipolytic action of the biological amines predominated; after alpha 2-adrenoceptor blockade by yohimbine or idazoxan, the amines exhibited an intrinsic activity similar to that of isoproterenol. When adenosine was prevented from accumulating in the incubation medium by inclusion of adenosine deaminase, low concentrations of epinephrine and norepinephrine preferentially exerted an antilipolytic action. We conclude that: he lipolytic response in abdominal human subcutaneous fat cells to physiological amines results from the interplay between beta 1-and alpha 2-adrenoceptor stimulation; alpha 2 adrenoceptors, with their higher number and higher affinity for the physiological amines, and the adrenoceptor population involved at the lowest (i.e. physiological) concentrations of the amines.(ABSTRACT TRUNCATED AT 250 WORDS)