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At least 1,063 records · Page 59Linked to original sources
PubMed1993

Lactotransferrin binding to its platelet receptor inhibits platelet aggregation.

A fluorescent lactotransferrin probe was prepared by coupling 5-(([2-(carbhydrazino)methyl]-thio)acetyl)amino fluorescein to aldehyde groups that were produced by a mild periodic-acid oxidation of the glycan moieties of lactotransferrin. In this manner, the receptor-binding site of the lactotransferrin remains active in contrast to the binding site of the lactotransferrin derivatized with fluorescein isothiocyanate. The fluorescent probe allowed us to characterize, by flow cytometry, the binding of lactotransferrin to non-activated human platelets. The putative lactotransferrin platelet receptor was purified and its immunological and physico-chemical properties were found to be very similar to those of the receptor previously isolated from activated human lymphocytes. Lactotransferrin inhibits ADP-induced platelet aggregation at concentrations down to 5 nM, which can be reached in the plasma after leukocyte degranulation. Inhibition of platelet aggregation was also observed with the N-terminal fragment of lactotransferrin (residues 3-281; 50% inhibition = 2 microM) and with CFQWQRNMRKVRGPPVSC synthetic octodecapeptide (residues 20-37; 50% inhibition = 20 microM) corresponding to one of the two external loops (residues 28-34 and 39-42) where we recently located the receptor-binding site. The activity (50% inhibition = 500 microM) of the tetrapeptide KRDS (residues 39-42), which has already been described, was at least 25-times and 16000-times lower than the activity of the octodecapeptide and of the lactotransferrin molecules, respectively. Finally, the inhibition was demonstrated to be mediated by a mechanism which requires the binding of lactotransferrin to its putative receptor and not to platelet glycoprotein IIb-IIIa.

Adenosine Diphosphate↗
PubMed1994

Inhibition of platelet aggregation by adenosine receptor agonists.

2-(Ar)alkoxyadenosines, which are agonists selective for the A2AAR in PC 12 cell and rat striatum membranes, are also agonists at the A2AR coupled to adenylate cyclase (AC) that mediates the inhibition of platelet aggregation. A panel of twelve well-characterized adenosine analogues stimulated human platelet AC and inhibited ADP-induced platelet aggregation at sub- to low-micromolar concentrations with a potency ranking CGS 21680 > adenosine > R-PIA. There were significant correlations between the EC50 of anti-aggregatory activity and either the EC50 of stimulation of platelet and PC 12 cell AC (r2 = 0.66 and 0.67, respectively) or the Ki of inhibition of [3H]NECA binding to the rat striatum membranes (r2 = 0.75). Likewise, platelet AC stimulation correlated well with stimulation of PC 12 cell AC and with [3H]NECA binding (r2 = 0.94 and 0.91, respectively). Ten 2-(ar)alkoxyadenosines stimulated platelet AC at EC50s ranging between 0.16 and 2.3 microM and inhibited platelet aggregation at EC50s ranging between 2 and 30 microM. There were no correlations between the EC50s of anti-aggregatory activity and either the EC50s of the stimulation of platelet or PC 12 AC (r2 = 0.08 and 0.06, respectively) or with the Ki of the inhibition of [3H]NECA binding to the A2aAR in rat striatum (r2 = 0.02). The EC50s of the stimulation of platelet AC correlated with those of the stimulation of PC 12 AC (r2 = 0.48), and also with the Ki of [3H]NECA binding (r2 = 0.71). Each of the 23 adenosines completely inhibited platelet aggregation and thus, functionally, all behaved as full agonists.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine↗
PubMed1990

Evidence for melanin concentrating hormone (MCH) receptors mediating melanosome aggregation in Labeo melanophores.

Melanin concentrating hormone (MCH: 5 x 10(-12)-5 x 10(-8) M) induced a concentration related, rapid and reversible pigment aggregation in innervated melanophores of Labeo rohita. In inducing melanosome aggregation MCH was found to be 10(4) times more potent than norepinephrine. Experiments employing phentolamine and propranolol suggest that MCH acts through its own specific receptors on the melanophores unrelated to adrenoceptors. MCH was able to aggregate the melanosomes even in the absence of extracellular Ca2+.

Animals↗
PubMed2001

Fc-receptor dependent platelet aggregation induced by monoclonal antibodies against platelet glycoprotein Ib or von Willebrand factor.

In this paper we describe two pathways leading to platelet activation by crosslinking glycoprotein (GP) Ibalpha to the platelet Fc-receptor (FcgammaRII). First the monoclonal antibody (MoAb) 9C8, raised against human platelet GPIbalpha, dose-dependently induced platelet aggregation of citrate-anticoagulated platelet-rich plasma, an effect that can be inhibited by several activation inhibitors. The FcgammaRII-inhibitory MoAb IV.3 was able to prevent the aggregatory effects of MoAb 9C8, indicating that crosslinking of the antigen GPIbalpha to the FcgammaII-receptor is necessary for the activating effect. Secondly we observed a synergistic activating effect of two anti-von Willebrand factor (vWF) MoAbs IC1E7 and B724, both known to enhance vWF binding to GPIbalpha in the presence of shear or ristocetin. When these antibodies are added together to PRP, platelet aggregation is induced without further need for an additional modulator. This effect can be blocked by either MoAb IV.3 or an inhibitory anti-GPIb MoAb, indicating that again the platelet activation results from signaling through FcgammaRII crosslinked to vWF bound to GPIbalpha. In addition, both the anti-GPIb MoAb 9C8, or the two anti-vWF MoAbs 1C1E7 and B724 induce genuine platelet activation, as evidenced by the secretion of ATP and protein tyrosine phosphorylation. These findings with both anti-GPIb and anti-vWF MoAbs add further proof to recent reports demonstrating an interaction between the platelet receptors GPIb and FcgammaRII, suggesting a role for the FcgammaII-receptor in GPIb-related signaling.

Adenosine Triphosphate↗
PubMed1995

Aggregation of IgE receptors in rat basophilic leukemia 2H3 cells induces tyrosine phosphorylation of the cytosolic protein-tyrosine phosphatase HePTP.

The cDNA encoding the rat equivalent of the human hematopoietic tyrosine phosphatase, also known as leukocyte phosphatase, was isolated from a rat basophilic leukemia mast cell cDNA library. By two-dimensional electrophoresis, the protein expressed in the mast cells was of a size (40 kDa) and pI (6.9) predicted from the deduced amino acid sequence. Thus, although previously shown to be preferentially expressed in T cells and B cells, the phosphatase is also found in mast cells. By immunofluorescence microscopy, rat hematopoietic tyrosine phosphatase localized to discrete, globular compartments within the cytoplasm and was not found either in the nucleus or associated with the cell surface membrane. Aggregation of high affinity IgE receptors in the mast cells induced tyrosine phosphorylation of the phosphatase. The tyrosine phosphorylation was mimicked by stimulation with calcium ionophore A23187 but not by direct activation of protein kinase C. Since phosphorylation of the phosphatase was dramatically reduced when the cells were activated in Ca(2+)-free media, it is dependent on a rise in intracellular Ca2+. These data strongly suggest that hematopoietic tyrosine phosphatase may be involved in the IgE receptor-mediated signaling cascade.

Amino Acid Sequence↗
PubMed1994

Dynamics of signal transduction after aggregation of cell-surface receptors: studies on the type I receptor for IgE.

Many ligands stimulate cellular responses by aggregating the cell-surface receptors to which they are bound. We investigated several mechanistic questions related to aggregation of receptors by using the high-affinity receptor for IgE (Fc epsilon RI) on mast cells as a model system. We briefly exposed cells to covalently cross-linked oligomers of IgE and then added excess monomeric IgE to prevent further aggregation. Early events were examined by monitoring the phosphorylation of protein tyrosines; later events were examined by monitoring secretion. We found that aggregated receptors continue to signal both late and early events in the absence of formation of new aggregates. Additional experiments suggested that the clustered receptors undergo a dynamic process of phosphorylation and dephosphorylation. Our findings suggest that for these and related receptors that function by aggregation, the persistence of signal transduction is directly related to the intrinsic affinity of the ligand for the individual receptor.

Animals↗
PubMed2005

Regulation of allergy by Fc receptors.

The aggregation of high-affinity IgE receptors (FcepsilonRI) on mast cells and basophils has long been known as the critical event that initiates allergic reactions. Monomeric IgE was recently found to induce a variety of effects when binding to FcepsilonRI. Upregulation of FcepsilonRI only requires binding, whereas other responses require FcepsilonRI aggregation. Interestingly, FcepsilonRI aggregation has recently been understood to generate a mixture of positive and negative intracellular signals. Mast cells and basophils also express low-affinity and, under specific conditions, high-affinity IgG receptors. When co-engaging these receptors with FcepsilonRI, IgG antibodies can amplify or dampen IgE-induced mast cell activation. On the basis of these findings, it has been proposed that FcRs can be used as targets and/or tools for new therapeutic approaches to allergies.

Animals↗
PubMed2005

Dlgh1 coordinates actin polymerization, synaptic T cell receptor and lipid raft aggregation, and effector function in T cells.

Lipid raft membrane compartmentalization and membrane-associated guanylate kinase (MAGUK) family molecular scaffolds function in establishing cell polarity and organizing signal transducers within epithelial cell junctions and neuronal synapses. Here, we elucidate a role for the MAGUK protein, Dlgh1, in polarized T cell synapse assembly and T cell function. We find that Dlgh1 translocates to the immune synapse and lipid rafts in response to T cell receptor (TCR)/CD28 engagement and that LckSH3-mediated interactions with Dlgh1 control its membrane targeting. TCR/CD28 engagement induces the formation of endogenous Lck-Dlgh1-Zap70-Wiskott-Aldrich syndrome protein (WASp) complexes in which Dlgh1 acts to facilitate interactions of Lck with Zap70 and WASp. Using small interfering RNA and overexpression approaches, we show that Dlgh1 promotes antigen-induced actin polymerization, synaptic raft and TCR clustering, nuclear factor of activated T cell activity, and cytokine production. We propose that Dlgh1 coordinates TCR/CD28-induced actin-driven T cell synapse assembly, signal transduction, and effector function. These findings highlight common molecular strategies used to regulate cell polarity, synapse assembly, and transducer organization in diverse cellular systems.

Actins↗
PubMed1995

Testosterone increases human platelet thromboxane A2 receptor density and aggregation responses.

BACKGROUND: The incidence of thrombotic cardiovascular disease is greater in men than in premenopausal women. Testosterone has been implicated as a significant risk factor for cardiovascular disease and for acute myocardial infarctions and strokes in young male athletes who abuse anabolic steroids. Thromboxane A2 (TXA2) is a vasoconstrictor and platelet proaggregatory agent that has been implicated in the pathogenesis of cardiovascular disease. We therefore tested the hypothesis that testosterone regulates the expression of human platelet TXA2 receptors. METHODS AND RESULTS: In a double-blind, placebo-controlled, randomized, parallel-group study, we determined the effects of testosterone cypionate 200 mg IM given twice, 2 weeks apart, or saline placebo in 16 healthy men. Platelet TXA2 receptor density (Bmax) and dissociation constant (Kd) were measured by use of the TXA2 mimetic 125I-BOP. Platelet aggregation responses to I-BOP and to thrombin and plasma testosterone concentrations were measured before treatment (pretreatment phase), at 2 and 4 weeks (active phase), and again at 8 weeks (recovery phase). Treatment with testosterone was associated with an increase in the Bmax value from 0.95 +/- 0.13 to 2.10 +/- 0.4 pmol/mg protein (n = 9), with a peak effect at 4 weeks (P = .001), returning to baseline by 8 weeks. There was no significant change in Bmax values in the saline-treated group. The Kd values were unchanged. Testosterone treatment was associated with a significant increase in the maximum platelet aggregation response to I-BOP (P < .001) at 4 weeks and returned to baseline at 8 weeks. The EC50 values were not significantly changed. Platelet TXA2 receptor density was positively correlated (r = .56, P < .001, n = 32 measurements) with pretreatment (endogenous) plasma testosterone levels (range, 215 to 883 ng/dL) but not Kd. CONCLUSIONS: Testosterone regulates the expression of platelet TXA2 receptors in humans. This may contribute to the thrombogenicity of androgenic steroids.

Adult↗
PubMed2000

Cbl-b is a negative regulator of receptor clustering and raft aggregation in T cells.

Stimulation of T cells via the antigen and costimulatory receptors leads to the organization of a supramolecular activation cluster called the immune synapse. We report that loss of the molecular adaptor Cbl-b in T cells frees antigen receptor-triggered receptor clustering, lipid raft aggregation, and sustained tyrosine phosphorylation from the requirement for CD28 costimulation. Introduction of the cbl-b mutation into a vav1-/- background relieved the functional defects of vav1-/- T cells and caused spontaneous autoimmunity. Wiscott Aldrich Syndrome protein (WASP) was found to be essential for deregulated proliferation and membrane receptor reorganization of cbl-b mutant T cells. Antigen receptor-triggered Ca2+ mobilization, cytokine production, and receptor clustering can be genetically uncoupled in cbl-b mutant T cells. Thus, Cbl-b functions as a negative regulator of receptor clustering and raft aggregation in T cells.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Crystal structure of an NK cell immunoglobulin-like receptor in complex with its class I MHC ligand.

Target cell lysis is regulated by natural killer (NK) cell receptors that recognize class I MHC molecules. Here we report the crystal structure of the human immunoglobulin-like NK cell receptor KIR2DL2 in complex with its class I ligand HLA-Cw3 and peptide. KIR binds in a nearly orthogonal orientation across the alpha1 and alpha2 helices of Cw3 and directly contacts positions 7 and 8 of the peptide. No significant conformational changes in KIR occur on complex formation. The receptor footprint on HLA overlaps with but is distinct from that of the T-cell receptor. Charge complementarity dominates the KIR/HLA interface and mutations that disrupt interface salt bridges substantially diminish binding. Most contacts in the complex are between KIR and conserved HLA-C residues, but a hydrogen bond between Lys 44 of KIR2DL2 and Asn 80 of Cw3 confers the allotype specificity. KIR contact requires position 8 of the peptide to be a residue smaller than valine. A second KIR/HLA interface produced an ordered receptor-ligand aggregation in the crystal which may resemble receptor clustering during immune synapse formation.

Amino Acid Sequence↗
PubMed1983

Inhibition of fibrinogen receptor-mediated platelet aggregation by heterologous anti-human platelet membrane antibody. Significance of an Mr = 66,000 protein derived from glycoprotein IIIa.

Heterologous anti-human platelet membrane antisera were raised in rabbits against membranes prepared from human intact or chymotrypsin- or pronase-treated platelets. Anti-intact, anti-chymotrypsin, and anti-pronase-treated platelet membrane antibodies (IgG and Fab fragments) inhibited the fibrinogen-induced aggregation of ADP-stimulated platelets and of chymotrypsin-treated platelets. The specific binding of 125I-fibrinogen to these platelets was also inhibited. As revealed by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 125I-surface-radiolabeled platelet proteins and by the electrophoresis of the immunoprecipitates prepared from these labeled proteins, predominant components on the surface of intact platelets were glycoproteins IIb and IIIa. These proteins were immunoprecipitated by all three antibodies. In addition, chymotrypsin-treated platelets contained an Mr = 66,000 protein on their surface that was also immunoprecipitated by the three types of anti-platelet membrane antibodies. The appearance of this Mr = 66,000 protein on the surface of chymotrypsin-treated platelets correlated with the exposure of fibrinogen receptors on the platelet surface as evidenced by the increased platelet aggregation and the enhanced 125I-fibrinogen binding shown by chymotrypsin-treated platelets. The origin of the Mr = 66,000 protein labeled on chymotrypsin-treated platelets was studied by first labeling intact platelets with 125I and then treating these platelets with chymotrypsin. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the total labeled proteins present after chymotrypsin treatment and those immunoprecipitated by anti-pronase-treated platelet membrane antibody from detergent extracts of chymotrypsin-treated platelets suggested that the Mr = 66,000 protein was a proteolytic cleavage product of glycoprotein IIIa. Analysis on sodium dodecyl sulfate gels of the major chymotryptic cleavage products of partially purified glycoprotein IIIa and analysis of the peptide of glycoprotein IIIa immunoprecipitated by anti-pronase-treated platelet membrane antibody revealed that the Mr = 66,000 protein produced by chymotrypsin on the platelet surface was the major chymotryptic cleavage product of glycoprotein IIIa. We propose a hypothesis that the Mr = 66,000 is a part of glycoprotein IIIa present on the surface of proteolytically treated platelets and it may function in fibrinogen binding and fibrinogen-induced platelet aggregation and that the 66,000-dalton region of glycoprotein IIIa in intact platelets may represent the fibrinogen-binding domain of glycoprotein IIIa.

Adenosine Diphosphate↗
PubMed2006

Effects of escalating doses of tirofiban on platelet aggregation and major receptor expression in diabetic patients: hitting the TARGET in the TENACITY trial?

INTRODUCTION: Ongoing search for the optimal dosing regimens, and valid concerns that some GPIIb/IIIa inhibitors may cause rebound platelet activation are limiting the use of these agents in patients with acute vascular events. MATERIALS AND METHODS: We assessed the in vitro effects of preincubation with escalating (12.5-200 ng/mL) concentrations of tirofiban on platelet biomarkers in 20 diabetic patients. Platelet activity was assessed by ADP-, and collagen-induced conventional plasma aggregometry, and by whole blood flow cytometry measuring expression of PECAM-1, GPIb, GP IIb/IIIa antigen and activity, vitronectin, P-selectin, LAMP-1, GP 37, LAMP-3, activated and intact PAR-1 thrombin receptors, GPIV, and platelet-monocyte formation. All patients were treated with aspirin (at least 81 mg daily for 1 month); other antiplatelet agents were not allowed. RESULTS: Significant decrease of ADP-induced platelet aggregation was observed starting at the low 12.5 ng/mL concentration (p=0.0001), with total inhibition occurring at 50 ng/mL of tirofiban dose. Inhibition of collagen-induced platelet aggregability requires 25 ng/ml of tirofiban (p=0.002), and was complete at 100 ng/mL. Dose-dependent blockade of GP IIb/IIIa activity was observed with tirofiban concentrations over 50 ng/mL (p=0.003). Other receptors were unaffected even with the high doses of tirofiban (100-200 ng/mL). CONCLUSION: Tirofiban completely inhibits ADP- and, with the higher dose, collagen-induced platelet aggregation. Higher loading dose of tirofiban used in the ongoing TENACITY trial (100 ng/mL) may be superior with regard to clinical outcomes to the regimens used in PRISM-PLUS (25 ng/mL), or TARGET (50 ng/mL). Selective inhibition of GPIIb/IIIa activity, and lack of alternative platelet activation beyond the GP IIb/IIIa blockade may represent the therapeutic advantage of tirofiban over other agents.

Aged↗
PubMed1979

Structure activity relationships between catecholamines and the alpha-adrenergic receptor responsible for the aggregation of human platelets by epinephrine.

PEA is a potent inhibitor (Ki approximately 13 microM) of human platelet aggregation induced by epinephrine. This led us to perform an SAR study of a congeneric series of compounds in an effort to identify the molecular components of epinephrine critical to its aggregating effect upon human platelets. Phenylethanolamine was similar to PEA in inhibitory potency. However, hydroxylation of the phenyl ring diminished the inhibitory effect (Ki tyramine approximately 87 microM; Ki octopamine approximately 88 microM). Dopamine, the weakest inhibitor (Ki approximately 150 microM), was a partial agonist capable of inducing platelet aggregation in some samples of PRP. The order of potency of catecholamines as aggregating agents was epinephrine greater than norepinephrine greater than Epinine greater than dopamine. Phenylephrine, the prototype alpha-agonist, did not induce aggregation but was a potent inhibitor (Ki approximately 12 microM) of the aggregation induced by epinephrine. Isoproterenol, the prototype beta-agonist, was neither an aggregant nor an inhibitor of epinephrine-induced platelet aggregation. These findings suggest that the binding of epinephrine to the alpha-adrenergic receptor responsible for platelet aggregation is accomplished by the N-methyl amino group whereas intrinsic aggregating activity is a function of the catechol moiety.

2-Hydroxyphenethylamine↗
PubMed1984

Interaction between electromagnetic fields and cells: microelectrophoretic effect on ligands and surface receptors.

The aggregation between lectins and lymphocyte surface receptors can be affected strongly by a low-level electric field induced in the cell suspension by a time-varying magnetic field. One of the possible mechanisms is the microelectrophoretic effect due to the electric field, which influences the distance (in the mean square sense) between charged ligands and receptors when they are about to separate. On a purely theoretical basis, it is shown that, at low frequencies, an externally induced periodic electric field always decreases the mean lifetime of ligand-receptor complexes. As a consequence, the mitogenic gain obtained by lectin addition to cell suspension is decreased. These results suggest that such a mechanism, if effective, reduces the lectin mitogenic capability and offers a way of handling similar phenomena which have been described for other biological systems.

Electromagnetic Fields↗
PubMed1991

A place for free radicals in platelet-derived histamine releasing factor (PDHRF) and evidence that histaminergic receptors modulate platelet aggregation.

The release of histamine from rat serosal mast cells induced by coincubation with resting and activated human platelets, or by exposure of mast cells to supernatants obtained from activated platelets, is significantly reduced by anti-free radical interventions, and is coupled with the generation of membrane lipid peroxidation products. These results suggest free radical participation in the activity of PDHRF. Human platelets possess specific binding sites for an H1-receptor antagonist, suggesting that H1-receptors could modulate the intracellular calcium levels in a pro-aggregatory fashion.

Animals↗
PubMed2004

Nongenomic effects of 17beta-estradiol in human platelets: potentiation of thrombin-induced aggregation through estrogen receptor beta and Src kinase.

The impact of estrogens on the cardiovascular system and their ability to regulate platelet function are matters of controversy. The recent finding that estrogen receptors are expressed in human platelets renders these cells an excellent model for studying the nongenomic effects of these hormones. In this work, we investigated 17beta-estradiol-dependent signaling in platelets from adult healthy men. 17beta-estradiol caused the rapid phosphorylation of the tyrosine kinases Src and Pyk2 and the formation of a signaling complex, which included Src, Pyk2, and the phosphatidylinositol 3-kinase. Both these events were dependent on estrogen receptor beta engagement. We found that estrogen receptor beta was membrane-associated in platelets. On treatment with 17beta-estradiol, Src and Pyk2 activation occurred in the membrane fraction but not in the cytosol. In contrast, no significant activation of phosphatidylinositol 3-kinase was detected in estrogen-treated platelets. 17beta-estradiol did not induce any platelet response directly, but it strongly potentiated the activation of integrin alpha(IIb)beta3 and the platelet aggregation induced by subthreshold concentrations of thrombin. These effects were dependent on estrogen receptor beta recruitment and were associated with a strong synergistic effect with thrombin on Src activation. Taken together, these results indicate that 17beta-estradiol can modulate platelet function by exercising a proaggregating role.

Adult↗