Thromboxane A 2 is a positive feedback lipid mediator produced following platelet activation. The G q -coupled thromboxane A 2 receptor subtype, TP␣, and G i -coupled TP subtype have been shown in human platelets. ADPinduced platelet aggregation requires concomitant signaling from two P2 receptor subtypes, P2Y1 and P2T AC , coupled to G q and G i , respectively. We investigated whether the stable thromboxane A 2 mimetic, (15S)-hydroxy-9,11-epoxymethanoprosta-5Z,13E-dienoic acid (U46619), also causes platelet aggregation by concomitant signaling through G q and G i , through co-activation of TP␣ and TP receptor subtypes. Here we report that secretion blockade with Ro 31-8220, a protein kinase C inhibitor, completely inhibited U46619-induced, but not ADP-or thrombin-induced, platelet aggregation. Ro 31-8220 had no effect on U46619-induced intracellular calcium mobilization or platelet shape change. Furthermore, U46619-induced intracellular calcium mobilization and shape change were unaffected by A3P5P, a P2Y1 receptor-selective antagonist, and/or cyproheptadine, a 5-hydroxytryptamine subtype 2A receptor antagonist. Either Ro 31-8220 or AR-C66096, a P2T AC receptor selective antagonist, abolished U46619-induced inhibition of adenylyl cyclase. In addition, AR-C66096 drastically inhibited U46619-mediated platelet aggregation, which was further inhibited by yohimbine, an ␣ 2A -adrenergic receptor antagonist. Furthermore, inhibition of U46619-induced platelet aggregation by Ro 31-8220 was relieved by activation of the G i pathway by selective activation of either the P2T AC receptor or the ␣ 2A -adrenergic receptor. We conclude that whereas thromboxane A 2 causes intracellular calcium mobilization and shape change independently, thromboxane A 2 -induced inhibition of adenylyl cyclase and platelet aggregation depends exclusively upon secretion of other agonists that stimulate G i -coupled receptors.Upon exposure to activating agonists (e.g. thrombin, ADP, and collagen), platelets liberate arachidonic acid stored as phospholipid in the platelet plasma membrane that is converted into thromboxane A 2 by sequential oxygenation of arachidonic acid by cycloxygenase and thromboxane A 2 synthase (1). The released thromboxane A 2 acts as a positive feedback mediator in the activation and recruitment of more platelets to the primary hemostatic plug (2). Thromboxane A 2 exerts its actions via specific G protein-coupled receptors and has been described as either a potent platelet agonist (2, 3) or as a weak agonist with an important role in amplifying the response of platelets to more potent agonists (4).Pharmacological studies indicate the presence of two potential thromboxane A 2 receptor (TP receptor) 1 subtypes on human platelets (5, 6). The TP receptor gene has been cloned and encodes two subtypes of the TP receptor that result from alternative splicing of the primary transcript (7). The subtypes share the identical first 293 amino acids but possess different carboxyl-terminal domains. A complete cDNA of the 343 amino acid T...
Platelets undergo shape change upon activation with agonists. During shape change, disc-shaped platelets turn into spiculated spheres with protruding filopodia. When agonist-induced cytosolic Ca(2+) increases were prevented using the cytosolic Ca(2+) chelator, 5, 5'-dimethyl-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (5, 5'-dimethyl-BAPTA), platelets still underwent shape change, although the onset was delayed and the initial rate was dramatically decreased. In the absence of cytosolic Ca(2+), agonist-stimulated myosin light chain phosphorylation was significantly inhibited. The myosin light chain was maximally phosphorylated at 2 s in control platelets compared with 30 s in 5,5'-dimethyl-BAPTA-treated platelets. ADP, thrombin, or U46619-induced Ca(2+)-independent platelet shape change was significantly reduced by staurosporine, a nonselective kinase inhibitor, by the selective p160 Rho-associated coiled-coil-containing protein kinase inhibitor Y-27632, or by HA 1077. Both Y-27632 and HA 1077 reduced peak levels of ADP-induced platelet shape change and myosin light chain phosphorylation in control platelets. In 5,5'-dimethyl-BAPTA-treated platelets, Y-27632 and HA 1077 completely abolished both ADP-induced platelet shape change and myosin light chain phosphorylation. Our results indicate that Ca(2+)/calmodulin-stimulated myosin light chain kinase and p160 Rho-associated coiled-coil-containing protein kinase independently contribute to myosin light chain phosphorylation and platelet shape change, through Ca(2+)-sensitive and Ca(2+)-insensitive pathways, respectively.
Previous studies of in vitro infection by human T-cell lymphoma/leukemia virus type I (HTLV-I) have required cocultivation of target cells with HTLV-I cell lines or vesicular stomatitis virus pseudotypes containing HTLV-I envelope proteins. We report here the development of a cell-free infection assay for HTLV-I. Target cells were incubated with purified, DNase-treated HTLV-I virions for 4 h at 37°C. Target cell DNA was then analyzed for the presence of newly synthesized HTLV-I proviral DNA by the highly sensitive polymerase chain reaction. Using this assay system, we have been able to consistently detect in vitro infection of a variety of cellular targets by different HTLV-I isolates. Optimal infection required the presence of 10 ,ug of DEAEdextran per ml. The assay was dose dependent with respect to virus input. In general, the amount of proviral DNA detected correlated with the level of HTLV-I receptors present on the surface of the target cells, as measured by fluorochrome-labelled HTLV-I binding. Finally, the specificity of the assay was confirmed by demonstrating that the cell line, Llq, a somatic cell hybrid containing human chromosome 17q, to which the gene for the HTLV-I receptor has been mapped, was susceptible to infection by HTLV-I, while the parental mouse cell line from which it was derived, LMTK-, which lacks human chromosome 17q, was not.
Summary.Prostaglandins act through specific receptors to stimulate cyclic AMP formation which inhibits platelet activation and relaxes vascular smooth muscle. We have used RT-PCR combined with Southern blot analysis to determine the subtypes of prostaglandin receptor on platelets. Platelets expressed the EP4 rather than the EP2 prostaglandin EP receptor subtype, whereas vascular smooth muscle cells predominantly expressed the EP2 receptor. The IP receptor, which binds prostacyclin and couples to stimulation of adenylyl cyclase, and three isoforms of the inhibitory EP3 receptor were equally expressed in platelets, HEL cells and umbilical artery smooth muscle cells. The EP3-II isoform showed variation in level of expression among the three cell types. As a positive control for the presence of platelet RNA, PCR was performed using primers specific for the alpha chain of the platelet membrane glycoprotein Ib. As a negative control for the absence of T and B cell contamination in the platelet RNA, PCR was performed using primers specific for the cell specific cluster determinants CD2 (a T-cell marker) and CD20 (a B-cell marker). The finding that platelets express both stimulatory and inhibitory prostaglandin receptors provides confirmation of a homeostatic model of regulation of platelet adenylyl cyclase previously proposed.
Summary. The integrin avb3 mediates platelet adhesion to the matrix protein osteopontin and likely is the predominant integrin mediating platelet adhesion to the matrix protein vitronectin. To address the mechanism that regulates avb3 activity in platelets, we measured the effect of the P2Y 1 antagonist adenosine 3 H -phosphate-5 H -phosphate (A3P5P) and the P2Y 12 antagonist AR-C66096 on ADP-stimulated platelet adhesion to osteopontin and vitronectin. Each antagonist completely inhibited platelet adhesion, implying that concurrent stimulation of P2Y 1 and P2Y 12 was required to activate avb3. The reducing agent dithiothreitol and Mn 2 also induced platelet adhesion to osteopontin, but did so without stimulating platelet activation. Thus, these data suggest that ADP stimulation regulates avb3 activity by perturbing the conformation of its extracellular domain. The actin polymerization inhibitors cytochalasin D and latrunculin A also induced platelet adhesion to osteopontin and vitronectin. Thus, avb3 activity in resting platelets appears to be constrained by the platelet cytoskeleton. Moreover, the effect of these agents was inhibited by A3P5P and AR-C66096 at micromolar and subnanomolar concentrations, respectively, suggesting that subthreshold platelet stimulation by ADP was required. Our data suggest that signals from both Ga q -and Ga icoupled receptors converge to release cytoskeletal constraints on avb3. We propose that the release of cytoskeletal constraints and a concurrent increase in af®nity for ligands is responsible for avb3-mediated platelet adhesion.Keywords: ADP receptors, osteopontin, vitronectin receptors, vitronectin.Platelets express both known b3 integrins, aIIbb3 and avb3. Although these integrins readily interact with the macromolecular ligands ®brinogen, von Willebrand factor (VWF), ®bro-nectin, and vitronectin (VN) [1], aIIbb3 does not interact with the matrix protein osteopontin (OPN), an Arg-Gly-Asp (RGD)-containing ligand for avb3 and other av-containing integrins [2,3]. Nonetheless, despite the presence of 50-to 500-fold fewer copies of avb3 compared with aIIbb3 on platelets [4,5], avb3 mediates platelet adhesion to OPN-coated surfaces [6].The ability of aIIbb3 to interact with ligands requires platelet stimulation by agonists such as thrombin, ADP, thromboxane A 2 , serotonin, and epinephrine [1]. How these agonists regulate aIIbb3 function is uncertain. ADP binding to the Ga qcoupled purinergic receptor P2Y 1 stimulates platelet shape change, activates phospholipase Cb i and induces the release of calcium from intracellular stores [7], whereas ADP binding to Ga i -coupled purinergic receptor P2Y 12 inhibits the activity of the enzyme adenylyl cyclase [8]. Concurrent stimulation of both P2Y 12 and P2Y 1 is required for ADP-stimulated platelet aggregation [9], but how signals emanating from these receptors cooperate to induce platelet aggregation is not known. We have reported that incubating platelets previously exposed to subthreshold concentrations of ADP with cytochalasin D and la...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.