RESUMEN
The role of fibrinogen as a cofactor for platelet aggregation was examined by measuring the binding of 125I-labeled human fibrinogen to gel-filtered human platelets both before and after platelet stimulation by ADP and epinephrine. Platelet stimulation by ADP resulted in the rapid, reversible binding of fibrinogen to receptors on the platelet surface. Fibrinogen binding increased as the concentration of ADP was increased from 0.1 to 2 microM, reaching a plateau at higher ADP concentrations. Binding occurred only after platelet stimulation and in the presence of divalent cations. However, fibrinogen binding did not occur to ADP-stimulated platelets from three patients with Glanzmann's thrombasthenia. Analysis of fibrinogen binding as a function of increasing fibrinogen concentration demonstrated that maximal platelet stimulation exposed approximately or equal to 45,000 binding sites per platelet with a dissociation constant of 80--170 nM. These fibrinogen binding parameters were essentially the same whether ADP or epinephrine was the platelet-stimulating agent. Thus, these studies demonstrate that platelet stimulation by ADP and epinephrine exposes a limited number of fibrinogen receptors on the platelet surface. Furthermore, these data suggest that the fibrinogen molecules bound to the platelet as a consequence of platelet stimulation are directly involved in the platelet aggregation response.
Asunto(s)
Adenosina Difosfato/farmacología , Plaquetas/fisiología , Epinefrina/farmacología , Fibrinógeno/metabolismo , Sitios de Unión , Plaquetas/efectos de los fármacos , Calcio/farmacología , Hormonas/farmacología , Humanos , Radioisótopos de Yodo , Marcaje Isotópico , Magnesio/farmacología , Agregación Plaquetaria , Púrpura Trombocitopénica/sangre , Estimulación QuímicaRESUMEN
Exposure of fibrinogen receptors by a variety of agonists is a prerequisite for platelet aggregation. Because the synthesis of prostaglandins and thromboxane A2 also occurs during platelet aggregation we wondered whether these agents participate in the exposure of platelet fibrinogen receptors. Therefore, we measured the binding of human 125I-fibrinogen to gel-filtered normal human platelets after prostaglandin and thromboxane synthesis had been inhibited by aspirin or indomethacin. The fibrinogen binding assay was performed at 37 degrees C but without stirring to prevent the formation of platelet aggregates. Platelet secretion, measured with [14C]serotonin, did not occur during the procedure. Aspirin or indomethacin inhibited fibrinogen binding stimulated by 10 microM epinephrine by 53%, and inhibited fibrinogen binding stimulated by 1-2 microM ADP by 37.1%. However, ADP at concentrations greater than 2 microM returned fibrinogen binding toward control values. Scatchard analysis demonstrated that aspirin decreased the number but not the affinity of the exposed fibrinogen receptors. To determine whether prostaglandins are capable of directly exposing fibrinogen receptors, prostaglandin H2 was used to stimulate platelets in the fibrinogen binding assay. Prostaglandin H2 exposed approximately 54,000 fibrinogen receptors/platelet and corrected the deficit in receptor exposure induced by aspirin. These studies demonstrate that platelet prostaglandins or thromboxane A2 can play a direct role in the exposure of platelet fibrinogen receptors. In addition, they suggest that the synthesis of prostaglandins and thromboxane A2 by stimulated platelets may be all that is required for optimal secondary platelet aggregation.
Asunto(s)
Plaquetas/efectos de los fármacos , Fibrinógeno/metabolismo , Prostaglandinas/farmacología , Receptores de Superficie Celular/efectos de los fármacos , Tromboxanos/farmacología , Adenosina Difosfato/farmacología , Aspirina/farmacología , Epinefrina/farmacología , Humanos , Indometacina/farmacología , Agregación Plaquetaria/efectos de los fármacos , Glicoproteínas de Membrana Plaquetaria , Prostaglandinas A/farmacología , Prostaglandinas H/farmacologíaRESUMEN
The platelet membrane glycoprotein IIb/IIIa complex is a member of a family of alpha/beta heterodimers that function as receptors for adhesive proteins. In this report we describe the structure of the human beta subunit GPIIIa deduced from an analysis of 4.0 kb of overlapping cDNA sequences isolated from a human erythroleukemia (HEL) cell cDNA expression library. A continuous open reading frame encoding all 788 amino acids for GPIIIa was present. The deduced amino acid sequence included a 26-residue amino-terminal signal peptide, a 29-residue transmembrane domain near the carboxy terminus, and four tandemly repeated cysteine-rich domains of 33-38 residues. An exact correspondence of 128 amino acids from seven human platelet GPIIIa fragments with HEL GPIIIa indicates that HEL and platelet GPIIIa are the same gene product. The HEL GPIIIa sequence was compared with the sequences of the beta subunit for the human LFA-1/Mac-1/p150.95 complex and human endothelial cell GPIIIa, revealing a 38% similarity with the former and virtual identity with the latter. Northern blot analysis using RNA from both HEL and endothelial cells revealed two GPIIIa transcripts of 5.9 and 4.1 kb. However, HEL RNA, but not endothelial cell RNA, contained a transcript for GPIIb. This indicates that the GPIIIa-containing heterodimers in platelets and endothelial cells are not identical structures, but are members of a subfamily within the human family of adhesion protein receptors sharing an identical beta subunit.
Asunto(s)
Glicoproteínas de Membrana Plaquetaria/genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN/genética , Endotelio Vascular/citología , Humanos , Leucemia Eritroblástica Aguda , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Señales de Clasificación de Proteína/genética , ARN/genética , Secuencias Repetitivas de Ácidos Nucleicos , Transcripción Genética , Células Tumorales CultivadasRESUMEN
The integrin alphavbeta3 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 alphavbeta3 activity in platelets, we measured the effect of the P2Y1 antagonist adenosine 3'-phosphate-5'-phosphate (A3P5P) and the P2Y12 antagonist AR-C66096 on ADP-stimulated platelet adhesion to osteopontin and vitronectin. Each antagonist completely inhibited platelet adhesion, implying that concurrent stimulation of P2Y1 and P2Y12 was required to activate alphavbeta3. The reducing agent dithiothreitol and Mn2+ also induced platelet adhesion to osteopontin, but did so without stimulating platelet activation. Thus, these data suggest that ADP stimulation regulates alphavbeta3 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, alphavbeta3 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 Galphaq- and Galphai-coupled receptors converge to release cytoskeletal constraints on alphavbeta3. We propose that the release of cytoskeletal constraints and a concurrent increase in affinity for ligands is responsible for alphavbeta3-mediated platelet adhesion.
Asunto(s)
Plaquetas/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Integrina alfaVbeta3/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal , Actinas/metabolismo , Citoesqueleto/metabolismo , Subunidad alfa de la Proteína de Unión al GTP Gi2 , Subunidades alfa de la Proteína de Unión al GTP Gq-G11 , Humanos , Osteopontina , Sialoglicoproteínas/metabolismo , Vitronectina/metabolismoRESUMEN
Function of the platelet integrin alphaIIbbeta3 is regulated by agonist-generated signals interacting with its cytoplasmic tails. When alphaIIbbeta3 is expressed in Epstein-Barr virus-transformed B lymphocytes, stimulation of the cells with phorbol 12-myristate 13-acetate results in alphaIIbbeta3-mediated lymphocyte adherence to immobilized fibrinogen, as well as soluble fibrinogen binding to alphaIIbbeta3, indicating that agonists increase the affinity of alphaIIbbeta3 for fibrinogen in these cells. To address the contribution of the alphaIIb and beta3 cytoplasmic tails to this process, we mutated each tail and expressed the mutants in B lymphocytes. Truncation of the alphaIIb tail did not impair unstimulated or stimulated lymphocyte adherence to fibrinogen, regardless whether the truncation was proximal or distal to the conserved GFFKR sequence. However, deleting GFFKR or replacing it with alanines markedly reduced alphaIIbbeta3 expression due to impaired intracellular assembly of alphaIIbbeta3 heterodimers, probably due to a mutation-induced change in the conformation of alphaIIb. Introducing beta3 mutations known to impair alphaIIbbeta3 function in platelets into the cytoplasmic tail of beta3 in lymphocytes also impaired alphaIIbbeta3 function in these cells. These studies demonstrate that the cytoplasmic tail of alphaIIb is not required for alphaIIbbeta3 function in lymphocytes, although the presence of GFFKR in the alphaIIb tail is required for alphaIIb to interact with beta3. Additionally, they indicate that signals interacting with the beta3 cytoplasmic tail are responsible for the ability of agonists to stimulate alphaIIbbeta3 function.
Asunto(s)
Citoplasma/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Secuencia de Aminoácidos , Adhesión Celular , Fibrinógeno/metabolismo , Humanos , Ligandos , Linfocitos/citología , Linfocitos/metabolismo , Datos de Secuencia Molecular , Mutagénesis , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/agonistas , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/genética , Unión ProteicaRESUMEN
Fibrinogen binding to receptors on stimulated platelets is a prerequisite for platelet aggregation. In order to identify the platelet fibrinogen receptor, we modified fibrinogen with the photoreactive, heterobifunctional cross-linking reagent methyl 4-azidobenzoimidate (MABI). MABI-fibrinogen was fully clottable and able to support platelet aggregation. To photoaffinity label the fibrinogen receptor, gel-filtered human platelets were incubated at 37 degrees C in the dark with 200 micrograms/ml of MABI-fibrinogen, 10 microM ADP, and 0.5 mM calcium. Irradiation of these platelets with ultraviolet light resulted in the incorporation of MABI-fibrinogen into the platelet surface. Incorporation could be prevented by excess native fibrinogen suggesting that MABI-fibrinogen had interacted with the fibrinogen receptor before photolysis. Examination of the irradiated platelets by sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the photoactivated MABI-fibrinogen had been incorporated into a 105,000 molecular weight membrane polypeptide that also contained the PlA1 antigen. Thus, this polypeptide has the characteristics of the membrane glycoprotein IIIa. Previous studies have shown that thrombasthenic platelets lack this glycoprotein and fail to bind fibrinogen after stimulation by ADP. Consequently, our data suggest that glycoprotein IIIa constitutes at least one component of the platelet fibrinogen receptor.
Asunto(s)
Marcadores de Afinidad/farmacología , Azidas , Plaquetas/metabolismo , Fibrinógeno/metabolismo , Imidoésteres/farmacología , Receptores de Superficie Celular/metabolismo , Membrana Celular/metabolismo , Humanos , Sueros Inmunes , Peso Molecular , Glicoproteínas de Membrana Plaquetaria , Receptores de Superficie Celular/efectos de los fármacos , Receptores de Superficie Celular/aislamiento & purificaciónRESUMEN
We studied the function of the platelet integrin alphaIIb beta3 using a B lymphocyte model in which alphaIIb beta3 can be induced to interact with fibrinogen using phorbol myristate acetate (PMA). To determine whether a G protein-coupled receptor could also activate alphaIIb beta3 in lymphocytes, we coexpressed the human formyl peptide receptor (fPR) and alphaIIb beta3, finding that the fPR agonist formyl Met-Leu-Phe (fMLP)-stimulated lymphocyte adherence to immobilized fibrinogen and binding of soluble fibrinogen to the lymphocyte surface. The response to fMLP, but not PMA, was abrogated by pertussis toxin, indicating that the fPR was coupled to the G-protein Galphai, whereas the protein kinase C inhibitor bisindolylmaleimide I inhibited the response to both fMLP and PMA, indicating that signaling from the fPR included protein kinase C. On the other hand, the tyrosine kinase inhibitor genistein, the Syk inhibitor piceatannol, and the RhoA inhibitor C3 exoenzyme had no effect, implying that neither tyrosine phosphorylation nor the GTPase RhoA were involved. Furthermore, whereas micromolar concentrations of cytochalasin D inhibited the PMA-stimulated interaction of alphaIIb beta3 with fibrinogen, nanomolar concentrations actually induced fibrinogen binding to unstimulated cells. Our studies demonstrate that alphaIIb beta3 expressed in B lymphocytes can be activated by a physiologic agonist and outline an activating pathway that includes Galphai, protein kinase C, and the actin cytoskeleton.
Asunto(s)
Linfocitos B/fisiología , Regulación de la Expresión Génica/genética , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/fisiología , Adhesión Celular/efectos de los fármacos , Citocalasina D/farmacología , Inhibidores Enzimáticos/farmacología , Fibrinógeno/metabolismo , Citometría de Flujo , Proteínas de Unión al GTP/fisiología , Genisteína/farmacología , Humanos , Indoles/farmacología , Maleimidas/farmacología , N-Formilmetionina Leucil-Fenilalanina/farmacología , Toxina del Pertussis , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/fisiología , Receptores de Formil Péptido , Receptores Inmunológicos/genética , Receptores de Péptidos/genética , Transducción de Señal , Acetato de Tetradecanoilforbol/farmacología , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
Cleavage of osteopontin by thrombin has been reported to enhance cell adhesion. We asked whether thrombin could regulate the alpha(v)beta(3)-mediated adhesion of platelets and B lymphocytes to this substrate. Although there was no difference in the extent or the avidity of thrombin- and ADP-stimulated platelet adhesion to intact or thrombin-cleaved human osteopontin, both the extent and avidity of phorbol ester-stimulated B cell adhesion to thrombin-cleaved osteopontin was significantly increased. Thus, these data suggest that the ability of alpha(v)beta(3) to recognize osteopontin can be differentially regulated in a cell-specific manner. To localize the alpha(v)beta(3) binding site on osteopontin, we measured cell adhesion to the two thrombin cleavage products of osteopontin and to a series of nested RGD-containing osteopontin peptides cross-linked to albumin. Whereas ADP-stimulated platelets adhered to the amino-terminal but not the carboxyl-terminal osteopontin fragment and to the osteopontin peptide RGDSVVYGLR, phorbol ester-stimulated B cells did not adhere to this peptide, although they did so in the presence of 1 mm Mn(2+). Thus, our data confirm that thrombin cleavage enhances the accessibility of the binding motif for alpha(v)beta(3) on osteopontin, but this enhancement is also a function of the activation state of alpha(v)beta(3). Moreover, they indicate that the sequence RGDSVVYGLR contains sufficient information to specify activation-dependent alpha(v)beta(3)-mediated platelet and lymphocyte adhesion.
Asunto(s)
Linfocitos B/fisiología , Adhesión Celular , Adhesividad Plaquetaria , Receptores de Vitronectina/fisiología , Sialoglicoproteínas/metabolismo , Trombina/metabolismo , Adenosina Difosfato/farmacología , Plaquetas/efectos de los fármacos , Dicroismo Circular , Electroforesis en Gel de Poliacrilamida , Herpesvirus Humano 4 , Humanos , Magnesio/farmacología , Espectroscopía de Resonancia Magnética , Osteopontina , Conformación ProteicaRESUMEN
The phosphorylated acidic glycoprotein osteopontin is present in the extracellular matrix of atherosclerotic plaques and the wall of injured but not normal arteries. To determine if osteopontin could serve as a substrate for platelet adhesion, we measured the adherence of resting and agonist-stimulated human platelets to immobilized recombinant human osteopontin. Agonist-stimulated but not resting platelets bound to osteopontin by a process that was mediated primarily by alphavbeta3. alphavbeta3-mediated adherence occurred at physiologic concentrations of calcium and was inhibited by an alphavbeta3-selective cyclic peptide. Assays using phorbol myristate acetate-stimulated transfected B lymphocytes expressing both alphavbeta3 and alphaIIbbeta3 confirmed that activated alphavbeta3 not activated alphaIIbbeta3 was responsible for the cellular adherence we measured. These studies indicate that alphavbeta3 can reside on the cell surface in an inactive state and can be converted to a ligand binding conformation by cellular agonists. Moreover, they suggest that platelet adherence to osteopontin mediated by activated alphavbeta3 could play a role in anchoring platelets to disrupted atherosclerotic plaques and the walls of injured arteries. By inhibiting alphavbeta3 function, it may be possible to inhibit platelet-mediated vascular occlusion with a minimal effect on primary hemostasis.
Asunto(s)
Plaquetas/metabolismo , Citocinas/metabolismo , Linfocitos/metabolismo , Fosfoproteínas/metabolismo , Receptores de Vitronectina/metabolismo , Sialoglicoproteínas/metabolismo , Adenosina Difosfato/farmacología , Angioplastia Coronaria con Balón , Calcio/metabolismo , Adhesión Celular , Fibrinógeno/metabolismo , Humanos , Magnesio/metabolismo , Osteopontina , Adhesividad Plaquetaria , Proteínas Recombinantes/metabolismoRESUMEN
Agonist-generated inside-out signals enable the platelet integrin alpha(IIb)beta(3) to bind soluble ligands such as fibrinogen. We found that inhibiting actin polymerization in unstimulated platelets with cytochalasin D or latrunculin A mimics the effects of platelet agonists by inducing fibrinogen binding to alpha(IIb)beta(3). By contrast, stabilizing actin filaments with jasplakinolide prevented cytochalasin D-, latrunculin A-, and ADP-induced fibrinogen binding. Cytochalasin D- and latrunculin A-induced fibrinogen was inhibited by ADP scavengers, suggesting that subthreshold concentrations of ADP provided the stimulus for the actin filament turnover required to see cytochalasin D and latrunculin A effects. Gelsolin, which severs actin filaments, is activated by calcium, whereas the actin disassembly factor cofilin is inhibited by serine phosphorylation. Consistent with a role for these factors in regulating alpha(IIb)beta(3) function, cytochalasin D- and latrunculin A-induced fibrinogen binding was inhibited by the intracellular calcium chelators 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid acetoxymethyl ester and EGTA acetoxymethyl ester and the Ser/Thr phosphatase inhibitors okadaic acid and calyculin A. Our results suggest that the actin cytoskeleton in unstimulated platelets constrains alpha(IIb)beta(3) in a low affinity state. We propose that agonist-stimulated increases in platelet cytosolic calcium initiate actin filament turnover. Increased actin filament turnover then relieves cytoskeletal constraints on alpha(IIb)beta(3), allowing it to assume the high affinity conformation required for soluble ligand binding.
Asunto(s)
Actinas/metabolismo , Plaquetas/metabolismo , Plaquetas/ultraestructura , Citoesqueleto/metabolismo , Fibrinógeno/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Citoesqueleto/ultraestructura , HumanosRESUMEN
The ligand binding activity of the platelet integrin alpha IIb beta 3 is initiated by agonist-generated intraplatelet signals. We studied this process in vitro by expressing recombinant alpha IIb beta 3 in Epstein-Barr virus-immortalized B lymphocytes. We found that phorbol ester stimulation induced the adhesion of lymphocytes expressing alpha IIb beta 3 to immobilized fibrinogen. Moreover, replacement of the transmembrane and cytoplasmic domains of the alpha and beta subunits of alpha IIb beta 3 with those of alpha L beta 2 significantly increased adherence, whereas replacement of only the cytoplasmic domains significantly decreased adherence. This suggests that transmembrane segments are involved in the agonist-induced modulation of alpha IIb beta 3 activity. Similar results were seen when the alpha IIb beta 3 activation-dependent monoclonal antibody PAC-1 was substituted for immobilized fibrinogen. We also found that the adherence of lymphocytes expressing beta 3 with either of the two alpha IIb/alpha L chimeras was similar to that of cells expressing alpha IIb beta 3, whereas the adherence of cells expressing alpha IIb with either of the two beta 3/beta 2 chimeras was substantially decreased, suggesting that the identity of the cytoplasmic domain of beta 3, but not of alpha IIb, is critical for alpha IIb beta 3 function. This report indicates that B lymphocytes contain signal transduction pathways involving protein kinase C that can increase the ligand binding activity of alpha IIb beta 3 and demonstrates the utility of these cells as an expression system for the study of agonist-stimulated alpha IIb beta 3 function.
Asunto(s)
Linfocitos B/metabolismo , Adhesión Celular/fisiología , Integrinas/metabolismo , Antígeno-1 Asociado a Función de Linfocito/metabolismo , Transducción de Señal , Secuencia de Aminoácidos , Linfocitos B/efectos de los fármacos , Secuencia de Bases , Plaquetas/química , Relación Dosis-Respuesta a Droga , Fibrinógeno/metabolismo , Humanos , Integrinas/agonistas , Integrinas/genética , Antígeno-1 Asociado a Función de Linfocito/genética , Datos de Secuencia Molecular , Oligopéptidos/farmacología , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria , Unión Proteica , Conformación Proteica , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Relación Estructura-Actividad , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
We have isolated from an HEL cell cDNA library an alternatively spliced transcript for the platelet membrane glycoprotein IIb (GPIIb) that resulted from the deletion of the 34 amino acids of exon 28 of the GPIIb gene. Confirming an earlier report, we also detected this transcript in platelet mRNA. To determine the consequences of exon 28 deletion on the expression of the GPIIb/IIIa heterodimer, we expressed cDNA for GPIIb-28 in COS-1 cells, either individually or simultaneously with a cDNA for GPIIIa. When recombinant GPIIb-28 was expressed alone, it did not acquire resistance to the enzyme endo-beta-N-acetylglucosaminidase H, was not cleaved into heavy and light chains, and was not transported to the cell surface. However, when recombinant GPIIb-28 was coexpressed with recombinant GPIIIa, GPIIb/IIIa heterodimers were assembled. Nevertheless, these heterodimers failed to complete posttranslational processing and were degraded intracellularly. Exon 28 contains one site for Asn-linked glycosylation. To determine if loss of this glycosylation site was responsible for the effects of exon 28 deletion, we removed the site from the exon 28 of intact GPIIb by oligonucleotide-mediated mutagenesis. However, absence of the carbohydrate appended to exon 28 did not prevent normal GPIIb/IIIa heterodimer expression. Our studies indicate that absence of the amino acids encoded by GPIIb exon 28 sufficiently perturbs the quaternary configuration of the GPIIb/IIIa heterodimer to impair its subsequent intracellular transport and processing. They also indicate that this alternatively spliced form of GPIIb mRNA, although present in megakaryocytes, is unlikely to make a significant contribution to the GPIIb/IIIa complexes expressed on platelets.
Asunto(s)
Deleción Cromosómica , Exones/genética , Expresión Génica , Glicoproteínas de Membrana Plaquetaria/genética , Secuencia de Bases , Sitios de Unión , Línea Celular , Glicosilación , Humanos , Técnicas de Inmunoadsorción , Sustancias Macromoleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Glicoproteínas de Membrana Plaquetaria/metabolismo , Procesamiento Proteico-Postraduccional , Empalme del ARN , ARN Mensajero/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
The platelet membrane glycoproteins IIb (GpIIb) and GpIIIa form calcium-dependent heterodimers containing binding sites for fibrinogen, von Willebrand factor, and fibronectin. Although GpIIb and GpIIIa are distinct proteins, both GpIIb and GpIIIa are deficient in platelets from individuals with the recessive disorder Glanzmann's thrombasthenia. To gain a better understanding of the genetic basis for GpIIb and GpIIIa synthesis, we studied their synthesis by two human leukemia cell lines, HEL and K562. HEL cells contained complexes of GpIIb and GpIIIa, and K562 cells expressed GpIIIa, but not GpIIb, when stimulated with phorbol-12-myristate-13-acetate (PMA). RNA from HEL cells directed the in vitro synthesis of a 110,000-Mr precursor for GpIIb and a 92,000-Mr precursor for GpIIIa, which indicates that the synthesis of GpIIb and GpIIIa by HEL cells is directed by separate mRNAs. In contrast, RNA from PMA-stimulated K562 cells only directed the synthesis of a 92,000-Mr precursor for GpIIIa. The dissociation of GpIIb and GpIIIa synthesis in K562 cells suggests that GpIIb and GpIIIa may be the products of separate genes.
Asunto(s)
Glicoproteínas de Membrana Plaquetaria/biosíntesis , Plaquetas/fisiología , Adhesión Celular , Sistema Libre de Células , Células Cultivadas , Regulación de la Expresión Génica , Humanos , Técnicas de Inmunoadsorción , Sustancias Macromoleculares , Peso Molecular , Glicoproteínas de Membrana Plaquetaria/genética , ARN Mensajero/genéticaRESUMEN
The platelet glycoproteins GPIIb and GPIIIa are integral membrane proteins and form calcium-dependent heterodimers in the endoplasmic reticulum (ER). In the absence of heterodimer formation, GPIIb and GPIIIa are retained in the ER and degraded. To produce soluble forms of these proteins, we truncated each at a site just proximal to its transmembrane anchor and expressed the mutants in COS-1 cells. We found that both truncated GPIIIa (GPIIIatr) and GPIIIatr were secreted by the transfected cells. However, GPIIbtr was retained by the cells and was immunoprecipitated as a doublet with a 115,000 molecular weight protein. Incubation of transfected cells with the calcium ionophore A23187 or the calcium chelator 1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl) ester (BAPTA-AM) failed to induce appreciable GPIIbtr secretion, suggesting that formation of intracellular calcium complexes was not a factor in GPIIbtr retention. Further, immunoblotting of immunoprecipitated GPIIbtr and GPIIIatr revealed that the chaperone binding protein (BiP) was associated with each, arguing that BiP alone was not responsible for GPIIbtr retention. These studies indicate that the intracellular retention of GPIIIa involves sequences located in the transmembrane or cytoplasmic domains of the molecule. GPIIb contains an additional retention signal located in the extracellular portion of the molecule whose effect is abrogated by formation of a GPIIb-IIIa heterodimer. This signal may be involved in the fate of nascent GPIIb monomers and the generation of correctly configured GPIIb-IIIa heterodimers.
Asunto(s)
Mutagénesis Sitio-Dirigida , Glicoproteínas de Membrana Plaquetaria/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Calcimicina/farmacología , Línea Celular , Membrana Celular/metabolismo , ADN/genética , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Retículo Endoplásmico/metabolismo , Cinética , Sustancias Macromoleculares , Datos de Secuencia Molecular , Plásmidos , Glicoproteínas de Membrana Plaquetaria/genética , Glicoproteínas de Membrana Plaquetaria/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
Fibrinogen binding to receptors on stimulated platelets is a prerequisite for platelet aggregation. To gain further insight into the role of fibrinogen in platelet aggregation and to identify the platelet fibrinogen receptor, we developed a monoclonal anti-platelet antibody that inhibited platelet aggregation. The purified antibody, designated A2A9, inhibited platelet aggregation stimulated by 10 microM ADP, 10 microM epinephrine, and thrombin at 1 unit/ml without inhibiting platelet shape change or platelet secretion. A2A9 was also a competitive inhibitor of fibrinogen binding to ADP-stimulated platelets. Fifty percent inhibition of fibrinogen binding occurred at 65 nM A2A9. Direct binding studies using radiolabeled A2A9 demonstrated 47,000 A2A9 binding sites on unstimulated platelets, with a dissociation constant of 60 nM. Platelets from two individuals with Glanzmann thrombasthenia bound essentially no A2A9. Therefore, these data support the hypothesis that receptor-bound fibrinogen mediates platelet aggregation. In order to identify the platelet fibrinogen receptor, A2A9 immobilized on agarose was used for affinity chromatography. Two platelet polypeptides with Mr = 140,000 and 93,000 were recovered from the immobilized A2A9. After disulfide reduction, these Mr values were altered to 125,000 and 116,000. The smaller polypeptide was also found to contain the PlA1 antigen. These data localize the epitope recognized by A2A9 to the platelet membrane glycoprotein IIb-IIIa complex and suggest that this complex forms the physiologic platelet fibrinogen receptor.
Asunto(s)
Anticuerpos Monoclonales , Plaquetas/metabolismo , Fibrinógeno/metabolismo , Adenosina Difosfato/farmacología , Animales , Cromatografía de Afinidad , Glicoproteínas/inmunología , Humanos , Ratones , Ratones Endogámicos C57BL , Peso Molecular , Agregación Plaquetaria , Glicoproteínas de Membrana Plaquetaria , Receptores de Superficie Celular/metabolismoRESUMEN
The precursor of platelet membrane glycoprotein IIb (GPIIb) undergoes endoproteolytic cleavage into heavy and light chains post-translation. Endoproteolysis occurs within a 17-amino acid stretch of the precursor that contains 4 arginine residues, 3 in dibasic sequences [Lys-Arg (855-856) and Arg-Arg (858-859)] and a single arginine at 871. To determine the site of GPIIb cleavage and its role in the function of the glycoprotein IIb/IIIa heterodimer, we mutated arginine 856, the di-arginine sequence 858-859, and arginine 871 and coexpressed the mutants with glycoprotein IIIa (GPIIIa) in COS-1 cells. Each GPIIb mutant formed recombinant GPIIb-IIIa heterodimers, but mutants lacking arginine at 856 or 858-859 failed to undergo cleavage. Nevertheless, heterodimers containing the uncleaved GPIIb were expressed on the cell surface. Because endoproteolysis most often occurs after arginines in dibasic sequences, we next expressed GPIIb mutants containing lysine at 856 or aspartic acid at 855 with GPIIIa. Both mutants were cleaved and surface-expressed, indicating that the dibasic sequence at 858-859, but not at 855-856, is required for GPIIb cleavage. Lastly, we tested the function of GPIIb-IIIa containing uncleaved GPIIb by measuring adhesion of transfected cells to immobilized fibrinogen. We found no difference in the adhesion of cells expressing either wild-type or mutant GPIIb, indicating GPIIb-IIIa heterodimers containing uncleaved GPIIb maintain their ability to interact with fibrinogen.
Asunto(s)
Glicoproteínas de Membrana Plaquetaria/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Escherichia coli/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Glicoproteínas de Membrana Plaquetaria/genética , Procesamiento Proteico-Postraduccional , TransfecciónRESUMEN
The platelet integrin, glycoprotein IIb-IIIa (GPIIb-IIIa), is a calcium-dependent heterodimer that binds fibrinogen, von Willebrand factor, and fibronectin after platelet activation. We examined GPIIb-IIIa alone and bound to these ligands by electron microscopy after rotary shadowing with platinum/tungsten. We found, as observed previously, that in the presence of detergent and 2 mM Ca2+, GPIIb-IIIa consists of an 8 x 12-nm globular head with two 18-nm flexible tails extending from one side. We also found that in the presence of EDTA, GPIIb-IIIa dissociates into two similar comma-shaped subunits, each containing a portion of the globular head and a single tail. Using monoclonal antibodies to GPIIb, GPIIIa, and the GPIIb-IIIa heterodimer, we found that the tails contained the carboxyl termini of each subunit, while the nodular head was composed of amino-terminal segments of both subunits. Electron microscopy of GPIIb-IIIa bound to fibrinogen revealed a highly specific interaction of the nodular head of GPIIb-IIIa with the distal end of the trinodular fibrinogen molecule and with the tails of GPIIb-IIIa extended laterally at an angle of approximately 98 degrees with respect to the long axis of fibrinogen. When a GPIIb-IIIa was bound to each end of a single fibrinogen, the tails were oriented to opposite sides of fibrinogen, enabling fibrinogen to bridge two adjacent platelets. Electron microscopy of GPIIb-IIIa bound to fibronectin revealed GPIIb/IIIa-binding sites approximately two-thirds of the distance from the amino terminus of each end of the fibronectin molecule, while GPIIb-IIIa was found to bind to von Willebrand factor protomers along a rod-like region near the central nodule of the molecule.
Asunto(s)
Fibrinógeno/metabolismo , Glicoproteínas de Membrana Plaquetaria/metabolismo , Detergentes/farmacología , Ácido Edético/farmacología , Electroforesis en Gel de Poliacrilamida , Fibrinógeno/aislamiento & purificación , Fibrinógeno/ultraestructura , Humanos , Ligandos , Sustancias Macromoleculares , Microscopía Electrónica , Modelos Moleculares , Peso Molecular , Glicoproteínas de Membrana Plaquetaria/aislamiento & purificación , Glicoproteínas de Membrana Plaquetaria/ultraestructura , Unión Proteica , Conformación Proteica , Factor de von Willebrand/metabolismo , Factor de von Willebrand/ultraestructuraRESUMEN
We report a case of Glanzmann thrombasthenia in a Pakistani child whose platelets express less than 10% of the normal amount of alphaIIb beta3 on their surface. Single-stranded conformation polymorphism analysis of the exons of the patient's alphaIIb and beta3 genes showed an abnormality in exon 4 of the beta3 gene. Direct sequence analysis showed that the patient was homozygous for a T --> G nucleotide substitution in this exon, resulting in the replacement of a highly conserved Leu at position 117 with Trp. Heterologous expression of alphaIIb beta3 containing the beta3 mutation in COS-1 cells confirmed the pathogenicity of the Leu117 --> Trp substitution and showed that it resulted in the intracellular retention of malfolded alphaIIb beta3 heterodimers. Additional site-directed mutagenesis at position 117 indicated that, although the smaller hydrophobic amino acid Val could be substituted for the wild-type Leu, the larger hydrophobic amino acids Trp and Phe or the charged amino acids Asp and Lys were not tolerated. These studies indicate that Leu117 in beta3 plays a critical role in attaining the correct folded conformation of alphaIIb beta3. These studies also suggest that the hydrophobic side chain of Leu117 is likely folded into the interior of beta3, where it serves to stabilize internal packing of the protein and determines its overall shape.
Asunto(s)
Plaquetas/metabolismo , Leucina/genética , Mutación , Oligopéptidos/genética , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Trombastenia/genética , Triptófano/genética , Alelos , Secuencia de Aminoácidos , Animales , Células COS , Citometría de Flujo , Humanos , Lactante , Datos de Secuencia Molecular , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/genética , Conformación Proteica , Propiedades de SuperficieRESUMEN
The integrin alpha IIb beta 3, a calcium-dependent heterodimer, plays a critical role in platelet aggregation. The alpha IIb subunit of the heterodimer contains four highly conserved putative calcium-binding domains in its extracellular portion. During studies of the molecular basis of Glanzmann thrombasthenia in a child of mixed Caucasian background whose platelets expressed little alpha IIb beta 3 on their surface, we found the patient heterozygous for a two amino acid deletion in the fourth alpha IIb calcium-binding domain. When this alpha IIb mutant was expressed in COS-1 cells, we found that the deletion did not interfere with the assembly of alpha IIb beta 3 heterodimers, but altered their conformation such that they were neither recognized by the heterodimer-specific antibody A2A9 nor able to undergo further intracellular processing or transport to the cell surface. These results suggest that the calcium-binding domains in alpha IIb play an important role maintaining the overall conformation of alpha IIb beta 3. To confirm this suggestion, we deleted each of the four 12 amino acid calcium-binding domains in alpha IIb by in vitro mutagenesis and expressed the mutants along with beta 3 in COS-1 cells. Each construct formed a heterodimer with beta 3, but none of the heterodimers interacted with A2A9 or underwent further intracellular processing. These data indicate that the calcium-binding domains in alpha IIb are not involved in alpha IIb beta 3 heterodimer formation, but their presence is required for the intracellular transport of alpha IIb beta 3 to the cell surface.
Asunto(s)
Calcio/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/genética , Conformación Proteica , Eliminación de Secuencia , Trombastenia/genética , Adulto , Alelos , Secuencia de Aminoácidos , Animales , Antígenos de Plaqueta Humana/química , Antígenos de Plaqueta Humana/genética , Antígenos de Plaqueta Humana/inmunología , Secuencia de Bases , Sitios de Unión , Transporte Biológico , Plaquetas/metabolismo , Línea Celular Transformada , Chlorocebus aethiops , Clonación Molecular , Epítopos/química , Epítopos/inmunología , Femenino , Citometría de Flujo , Humanos , Recién Nacido , Masculino , Datos de Secuencia Molecular , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/química , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/inmunología , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
A G273D mutation immediately proximal to the first calcium binding domain of platelet GPIIb impairs the export of GPIIb-IIIa heterodimers to the platelet surface. To examine how this mutation might alter the structure of GPIIb, G273 was replaced by other amino acids and the resulting mutants were coexpressed with GPIIIa in COS-1 cells. Although replacement with Ala or Val had no effect on GPIIb-IIIa expression, replacement with Glu, Lys, Pro, or Asn caused intracellular retention of GPIIb-IIIa. Concurrently, the consequences of these replacements were examined by comparative modeling by introducing them into the analogous position of the first helix-loop-helix (HLH) motif of calmodulin, based on homology between the calcium binding domains of GPIIb and the calcium binding loops of HLH-containing proteins. The modeling revealed that as the side chain of the introduced amino acid increased in size, it progressively interfered with hydrophobic interactions between the incoming and outgoing helices of the motif. To test whether this observation also applies to GPIIb, V286, located immediately distal to the first GPIIb calcium binding domain, was replaced by Asp and Phe. Expression of these mutants in COS-1 cells also resulted in the intracellular retention of GPIIb-IIIa, suggesting that interactions between sequences that flank the first calcium binding domain of GPIIb affect its folding. Finally, the endoplasmic reticulum chaperone BiP was detected in immunoprecipitates of GPIIb-IIIa containing GPIIb with Ala, Val, Lys, or Pro, but not Gly, at position 273. This suggests that although BiP binding is a sensitive indication of the fidelity of GPIIb-IIIa folding, it is not sufficient to account for the intracellular retention of the heterodimer.