RESUMO
The effect of the protease-activated receptor-1 (PAR-1) antagonist vorapaxar on human bleeding time is not known. This was a randomized, two-period, open-label trial in healthy men (n = 31) and women (n = 5). In period 1, subjects received 81 mg aspirin q.d. or a vorapaxar regimen achieving steady-state plasma concentrations equivalent to chronic 2.5 mg q.d. doses, for 7 days. In period 2, each group added 7 days of the therapy alternate to that of period 1 without washout. Bleeding time and platelet aggregation using arachidonic acid, ADP, and TRAP agonists were assessed. Bleeding time geometric mean ratio (90% CI) for vorapaxar/baseline was 1.01 (0.88-1.15), aspirin/baseline was 1.32 (1.15-1.51), vorapaxar + aspirin/vorapaxar was 1.47 (1.26-1.70), and vorapaxar + aspirin/aspirin was 1.12 (0.96-1.30). Unlike aspirin, vorapaxar did not prolong bleeding time compared with baseline. Bleeding time following administration of vorapaxar with aspirin was similar to that following aspirin alone.
Assuntos
Aspirina/farmacologia , Voluntários Saudáveis , Lactonas/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Piridinas/farmacologia , Difosfato de Adenosina/farmacologia , Adulto , Ácido Araquidônico/farmacologia , Aspirina/administração & dosagem , Aspirina/efeitos adversos , Tempo de Sangramento , Testes de Coagulação Sanguínea , Quimioterapia Combinada , Feminino , Humanos , Lactonas/administração & dosagem , Lactonas/sangue , Lactonas/farmacocinética , Masculino , Pessoa de Meia-Idade , Piridinas/administração & dosagem , Piridinas/sangue , Piridinas/farmacocinética , Receptores de Trombina/agonistas , Adulto JovemRESUMO
UNLABELLED: Agonist-induced inside-out signaling activates platelet integrin alpha(IIb)beta(3), rendering it to bind plasma fibrinogen (Fg). Fg binding induces outside-in signaling that culminates in platelet aggregation, leading to physiological hemostasis and pathological thrombosis. How outside-in signaling through alpha(IIb)beta(3) regulates hemostasis and thrombosis is not well understood. We have previously shown that CIB1 is involved in regulating alpha(IIb)beta(3) function. OBJECTIVE: To determine the in vivo role of CIB1 in the process of hemostasis and thrombosis. METHODS AND RESULTS: Genetic ablation of Cib1 significantly increased mouse tail bleeding time. Greater than 50% of the Cib1 null mice showed a rebleeding phenotype. Time taken for complete occlusion of carotid artery upon 10% FeCl(3)-induced injury was significantly delayed in the absence of Cib1. This was also associated with unstable thrombus formation. The inside-out signaling appears normal as ADP-, collagen- and PAR4 peptide-induced aggregation and fibrinogen binding was unaffected. The absence of Cib1 also affected the ability of platelets to spread on immobilized Fg, but not filopodia formation. Spreading could be restored in Cib1 null platelets by the addition of exogenous ADP. Outside-in signaling-dependent tyrosine phosphorylation of the integrin beta(3) subunit was significantly reduced in the absence of Cib1 as determined by Western blot analysis. CONCLUSION: Using gene knockout mice, we show for the first time that lack of Cib1 results in impaired thrombosis. CIB1 regulates these processes by affecting platelet spreading, but not platelet filopodia formation. These in vivo and in vitro results clearly show that CIB1 is a key regulator of thrombosis.
Assuntos
Proteínas de Ligação ao Cálcio/deficiência , Proteínas de Ligação ao Cálcio/fisiologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Transdução de Sinais , Trombose/etiologia , Animais , Plaquetas/patologia , Forma Celular , Fibrinogênio/metabolismo , Hemostasia , Camundongos , Camundongos Knockout , Adesividade Plaquetária , Trombose/patologiaRESUMO
Regulation of protein-protein interactions because of their spatial organisation in cells often shapes cell signalling responses to external stimuli, yet most current cell signalling models do not include spatial segregation of proteins beyond coarse control volumes like the cytosol or nucleus. A significant hindrance to spatial modelling of cell signalling is a lack of data describing the spatial organisation of proteins in cells. One signalling system in which spatial organisation is critical is integrin signalling, where protein interactions are restricted to small, micron-sized protein complexes that form on clusters of transmembrane integrin proteins. Using confocal microscopy and image analysis to quantify the size, shape and location of integrin clusters, the authors observed that cells exhibit large variability in these integrin cluster properties. To describe these heterogeneous populations of clusters quantitatively, the authors identified appropriate probability models to characterise the size, shape and location of integrin clusters in a population of adherent cells. The authors determined that integrin cluster sizes are lognormally distributed, integrin cluster eccentricities are beta distributed, and the distances of integrin clusters from the cell centre are gamma distributed. The authors estimated the parameters corresponding to these probability models from empirical data describing integrin clusters in a population of cells, and the resulting probability models describe the heterogeneous populations of clusters. These population models provide the means to create an accurate mathematical description of spatially localised integrin signalling compartments for use in computational models of integrin signalling.
Assuntos
Integrinas/química , Integrinas/metabolismo , Modelos Biológicos , Animais , Células CHO , Adesão Celular , Cricetinae , Cricetulus , Processamento de Imagem Assistida por Computador , Microscopia Confocal , Modelos Estatísticos , Complexos Multiproteicos , Domínios e Motivos de Interação entre Proteínas , Transdução de Sinais , Biologia de SistemasRESUMO
Junctional adhesion molecule 1 (JAM-1) was the first of a family of related proteins (JAM family) to be discovered. Two proteins with structural and sequence similarities to JAM-1, named JAM-2 and JAM-3, have been identified more recently. JAM-1 is specifically localized at the tight junctions of epithelial and endothelial cells and is involved in the regulation of junctional integrity and permeability. This function is attributed to its ability to interact in a homophilic manner. JAM-1 can also bind in a heterophilic manner as it serves as a ligand for integrin LFA-1 (CD11a/CD18), and plays a key role in the process of leukocyte transmigration. In addition, JAM-1 is also a receptor for reovirus, and is a platelet receptor involved in platelet adhesion and antibody-induced platelet aggregation. Further study of the mechanism of JAM-1 action within these diverse systems may demonstrate that JAM-1 is a key player in many different cellular functions.
Assuntos
Moléculas de Adesão Celular/fisiologia , Receptores de Superfície Celular/fisiologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Plaquetas/metabolismo , Moléculas de Adesão Celular/química , Movimento Celular , Mapeamento Cromossômico , Humanos , Leucócitos/metabolismo , Camundongos , Dados de Sequência Molecular , Conformação Proteica , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Ratos , Receptores de Superfície Celular/química , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Junções Íntimas , Distribuição TecidualRESUMO
A number of non-peptide orally active RGD mimetic prodrug such as Orbofiban, Sibrafiban, SR121566, Roxifiban and others entered into the clinical evaluation stage. Some of these agents were terminated and some are still in clinical trials. The present study examined the platelet GPIIb/IIIa binding profiles for the active form of Roxifiban, Sibrafiban, SR121566 and Orbofiban using 3H-Roxifiban active form (XV459), 3H-DMP728, 125I-Echistatin, and 125I-Fibrinogen. Either DMP728, Orbofiban, Sibrafiban, SR121566 or Roxifiban active form as well as other RGD mimetic bind to the same binding site(s) on human platelets as evident from the competitive inhibition of binding of each other to human platelet. Additionally, Roxifiban active form competed with FITC labeled GPIIb/IIIa antagonist cyclic RGD peptidomimetic (XL086) as demonstrated using confocal microscopy technique. Roxifiban active form (XV459) demonstrated the highest potency in inhibiting 3H-XV459, 3H-DMP728, 125I-Echistatin, and 125I-Fibrinogen binding to human platelets as compared to the others. Structure activity relationship within the isoxazoline Roxifiban series showed that substituent at the alpha-carbon next to the carboxy terminal represents an exosite for the affinity binding to human platelets leading to slow platelet dissociation rate. These data indicated a distinct binding profile for Roxifiban (high affinity to both activated and resting platelets associated with a relatively slow K(off)) as compared to others. These differences might determine the pharmacodynamics and pharmackokinetics of the different GPIIb/IIIa antagonists.
Assuntos
Amidinas/química , Amidinas/metabolismo , Plaquetas/metabolismo , Isoxazóis/química , Isoxazóis/metabolismo , Mimetismo Molecular , Oligopeptídeos/química , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/antagonistas & inibidores , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Ligação Competitiva , Colágeno/metabolismo , Fibrinogênio/metabolismo , Fibrinolíticos/química , Fibrinolíticos/metabolismo , Humanos , Concentração Inibidora 50 , Cinética , Ligantes , Microscopia Confocal , Ativação Plaquetária , Inibidores da Agregação Plaquetária/metabolismo , Ligação Proteica , Relação Estrutura-Atividade , TermodinâmicaRESUMO
We have previously reported the purification and characterization of a 32 kDa platelet surface glycoprotein that is recognized by the stimulatory monoclonal antibody, F11. The cDNA has been cloned and found to encode the human homolog of the murine junctional adhesion molecule, JAM; we therefore named this human homolog JAM-1. Northern blot analysis indicated that JAM-1 mRNA is expressed as multiple species, the predominant transcript being approximately 4.0 kb in size. Genetic mapping analysis using fluorescence in situ hybridization (FISH) showed that it is localized to chromosome 1q21.1-21.3. Recombinant JAM-1, when expressed in Chinese hamster ovary (CHO) cells, localized to the cell membrane with intense staining where two adjacent cells actually made contact with each other, suggesting that, similar to murine JAM, human JAM-1 may also localize at the cell-cell junction. In well-spread cells, JAM-1 co-localized with F-actin at the cell-cell contacts and at the membrane ruffles, but not at the stress fibers. Interestingly, JAM-1 localizes only to the cell-cell junctions formed by two transfected cells and not to the cell-cell junctions formed by a transfected cell with an untransfected cell, suggesting that JAM-1 may facilitate cell adhesion through homophilic binding. In addition, human platelets specifically bind to a monolayer of CHO cells expressing human JAM-1, further supporting homophilic interactions. The results presented here indicate that JAM-1, a receptor for a platelet-activating antibody, is the human homolog of the junctional adhesion molecule. JAM-1 is a single copy gene, which is constitutively expressed on various tissues and cells, and may be involved in cell to cell adhesion through homophilic interaction.
Assuntos
Moléculas de Adesão Celular , Cromossomos Humanos Par 1 , Proteínas de Membrana/genética , Receptores de Superfície Celular , Sequência de Aminoácidos , Animais , Sequência de Bases , Plaquetas/citologia , Células CHO , Adesão Celular , Mapeamento Cromossômico , Cricetinae , DNA Complementar , Humanos , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Virais , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Homologia de Sequência de Aminoácidos , Frações Subcelulares/metabolismo , Células Tumorais CultivadasRESUMO
The alphaIIbbeta3 integrin receives signals in agonist-activated platelets, resulting in its conversion to an active conformation that binds fibrinogen, thereby mediating platelet aggregation. Fibrinogen binding to alphaIIbbeta3 subsequently induces a cascade of intracellular signalling events. The molecular mechanisms of this bi-directional alphaIIbbeta3-mediated signalling are unknown but may involve the binding of proteins to the integrin cytoplasmic domains. We reported previously the sequence of a novel 22-kDa, EF-hand-containing, protein termed CIB (calcium- and integrin-binding protein) that interacts specifically with the alphaIIb cytoplasmic domain in the yeast two-hybrid system. Further analysis of numerous tissues and cell lines indicated that CIB mRNA and protein are widely expressed. In addition, isothermal titration calorimetry indicated that CIB binds to an alphaIIb cytoplasmic-domain peptide in a Ca(2+)-dependent manner, with moderate affinity (K(d), 700 nM) and 1:1 stoichiometry. In aggregated platelets, endogenous CIB and alphaIIbbeta3 translocate to the Triton X-100-insoluble cytoskeleton in a parallel manner, demonstrating that the cellular localization of CIB is regulated, potentially by alphaIIbbeta3. Thus CIB may contribute to integrin-related functions by mechanisms involving Ca(2+)-modulated binding to the alphaIIb cytoplasmic domain and changes in intracellular distribution.
Assuntos
Plaquetas/metabolismo , Proteínas de Ligação ao Cálcio , Cálcio/metabolismo , Proteínas de Transporte/metabolismo , Citoplasma/metabolismo , Citoesqueleto/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Transporte Biológico Ativo , Plaquetas/ultraestrutura , Calorimetria , Detergentes , Humanos , Dados de Sequência Molecular , Octoxinol , Reação em Cadeia da Polimerase , Ligação Proteica , Conformação Proteica , SolubilidadeRESUMO
Platelet aggregation is mediated by the platelet fibrinogen receptor, the alpha IIb beta 3 integrin (glycoprotein IIb-IIIa). This integrin has served as a prototype for studies probing structure-function relationships, cellular modulation of integrin function, and integrin-mediated signaling. Use of new model systems and molecular approaches have helped investigators to make rapid progress in our understanding of these areas, as reviewed here.
Assuntos
Complexo Glicoproteico GPIIb-IIIa de Plaquetas/química , Animais , Humanos , Ligantes , Modelos Moleculares , Agregação Plaquetária , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
The mechanism by which platelets regulate the function of integrin alphaIIbbeta3 (or GPIIb/IIIa), the platelet fibrinogen receptor, is unknown but may involve the binding of proteins or other factors to integrin cytoplasmic domains. To identify candidate cytoplasmic domain binding proteins, we screened a human fetal liver cDNA library in the yeast two-hybrid system, using the alphaIIb cytoplasmic domain as "bait," and isolated a novel 855-base pair clone. The open reading frame encodes a novel 191-amino acid polypeptide (termed CIB for calcium- and integrin-binding protein) that appears to be specific for the cytoplasmic domain of alphaIIb, since it does not interact with the alphav, alpha2, alpha5, beta1, or beta3 integrin cytoplasmic domains in the yeast two-hybrid system. This protein has sequence homology to two known Ca2+-binding regulatory proteins, calcineurin B (58% similarity) and calmodulin (56% similarity), and has two EF-hand motifs corresponding to the two C-terminal Ca2+ binding domains of these proteins. Moreover, recombinant CIB specifically binds 45Ca2+ in blot overlay assays. Using reverse transcriptase-polymerase chain reaction and Western blot analysis, we detected CIB mRNA and protein ( approximately 25 kDa), respectively, in human platelets. An enzyme-linked immunosorbent assay performed using either immobilized recombinant CIB or monoclonal antibody-captured alphaIIbbeta3 indicates a specific interaction between CIB and intact alphaIIbbeta3. These results suggest that CIB is a candidate regulatory molecule for integrin alphaIIbbeta3.
Assuntos
Proteínas de Ligação ao Cálcio/genética , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Proteínas de Ligação ao Cálcio/isolamento & purificação , Proteínas de Ligação ao Cálcio/metabolismo , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Humanos , Dados de Sequência Molecular , Análise de SequênciaRESUMO
A novel stimulatory monoclonal antibody (Mab) termed Mab.F11 induces granular secretion and subsequent aggregation of human platelets. Mab.F11 recognizes a unique 32 and 35 kDa protein duplex on the platelet membrane surface, called the F11 receptor; binding of Mab.F11 to its receptor results in increased intracellular phosphorylation of P47, the known protein kinase C (PKC) substrate pleckstrin. In order to determine whether the mechanism of action of Mab.F11 involves direct activation of PKC, two types of functional assays for measuring PKC activity were performed. Measurement of PKC activity in digitonin-permeabilized platelets revealed that Mab.F11 produced a rapid, 2-3 fold increase in the control value in the phosphorylation of the PKC peptide substrate, PKC(19-31) Ser25. The increase in PKC activity induced by Mab.F11 was found to be associated with the platelet membrane; a 1.6-fold control value increase in membrane PKC activity occurred rapidly, within 10 s of the addition of Mab.F11. The translocation from the cytoplasm to the membrane induced by Mab.F11 in PKC isoenzymes alpha and zeta was reversible, whereas translocation of the PKC isoenzymes delta, beta, eta' and theta was irreversible, with PKC levels remaining elevated in the membrane for at least 15 min. Taken together, our results demonstrate that in the initial stages of platelet activation by this stimulatory antibody, the enhanced membrane PKC activity reflects the presence of all six isoenzymes. At later stages, PKC activity is reflective of four isoenzymes. These results demonstrate that separate groups of PKC isoenzymes must be involved in different aspects of platelet activation. The long lag period and prolonged activation time of platelets by Mab.F11 renders this agonist most suitable for identifying the isoenzymes and their specific endogenous protein substrates involved in platelet secretion and aggregation induced by platelet membrane protein antibodies.
Assuntos
Anticorpos Monoclonais/farmacologia , Plaquetas/enzimologia , Isoenzimas/sangue , Proteína Quinase C/sangue , Sequência de Aminoácidos , Plaquetas/efeitos dos fármacos , Plaquetas/imunologia , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Humanos , Immunoblotting , Proteínas de Membrana/química , Proteínas de Membrana/imunologia , Dados de Sequência Molecular , Fragmentos de Peptídeos , Fosforilação , Ativação Plaquetária/fisiologia , Agregação Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/fisiologia , Receptores de IgG/fisiologiaRESUMO
The mechanisms by which a stimulatory monoclonal antibody (mAb), called mAb F11, induces granular secretion and aggregation in human platelets have been characterized. Fab fragments of mAb F11, as well as an mAb directed against the platelet Fc gamma RII receptor (mAb IV.3) were found to inhibit mAb F11-induced platelet secretion and aggregation, indicating that the mAb F11 IgG molecule interacts with the Fc gamma RII receptor through its Fc domain and with its own antigen through its Fab domain. The mAb F11 recognized two platelet proteins of 32 and 35 kDa on the platelet membrane surface, as identified by Western blot analysis. We purified both proteins from human platelet membranes using DEAE-Sepharose chromatography followed by mAb F11 affinity chromatography. When added to platelet-rich plasma, the purified proteins dose-dependently inhibited mAb F11-induced platelet aggregation. The purified protein preparation also competitively inhibited the binding of 125I-labelled mAb F11 to intact platelets. The N-terminal 26 amino acid sequences of both the 32 and 35 kDa proteins were identical and contained a single unblocked serine in the N-terminal position. When digested with N-glycanase, the 32 and 35 kDa proteins were converted into a single approximately 29 kDa protein, indicating that these two proteins are derived from the same core protein but differ in their degree of glycosylation. Internal amino acid sequence analysis of the F11 antigen provided information concerning 68 amino acids and suggested two consensus phosphorylation sites for protein kinase C (PKC). The phosphorylation by PKC of the isolated F11 antigen was observed following stimulation by phorbol 12-myristate 13-acetate. Databank analysis of the N-terminal and internal amino acid sequences of the F11 antigen indicated that the N-terminal sequence exhibited the highest degree of similarity to the variable region of the alpha-chain of human T-cell receptors (TCR). In contrast, the F11 internal sequences did not exhibit any similarity to the TCR. Our results demonstrate that the F11 antigen is a novel platelet membrane surface glycoprotein which becomes cross-linked with the Fc gamma RII receptor when platelets are activated by the stimulatory mAb F11. These mechanisms may be relevant to the production of immune thrombocytopenia by platelet-activating antibodies.
Assuntos
Anticorpos Monoclonais/imunologia , Ativação Plaquetária/imunologia , Receptores de IgG/imunologia , Sequência de Aminoácidos , Antígenos/isolamento & purificação , Antígenos/metabolismo , Plaquetas/imunologia , Humanos , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional , Homologia de Sequência de AminoácidosRESUMO
We have detected in human platelets two protein kinase C isozymes that have not been reported previously. Using an anti-nPKC theta antibody and Western blotting, we calculated the molecular weight of platelet nPKC theta as 79K. This molecular weight is identical to that described for nPKC theta in skeletal muscle and in COS cells transfected with the nPKC theta-cDNA. Using an anti-nPKC eta antibody, we determined the molecular weight of an immunoreactive protein, which we called nPKC eta', to be 95K. This molecular weight is higher than that of nPKC eta found in lung and skin tissue of 82K and 78K, and it is higher than nPKC eta of COS cells transfected with the nPKC eta-cDNA expression plasmid. Together with previous reports, these findings make the total number of PKC isozymes in human platelets equal to six. These are the PKC isozymes: alpha, beta, delta and zeta, which have been previously described, and eta' and theta which we describe here. To assess the functionality of these new PKC isoforms, we stimulated platelets with PAF. We found a 200% and 175% increase in the levels of membrane-bound nPKC eta' and nPKC theta, respectively, in human platelets stimulated by PAF. A concomitant decrease in the level of these isoforms in the cytoplasm was observed. This PAF-induced translocation was time-dependent, and it reached its peak after a 1 minute incubation of human platelets with PAF for nPKC theta and 30 seconds for nPKC eta'.
Assuntos
Plaquetas/enzimologia , Isoenzimas/sangue , Fator de Ativação de Plaquetas/farmacologia , Proteína Quinase C/sangue , Transdução de Sinais/efeitos dos fármacos , Sequência de Aminoácidos , Anticorpos , Anticorpos Monoclonais , Plaquetas/efeitos dos fármacos , Plaquetas/fisiologia , Western Blotting , Membrana Celular/enzimologia , Citosol/enzimologia , Eletroforese em Gel de Poliacrilamida , Humanos , Técnicas In Vitro , Isoenzimas/isolamento & purificação , Cinética , Dados de Sequência Molecular , Peso Molecular , Peptídeos/síntese química , Peptídeos/imunologia , Proteína Quinase C/isolamento & purificação , Fatores de TempoRESUMO
We have determined that ADP-induced platelet aggregation and secretion are enhanced by preincubation of human platelets with the immunosuppressant cyclosporine A (CSA) (see accompanying article by Naik et al., 1993). In the present report we compare the effects of CSA with two other potent immunosuppressants, cyclosporine G (CSG), an analogue of CSA, and FK-506, on platelet activation. Preincubation of platelets with either CSA, CSG, or FK-506 resulted in platelets which exhibited hyperaggregability when stimulated by ADP. CSG produced the lowest degree of hyperaggregation, whereas FK-506 produced the highest at an equivalent concentration. All three compounds enhanced the secretion of serotonin, with FK-506 producing the highest degree of serotonin release and CSG producing the lowest amount. Platelet hyperaggregation and enhanced secretion were both time- and dose-dependent. Comparative studies performed with CSA and CSG indicated that a short preincubation period with CSA (2.5 min) resulted in a 90% enhancement of ADP-induced platelet aggregation, whereas CSG enhancement was only 20%. At a concentration of 600 ng/ml, CSA produced 120% enhancement of ADP-induced platelet aggregation, whereas CSG produced only a 30% enhancement. Several potential mechanisms responsible for the enhanced ADP-induced aggregation and secretion were investigated. Determination of the binding of two radiolabeled probes directed against the fibrinogen receptor, fibrinogen itself, and a monoclonal antibody (M.Ab.G10) revealed that the binding of fibrinogen to ADP-stimulated platelets was enhanced by 50% following the preincubation of platelets with 600 ng/ml CSA. Smaller, yet significant enhancement occurred in the presence of CSG. Similar results were obtained when M.Ab.G10 was used. Preincubation of platelets with 600 ng/ml CSA or CSG resulted in 60% and 30% increase in total protein kinase C activity, respectively, following the addition of ADP. In conclusion, this study has determined that preincubation of platelets with CSA or FK-506 (CSG, to a smaller extent) results in significant enhancement of ADP-induced platelet aggregation and secretion. The hyperaggregation and hypersecretion observed may be due to an enhanced expression of fibrinogen receptors on the platelet surface resulting from an increase in protein kinase C activity.
Assuntos
Ciclosporina/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Tacrolimo/farmacologia , Anticorpos Monoclonais , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Humanos , Técnicas In Vitro , Agregação Plaquetária/efeitos dos fármacos , Proteína Quinase C/metabolismo , Serotonina/metabolismoRESUMO
Use of the immunosuppressant drug cyclosporine A (CSA) has resulted in improved renal graft survival. However, an increased incidence of arterial and venous thrombotic diseases, hemolytic-uremic type syndrome, and findings resembling vasculitis in the kidneys of patients with CSA nephrotoxicity and accelerated atherogenesis have been reported. These disorders may be related to CSA-induced abnormalities in platelet function. We report here that CSA causes increased ADP-stimulated aggregation in isolated platelet suspensions indicating that CSA has a direct effect on platelet function, independent of CSA interactions with plasma factors. Maximal hyperaggregability of ADP-stimulated platelets occurred following a 1 h preincubation period with CSA. Hyperaggregability of platelets due to the presence of CSA was dose-dependent and approached plateau between 200-500 ng/ml CSA. We determined that CSA exerted its effects through a signal transduction pathway involving the phosphorylation of two intracellular proteins, a 40 kD substrate of PKC (p47) and the 20 kD light chain of myosin (p20), a substrate of calcium/calmodulin dependent kinase. Preincubation with CSA resulted in a 200% increase in the phosphorylation of these proteins in platelets stimulated with ADP. We conclude that CSA enhances ADP-induced platelet aggregation and secretion, in part, by potentiating the phosphorylative response of specific intracellular proteins to stimulation by agonists. This process may be responsible for the increased thrombosis and atherogenesis observed in CSA-treated patients.
Assuntos
Ciclosporina/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Difosfato de Adenosina/metabolismo , Humanos , Técnicas In Vitro , Cinética , Fosforilação/efeitos dos fármacos , Proteínas/metabolismo , Serotonina/metabolismo , Transdução de SinaisRESUMO
Several methodologies used for the identification and characterization of platelet receptors for antiplatelet monoclonal antibodies are compared. Two antiplatelet monoclonal antibodies, are investigated due to their potent effects on human platelet function. A platelet-activating monoclonal antibody, called M.Ab.F11, is able to induce platelet aggregation and granular secretion. A platelet-inhibitory monoclonal antibody, named G10, strongly blocks the platelet aggregation and granular secretion induced by M.Ab.F11, as well as by physiological agonists. In order to identify the specific antigens recognized by these monoclonal antibodies, we tested a number of immunostaining methods. Comparison of various procedures revealed that a high degree of nonspecific interactions with platelet proteins occurred when the commonly used secondary reagents, protein A and radiolabeled or enzyme-conjugated secondary antibodies interacted with the platelet proteins either in the presence or absence of primary monoclonal antibodies. On the other hand, we observed a high degree of specificity and selectivity when only radiolabeled anti-platelet monoclonal antibodies were used as single reagents. It is established that M.Ab.F11 interacts with the platelet membrane proteins of 32 and 35 Kd, and M.Ab.G10 recognizes 100 Kd protein, which corresponds to GPIIIa molecule.
Assuntos
Anticorpos Monoclonais , Plaquetas/química , Immunoblotting/métodos , Glicoproteínas da Membrana de Plaquetas/análise , Autorradiografia , Humanos , Radioisótopos do Iodo , Métodos , Sensibilidade e EspecificidadeRESUMO
We have characterized a novel ecto-protein kinase activity and a novel ecto-protein phosphatase activity on the membrane surface of human platelets. Washed intact platelets, when incubated with [gamma-32P]ATP in Tyrode's buffer, showed the phosphorylation of a membrane surface protein migrating with an apparent molecular mass of 42 kDa on 5-15% SDS polyacrylamide gradient gels. The 42 kDa protein could be further resolved on 15% SDS gels into two proteins of 39 kDa and 42 kDa. In this gel system, it was found that the 39 kDa protein became rapidly phosphorylated and dephosphorylated, whereas the 42 kDa protein was phosphorylated and dephosphorylated at a much slower rate. NaF inhibited the dephosphorylation of these proteins indicating the involvement of an ecto-protein phosphatase. The platelet membrane ecto-protein kinase responsible for the phosphorylation of both of these proteins was identified as a serine kinase and showed dependency on divalent cations Mg2+ or Mn2+ ions. Ca2+ ions potentiated the Mg(2+)-dependent ecto-protein kinase activity. The ecto-protein kinase rapidly phosphorylated histone and casein added exogenously to the extracellular medium of intact platelets. Following activation of platelets by alpha-thrombin, the incorporation of [32P]phosphate from exogenously added [gamma-32P]ATP by endogenous protein substrates was reduced by 90%, suggesting a role of the ecto-protein kinase system in the regulation of platelet function. The results presented here demonstrate that both protein kinase and protein phosphatase activities reside on the membrane surface of human platelets. These activities are capable of rapidly phosphorylating and dephosphorylating specific surface platelet membrane proteins which may play important roles in early events of platelet activation and secretion.
Assuntos
Plaquetas/metabolismo , Fosfoproteínas Fosfatases/sangue , Fosfoproteínas/sangue , Proteínas Quinases/sangue , Trifosfato de Adenosina/sangue , Plaquetas/efeitos dos fármacos , Cálcio/farmacologia , Caseínas/metabolismo , Membrana Celular/enzimologia , Eletroforese em Gel de Poliacrilamida , Histonas/metabolismo , Humanos , Cinética , Magnésio/farmacologia , Manganês/farmacologia , Peso Molecular , Fosforilação , Proteínas Serina-Treonina Quinases , Fluoreto de Sódio/farmacologia , Trombina/farmacologiaRESUMO
The clinical significance of the interaction of antibodies with circulating platelets is well documented, but the mechanisms underlying these interactions are not fully known. Here we describe the characterization of anti-human platelet membrane protein monoclonal antibody (mAb) termed F11. Interaction of mAb F11 with human platelets resulted in dose-dependent granular secretion, measured by [14C]serotonin and ATP release, fibrinogen binding and aggregation. Analysis of the specific binding of mAb F11 to platelets revealed a high affinity site with 8,067 +/- 1,307 sites per platelet with a dissociation constant (Kd) of 2.7 +/- 0.9 x 10(-8) M. Two membrane proteins of 32,000 and 35,000 daltons, identified by Western blotting, were recognized by mAb F11. Incubation of 32Pi-labeled platelets with mAb F11 resulted in rapid phosphorylation of intracellular 40,000- and 20,000-dalton proteins, followed by dephosphorylation of these proteins. Monovalent Fab fragments or Fc fragments of mAb F11 IgG did not induce platelet aggregation or secretion; however, Fab fragments of mAb F11 IgG blocked mAb F11-induced platelet aggregation and the binding of 125I-mAb F11 to platelets. The addition of an anti-GPIIIa monoclonal antibody (mAb G10), which inhibits 125I-fibrinogen binding and platelet aggregation, completely blocked mAb F11-induced [14C]serotonin secretion and aggregation but not the binding of 125I-mAb F11 to platelets. mAb G10 also inhibited the increase in the phosphorylation of the 40,000- and 20,000-dalton proteins induced by mAb F11. These results implicate the involvement of the GPIIIa molecule in the chain of biochemical events involved in the induction of granular secretion.