RESUMEN
Snake venoms are complex mixtures of biologically active proteins and peptides. Many of them affect hemostasis by activating or inhibiting coagulant factors or platelets, or by disrupting endothelium. Based on sequence, these snake venom components have been classified into various families, such as serine proteases, metalloproteinases, C-type lectins, disintegrins and phospholipases. The various members of a particular family act selectively on different blood coagulation factors, blood cells or tissues. For almost every factor involved in coagulation or fibrinolysis there is a venom protein that can activate or inactivate it. Venom proteins affect platelet function by binding or degrading vWF or platelet receptors, activating protease-activated receptors or modulating ADP release and thromboxane A2 formation. Some venom enzymes cleave key basement membrane components and directly affect capillary blood vessels to cause hemorrhaging. L-Amino acid oxidases activate platelets via H2O2 production.
Asunto(s)
Hemostasis , Venenos de Serpiente/metabolismo , Aminoácido Oxidorreductasas/metabolismo , Animales , Coagulación Sanguínea , Factores de Coagulación Sanguínea/metabolismo , Plaquetas/metabolismo , Endotelio/metabolismo , Endotelio Vascular/citología , Humanos , Peróxido de Hidrógeno/farmacología , Integrinas/química , Lectinas/química , Modelos Biológicos , Fosfolipasas/química , Unión Proteica , Tromboxano A2/químicaRESUMEN
Recent studies have implied that GPIb-IX-V as well as functioning as an adhesion receptor may also induce signaling to mediate binding of platelets to damaged vessel wall to prevent bleeding. Reorganization of the cytoskeleton and redistribution of platelet structural proteins and signaling molecules are thought to be important in this early activation process, though the molecular mechanisms remain to be fully defined. In this study, we have used mucetin, a snake venom lectin protein that activates platelets via GPIb, to study the redistribution of GPIb in platelets. In unstimulated platelets, a minor portion of GPIb localized to Triton-insoluble cytoskeleton fractions (TIC). This portion increased considerably after platelet activation by mucetin. We also find increased contents of the FcRgamma chain in TIC. Anti-GPIb antibodies, mocarhagin or cytochalasin D completely inhibited the cytoskeletal translocation. In addition, BAPTA-AM, a cytoplasmic calcium chelator, strongly inhibited this process. On the other hand, inhibitors of alphaIIbbeta3, PLCgamma, PKC, tyrosine kinases, ADP receptor, PI3-kinase or EDTA are effective in preventing GPIb relocation in convulxin- but not in mucetin-activated platelets. We propose that cytoskeletal translocation of GPIb is upstream of alphaIIbbeta3 activation and cross-linking of GPIb is sufficient to induce this event in mucetin-activated platelets.
Asunto(s)
Venenos de Crotálidos/farmacología , Citoesqueleto/metabolismo , Activación Plaquetaria/efectos de los fármacos , Complejo GPIb-IX de Glicoproteína Plaquetaria/metabolismo , Receptores de IgG/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Lectinas Tipo C , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Transporte de Proteínas , Venenos de Víboras/farmacologíaRESUMEN
Collagens are important platelet activators in the vascular subendothelium and vessel wall. Since the regulation of platelet activation is a key step in distinguishing normal haemostasis from pathological thrombosis, collagen interactions with platelets are important targets for pharmacological control. Platelets have two major receptors for collagens, the integrin alpha2beta1, with a major role in adhesion and platelet anchoring and the Ig superfamily member, GPVI, principally responsible for signalling and platelet activation. In addition, GPIb-V-IX, can be considered as an indirect collagen receptor acting via von Willebrand factor as bridging molecule and is essential for platelet interactions with collagen at high shear rates. There is some evidence for additional receptors, which may regulate the response to individual collagen types. This review discusses how these receptors work separately with specific agonists and proposes possible mechanisms for how they work together to regulate platelet activation by collagen, which remains controversial and poorly understood.
Asunto(s)
Integrinas/sangre , Glicoproteínas de Membrana Plaquetaria/fisiología , Humanos , Integrinas/química , Activación Plaquetaria/efectos de los fármacos , Complejo GPIb-IX de Glicoproteína Plaquetaria/química , Complejo GPIb-IX de Glicoproteína Plaquetaria/fisiología , Glicoproteínas de Membrana Plaquetaria/química , Receptores de Colágeno , Transducción de Señal/efectos de los fármacosRESUMEN
Aggretin, a potent platelet activator, was isolated from Calloselasma rhodostoma venom, and 30-amino acid N-terminal sequences of both subunits were determined. Aggretin belongs to the heterodimeric snake C-type lectin family and is thought to activate platelets by binding to platelet glycoprotein alpha(2)beta(1). We now show that binding to glycoprotein (GP) Ib is also required. Aggretin-induced platelet activation was inhibited by a monoclonal antibody to GPIb as well as by antibodies to alpha(2)beta(1). Binding of both of these platelet receptors to aggretin was confirmed by affinity chromatography. No binding of other major platelet membrane glycoproteins, in particular GPVI, to aggretin was detected. Aggretin also activates platelets from Fc receptor gamma chain (Fcgamma)-deficient mice to a greater extent than those from normal control mice, showing that it does not use the GPVI/Fcgamma pathway. Platelets from Fcgamma-deficient mice expressed fibrinogen receptors normally in response to collagen, although they did not aggregate, indicating that these platelets may partly compensate via other receptors including alpha(2)beta(1) or GPIb for the lack of the Fcgamma pathway. Signaling by aggretin involves a dose-dependent lag phase followed by rapid tyrosine phosphorylation of a number of proteins. Among these are p72(SYK), p125(FAK), and PLCgamma2, whereas, in comparison with collagen and convulxin, the Fcgamma subunit neither is phosphorylated nor coprecipitates with p72(SYK). This supports an independent, GPIb- and integrin-based pathway for activation of p72(SYK) not involving the Fcgamma receptor.
Asunto(s)
Plaquetas/fisiología , Precursores Enzimáticos/sangre , Integrinas/sangre , Integrinas/fisiología , Isoenzimas/sangre , Lectinas Tipo C , Lectinas/farmacología , Activación Plaquetaria/fisiología , Complejo GPIb-IX de Glicoproteína Plaquetaria/fisiología , Proteínas Tirosina Quinasas/sangre , Fosfolipasas de Tipo C/sangre , Venenos de Víboras/química , Venenos de Víboras/farmacología , Agkistrodon , Secuencia de Aminoácidos , Animales , Plaquetas/efectos de los fármacos , Cromatografía de Afinidad , Colágeno/farmacología , Venenos de Crotálidos/farmacología , Humanos , Técnicas In Vitro , Péptidos y Proteínas de Señalización Intracelular , Lectinas/química , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Fosfolipasa C gamma , Fosforilación , Fosfotirosina/sangre , Activación Plaquetaria/efectos de los fármacos , Complejo GPIb-IX de Glicoproteína Plaquetaria/efectos de los fármacos , Subunidades de Proteína , Receptores de Colágeno , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Quinasa Syk , Venenos de Víboras/aislamiento & purificaciónRESUMEN
Echicetin, a heterodimeric snake C-type lectin from Echis carinatus, is known to bind specifically to platelet glycoprotein (GP)Ib. We now show that, in addition, it agglutinates platelets in plasma and induces platelet signal transduction. The agglutination is caused by binding to a specific protein in plasma. The protein was isolated from plasma and shown to cause platelet agglutination when added to washed platelets in the presence of echicetin. It was identified as immunoglobulin Mkappa (IgMkappa) by peptide sequencing and dot blotting with specific heavy and light chain anti-immunoglobulin reagents. Platelet agglutination by clustering echicetin with IgMkappa induced P-selectin expression and activation of GPIIb/IIIa as well as tyrosine phosphorylation of several signal transduction molecules, including p53/56(LYN), p64, p72(SYK), p70 to p90, and p120. However, neither ethylenediaminetetraacetic acid nor specific inhibition of GPIIb/IIIa affected platelet agglutination or activation by echicetin. Platelet agglutination and induction of signal transduction could also be produced by cross-linking biotinylated echicetin with avidin. These data indicate that clustering of GPIb alone is sufficient to activate platelets. In vivo, echicetin probably activates platelets rather than inhibits platelet activation, as previously proposed, accounting for the observed induction of thrombocytopenia.
Asunto(s)
Inmunoglobulina M/metabolismo , Cadenas kappa de Inmunoglobulina/metabolismo , Agregación Plaquetaria/efectos de los fármacos , Proteínas/química , Transducción de Señal/efectos de los fármacos , Tirosina/análogos & derivados , Venenos de Víboras/química , Acetatos/farmacología , Animales , Aspirina/farmacología , Avidina/farmacología , Sitios de Unión , Biotinilación , Proteínas Sanguíneas/metabolismo , Proteínas Portadoras , Quelantes/farmacología , Ácido Edético/farmacología , Fibrinógeno/metabolismo , Lectinas/química , Lectinas/farmacología , Sustancias Macromoleculares , Selectina-P/biosíntesis , Fosforilación/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Unión Proteica , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Tirosina Quinasas/metabolismo , Proteínas/farmacología , Tirosina/farmacologíaRESUMEN
The snake venom C-type lectin alboaggregin A (or 50-kd alboaggregin) from Trimeresurus albolabris was previously shown to be a platelet glycoprotein (GP) Ib agonist. However, investigations of the signal transduction induced in platelets showed patterns of tyrosine phosphorylation that were different from those of other GPIb agonists and suggested the presence of an additional receptor. In this study, the binding of biotinylated alboaggregin A to platelet lysates, as well as affinity chromatography evaluations of platelet lysates on an alboaggregin A-coated column, indicated that this other receptor is GPVI. Additional experiments with reagents that inhibit either GPIb or GPVI specifically supported this finding. These experiments also showed that both GPIb and GPVI have a role in the combined signaling and that the overall direction this takes can be influenced by inhibitors of one or the other receptor pathway.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Venenos de Crotálidos/farmacología , Lectinas Tipo C , Proteínas de la Membrana , Activación Plaquetaria/efectos de los fármacos , Complejo GPIb-IX de Glicoproteína Plaquetaria/agonistas , Glicoproteínas de Membrana Plaquetaria/fisiología , Transducción de Señal/efectos de los fármacos , Adulto , Anexina A5/metabolismo , Biotinilación , Coagulación Sanguínea/efectos de los fármacos , Plaquetas/metabolismo , Proteínas Portadoras/metabolismo , Cromatografía de Afinidad , Venenos de Crotálidos/aislamiento & purificación , Venenos de Crotálidos/metabolismo , Gránulos Citoplasmáticos/metabolismo , Humanos , Fragmentos Fab de Inmunoglobulinas/farmacología , Isoenzimas/metabolismo , Fosfolipasa C gamma , Fosfoproteínas/metabolismo , Fosforilación/efectos de los fármacos , Activación Plaquetaria/fisiología , Adhesividad Plaquetaria/efectos de los fármacos , Glicoproteínas de Membrana Plaquetaria/efectos de los fármacos , Glicoproteínas de Membrana Plaquetaria/inmunología , Unión Proteica , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas/farmacología , Receptores de IgG/metabolismo , Fosfolipasas de Tipo C/metabolismo , Venenos de Víboras/químicaRESUMEN
A new snake protein, named bilinexin, has been purified from Agkistrodon bilineatus venom by ion-exchange chromatography and gel filtration chromatography. Under non-reducing conditions it has a mass of 110 kDa protein on SDS-PAGE. On reduction, it can be separated into five subunits with masses in the range 13-25 kDa. The N-terminal sequences of these subunits are very similar to those of convulxin or the alboaggregins, identifying bilinexin as a new member of the snake C-type lectin family, unusual in having multiple subunits. Bilinexin agglutinates fixed platelets. washed platelets and platelet rich plasma (PRP) without obvious activation (shape change) as confirmed by light microscope examination. Both inhibitory and binding studies indicate that antibodies against alpha2beta1 inhibit not only platelet agglutination induced by bilinexin, but also bilinexin binding to platelets. VM16d, a monoclonal anti-GPIbalpha antibody, completely inhibits platelet agglutination induced by bilinexin, and polyclonal antibodies against GPIbalpha prevent its binding to platelets. However, neither convulxin, polyclonal anti-GPVI antibodies, nor GPIIb/IIIa inhibitors affect its binding to and agglutination of platelets. Bilinexin neither activates GPIIb/IIIa integrin on platelets nor induces tyrosine phosphorylation of platelet proteins, nor increases intracellular Ca2+ in platelets. Like alboaggregin B, bilinexin agglutinates platelets, which makes it a good tool to investigate the differences in mechanism between snake C-type lectins causing platelet agglutination and those that induce full activation.
Asunto(s)
Plaquetas/efectos de los fármacos , Venenos de Crotálidos/metabolismo , Venenos de Crotálidos/farmacología , Integrina alfa2beta1/fisiología , Lectinas Tipo C/metabolismo , Agregación Plaquetaria/efectos de los fármacos , Complejo GPIb-IX de Glicoproteína Plaquetaria/fisiología , Secuencia de Aminoácidos , Animales , Anticuerpos/farmacología , Plaquetas/ultraestructura , Venenos de Crotálidos/química , Venenos de Crotálidos/aislamiento & purificación , Humanos , Integrina alfa2beta1/inmunología , Lectinas Tipo C/química , Lectinas Tipo C/aislamiento & purificación , Peso Molecular , Complejo GPIb-IX de Glicoproteína Plaquetaria/inmunología , Subunidades de ProteínaRESUMEN
Snake venoms contain a wide range of components, many of which affect haemostasis by activation or inhibition of platelets or coagulation factors. They can be classified into groups based on structure and mode of action. One group is the snake C-type lectins, so called because of the typical folding which closely resembles that found in classical C-type lectins, such as selectins and mannose-binding proteins. Unlike the classic C-type lectins, those from snakes are generally heterodimeric with two subunits, alpha and beta. Some are multimeric heterodimers. The subunits have homologous sequences and are generally linked by a disulphide bond as well as by swapping loops. One of the first C-type lectins with a defined function was echicetin which was demonstrated to bind to platelet GPIb and block several functions of this receptor. Since then, many proteins with similar structure have been reported to act on platelet receptors or coagulation factors and several have been crystallized. These proteins were thought to be specific for a single platelet receptor or coagulation factor, i.e. they had only one receptor per heterodimer. Recent studies show that most of these C-type lectins have binding sites for more than one ligand and have complex mechanisms of action.
Asunto(s)
Plaquetas/efectos de los fármacos , Lectinas Tipo C/química , Venenos de Serpiente/química , Animales , Sitios de Unión , Humanos , Lectinas Tipo C/metabolismo , Glicoproteínas de Membrana Plaquetaria/metabolismo , Venenos de Serpiente/metabolismo , Venenos de Serpiente/farmacología , Relación Estructura-ActividadRESUMEN
Platelets are known to contain platelet factor 4 and beta-thromboglobulin, alpha-chemokines containing the CXC motif, but recent studies extended the range to the beta-family characterized by the CC motif, including RANTES and Gro-alpha. There is also evidence for expression of chemokine receptors CCR4 and CXCR4 in platelets. This study shows that platelets have functional CCR1, CCR3, CCR4, and CXCR4 chemokine receptors. Polymerase chain reaction detected chemokine receptor messenger RNA in platelet RNA. CCR1, CCR3, and especially CCR4 gave strong signals; CXCR1 and CXCR4 were weakly positive. Flow cytometry with specific antibodies showed the presence of a clear signal for CXCR4 and weak signals for CCR1 and CCR3, whereas CXCR1, CXCR2, CXCR3, and CCR5 were all negative. Immunoprecipitation and Western blotting with polyclonal antibodies to cytoplasmic peptides clearly showed the presence of CCR1 and CCR4 in platelets in amounts comparable to monocytes and CCR4 transfected cells, respectively. Chemokines specific for these receptors, including monocyte chemotactic protein 1, macrophage inflammatory peptide 1alpha, eotaxin, RANTES, TARC, macrophage-derived chemokine, and stromal cell-derived factor 1, activate platelets to give Ca(++) signals, aggregation, and release of granule contents. Platelet aggregation was dependent on release of adenosine diphosphate (ADP) and its interaction with platelet ADP receptors. Part, but not all, of the Ca(++) signal was due to ADP release feeding back to its receptors. Platelet activation also involved heparan or chondroitin sulfate associated with the platelet surface and was inhibited by cleavage of these glycosaminoglycans or by heparin or low molecular weight heparin. These platelet receptors may be involved in inflammatory or allergic responses or in platelet activation in human immunodeficiency virus infection.
Asunto(s)
Adenosina Trifosfato/análogos & derivados , Plaquetas/metabolismo , Receptores CXCR4/biosíntesis , Receptores de Quimiocina/biosíntesis , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/farmacología , Adulto , Plaquetas/efectos de los fármacos , Western Blotting , Señalización del Calcio , Quimiocinas/farmacología , Condroitina ABC Liasa/farmacología , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Precursores Enzimáticos/metabolismo , Retroalimentación , Citometría de Flujo , Regulación de la Expresión Génica , Heparina/farmacología , Liasa de Heparina/farmacología , Heparina de Bajo-Peso-Molecular/farmacología , Humanos , Péptidos y Proteínas de Señalización Intracelular , Isoenzimas/metabolismo , Fragmentos de Péptidos/farmacología , Fosfolipasa C gamma , Fosforilación/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/fisiología , Reacción en Cadena de la Polimerasa , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Tirosina Quinasas/metabolismo , Antagonistas del Receptor Purinérgico P2 , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Receptores CCR1 , Receptores CCR3 , Receptores CCR4 , Receptores CXCR4/genética , Receptores de Quimiocina/genética , Receptores de IgG/metabolismo , Receptores Purinérgicos P2/fisiología , Quinasa Syk , Tromboxanos/biosíntesis , Transfección , Fosfolipasas de Tipo C/metabolismoRESUMEN
We have cloned the platelet collagen receptor glycoprotein (GP) VI from a human bone marrow cDNA library using rapid amplification of cDNA ends with platelet mRNA to complete the 5' end sequence. GPVI was isolated from platelets using affinity chromatography on the snake C-type lectin, convulxin, as a critical step. Internal peptide sequences were obtained, and degenerate primers were designed to amplify a fragment of the GPVI cDNA, which was then used as a probe to screen the library. Purified GPVI, as well as Fab fragments of polyclonal antibodies made against the receptor, inhibited collagen-induced platelet aggregation. The GPVI receptor cDNA has an open reading frame of 1017 base pairs coding for a protein of 339 amino acids including a putative 23-amino acid signal sequence and a 19-amino acid transmembrane domain between residues 247 and 265. GPVI belongs to the immunoglobulin superfamily, and its sequence is closely related to FcalphaR and to the natural killer receptors. Its extracellular chain has two Ig-C2-like domains formed by disulfide bridges. An arginine residue is found in position 3 of the transmembrane portion, which should permit association with Fcgamma and its immunoreceptor tyrosine-based activation motif via a salt bridge. With 51 amino acids, the cytoplasmic tail is relatively long and shows little homology to the C-terminal part of the other family members. The ability of the cloned GPVI cDNA to code for a functional platelet collagen receptor was demonstrated in the megakaryocytic cell line Dami. Dami cells transfected with GPVI cDNA mobilized intracellular Ca(2+) in response to collagen, unlike the nontransfected or mock transfected Dami cells, which do not respond to collagen.
Asunto(s)
Integrinas/genética , Lectinas Tipo C , Glicoproteínas de Membrana Plaquetaria/genética , Secuencia de Aminoácidos , Secuencia de Bases , Calcio/metabolismo , Línea Celular , Clonación Molecular , Venenos de Crotálidos/metabolismo , Humanos , Fragmentos Fab de Inmunoglobulinas/inmunología , Integrinas/química , Datos de Secuencia Molecular , Agregación Plaquetaria , Glicoproteínas de Membrana Plaquetaria/química , Receptores de Colágeno , Alineación de Secuencia , TransfecciónRESUMEN
Cardiovascular diseases involve abnormal cell-cell interactions leading to the development of atherosclerotic plaque, which when ruptured causes massive platelet activation and thrombus formation. Parts of a loose thrombus may detach to form an embolus, blocking circulation at a more distant point. The integrins are a family of adhesive cell receptors interacting with adhesive proteins or with counterreceptors on other cells. There is now solid evidence that the major integrin on platelets, the fibrinogen receptor alpha IIb beta 3, has an important role in several aspects of cardiovascular diseases and that its regulated inhibition leads to a reduction in incidence and mortality due to these disorders. The development of alpha IIb beta 3 inhibitors is an important strategy of many pharmaceutical companies which foresee a large market for the treatment of acute conditions in surgery, the symptoms of chronic conditions and, it is hoped, maybe even the successful prophylaxis of these conditions. Although all the associated problems have not been solved, the undoubted improvements in patient care resulting from the first of these treatments in the clinic have stimulated further research on the role of integrins on other vascular cells in these processes and in the search for new inhibitors. Both the development of specific inhibitors and of mice with specific integrin subunit genes ablated have contributed to a better understanding of the function of integrins in development of the cardiovascular system.
Asunto(s)
Enfermedades Cardiovasculares/fisiopatología , Integrinas/fisiología , Arteriosclerosis/fisiopatología , Plaquetas/fisiología , Enfermedades Cardiovasculares/tratamiento farmacológico , Endotelio Vascular/fisiología , Humanos , Leucocitos/fisiología , Monocitos/fisiología , Activación Plaquetaria/fisiología , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/antagonistas & inhibidores , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/fisiología , Trombosis/metabolismoRESUMEN
The snake C-type lectins are a major group of proteins present in venoms that fold to a structure with similarities to classic C-type lectins. The loop that would be involved in calcium and sugar binding is truncated and heterodimers are linked by a disulphide bond and by swapping loop domains between the subunits. M any of these C-type lectins interact with platelet receptors to inhibit or induce platelet activation. The use of these C-type lectins to investigate platelet function is discussed and illustrated with specific examples.
RESUMEN
Convulxin, a powerful platelet activator, was isolated from Crotalus durissus terrificus venom, and 20 amino acid N-terminal sequences of both subunits were determined. These indicated that convulxin belongs to the heterodimeric C-type lectin family. Neither antibodies against GPIb nor echicetin had any effect on convulxin-induced platelet aggregation showing that, in contrast to other venom C-type lectins acting on platelets, GPIb is not involved in convulxin-induced platelet activation. In addition, partially reduced/denatured convulxin only affects collagen-induced platelet aggregation. The mechanism of convulxin-induced platelet activation was examined by platelet aggregation, detection of time-dependent tyrosine phosphorylation of platelet proteins, and binding studies with 125I-convulxin. Convulxin induces signal transduction in part like collagen, involving the time-dependent tyrosine phosphorylation of Fc receptor gamma chain, phospholipase Cgamma2, p72(SYK), c-Cbl, and p36-38. However, unlike collagen, pp125(FAK) and some other bands are not tyrosine-phosphorylated. Convulxin binds to a glycosylated 62-kDa membrane component in platelet lysate and to p62/GPVI immunoprecipitated by human anti-p62/GPVI antibodies. Convulxin subunits inhibit both aggregation and tyrosine phosphorylation in response to collagen. Piceatannol, a tyrosine kinase inhibitor with some specificity for p72(SYK), showed differential effects on collagen and convulxin-stimulated signaling. These results suggest that convulxin uses the p62/GPVI but not the alpha2beta1 part of the collagen signaling pathways to activate platelets. Occupation and clustering of p62/GPVI may activate Src family kinases phosphorylating Fc receptor gamma chain and, by a mechanism previously described in T- and B-cells, activate p72(SYK) that is critical for downstream activation of platelets.
Asunto(s)
Venenos de Crotálidos/farmacología , Lectinas Tipo C , Activación Plaquetaria/efectos de los fármacos , Glicoproteínas de Membrana Plaquetaria/metabolismo , Transducción de Señal/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/metabolismo , Cromatografía Líquida de Alta Presión , Colágeno/farmacología , Venenos de Crotálidos/química , Crotalus , Humanos , Integrinas/metabolismo , Datos de Secuencia Molecular , Fosforilación , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Complejo GPIb-IX de Glicoproteína Plaquetaria/metabolismo , Unión Proteica , Desnaturalización Proteica , Receptores de Colágeno , Estilbenos/farmacología , Tirosina/metabolismoRESUMEN
Granzyme A is a serine protease stored in cytoplasmic granules of cytotoxic and helper T lymphocytes. This protease seems to elicit thrombin receptor-mediated responses in neural cells, thereby triggering neurite retraction and reversal of astrocyte stellation. Here we report that granzyme A does not cause platelet aggregation even at concentrations that are more than two orders of magnitude higher than the EC50 for granzyme A in causing morphological changes in neural cells. However, granzyme A blocks thrombin-induced platelet aggregation in a dose-dependent manner without affecting the response to either ADP or to the peptide agonist of the thrombin receptor SFLLRN that corresponds in sequence to the tethered ligand domain. The inability of granzyme A to cause aggregation and its inhibition of thrombin-induced aggregation were seen in platelets from man, rat and mouse. Granzyme A does not affect the catalytic activity of thrombin in cleaving a chromogenic substrate or the macromolecular substrate fibrinogen. However, granzyme A does seem to cleave the thrombin receptor on platelets to produce a weak Ca2+ signal and reduce the response to subsequent challenge with thrombin, but does not induce a signal in thrombin-stimulated platelets. It is proposed that granzyme A interacts with the thrombin receptor found on platelets in a manner that is insufficient to cause aggregation, but sufficient to compete with thrombin for the receptor. These results suggest that granzyme A cleaves the thrombin receptor at a rate that is insufficient to cause platelet aggregation but is sufficient to cause morphological changes in neural cells. Furthermore, these observations demonstrate that granzyme A release occurring during immune responses within blood vessels would not directly cause platelet aggregation.
Asunto(s)
Inhibidores de Agregación Plaquetaria/farmacología , Agregación Plaquetaria/efectos de los fármacos , Serina Endopeptidasas/farmacología , Trombina/farmacología , Secuencia de Aminoácidos , Animales , Calcio/metabolismo , Granzimas , Humanos , Técnicas In Vitro , Ratones , Datos de Secuencia Molecular , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/farmacología , Agregación Plaquetaria/fisiología , Complejo GPIb-IX de Glicoproteína Plaquetaria/metabolismo , Ratas , Receptores de Trombina/efectos de los fármacos , Receptores de Trombina/genética , Receptores de Trombina/metabolismo , Serina Endopeptidasas/metabolismo , Transducción de Señal/efectos de los fármacos , Subgrupos de Linfocitos T/enzimología , Trombina/metabolismoRESUMEN
In order to study the role of platelets in inflammation we constructed a cDNA library from human platelet mRNA. By polymerase chain reaction (PCR) analysis of the library we have shown that platelets express mRNAs for the following chemokines: connective tissue activating peptide-III (CTAP-III), epithelial-derived neutrophil activating factor-78 (ENA-78), RANTES and monocyte chemotactic protein-3 (MCP-3). Platelets also express mRNAs for interleukin 8 receptor A (IL-8RA) and a novel chemokine receptor K5.5. These results suggest that chemokines may not only play an important role in platelet activation but can also influence the nature of the leukocyte infiltrate to sites of inflammation and infection, by the production of multiple chemokines with overlapping specificities.
Asunto(s)
Plaquetas/metabolismo , Quimiocinas/genética , Biblioteca Genómica , Inflamación/sangre , ARN Mensajero/biosíntesis , Receptores de Quimiocina , Receptores de Citocinas/genética , Secuencia de Bases , Separación Celular , ADN Complementario/genética , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Receptores CCR4RESUMEN
In the early phase of primary hemostasis, platelets adhere to damaged vessel wall by binding via the platelet glycoprotein (GP) Ib-V-IX complex to von Willebrand factor (vWf) exposed on the subendothelium. The complex is composed of four glycoprotein subunits, GPIb alpha, GPIb beta, GPIX and GPV, each with a variable number of leucine-rich repeats. GPIb alpha and GPIb beta are linked by a disulphide bridge while GPIX and GPV associate noncovalently with the complex. The study of defects in the expression of the GPIb-V-IX complex at the platelet surface leading to pathological disorders, like Bernard-Soulier syndrome (BSS), or in the affinity of platelets for vWf, like pseudo-von Willebrand disease, has helped to delineate the binding site for vWf on GPIb alpha. However, the mechanism by which the complex binds to vWf has not yet been elucidated but it must involve changes in the conformation of the molecules as no interaction between platelets and vWf occurs in the plasma. The GPIb-V-IX complex has a binding site for thrombin on GPIb alpha which participates in the platelet activation by that agonist. GPV is also cleaved by thrombin but the function of this proteolysis is not clear. The platelet response to thrombin is slower and weaker when the thrombin binding site on GPIb alpha is blocked or cleaved or when the GPIb-V-IX complex is not expressed on the platelet surface as in classic BSS. At low doses of thrombin, the rapid activation of the platelets via the seven-transmembrane thrombin receptor is dependent on the presence of the GPIb-V-IX complex.
Asunto(s)
Hemorragia/fisiopatología , Glicoproteínas de Membrana Plaquetaria/química , Glicoproteínas de Membrana Plaquetaria/fisiología , Endotelio Vascular/metabolismo , Hemorragia/genética , Humanos , Glicoproteínas de Membrana Plaquetaria/metabolismo , Unión Proteica , Relación Estructura-ActividadRESUMEN
Genetic defects of the blood platelet membrane glycoproteins, GPIIb-IIIa (alpha IIb/beta 3; CD41/CD61) and GPIb-V-IX (CD42) are the origin of several rare bleeding disorders, the best known of which are Glanzmann's thrombasthenia, Bernard-Soulier syndrome, and platelet-type von Willebrand's disease. In Glanzmann's thrombasthenia, GPIIb-IIIa are missing or defective and platelet aggregation is lacking or reduced. Either gene can be affected and mutations leading to lack of expression or to expression of poorly functional forms have been described. In Bernard-Soulier syndrome, GPIb-V-IX are missing or defective, leading to poor platelet adhesion at high-shear stress to damaged vessel wall and reduced platelet response to thrombin. Mutations in both GPIb alpha (CD42b) and GPIX (CD42a) have been described. Mutations in GPIb alpha can also lead to platelet-type von Willebrand's disease in which GPIb-V-IX are expressed normally but bind von Willebrand's factor spontaneously, which leads to platelet aggregation and thrombocytopenia.
Asunto(s)
Síndrome de Bernard-Soulier/fisiopatología , Plaquetas , Trombastenia/fisiopatología , Enfermedades de von Willebrand/fisiopatología , Antígenos CD/química , Humanos , Integrina beta3 , Complejo GPIb-IX de Glicoproteína Plaquetaria/química , Glicoproteínas de Membrana Plaquetaria/químicaRESUMEN
We describe a new variant of Bernard-Soulier syndrome. The patient (W.K.) showed the classic bleeding symptoms together with absence of platelet agglutination to restocetin plus von Willebrand factor, whereas aggregation to ADP, collagen, and arachidonic acid was normal. Platelets were markedly larger than normal and the patient had life-long thrombocytopenia. Surface-labeling of the platelets and two-dimensional gel electrophoresis showed reduced but detectable amounts of glycoprotein (GP) Ib-IX-V present;however, there was markedly less GPIX (2% +/- 1% of normal) than GPIb alpha, Ib beta, or V (7% +/- 2% of normal). This disproportion was confirmed by Western blotting. Sequence analysis was performed after polymerase chain reaction amplification of the coding region of the GPIX and GPI b alpha genes from the patient. A point mutation (A-->G) was found in GPIX converting 45Asn to Ser within the leucine-rich domain. No mutations were found in GPIb alpha. Both alleles of GPIX contained the same defect, which was confirmed by the appearance of a new cleavage site for the restriction enzyme Fnu4HI. This substitution did not affect glycosylation at the neighboring 44Asn as judged by the distribution on two-dimensional gels but did appear to change the conformation of the leucine-rich domain, thus reducing surface expression of the complex. The relationship between GPIb and GPV was not affected, indicating that GPIX does not regulate this. This homozygous mutation in GPIX indicates that, among other possible functions, the leucine-rich domains present on all components of GPIb-IX-V may play a role in the assembly and surface expression of the complex.
Asunto(s)
Síndrome de Bernard-Soulier/sangre , Síndrome de Bernard-Soulier/genética , Plaquetas/fisiología , Variación Genética , Glicoproteínas de Membrana Plaquetaria/genética , Mutación Puntual , Adulto , Secuencia de Aminoácidos , Asparagina , Secuencia de Bases , Western Blotting , ADN/sangre , Cartilla de ADN , Femenino , Humanos , Leucina , Masculino , Datos de Secuencia Molecular , Linaje , Agregación Plaquetaria , Glicoproteínas de Membrana Plaquetaria/análisis , Glicoproteínas de Membrana Plaquetaria/biosíntesis , Reacción en Cadena de la Polimerasa , Valores de Referencia , SerinaRESUMEN
In order to detect novel glycophosphatidylinositol (GPI)-anchored platelet proteins, human platelets were incubated with PI-specific phospholipase C (PI-PLC) and the supernatant was analysed by PAGE and silver-staining for additional protein bands. PI-PLC treatment resulted in the appearance of at least two additional novel GPI-linked glycoproteins (GP), GP500 and GP175, in the supernatant. Their presence on the platelet plasma membrane surface was demonstrated by periodate/[3H]borohydride surface-labelling. Activation of platelets did not enhance the amount of GP500 and GP175 that could be cleaved by PI-PLC. In Triton X-114 phase partitioning of platelet membranes the membrane form of GP175, mfGP175, was in the Triton phase while mfGP500 was found in the water phase. Neither GP500 nor GP175 were present in the supernatant of surface-labelled platelets treated with PI-PLC from 4 patients, diagnosed as having paroxysmal nocturnal haemoglobinuria (PNH), but the supernatant from platelets from healthy volunteers treated the same way contained both.
Asunto(s)
Plaquetas/metabolismo , Glicosilfosfatidilinositoles/metabolismo , Hemoglobinuria Paroxística/sangre , Glicoproteínas de Membrana/metabolismo , Proteínas Sanguíneas/deficiencia , Cromatografía de Afinidad , Electroforesis en Gel de Poliacrilamida , Humanos , Activación Plaquetaria , Tinción con Nitrato de Plata , Fosfolipasas de Tipo C/metabolismoRESUMEN
The present investigation revealed the presence of lipocortins I and IV, but not lipocortins II and VI, in human platelets. Lipocortin I was found in the Triton-soluble fraction of both resting and thrombin-activated platelets and was not covalently bound to skeletal components. Without detergents, when resting platelets were lysed and fractionated in the absence of Ca2+, lipocortin I was found only in the cytosolic fraction, whereas, in the presence of Ca2+, lipocortin I was associated only with the crude particulate and not with the membrane nor the cytosolic fractions.