RESUMEN
Transforming growth factor beta 1 (TGF-beta 1) is a platelet-derived cytokine involved in both normal wound healing and scarring. We show that human platelets contain two pools of latent TGF-beta 1, which constitute more than 95% of the total TGF-beta assayed in whole platelets. During clotting, one pool, the large latent TGF-beta complex consisting of latent TGF-beta binding protein (LTBP), the latency-associated peptide (LAP) and the 25-kD mature TGF-beta 1 dimer is released into the serum. A second pool, which contains LAP but not LTBP, is retained in the clot, but can be released by RGD peptide. When the clot is dissolved by plasmin this bound TGF-beta 1 is gradually activated and released. If similar mechanisms operate in vivo, the clot will act as a slow-release capsule of TGF-beta 1 activity during wound healing.
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Plaquetas/metabolismo , Proteínas Portadoras/sangre , Fibrinolisina/farmacología , Fibrinólisis/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular , Fragmentos de Péptidos , Precursores de Proteínas , Proteínas/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Plaquetas/efectos de los fármacos , Compartimento Celular , Humanos , Proteínas de Unión a TGF-beta Latente , Sustancias Macromoleculares , Masculino , Persona de Mediana Edad , Peso Molecular , Oligopéptidos/farmacología , Trombina/farmacología , Factor de Crecimiento Transformador beta1RESUMEN
Essentials Glycoprotein VI (GPVI) binds collagen, starting thrombogenesis, and fibrin, stabilizing thrombi. GPVI-dimers, not monomers, recognize immobilized fibrinogen and fibrin through their D-domains. Collagen, D-fragment and D-dimer may share a common or proximate binding site(s) on GPVI-dimer. GPVI-dimer-fibrin interaction supports spreading, activation and adhesion involving αIIbß3. SUMMARY: Background Platelet collagen receptor Glycoprotein VI (GPVI) binds collagen, initiating thrombogenesis, and stabilizes thrombi by binding fibrin. Objectives To determine if GPVI-dimer, GPVI-monomer, or both bind to fibrinogen substrates, and which region common to these substrates contains the interaction site. Methods Recombinant GPVI monomeric extracellular domain (GPVIex ) or dimeric Fc-fusion protein (GPVI-Fc2 ) binding to immobilized fibrinogen derivatives was measured by ELISA, including competition assays involving collagenous substrates and fibrinogen derivatives. Flow adhesion was performed with normal or Glanzmann thrombasthenic (GT) platelets over immobilized fibrinogen, with or without anti-GPVI-dimer or anti-αIIbß3. Results Under static conditions, GPVIex did not bind to any fibrinogen substrate. GPVI-Fc2 exhibited specific, saturable binding to both D-fragment and D-dimer, which was inhibited by mFab-F (anti-GPVI-dimer), but showed low binding to fibrinogen and fibrin under our conditions. GPVI-Fc2 binding to D-fragment or D-dimer was abrogated by collagen type III, Horm collagen or CRP-XL (crosslinked collagen-related peptide), suggesting proximity between the D-domain and collagen binding sites on GPVI-dimer. Under low shear, adhesion of normal platelets to D-fragment, D-dimer, fibrinogen and fibrin was inhibited by mFab-F (inhibitor of GPVI-dimer) and abolished by Eptifibatide (inhibitor of αIIbß3), suggesting that both receptors contribute to thrombus formation on these substrates, but αIIbß3 makes a greater contribution. Notably, thrombasthenic platelets showed limited adhesion to fibrinogen substrates under flow, which was further reduced by mFab-F, supporting some independent GPVI-dimer involvement in this interaction. Conclusion Only dimeric GPVI interacts with fibrinogen D-domain, at a site proximate to its collagen binding site, to support platelet adhesion/activation/aggregate formation on immobilized fibrinogen and polymerized fibrin.
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Plaquetas/metabolismo , Colágeno/metabolismo , Fibrina/metabolismo , Fibrinógeno/metabolismo , Activación Plaquetaria , Glicoproteínas de Membrana Plaquetaria/metabolismo , Trombastenia/sangre , Trombosis/sangre , Sitios de Unión , Estudios de Casos y Controles , Fibrina/química , Productos de Degradación de Fibrina-Fibrinógeno/metabolismo , Fibrinógeno/química , Humanos , Adhesividad Plaquetaria , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/genética , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Glicoproteínas de Membrana Plaquetaria/química , Glicoproteínas de Membrana Plaquetaria/genética , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Transducción de Señal , Relación Estructura-Actividad , Trombastenia/genética , Trombosis/genéticaRESUMEN
Essentials Heat shock protein 47 (HSP47), a collagen specific chaperone is present on the platelet surface. Collagen mediated platelet function was reduced following blockade or deletion of HSP47. GPVI receptor regulated signalling was reduced in HSP47 deficient platelets. Platelet HSP47 tethers to exposed collagen thus modulating thrombosis and hemostasis. SUMMARY: Objective Heat shock protein 47 (HSP47) is an intracellular chaperone protein that is vital for collagen biosynthesis in collagen secreting cells. This protein has also been shown to be present on the surface of platelets. Given the importance of collagen and its interactions with platelets in triggering hemostasis and thrombosis, in this study we sought to characterize the role of HSP47 in these cells. Methods and Results The deletion of HSP47 in mouse platelets or its inhibition in human platelets reduced their function in response to collagen and the GPVI agonist (CRP-XL), but responses to thrombin were unaltered. In the absence of functional HSP47, the interaction of collagen with platelets was reduced, and this was associated with reduced GPVI-collagen binding, signalling and platelet activation. Thrombus formation on collagen, under arterial flow conditions, was also decreased following the inhibition or deletion of HSP47, in the presence or absence of eptifibatide, consistent with a role for HSP47 in enhancing platelet adhesion to collagen. Platelet adhesion under flow to von Willebrand factor was unaltered following HSP47 inhibition. Laser-induced thrombosis in cremaster muscle arterioles was reduced and bleeding time was prolonged in HSP47-deficient mice or following inhibition of HSP47. Conclusions Our study demonstrates the presence of HSP47 on the platelet surface, where it interacts with collagen, stabilizes platelet adhesion and increases collagen-mediated signalling and therefore thrombus formation and hemostasis.
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Plaquetas/metabolismo , Proteínas Portadoras/sangre , Colágeno/sangre , Proteínas HSP70 de Choque Térmico/sangre , Hemostasis , Activación Plaquetaria , Trombosis/sangre , Animales , Plaquetas/efectos de los fármacos , Señalización del Calcio , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Modelos Animales de Enfermedad , Proteínas HSP70 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP70 de Choque Térmico/deficiencia , Proteínas HSP70 de Choque Térmico/genética , Hemostasis/efectos de los fármacos , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Mitocondriales , Activación Plaquetaria/efectos de los fármacos , Adhesividad Plaquetaria , Inhibidores de Agregación Plaquetaria/farmacología , Glicoproteínas de Membrana Plaquetaria/metabolismo , Unión Proteica , Trombosis/genética , Trombosis/prevención & controlRESUMEN
In the platelet, it is well established that many G-protein- and tyrosine kinase-coupled receptors stimulate phospholipase-C-dependent Ca(2+) mobilization; however, the extent to which secondary activation of adenosine 5'-triphosphate (ATP)-gated P2X(1) receptors contributes to intracellular Ca(2+) responses remains unclear. We now show that selective inhibition of P2X(1) receptors substantially reduces the [Ca(2+)](i) increase evoked by several important agonists in human platelets; for collagen, thromboxane A(2), thrombin, and adenosine 5'-diphoshate (ADP) the maximal effect was a reduction to 18%, 34%, 52%, and 69% of control, respectively. The direct contribution of P2X(1) to the secondary Ca(2+) response was far greater than that of either P2Y receptors activated by co-released ADP, or via synergistic P2X(1):P2Y interactions. The relative contribution of P2X(1) to the peak Ca(2+) increase varied with the strength of the initial stimulus, being greater at low compared to high levels of stimulation for both glycoprotein VI and PAR-1, whereas P2X(1) contributed equally at both low and high levels of stimulation of thromboxane A(2) receptors. In contrast, only strong stimulation of P2Y receptors resulted in significant P2X(1) receptor activation. ATP release was detected by soluble luciferin:luciferase in response to all agonists that stimulated secondary P2X(1) receptor activation. However, P2X(1) receptors were stimulated earlier and to a greater extent than predicted from the average ATP release, which can be accounted for by a predominantly autocrine mechanism of activation. Given the central role of [Ca(2+)](i) increases in platelet activation, these studies indicate that ATP should be considered alongside ADP and thromboxane A(2) as a significant secondary platelet agonist.
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Plaquetas/efectos de los fármacos , Calcio/metabolismo , Agonistas del Receptor Purinérgico P2 , Adenosina Difosfato/farmacología , Bencenosulfonatos/farmacología , Plaquetas/citología , Plaquetas/metabolismo , Humanos , Luminiscencia , Receptores Purinérgicos P2X , Espectrometría de Fluorescencia , Tromboxano A2/farmacologíaRESUMEN
Comprehensive mapping of protein-binding sites within human collagen III has allowed the recognition motifs for integrin alpha(2)beta(1) and VWF A3 domain to be identified. Glycoprotein VI-binding sites are understood, although less well defined. This information, together with recent developments in understanding collagen fiber architecture, and crystal structures of the receptor collagen-binding domains, allows a coherent model for the interaction of collagen with the platelet surface to be developed. This complements our understanding of the orchestration of receptor presentation by membrane microdomains, such that the polyvalent collagen surface may stabilize signaling complexes within the heterogeneous receptor composition of the lipid raft. The ensuing interactions lead to the convergence of signals from each of the adhesive receptors, mediated by FcR gamma-chain and/or FcgammaRIIa, leading to concerted and co-operative platelet activation. Each receptor has a shear-dependent role, VWF/GpIb essential at high shear, and alpha(2)beta(1) at low and intermediate shear, whilst GpVI provides core signals that contribute to enhanced integrin affinity, tighter binding to collagen and consequent platelet activation.
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Plaquetas/citología , Colágeno/fisiología , Activación Plaquetaria/fisiología , Receptor Cross-Talk , Receptores de Superficie Celular/fisiologíaRESUMEN
BACKGROUND: Evidence suggests the wide variation in platelet response within the population is genetically controlled. Unraveling the complex relationship between sequence variation and platelet phenotype requires accurate and reproducible measurement of platelet response. OBJECTIVE: To develop a methodology suitable for measuring signaling pathway-specific platelet phenotype, to use this to measure platelet response in a large cohort, and to demonstrate the effect size of sequence variation in a relevant model gene. METHODS: Three established platelet assays were evaluated: mobilization of [Ca(2+)](i), aggregometry and flow cytometry, each in response to adenosine 5'-diphosphate (ADP) or the glycoprotein (GP) VI-specific crosslinked collagen-related peptide (CRP). Flow cytometric measurement of fibrinogen binding and P-selectin expression in response to a single, intermediate dose of each agonist gave the best combination of reproducibility and inter-individual variability and was used to measure the platelet response in 506 healthy volunteers. Pathway specificity was ensured by blocking the main subsidiary signaling pathways. RESULTS: Individuals were identified who were hypo- or hyper-responders for both pathways, or who had differential responses to the two agonists, or between outcomes. 89 individuals, retested three months later using the same methodology, showed high concordance between the two visits in all four assays (r(2) = 0.872, 0.868, 0.766 and 0.549); all subjects retaining their phenotype at recall. The effect of sequence variation at the GP6 locus accounted for approximately 35% of the variation in the CRP-XL response. CONCLUSION: Genotyping-phenotype association studies in a well-characterized, large cohort provides a powerful strategy to measure the effect of sequence variation in genes regulating the platelet response.
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Plaquetas/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Glicoproteínas de Membrana Plaquetaria/genética , Adulto , Proteínas Portadoras/química , Femenino , Citometría de Flujo , Genómica/métodos , Humanos , Masculino , Persona de Mediana Edad , Péptidos/química , Inhibidores de Agregación Plaquetaria/farmacología , Transducción de SeñalRESUMEN
Essentials Dimeric high-affinity collagen receptor glycoprotein VI (GPVI) is present on resting platelets. Spatio-temporal organization of platelet GPVI-dimers was evaluated using advanced microscopy. Upon platelet adhesion to collagenous substrates, GPVI-dimers coalesce to form clusters. Clustering of GPVI-dimers may increase avidity and facilitate platelet activation SUMMARY: Background Platelet glycoprotein VI (GPVI) binding to subendothelial collagen exposed upon blood vessel injury initiates thrombus formation. Dimeric GPVI has high affinity for collagen, and occurs constitutively on resting platelets. Objective To identify higher-order oligomerization (clustering) of pre-existing GPVI dimers upon interaction with collagen as a mechanism to initiate GPVI-mediated signaling. Methods GPVI was located by use of fluorophore-conjugated GPVI dimer-specific Fab (antigen-binding fragment). The tested substrates include Horm collagen I fibers, soluble collagen III, GPVI-specific collagen peptides, and fibrinogen. GPVI dimer clusters on the platelet surface interacting with these substrates were visualized with complementary imaging techniques: total internal reflection fluorescence microscopy to monitor real-time interactions, and direct stochastic optical reconstruction microscopy (dSTORM), providing relative quantification of GPVI cluster size and density. Confocal microscopy was used to locate GPVI dimer clusters, glycoprotein Ib, integrin α2 ß1 , and phosphotyrosine. Results Upon platelet adhesion to all collagenous substrates, GPVI dimers coalesced to form clusters; notably clusters formed along the fibers of Horm collagen. dSTORM revealed that GPVI density within clusters depended on the substrate, collagen III being the most effective. Clusters on fibrinogen-adhered platelets were much smaller and more numerous; whether these are pre-existing oligomers of GPVI dimers or fibrinogen-induced is not clear. Some GPVI dimer clusters colocalized with areas of phosphotyrosine, indicative of signaling activity. Integrin α2 ß1 was localized to collagen fibers close to GPVI dimer clusters. GPVI clustering depends on a dynamic actin cytoskeleton. Conclusions Platelet adhesion to collagen induces GPVI dimer clustering. GPVI clustering increases both avidity for collagen and the proximity of GPVI-associated signaling molecules, which may be crucial for the initiation and persistence of signaling.
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Plaquetas/metabolismo , Colágeno/metabolismo , Glicoproteínas de Membrana Plaquetaria/metabolismo , Actinas/metabolismo , Vasos Sanguíneos/lesiones , Adhesión Celular , Citoesqueleto/metabolismo , Humanos , Microscopía Confocal , Activación Plaquetaria , Adhesividad Plaquetaria , Multimerización de Proteína , Transducción de SeñalRESUMEN
BACKGROUND: By site-directed mutagenesis of recombinant receptor fragments, we have previously identified residue lysine59 of the platelet collagen receptor glycoprotein VI (GPVI) as being critical for its interaction with the synthetic ligand collagen-related peptide (CRP) and the inhibitory phage antibody 10B12. Lysine59 is proposed to lie on the apical surface of the receptor near the linker joining the two immunoglobulin (Ig)-like extracellular domains. Recently, others have postulated the involvement of a portion of the first domain distant from the interdomain hinge as being involved in an extended collagen-binding site. AIM AND METHODS: To extend our knowledge of the primary collagen-binding site of GPVI, a number of neighboring residues on the apical surface of recombinant soluble GPVI were mutated to alanine and binding of these mutants, as well as the lysine59 mutant, to fibrillar collagen was measured. RESULTS: Binding of recombinant GPVI to collagen, like CRP, was dramatically reduced by the mutation of residue lysine59 to glutamate. Remarkably, the mutation of residues arginine60 in domain one and arginine166 in domain two, individually to alanine, which had no significant affect on CRP binding, reduced binding of recombinant GPVI to collagen. Mutation of the residue lysine41 to alanine dramatically increased binding to both CRP and collagen. This mutation abolished 10B12 binding, confirming its position in the epitope of our inhibitory phage antibody. CONCLUSIONS: Residues lysine59, arginine60, and arginine166, from both Ig-like domains of GPVI, are critical for collagen binding by the receptor. This provides additional evidence for a basic patch on the apical surface of the receptor as the primary collagen-binding site of GPVI.
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Colágeno/química , Mutación , Glicoproteínas de Membrana Plaquetaria/química , Glicoproteínas de Membrana Plaquetaria/genética , Sitios de Unión , Proteínas Portadoras/química , Colágeno/genética , Mapeo Epitopo , Humanos , Inmunoglobulinas/química , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Péptidos/química , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/químicaRESUMEN
BACKGROUND: Common genetic variants of cell surface receptors contribute to differences in functional responses and disease susceptibility. We have previously shown that single nucleotide polymorphisms (SNPs) in platelet glycoprotein VI (GP6) determine the extent of response to agonist. In addition, SNPs in the GP6 gene have been proposed as risk factors for coronary artery disease. METHODS: To completely characterize genetic variation in the GP6 gene we generated a high-resolution SNP map by sequencing the promoter, exons and consensus splice sequences in 94 non-related Caucasoids. In addition, we sequenced DNA encoding the ligand-binding domains of GP6 from non-human primates to determine the level of evolutionary conservation. RESULTS: Eighteen SNPs were identified, six of which encoded amino acid substitutions in the mature form of the protein. The single non-synonymous SNP identified in the exons encoding the ligand-binding domains, encoding for a 103Leu > Val substitution, resulted in reduced ligand binding. Two common protein isoforms were confirmed in Caucasoid with frequencies of 0.82 and 0.15. Variation at the GP6 locus was characterized further by determining SNP frequency in over 2000 individuals from different ethnic backgrounds. CONCLUSIONS: The SNPs were polymorphic in all populations studied although significant differences in allele frequencies were observed. Twelve additional GP6 protein isoforms were identified from the genotyping results and, despite extensive variation in GP6, the sequence of the ligand-binding domains is conserved. Sequences from non-human primates confirmed this observation. These data provide valuable information for the optimal selection of genetic variants for use in future association studies.
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Exones , Frecuencia de los Genes , Glicoproteínas de Membrana Plaquetaria/genética , Polimorfismo de Nucleótido Simple , Secuencia de Aminoácidos , Animales , Plaquetas/metabolismo , Proteínas Portadoras/metabolismo , Línea Celular , Drosophila/genética , Genotipo , Haplotipos , Humanos , Desequilibrio de Ligamiento , Datos de Secuencia Molecular , Péptidos/metabolismo , Glicoproteínas de Membrana Plaquetaria/metabolismo , Primates/genética , Regiones Promotoras Genéticas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
Pulsed magnetic fields, similar to those used clinically to promote bone repair, have been applied to cultured fibroblasts obtained from chick embryo tendons and rabbit bone marrow. Cyclic adenosine monophosphate (cAMP) levels were found to be lower in field-treated cultures in response to hormones such as prostaglandin E2 and isoproterenol, and the fibroblasts appear less sensitive to environmental perturbation prior to hormone incubation. We propose that the adenylate cyclase complex is temporarily inactivated by prolonged exposure to pulsed magnetic fields, and that this effect might be analogous to desensitisation phenomena.
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AMP Cíclico/análisis , Fibroblastos/análisis , Magnetismo , Animales , Médula Ósea/efectos de los fármacos , Células de la Médula Ósea , Células Cultivadas , Pollos , Dinoprostona , Fibroblastos/efectos de los fármacos , Indometacina/farmacología , Prostaglandinas E/farmacología , Conejos , Tendones/citología , Tendones/efectos de los fármacosRESUMEN
Primary cultures of chicken tendon fibroblasts have been exposed for various periods to a low-frequency, pulsed magnetic field, and the effects on protein and collagen synthesis have been examined by radioisotopic incorporation. Total protein synthesis was increased in confluent cells treated with a pulsed magnetic field for the last 24 h of culture as well as in cells treated for a total of 6 days. However, in 6 day-treated cultures, collagen accumulation was specifically enhanced as compared to total protein, whereas after short-term exposure, collagen production was increased only to the same extent as total protein. Levels of cyclic AMP were significantly decreased after 6-day pulsed magnetic field treatment, probably as a consequence of diminished adenylate cyclase activity. Exposure to pulsed magnetic field had no effect on cell proliferation or collagen phenotype. These results indicate that a pulsed magnetic field can specifically increase production of collagen, the major differentiated function of fibroblasts, possibly by altering cyclic-AMP metabolism.
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Colágeno/biosíntesis , Campos Electromagnéticos , Fenómenos Electromagnéticos , Fibroblastos/metabolismo , Animales , División Celular , Células Cultivadas , Embrión de Pollo , AMP Cíclico/metabolismo , Hidroxiprolina/metabolismo , Prolina/metabolismoRESUMEN
The dimethylmethylene blue assay for sulphated glycosaminoglycans has found wide acceptance as a quick and simple method of measuring the sulphated glycosaminoglycan content of tissues and fluids. The available assay methods have lacked specificity for sulphated glycosaminoglycans in the presence of other polyanions, however, and have not discriminated between the different sulphated glycosaminoglycans. We now describe a modified form of the dimethylmethylene blue assay that has improved specificity for sulphated glycosaminoglycans, and we show that in conjunction with specific polysaccharidases, the dimethylmethylene blue assay can be used to quantitate individual sulphated glycosaminoglycans.
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Glicosaminoglicanos/análisis , Glicósido Hidrolasas , Azul de Metileno/análogos & derivados , Condroitín Liasas/metabolismo , Espectrofotometría , Sulfatos , beta-Galactosidasa/metabolismoRESUMEN
Mechanical deformation of bone cells was thought to be mediated via prostaglandin production and the cyclic AMP pathway. We present evidence that the phosphoinositide pathway is also activated by mechanical stress. We find that inositol phosphate production, but not glycerophosphoinositol production, is elevated, and the activation of adenylate cyclase is relatively small. These results are not compatible with the proposal that mechanical deformation of bone cells acts solely via prostaglandin synthesis.
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AMP Cíclico/metabolismo , Fosfatos de Inositol/metabolismo , Osteoblastos/citología , Fosfatos de Azúcar/metabolismo , Animales , Células Cultivadas , Inositol/metabolismo , Cinética , Ratones , Osteoblastos/metabolismo , Estrés MecánicoRESUMEN
We have investigated the actions of the PLC inhibitor, U73122, and its close analogue, U73343, which does not inhibit PLC, in Fura-2-loaded human platelets. Rises in [Ca2+]i evoked by thrombin and collagen, and the TxA2-dependent rise in [Ca2+]i evoked by thapsigargin, were abolished by U73122, indicating that it inhibits the activity of both beta and gamma isoforms of PLC. The supposed control compound U73343, was found to inhibit TxA2 formation; it therefore partially inhibited the rise in [Ca2+]i evoked by low concentrations of thrombin, by thapsigargin or by collagen. U73343 had a greater effect than aspirin on the action of collagen, indicating an action on the TxA2-independent component of the signal, via PLC gamma-U73343 lowered TxA2 production by inhibiting the activation of cPLA2, probably at a tyrosine phosphorylation step. U73343 seems to inhibit only the tyrosine kinases involved in the activation of PLC gamma and the generation of TxA2. In contrast, U73122 increased tyrosine phosphorylation of platelet proteins, perhaps by inhibiting receptor independent tyrosine phosphatases, but inhibited all further tyrosine phosphorylation on addition of thrombin or other agonists.
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Plaquetas/metabolismo , Calcio/metabolismo , Estrenos/farmacología , Fosfotirosina/metabolismo , Pirrolidinonas/farmacología , Transducción de Señal/efectos de los fármacos , Ácido Araquidónico/farmacología , Aspirina/farmacología , Plaquetas/efectos de los fármacos , Proteínas Sanguíneas/metabolismo , Colágeno/farmacología , Humanos , Inhibidores de Fosfodiesterasa/farmacología , Fosforilación , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Tapsigargina/farmacología , Trombina/farmacología , Tromboxano A2/metabolismo , Fosfolipasas de Tipo C/antagonistas & inhibidoresRESUMEN
Adhesion to von Willebrand factor (VWF) induces platelet spreading, whereas adhesion to collagen induces aggregation. Here we report that cholesterol-rich domains (CRDs) or rafts play a critical role in clustering of receptors that control these responses. Platelets adhered to VWF and collagen show CRDs concentrated in filopodia which contain both the VWF receptor glycoprotein (GP) Ibalpha and the collagen receptor GPVI. Biochemical analysis of CRDs shows a threefold enrichment of GPIbalpha (but not GPVI) in VWF-adhered platelets and a fourfold enrichment of GPVI (but not GPIbalpha) in collagen-adhered platelets. Depletion of cholesterol (i) leaves the initial adhesion unchanged, (ii) inhibits spreading on VWF and aggregate formation on collagen, (iii) leaves filopodia formation intact, and (iv) reduces the localization in filopodia of GPIbalpha but not of GPVI. These data show that the adhesive substrate determines the composition of CRDs, and that cholesterol is crucial for redistribution of GPIbalpha but not of GPVI.
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Plaquetas/química , Microdominios de Membrana/química , Adhesividad Plaquetaria/fisiología , Seudópodos/química , Plaquetas/metabolismo , Plaquetas/ultraestructura , Células Cultivadas , Colesterol/deficiencia , Colesterol/metabolismo , Colágeno/metabolismo , Humanos , Glicoproteínas de Membrana , Microdominios de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Perfusión , Agregación Plaquetaria/fisiología , Complejo GPIb-IX de Glicoproteína Plaquetaria , Glicoproteínas de Membrana Plaquetaria/metabolismo , Seudópodos/metabolismo , Reología , Transducción de Señal/fisiología , Propiedades de Superficie , Factor de von Willebrand/metabolismoRESUMEN
In the final stages of activation, platelets express coagulation-promoting activity by 2 simultaneous processes: exposure of aminophospholipids, eg, phosphatidylserine (PS), at the platelet surface, and formation of membrane blebs, which may be shed as microvesicles. Contact with collagen triggers both processes via platelet glycoprotein VI (GPVI). Here, we studied the capacity of 2 GPVI ligands, collagen-related peptide (CRP) and the snake venom protein convulxin (CVX), to elicit the procoagulant platelet response. In platelets in suspension, either ligand induced full aggregation and high Ca(2+) signals but little microvesiculation or PS exposure. However, most of the platelets adhering to immobilized CRP or CVX had exposed PS and formed membrane blebs after a prolonged increase in cytosolic [Ca(2+)](i). Platelets adhering to fibrinogen responded similarly but only when exposed to soluble CRP or CVX. By scanning electron microscopic analysis, the bleb-forming platelets were detected as either round, spongelike structures with associated microparticles or as arrays of vesicular cell fragments. The phosphorylation of p38 mitogen-activated protein kinase (MAPK) elicited by CRP and CVX was enhanced in fibrinogen-adherent platelets compared with that in platelets in suspension. The p38 inhibitor SB203580 and the calpain protease inhibitor calpeptin reduced only the procoagulant bleb formation, having no effect on PS exposure. Inhibition of p38 also downregulated calpain activity. We conclude that the procoagulant response evoked by GPVI stimulation is potentiated by platelet adhesion. The sequential activation of p38 MAPK and calpain appears to regulate procoagulant membrane blebbing but not PS exposure.
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Plaquetas/fisiología , Proteínas Portadoras , Lectinas Tipo C , Activación Plaquetaria/fisiología , Adhesividad Plaquetaria/fisiología , Plaquetas/citología , Plaquetas/efectos de los fármacos , Western Blotting , Calcio/metabolismo , Calcio/fisiología , Calpaína/metabolismo , Calpaína/farmacología , Venenos de Crotálidos/metabolismo , Venenos de Crotálidos/farmacología , Activación Enzimática , Citometría de Flujo , Humanos , Microscopía Electrónica de Rastreo , Microscopía Fluorescente , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/farmacología , Fosfatidilserinas/metabolismo , Fosfatidilserinas/farmacología , Fosforilación , Activación Plaquetaria/efectos de los fármacos , Adhesividad Plaquetaria/efectos de los fármacos , Glicoproteínas de Membrana Plaquetaria/metabolismo , Glicoproteínas de Membrana Plaquetaria/farmacología , Proteínas/metabolismo , Proteínas/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
OBJECTIVE: Peptides consisting of a repeat Gly-Pro-Hyp sequence are potent platelet agonists. The aim of this study was: (1) to examine the specificity of this sequence for platelet activation; (2) to confirm its recognition by platelet glycoprotein VI; and (3) to assess with suitable peptides the relative importance of glycoprotein VI and integrin alpha 2 beta 1 in platelet activation by collagen. METHODS: Peptides were synthesized by standard Fmoc chemistry and tested for their ability to support adhesion of human platelets and HT 1080 cells, induce platelet aggregation, bind integrin alpha 2 subunit A-domain and to cause tyrosine phosphorylation of platelet proteins. RESULTS: (1) Peptides consisting of a repeat Gly-Pro-Pro, Gly-Pro-Ala or Gly-Pro-Arg sequence exhibited little if any platelet-reactivity. (2) The platelet-reactive peptide consisting of a repeating Gly-Pro-Hyp sequence failed to induce tyrosine phosphorylation in glycoprotein VI-deficient platelets. Platelet adhesion to this peptide was inhibited by intact anti-glycoprotein VI antibody and its Fab fragment. The latter inhibited aggregation by the peptide and fibres of both collagens I and III. (3) A peptide containing a 15-mer alpha 2 beta 1-binding sequence in a repeat Gly-Pro-Pro structure supported alpha 2 beta 1-mediated platelet and HT 1080 cell adhesion and bound alpha 2 A-domain, but failed to activate platelets or to induce tyrosine phosphorylation. Conversely, a peptide containing this sequence but with an essential Glu replaced by Ala and inserted in a repeat Gly-Pro-Hyp structure did not recognize alpha 2 beta 1, but was highly platelet activatory. CONCLUSIONS: Platelet activation by collagen involves the highly-specific recognition of the Gly-Pro-Hyp sequence by platelet glycoprotein VI. Recognition of alpha 2 beta 1 is insufficient to cause activation. Interaction between collagen and glycoprotein VI is unique since Gly-Pro-Hyp is common in collagens but occurs rarely in other proteins, and glycoprotein VI may be expressed solely by platelets. This sequence could provide a basis for a highly-specific anti-thrombotic reagent to control thrombosis associated with plaque rupture.
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Plaquetas/fisiología , Colágeno/metabolismo , Activación Plaquetaria/fisiología , Glicoproteínas de Membrana Plaquetaria/química , Trombosis/metabolismo , Humanos , Integrinas/metabolismo , Fragmentos de Péptidos , Glicoproteínas de Membrana Plaquetaria/metabolismo , Unión Proteica , Conformación Proteica , Receptores de ColágenoRESUMEN
BACKGROUND: Atherothrombosis underlies acute coronary syndromes, including unstable angina and acute myocardial infarction. Within the unstable plaque, monocytes express collagenolytic matrix metalloproteinases (MMPs), including MMP-13, which degrades fibrous collagen. Following rupture, vessel wall components including degraded collagen are exposed to circulating platelets. Platelet receptors then mediate the recruitment and activation of platelets to form a thrombus, blocking blood flow and resulting in myocardial infarction and sudden death. OBJECTIVES: Here we aim to provide information on the effects of collagen degradation on platelet adhesion and thrombus formation. METHODS: Using increasing concentrations of MMP-13, we induced progressive degradation of fibrous and monomeric collagen I, visualized by electrophoresis, and then investigated the capacity of the resulting fragments to support static platelet adhesion and thrombus formation in whole flowing blood. RESULTS: Both integrin and glycoprotein VI-dependent interactions with fibrous collagen underpin high levels of platelet adhesion under both conditions, with little obvious effect of MMP-13 treatment. Static platelet adhesion to monomeric collagen was strongly α2ß1-dependent regardless of degradation status. Under flow conditions, partially degraded monomeric collagen supported increased thrombus deposition at 10 µg mL(-1) MMP-13, falling close to background when collagen degradation was complete (100 µg mL(-1) MMP-13). CONCLUSIONS: New binding activities come into play after partial digestion of collagen monomers, and net platelet-reactivity through all axes is abolished as degradation becomes more complete.
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Plaquetas/metabolismo , Colágeno Tipo I/metabolismo , Metaloproteinasa 13 de la Matriz/metabolismo , Adhesividad Plaquetaria , Trombosis/metabolismo , Electroforesis en Gel de Poliacrilamida , Humanos , Integrina alfa2beta1/metabolismo , Proteolisis , Trombosis/sangre , Trombosis/enzimologíaRESUMEN
We provide evidence to show that the standard reactant concentrations used in tissue engineering to cross-link collagen-based scaffolds are up to 100 times higher than required for mechanical integrity in service, and stability against degradation in an aqueous environment. We demonstrate this with a detailed and systematic study by comparing scaffolds made from (a) collagen from two different suppliers, (b) gelatin (a partially denatured collagen) and (c) 50% collagen-50% gelatin mixtures. The materials were processed, using lyophilisation, to produce homogeneous, highly porous scaffolds with isotropic architectures and pore diameters ranging from 130 to 260 µm. Scaffolds were cross-linked using a carbodiimide treatment, to establish the effect of the variations in crosslinking conditions (down to very low concentrations) on the morphology, swelling, degradation and mechanical properties of the scaffolds. Carbodiimide concentration of 11.5mg/ml was defined as the standard (100%) and was progressively diluted down to 0.1%. It was found that 10-fold reduction in the carbodiimide content led to the significant increase (almost 4-fold) in the amount of free amine groups (primarily on collagen lysine residues) without compromising mechanics and stability in water of all resultant scaffolds. The importance of this finding is that, by reducing cross-linking, the corresponding cell-reactive carboxylate anions (collagen glutamate or aspartate residues) that are essential for integrin-mediated binding remain intact. Indeed, a 10-fold reduction in carbodiimide crosslinking resulted in near native-like cell attachment to collagen scaffolds. We have demonstrated that controlling the degree of cross-linking, and hence retaining native scaffold chemistry, offers a major step forward in the biological performance of collagen- and gelatin-based tissue engineering scaffolds. STATEMENT OF SIGNIFICANCE: This work developed collagen and gelatine-based scaffolds with structural, material and biological properties suitable for use in myocardial tissue regeneration. The novelty and significance of this research consist in elucidating the effect of the composition, origin of collagen and crosslinking concentration on the scaffold physical and cell-binding characteristics. We demonstrate that the standard carbodiimide concentrations used to crosslink collagenous scaffolds are up to 100 times higher than required for mechanical integrity in service, and stability against dissolution. The importance of this finding is that, by reducing crosslinking, the corresponding cell-reactive carboxylate anions (essential for integrin-mediated binding) remain intact and the native scaffold chemistry is retained. This offers a major step forward in the biological performance of tissue engineered scaffolds.
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Colágeno/química , Reactivos de Enlaces Cruzados/química , Fenómenos Mecánicos , Andamios del Tejido/química , Aminas/análisis , Animales , Carbodiimidas/química , Bovinos , Comunicación Celular , Línea Celular Tumoral , Humanos , Microscopía Confocal , Microscopía Electrónica de Rastreo , Péptidos/química , Porosidad , Reología , Solubilidad , Suspensiones , Viscosidad , Agua/químicaRESUMEN
Recent work has established that various bone-resorbing hormones are able to activate phosphoinositide metabolism as well as eicosanoid production in osteoblast-like cells, although the relationship between these pathways is unclear. We used pertussis toxin and indomethacin to inhibit the stimulation of [3H]arachidonic acid release and [3H]phosphoinositide turnover caused by treating primary cultures of mouse osteoblasts with fetal calf serum. We found (1) that pertussis toxin and indomethacin each inhibited both pathways and (2) that although pertussis toxin inhibited [3H]arachidonic acid release to a greater extent than indomethacin, [3H]inositol phosphate accumulation was inhibited rather more effectively by indomethacin. These data suggest that whereas ligands in fetal calf serum activate [3H]arachidonic acid release largely directly via the action of a pertussis-sensitive G protein, activation of phosphoinositidase C is indirect, being substantially dependent upon eicosanoid production. These experiments suggest that serial activation of phospholipase A2 and phosphoinositidase C may occur in osteoblasts and that only the former enzyme is regulated by a pertussis toxin-sensitive G protein.