RESUMO
We report size reduction-induced chain breaking in the spin-1 Haldane-chain SrNi2-x Mg x V2O8 (x = 0 and 0.1) by magnetization and electron spin resonance measurements. For x = 0.0, the magnetic susceptibility of all samples can be well described by a temperature-independent term, a Curie-Weiss term and a Haldane-gap term. This implies that a reduced sample grain size breaks the long chain and creates a considerable number of S = 1/2 edge spins, resulting in the enhancement of magnetization and the decrease of Haldane gap in the samples. These edge spins as well as the other paramagnetic ions at grain boundary and surface might be weakly coupled with each other. For the Mg-doped sample with x = 0.1, there are more S = 1/2 spins creased in relative to x = 0.0 because of a combined effect of lattice defects, Mg-doping and reduced size. In addition, the antiferromagnetic resonance of x = 0.1 is also presented.
RESUMO
High-field electron spin resonance measurements of an antiferromagnet Ca3ZnMnO6 isostructure, with the Ising-chain multiferroic Ca3CoMnO6, have been carried out. Two distinct resonance modes were observed below TN = 25 K, which is well explained by conventional antiferromagnetic resonance theory with easy-plane anisotropy. The zero-field spin gap is derived to be about 166 GHz, originating from the easy-plane anisotropy and exchange interaction. Our result suggests that the Dzyaloshinsky-Moriya interaction, which may induce spin canting, is absent. Disappearance of Ising anisotropy in Ca3ZnMnO6 suggests that the Co(4+) ion, as well as the Co-Mn superexchange, plays an important role for the Ising nature in Ca3CoMnO6.
Assuntos
Compostos de Cálcio/química , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Compostos de Manganês/química , Óxidos/química , Compostos de Zinco/química , Anisotropia , Simulação por Computador , Magnetismo , Modelos Químicos , Estrutura Molecular , Marcadores de SpinRESUMO
Smooth muscle cell proliferation plays a major role in the genesis of restenosis after angioplasty or vascular injury. Controlled release of appropriate drugs alone and in combinations is one approach for treating coronary obstructions, balloon angioplasty, restenosis associated with thrombosis, and calcification. We demonstrated the possibility of encapsulating taxol-loaded polylactic acid (PLA) microspheres within heparin-chitosan spheres to develop a prolonged release co-matrix form. The in vitro release profile of taxol and heparin from this co-matrix system was monitored in phosphate buffered saline pH 7.4, using an ultraviolet spectrophotometer. The amount of taxol/heparin release was initially much higher, followed by a constant slow release profile for a prolonged period. The initial burst release of taxol (15.8%) and heparin (32.7%) from the co-matrix was modified with polyethylene glycol coatings (13.5% and 25.4%, respectively, for 24 hr). From scanning electron microscopy studies, it appears that these drugs diffuse out slowly to the dissolution medium through the micropores of the co-matrix. However, the surface micropores were modified with polyethylene glycol (PEG) coatings for a constant slow release profile. This PEG-coated PLA/chitosan co-matrix may target drug combinations having synergestic effects for prolonged periods to treat restenosis.
Assuntos
Anticoagulantes/administração & dosagem , Antineoplásicos Fitogênicos/administração & dosagem , Quitina/química , Sistemas de Liberação de Medicamentos , Excipientes , Oclusão de Enxerto Vascular/prevenção & controle , Heparina/administração & dosagem , Ácido Láctico/química , Paclitaxel/administração & dosagem , Polímeros/química , Anticoagulantes/uso terapêutico , Antineoplásicos Fitogênicos/uso terapêutico , Quitina/análogos & derivados , Quitosana , Preparações de Ação Retardada , Composição de Medicamentos , Heparina/uso terapêutico , Microscopia Eletrônica de Varredura , Microesferas , Paclitaxel/uso terapêutico , Tamanho da Partícula , Poliésteres , Solubilidade , Tempo de TrombinaRESUMO
Human platelets undergo agglutination when stirred with bovine plasma (BP), but bovine platelets do not. The present study has shown that exposure of washed bovine platelets to subthreshold concentrations of adenosine diphosphate or thrombin before stirring restores their sensitivity to BP, and the cells undergo rapid agglutination. This agglutination was prevented by a monoclonal antibody, to glycoprotein GPIb. Flow cytometry studies revealed that exposure of bovine platelets to thrombin caused an increase in their ability to bind antibodies known to react with human GPIb or GPIIb-IIIa receptors. Interaction of bovine and human platelets with vascular subendothelium revealed additional differences in reactivity. Bovine platelets in citrate anticoagulant reacted poorly with subendothelium under flow conditions compared with human platelets. In contrast, bovine platelets in blood with low molecular weight heparin as anticoagulant adhered more readily than human cells. These findings suggest that different mechanisms are involved in hemostasis in human and bovine species.
Assuntos
Plaquetas/efeitos dos fármacos , Bovinos/sangue , Endotélio Vascular/fisiologia , Fator de von Willebrand/farmacologia , Difosfato de Adenosina/farmacologia , Animais , Plaquetas/metabolismo , Plaquetas/fisiologia , Endotélio Vascular/metabolismo , Humanos , Adesividade Plaquetária , Agregação Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/efeitos dos fármacos , Complexo Glicoproteico GPIb-IX de Plaquetas/efeitos dos fármacos , Trombina/farmacologia , Fator de von Willebrand/agonistasRESUMO
The development of injectable microspheres for anticancer drug delivery into the brain is a major challenge. The possibility of entrapping 5-fluorouracil (5-FU) in chitosan coated monodisperse biodegradable microspheres with a mean diameter of 10-25 um was demonstrated. An emulsion of 5-FU (in water) and polylactic acid (PLA) dissolved in acetone-dichloromethane mixture was poured into an aqueous solution of chitosan (or poly-vinyl alcohol) with stirring using a high-speed homogenizer, for the formation of microspheres. 5-FU recovery in microspheres ranged from 44-66% depending on the polymer and emulsification systems used for the preparation. Scanning electron microscopy revealed that the chitosan coated microspheres had less surface micropores compared to PVA based preparations. The drug release behaviour from microspheres suspended in phosphate buffered saline exhibited a biphasic pattern. The amount of drug release was much higher initially (approximately 25%), followed by a constant slow release profile for a 30 days period of study. This chitosan coated PLA/PLGA microsphere formulation may have potential for the targeted delivery of 5-FU to treat cerebral tumours.
Assuntos
Antimetabólitos Antineoplásicos/administração & dosagem , Neoplasias Encefálicas/tratamento farmacológico , Fluoruracila/administração & dosagem , Antimetabólitos Antineoplásicos/uso terapêutico , Biodegradação Ambiental , Cápsulas , Quitina/análogos & derivados , Quitosana , Portadores de Fármacos , Composição de Medicamentos/métodos , Fluoruracila/uso terapêutico , Glioma/tratamento farmacológico , Humanos , Ácido Láctico , Microscopia Eletrônica de Varredura , Microesferas , Tamanho da Partícula , Poliésteres , PolímerosRESUMO
Smooth muscle cell proliferation plays a major role in the genesis of restenosis after angioplasty or vascular injury. Local delivery of agents capable of modulating vascular responses have the potential to prevent restenosis. However, the development of injectable microspheres for maintaining high tissue levels of drugs at the site of vascular injury is a major challenge. We demonstrated the possibility of entrapping an antiproliferative agent, colchicine, in polyethylene glycol (PEG)-coated biodegradable microspheres composed of poly(lactic acid)/poly(epsilon-caprolactone) blends, with a mean diameter of 3-6 microm. A solution of colchicine and blends of polylactic acid (PLA)/polycaprolactone (PCL) dissolved in acetone-dichloromethane mixture was poured into an aqueous solution of PEG (or polyvinyl alcohol) with stirring by a high-speed homogenizer to form microspheres. Colchicine recovery in microspheres ranged from 30-50% depending on the emulsification system and the ratio of polymer blends used for the preparations. Scanning electron microscopy revealed that the PLA/PCL microspheres were spherical in shape and had a smooth surface texture. Results of in vitro release studies showed that it is possible to control the colchicine release by choosing the appropriate particle size, loading, and PLA/PCL composition. Water permeability through the PLA membrane was greater, when compared with PCL blends. The amount of drug release also was much higher (58.3%) in PLA compared with PCL (39.3%) microspheres, for 30 days. Therefore, we concluded that the drug release from the microspheres followed a diffusion mechanism where bulk erosion and surface deposition were negligible. These PEG-coated PLA/PCL microspheres may have potential for targeting antiproliferative agents for prolonged periods to treat restenosis.
Assuntos
Colchicina/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Excipientes/administração & dosagem , Supressores da Gota/administração & dosagem , Polietilenoglicóis/administração & dosagem , Caproatos/administração & dosagem , Caproatos/síntese química , Cápsulas , Colchicina/síntese química , Excipientes/síntese química , Supressores da Gota/síntese química , Ácido Láctico/administração & dosagem , Ácido Láctico/síntese química , Lactonas/administração & dosagem , Lactonas/síntese química , Poliésteres , Polietilenoglicóis/síntese química , Polímeros/administração & dosagem , Polímeros/síntese químicaRESUMO
The search for a nonthrombogenic material having patency to be used for small diameter vascular graft applications continues to be a field of extensive investigation. The purpose of the present study was to examine whether surface modification of polytetra fluoroethylene (PTFE, Teflon) and polyethylene-terephthalate (Dacron) vascular grafts might extend graft biocompatibility without modifying the graft structure. A series of surface coatings were prepared by modifying the argon plasma-treated PTFE and Dacron grafts with collagen IV and laminin and subsequently immobilizing bioactive molecules like PGE1, heparin or phosphatidyl choline via the carbodiimide functionalities. Surface analysis by Fourier transform infrared spectroscopy-attenuated total reflectance revealed the presence of new functional groups on the modified graft surfaces. In vitro studies showed that fibrinogen adsorption and platelet adhesion on modified grafts were significantly reduced. This study proposes that surface grafting of matrix components (collagen-type IV and laminin) and subsequent immobilization of bioactive molecules (PGE1, heparin or phosphatidyl choline) changed the surface conditioning of vascular grafts and subsequently improved their biocompatibility. However, more detailed in vivo studies are needed to confirm these observations.
Assuntos
Materiais Biocompatíveis , Biotecnologia , Prótese Vascular , Sangue , Polietilenotereftalatos , Politetrafluoretileno , Plaquetas/citologia , Plaquetas/ultraestrutura , Adesão Celular , Ensaio de Imunoadsorção Enzimática , Humanos , Microscopia Eletrônica de Varredura , Espectroscopia de Infravermelho com Transformada de Fourier , Propriedades de SuperfícieRESUMO
A technique is described to encapsulate activated charcoal for hemoperfusion to be used in an artificial liver support. Activated charcoal was encapsulated within chitosan-PEG matrix and subsequently surface modified with PGE1 or heparin (hep-AC-PEGCB) via the glutaraldehyde functionalities. This novel matrix was used as the supports for perfusion of endotoxin, under a flow rate of 30 ml/mt. Endotoxin adsorption was quantitatively measured by the method of Limulus Amebocyte lysate test. It seems, the hep-AC-PEGCB may be a good adsorbent system for the removal of toxic endotoxin, and the system may be useful for detoxification of blood. The hep-AC-PEGCB matrix had improved biocompatibility as demonstrated from their hemolytic potential and charcoal release. However, further studies are needed to determine their behaviour under clinical conditions.
Assuntos
Carvão Vegetal/farmacocinética , Quitina/análogos & derivados , Composição de Medicamentos/métodos , Endotoxinas/farmacocinética , Hemoperfusão/métodos , Heparina/metabolismo , Polietilenoglicóis/farmacocinética , Alprostadil/metabolismo , Materiais Biocompatíveis/metabolismo , Materiais Biocompatíveis/farmacocinética , Carvão Vegetal/metabolismo , Quitina/metabolismo , Quitina/farmacocinética , Quitosana , Composição de Medicamentos/normas , Estudos de Avaliação como Assunto , Fibrinolíticos , Humanos , Polietilenoglicóis/metabolismo , Trombose/prevenção & controleRESUMO
A bioartificial pancreas, a medical device entrapping islets of Langerhans (islets) in an immunoisolative membrane, has been regarded as one of the most promising approaches to treat insulin-dependent diabetic patients. In this study, various modifications of alginate-chitosan microcapsules were made such as the inclusion of polyethylene glycol (PEG) and the use of crosslinkers such as carbodiimide (EDC) and glutaraldehyde (GA) in the core and onto the microcapsule membrane surface. A characterization of the modified microcapsules in terms of mechanical stability and albumin diffusion as well as their surface properties using SEM was performed. A mild GA treatment greatly enhanced the mechanical stability of the microcapsules, and this treatment did not affect the coating process of chitosan or PEG. The biological response to such microcapsules was evaluated by microencapsulation of red blood cells (RBC) and subsequent observation of their hemoglobin release. The encapsulated RBC in the PEG-GA coated microcapsules were found to be less hemolytic and had improved stability and biocompatibility. The results suggest the possibility of developing biological assist organs by microencapsulation of mammalian cells such as islets or liver cells in immunoisolative microcapsules in the near future.
Assuntos
Alginatos/química , Materiais Biocompatíveis/química , Biopolímeros/química , Quitina/análogos & derivados , Ilhotas Pancreáticas/citologia , Membranas Artificiais , Pâncreas Artificial , Polietilenoglicóis/química , Albuminas/química , Cápsulas , Carbodi-Imidas/química , Quitina/química , Quitosana , Reagentes de Ligações Cruzadas/química , Diabetes Mellitus Tipo 1/terapia , Difusão , Composição de Medicamentos , Eritrócitos/citologia , Estudos de Avaliação como Assunto , Glutaral/química , Hemoglobinas/metabolismo , Hemólise , Humanos , Microscopia Eletrônica de Varredura , Estresse Mecânico , Propriedades de SuperfícieRESUMO
The search for a noncalcifying tissue material to be used for valve replacement application continues to be a field of extensive investigation. A series of porcine pericardial membranes was prepared by modifying the glutaraldehyde--treated tissues with albumin and subsequently immobilizing bioactive molecules like PGE1, PGI2 or heparin via the carbodiimide functionalities. The in vitro calcification and collagenase degradation of these modified tissues were studied as a function of exposure time. Furthermore, the biocompatibility aspects of such novel interfaces were established by platelet adhesion and fibrinogen adsorption. The results reported in this article propose that the treatment with antiplatelet agents such as albumin, heparin and prostaglandins (PGE1 or PGI2) change the surface conditioning of pericardial tissues, suggesting a possible role of deposited serum components in affecting mineralization process on bioprosthesis. Therefore, it is worthy to hypothesize that besides inhibiting the accumulation of calcium in the devitalized cells, the early formation of a conditioning layer on the bioprosthesis surface may affect salt precipitations, determining the propensity of the implant to calcify. More detailed studies are needed to understand the involvement of plasma proteins and cellular components of the recipient blood in tissue-associated calcification.
Assuntos
Materiais Biocompatíveis , Calcinose/prevenção & controle , Cardiomiopatias/prevenção & controle , Teste de Materiais , Pericárdio/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Trombose/prevenção & controle , Albuminas/farmacologia , Alprostadil/farmacologia , Animais , Calcinose/patologia , Cardiomiopatias/patologia , Técnicas de Cultura , Ensaio de Imunoadsorção Enzimática , Epoprostenol/farmacologia , Fibrinogênio/análise , Heparina/farmacologia , Humanos , Pericárdio/ultraestrutura , Adesividade Plaquetária/efeitos dos fármacos , Probabilidade , Propriedades de Superfície , SuínosRESUMO
Dual color flow cytometric techniques were applied to micro-aliquots of whole blood obtained from bleeding time (BT) wounds. Modifications in platelet activation markers (P-selectin [CD62P]) and lysosomal related protein (UMPS [CD63]), presence of membrane glycoproteins (GPIb [CD42b], GPIIb-IIIa [CD41a], GDIV [CD36], binding of von Willebrand factor (vWF), fibrinogen (Fg) and factor V [FV]) were analyzed in normal donors (n = 10) and in a severe von Willebrand patient (type 3) von Willebrand disease [vWD]). Samples of blood (20 microl) were sequentially removed from BT wound edges for up to 5 min and fixed with paraformaldehyde. Antigens were detected using the corresponding tagged monoclonal antibodies (moAbs) and quantitative results were referred to those found on platelet samples obtained from venous blood obtained from the same individuals. A progressive increase in % of platelets positive for activation dependent antigens (CD62 from 7 +/- 2 to 48 +/- 19% and CD63 from 9 +/- 1 to 44 +/- 8%; initial vs. 4 min) was observed. Accessibility of GPIIb-IIIa epitopes on platelets from BT wounds remained slightly above levels observed in venous blood platelets, despite a progressive increase in the presence of platelets positive for Fgn. Binding of MoAb to GPIV increased at late stages of BT. A moderate decrease in the binding of a moAb to GPIb was observed on platelets obtained at late stages of the BT (14 +/- 9% and 20 +/- 6% at 4 and 5 min, respectively). This apparent decrease in GPIb epitopes paralleled an increased presence of platelets positive for vWF (26 +/- 12 and 38 +/- 15%). Binding of moAb to GPIb always remained above basal levels in platelets obtained from BTs performed in the patient with type 3 vWD. FV levels on platelets coming from the BT persisted at background levels in all the individuals and at all times studied.
Assuntos
Tempo de Sangramento , Fator V/metabolismo , Ativação Plaquetária/fisiologia , Glicoproteínas da Membrana de Plaquetas/metabolismo , Doenças de von Willebrand/sangue , Anticorpos Monoclonais/metabolismo , Antígenos CD36/imunologia , Antígenos CD36/metabolismo , Fibrinogênio/metabolismo , Humanos , Contagem de Plaquetas , Fator de von Willebrand/metabolismoRESUMO
Platelet attachment and spreading were monitored on glass and various protein coated glass, under shear with washed platelets, platelet rich plasma (PRP) and whole blood, using fluorescence Optimas imaging system and software. Results showed that the platelet adhesion and spreading were sensitive to the nature of precoated proteins and the type of medium used for introducing platelet suspension for the study. In general, the cell adhesion and spreading were higher with fibrinogen (Fg), fibronectin (Fn), von Willebrand Factor (vWF), and collagen precoated surfaces. In the presence of albumin on the surface, however, platelets could not attach and spread fully when using washed cells. But, the surface attachment and spreading of the cells were higher on albumin substrates on exposure to PRP or whole blood. This may be due to the replacement of precoated albumin by other plasma proteins, like Fg to facilitate the platelet-surface attachment. The composition of this layer determines the extent of platelet activation and the adhesive strength between platelets and polymer surface. These results indicate that multiple adhesion receptors can mediate platelet adhesion and spread to matrix proteins immobilized on surfaces. Further, these studies combined with some of our earlier observations and suggestions propose the need for developing in vitro tests that resemble in vivo conditions.
Assuntos
Plaquetas/fisiologia , Proteínas Sanguíneas/farmacologia , Plasma/fisiologia , Adesividade Plaquetária/efeitos dos fármacos , Adsorção , Vidro , Humanos , Técnicas In Vitro , Microscopia de Vídeo/instrumentação , Microscopia de Vídeo/métodos , Reologia/instrumentação , Reologia/métodos , Reologia/estatística & dados numéricos , Propriedades de Superfície , Fatores de TempoRESUMO
To develop tissue valves for prolonged use in the cardiovascular system, the complicated process of surface induced calcification must be better understood. Calcification was examined for 60 days on glutaraldehyde treated bovine pericardium (GABP) and enzyme extracted tissues fixed in glutaraldehyde (GATBP) incubated in metastable solutions of calcium phosphate, and the roles of aspirin and persantine in conjunction with vitamins C, B, or E, gentamycin (antibiotic), or pentothal sodium (anesthetic) in the medium were examined. Further, the diffusion of calcium across the GATBP was evaluated using a diffusion cell with 2 compartments. Pericardial calcification was also observed using scanning electron microscopy (SEM) techniques. It seems that the examined antiplatelet agents can modify the pericardial surfaces and subsequently their mineralization processes (GATBP, 31.7 micrograms/mg tissue; in the presence of 5 mg% vitamin C, 13.1 micrograms/mg tissue; in 1.5 mg% aspirin, 17.2 micrograms/mg tissue; and 1 mg% gentamycin, 14.8 micrograms/mg tissue) on exposure with the metastable calcium phosphate solution for 60 days. In addition, these agents may modify calcium transport and interfere with the adsorption at the surface, hence reducing calcium nodulation on GATBP. Scanning electron micrographs also revealed a reduction in calcium deposition on the pericardium due to these antiplatelet agents. It may be hypothesized that the influx of calcium on GATBP may be due to the cellular components or the involvement of plasma proteins like the fibrinogen molecule. The exact mechanism of these changes in the calcification of the pericardium are still unknown. From these in vitro findings, it appears that a combined vitamin therapy with low doses of aspirin may be beneficial for platelet suppression and thereby for prevention of thrombosis and calcification. However, more in vivo studies are needed to develop applications.
Assuntos
Bioprótese , Calcinose/patologia , Pericárdio/patologia , Inibidores da Agregação Plaquetária/farmacologia , Anestésicos Intravenosos/farmacologia , Animais , Antibacterianos/farmacologia , Aspirina/farmacologia , Bovinos , Dipiridamol/farmacologia , Gentamicinas/farmacologia , Glutaral , Técnicas In Vitro , Tiopental/farmacologia , Fixação de Tecidos , Vitaminas/farmacologiaRESUMO
The discoid form of blood platelets is important to their function in hemostasis. Recent studies have suggested that the spectrin-rich surface membrane cytoskeleton and the cytoplasmic, actin-rich cytoskeleton are responsible for discoid shape, shape change, and recovery after activation or chilling. Earlier studies had suggested that circumferential coils of microtubules supported the disc shape of resting platelets and that their repositioning or reassembly restored disc shape after exposure to low temperature. The present study has used the chilling-rewarming model, together with microtubule stabilizing (taxol) and disassembling (vincristine) agents to retest the relative importance of the surface membrane cytoskeleton and circumferential microtubules in platelet discoid shape and its restoration. Washed platelet samples were rested at 37 degrees C and chilled to 4 degrees C; chilled and rewarmed to 37 degrees C for 60 minutes; or chilled, rewarmed, and exposed to the same cycle in the presence or absence of vincristine or taxol and fixed for study by disseminated interference phase contrast microscopy and electron microscopy. Rhodamine-phalloidin and flow cytometry were used to measure changes in actin filament assembly. Chilling caused loss of disc shape, pseudopod extension, disassembly of microtubule coils, and assembly of new actin filaments. Rewarming resulted in restoration of disc shape, pseudopod retraction, disassembly of new actin filaments, and reassembly of circumferential microtubule coils. Vincristine converted discoid platelets to rounded cells that extended pseudopods when chilled and retracted them when rewarmed, leaving spheres that could undergo the same sequence of changes when chilled and rewarmed again. Taxol prevented cold-induced disassembly of microtubules and limited pseudopod formation. Rewarming caused retraction of pseudopods on taxol-treated, discoid cells. Cytochalasin B, an agent that blocks new actin filament assembly, alone or in combination with taxol, inhibited the cold-induced shape change but not dilation of the open canalicular system. Rewarming eliminated open canalicular system dilation and restored lentiform appearance. The results indicate that microtubule coils are the major structural elements responsible for disc shape and its restoration after submaximal stimulation or rewarming of chilled platelets.
Assuntos
Plaquetas/citologia , Citoesqueleto/fisiologia , Microtúbulos/fisiologia , Actinas/antagonistas & inibidores , Actinas/fisiologia , Plaquetas/fisiologia , Plaquetas/ultraestrutura , Membrana Celular/fisiologia , Colchicina/farmacologia , Temperatura Baixa , Citocalasina B/farmacologia , Temperatura Alta , Humanos , Microtúbulos/efeitos dos fármacos , Nocodazol/farmacologia , Paclitaxel/farmacologia , Valores de Referência , Temperatura , Vincristina/farmacologiaRESUMO
The receptor for von Willebrand factor (vWF) on human platelets, glycoprotein (GP) Ib/IX, has been shown in our studies to be an immobile complex when stimulated in suspension or on surfaces. Recent investigations have revealed that GP Ib/IX remains immobile on platelets activated in suspension followed by exposure to formvar surfaces that cause the cells to spread. However, since channels of the open canalicular system (OCS) are evaginated back on to the exposed surface during spreading, it was suggested that our study missed the clearance of GP Ib/IX from the exposed surface to internal membranes. The present study has added cytochalasin B after exposure of platelets to thrombin or TRAP in suspension in order to prevent spreading and movement of GP Ib/IX during subsequent exposure to surface activation on formvar grids. Results indicate that GP Ib/IX receptors remain randomly dispersed from edge to edge on platelets activated by thrombin or TRAP in suspension 10 minutes before treatment with CB followed by surface activation. Statistical analysis of the frequency of immunogold particles binding to monoclonal antibodies attached to GP Ib/IX revealed no significant reduction in frequency, translocation from cell edges or concentration of GP Ib/IX receptors in or around channels of the OCS. Results support the concept that GP Ib/IX is not cleared from exposed surfaces to the OCS of platelets activated by thrombin or TRAP and surface activation.
RESUMO
The influence of ionized calcium on the down-regulation of GPIb/IX receptors on human platelets was evaluated by flow cytometry using monoclonal antibodies. Addition of EDTA alone to a washed platelet suspension did not cause decreased monoclonal antibody binding to the cells. However, introduction of thrombin to the washed platelets containing EDTA resulted in a marked decrease in binding of monoclonal antibodies to the GPIb/IX receptors. If calcium at 1-3 mmol/L was added to buffered platelets instead of EDTA before thrombin, down-regulation was prevented or significantly reduced. Restoring calcium to EDTA platelets after the thrombin-stimulated down-regulation had been in progress for 1-3 minutes caused reversal of decreased antibody binding by GPIb/IX to near resting levels. Results demonstrate that extracellular calcium is a major factor regulating thrombin-induced down-regulation.
Assuntos
Plaquetas/metabolismo , Cálcio/metabolismo , Complexo Glicoproteico GPIb-IX de Plaquetas , Glicoproteínas da Membrana de Plaquetas , Receptores de Superfície Celular/metabolismo , Trombina/farmacologia , Regulação para Baixo , Ácido Edético , Citometria de Fluxo , HumanosRESUMO
The present investigation has used an aggregation-disaggregation-reaggregation model to determine if exposure to potent aggregating agents causing a refractory state in deaggregated platelets results in down-regulation and clearance of GPIb/IX, the receptor for von willebrand factor. Thrombin, the thrombin receptor activating peptide (TRAP) and the thromboxane A2 mimic, U46619, caused irreversible aggregation and secretion when stirred on an aggregometer with plateletrich plasma (PRP). Addition of prostaglandin E1 (PGE1) after the plateau of maximum aggregation was reached caused rapid dissociation of platelet aggregates. Dissociated platelets were refractory to a second exposure to the primary stimulus or to other aggregating agents. Exposure of the refractory cells to epinephrine before the primary agent restored sensitivity resulting in a second wave of irreversible aggregation. Deaggregated, refractory platelets, however, retained their sensitivity to ristocetin. Amounts of the antibiotic causing agglutination of resting PRP also caused agglutination of disaggregated, refractory platelets. Addition of epinephrine to samples of refractory platelets less sensitive to low concentrations of the antibiotic restored their capacity to undergo irreversible ristocetin-induced agglutination. Analysis of the frequency of gold particles associated with monoclonal antibodies directed against GPIb/IX on control platelets and disaggregated, refractory platelets showed no significant difference in the number of receptors. The findings indicate that significant numbers of GPIb/IX receptors remain on platelet surfaces following exposure to potent aggregating agents.
Assuntos
Plaquetas/efeitos dos fármacos , Agregação Plaquetária , Ristocetina/farmacologia , Adulto , Alprostadil/farmacologia , Regulação para Baixo , Sinergismo Farmacológico , Epinefrina/farmacologia , Humanos , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Trombina/farmacologiaRESUMO
Based on the premise of achieving blood compatibility through mimicking the chemical constitutents of the biologically insert surface of the unactivated platelet membrane, a process was developed that entails the covalent grafting of modified phosphatidylcholine molecules to materials including silica, polypropylene, and polytetrafluoroethylene (PTFE) polymer films. These materials were characterized using x-ray photoelectron spectroscopy (XPS) and contactangle measurements. The phosphatidylcholine-containing materials (PC materials) were used as substrates in the plateletadhesion assays and were subjected to enzymatic degradation evaluation. Phosphatidylcholine-grafted silica materials do not support platelet adhesion. In addition the number of adherent platelets correlate with the amount of grafted phospholipid present, as indicated by the phosphorus/ carbon ratio obtained by XPS analysis. Platelet adhesion to phosphatidylcholine-grafted polypropylene and PTFE was inhibited 80% and 90%, respectively, when compared with platelet adhesion to unmodified polypropylene and PTFE.