RESUMO
In 22 cases of chronic ITP, the platelet 5-HT storage organelles were counted by examination of platelets loaded with mepacrine and correlated with the size and volume of the platelets. Statistical analysis showed that the mean volume and the number of granules increased in ITP without increase in the mean number of granules per unit volume. A strong correlation was found between platelet long diameter and number of dense bodies in controls (44 healthy subjects) (r = 0.94; y = 2.826 x - 0.699) and in ITP (r = 0.92; y = 2.587 x + 0.06). This study demonstrated in chronic ITP the presence both of platelets without granules and others rich in granules. The anomalies were present no matter what the count of platelets and did not change the mean values for granules and for ADP in most cases. Most platelets remain morphologically normal.
Assuntos
Plaquetas/patologia , Púrpura Trombocitopênica/sangue , Quinacrina/sangue , Difosfato de Adenosina/sangue , Adolescente , Adulto , Idoso , Plaquetas/análise , Criança , Grânulos Citoplasmáticos/análise , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Although it is recognized that glycoprotein (GP) IIb-IIIa complexes are deficient in platelets in Glanzmann's thrombasthenia, little is known of the origin of the defect. We have examined the megakaryocytes in a bone marrow aspirate obtained from a thrombasthenia patient during surgery. Analysis of platelet proteins by SDS-polyacrylamide gel electrophoresis confirmed the patient to be of the type I subgroup. The megakaryocytes were examined by immunofluorescence or by immunocytochemical procedures combined with electron microscopy. Antibodies used included the murine monoclonal antibody, AP-2 and the human allo-antibody, IgG L, both of which recognize determinants on GP IIb-IIIa complexes. Bound antibody was detected by anti-IgG antibodies coupled to fluorescein isothiocyanate or absorbed on gold particles. In the immunofluorescence studies, permeabilized megakaryocytes were identified by double staining using an antibody to von Willebrand factor (vWF). Whereas mature megakaryocytes and their small precursor cells from normal individuals were strongly fluorescent with AP-2 and IgG L, most vWF positive cells from the Glanzmann's thrombasthenia patient were negative and the remainder gave but a weak background fluorescence. Immunogold staining on the surface of marrow cells was severely reduced. Our results confirm a deficiency of GP IIb-IIIa complexes in megakaryocytes in thrombasthenia.
Assuntos
Transtornos Plaquetários/patologia , Medula Óssea/patologia , Megacariócitos/citologia , Glicoproteínas da Membrana de Plaquetas/efeitos dos fármacos , Trombastenia/patologia , Plaquetas/análise , Imunofluorescência , Humanos , Megacariócitos/ultraestrutura , Microscopia Eletrônica , Glicoproteínas da Membrana de Plaquetas/análise , Valores de Referência , Trombastenia/sangueRESUMO
5-HT storage organelles were observed by electron microscope analysis in human megakaryocytes. They were less numerous per unit of surface than in platelets. Their number depended on the visualization technique employed. Thus after fixation with calcium-enriched glutaraldehyde a higher number of very opaque organelles was observed than of uranaffin-positive organelles after the cytochemical uranaffin reaction. With conventional electron microscopy deep black granules characteristic of dense bodies were not observed. Fluorescent microscopy showed greenish-yellow granules distributed throughout the whole cytoplasm in 96 +/- 1.4% of normal megakaryocytes incubated with mepacrine. In 85.6 +/- 5%, 5.28 +/- 1.28 granules per 10 microns 2 were observed. With the mepacrine labelling test, 74% of the megakaryocytes of a patient with Hermansky-Pudlak syndrome contained no granules. A similar finding was made in the platelets of the same patient. This suggests that mepacrine also stains the dense bodies in the megakaryocytes and that in the Hermansky-Pudlak syndrome the platelet anomaly is secondary to a megakaryocyte anomaly.
Assuntos
Transtornos Plaquetários/diagnóstico , Fluoresceínas , Megacariócitos/metabolismo , Quinacrina , Adulto , Transtornos Plaquetários/sangue , Plaquetas/metabolismo , Plaquetas/ultraestrutura , Grânulos Citoplasmáticos/metabolismo , Grânulos Citoplasmáticos/ultraestrutura , Feminino , Fluoresceína , Humanos , Megacariócitos/ultraestrutura , Serotonina/metabolismo , SíndromeRESUMO
A rapid method is described for the production of a human marrow cell suspension highly enriched in megakaryocytes. These concentrates were incubated with radiolabelled amino-acids, and cell lysates were then analysed for fibrinogen synthesis. Neosynthesized proteins were detected by immunoprecipitation, immuno-affinity chromatography and electrophoresis. Fluorography of the electrophoresis gels showed three radioactive bands corresponding to the three chains of cold fibrinogen. Immunoblotting and autoradiography of bidimensional, nonreduced-reduced electrophoresis gels showed that these three proteins were joined by disulfide bonds in the cell. These results suggest that megakaryocytes synthesize fibrinogen, and imply that platelet fibrinogen is of megakaryocytic origin.
Assuntos
Células da Medula Óssea , Fibrinogênio/biossíntese , Megacariócitos/metabolismo , Autorradiografia , Separação Celular , Eletroforese em Gel de Poliacrilamida , Fluorometria , Humanos , Técnicas de Imunoadsorção , MétodosRESUMO
The distribution of glycoprotein (GP) IIb-IIIa complexes in the surface membranes of human platelets and megakaryocytes was investigated by transmission electron microscopy of cells that had been incubated with Fab fragments of a human alloantibody (IgG L) specific for the complex. Binding was visualized by a second antibody conjugated to peroxidase or adsorbed onto gold particles. Initial studies showed that the peroxidase reaction product and the gold particles were to be found at the outer surface of unactivated platelets. The occasional small cluster of particles was present. A positive reaction, more apparent with peroxidase labelling, was also seen in the channels of the platelet open canalicular system. Gold particles were abundant on the outer surface of mature megakaryocytes, and their distribution resembled that on unactivated platelets. As with platelets, peroxidase-labelled antibodies penetrated better, and revealed GP IIb-IIIa in the demarcation membrane system. A double immunofluorescence study, involving Fab fragments of IgG L and rhodamine-conjugated antibodies to factor VIII R:Ag, demonstrated the presence of GP IIb-IIIa in megakaryocyte precursor cells. Our results show that the GP IIb-IIIa complex is present in megakaryocyte membranes and that it appears at the same time as the other platelet antigens.
Assuntos
Plaquetas/análise , Glicoproteínas/sangue , Megacariócitos/análise , Proteínas de Membrana/sangue , Antígenos de Superfície/análise , Plaquetas/imunologia , Plaquetas/ultraestrutura , Membrana Celular/análise , Células Cultivadas , Imunofluorescência , Humanos , Fragmentos Fab das Imunoglobulinas/metabolismo , Megacariócitos/imunologia , Megacariócitos/ultraestrutura , Microscopia Eletrônica , Glicoproteínas da Membrana de PlaquetasRESUMO
We have used immunogold staining to locate thrombospondin (TSP) on thrombin-activated human platelets, and have compared its distribution with that of fibrinogen (or fibrin) on thrombin- and ADP-stimulated platelets. To do this, isolated platelets were incubated with monospecific antibodies to TSP or fibrinogen (fib) and the bound IgG located with a second antibody adsorbed to gold particles. Thrombin-induced secretion in Tyrode-Ca2+ was followed by both anti-TSP and anti-fib binding, with large clusters of gold particles observed on the platelet surface. Little or no labeling was observed on unstimulated platelets with either antibody. When secretion was effected in Tyrode-EDTA, anti-TSP IgG still bound to the activated platelets, but the number of particle clusters was significantly reduced. Little binding of anti-fib IgG now occurred. Platelets activated with ADP in the presence of added fib, and subsequently incubated with anti-fib IgG, showed small particle clusters over the whole platelet surface. Thrombin-stimulated platelets from two patients with thrombasthenia bound anti-TSP IgG similarly to normal platelets activated in Tyrode-EDTA. No anti-fib binding occurred. Our results suggest that fib and TSP bind to specific domains on the stimulated platelet membrane. Such sites may be responsible for the mediation of platelet surface contact interactions.
Assuntos
Plaquetas/imunologia , Fibrinogênio/metabolismo , Glicoproteínas/metabolismo , Difosfato de Adenosina/farmacologia , Reações Antígeno-Anticorpo , Plaquetas/efeitos dos fármacos , Plaquetas/ultraestrutura , Grânulos Citoplasmáticos/metabolismo , Ouro , Humanos , Coloração e Rotulagem , Trombina/farmacologia , TrombospondinasRESUMO
Protein synthesis was investigated in human platelets by measuring incorporation of radio actively labelled amino acids into trichloroacetic acid (TCA) precipitable material. Platelet polysomes were characterized by their sedimentation rate in a sucrose gradient. It was confirmed that platelets synthesize proteins in their cytoplasm and that a part of their polysomes are bound to the skeletal framework. A higher level of protein synthesis was found in a population of small platelets separated on Ficoll (2%-4%) density gradient. Moreover, small platelets contained more polysomes than larger platelets. These results show that small platelets are more active in protein turnover or de novo synthesis. These findings can be related to peripheral maturation of platelets.
Assuntos
Plaquetas/metabolismo , Proteínas Sanguíneas/biossíntese , Plaquetas/classificação , Separação Celular , Células Cultivadas , Centrifugação com Gradiente de Concentração/métodos , Humanos , Polirribossomos/análise , Polirribossomos/metabolismoRESUMO
Previous studies have shown a decreased binding of monoclonal antibodies (MoAbs) to glycoprotein (GP) Ib-IX complexes on thrombin-stimulated platelets, but the reason for this is poorly understood. We have used (1) immunofluorescence procedures and flow cytometry, and (2) immunogold staining and electron microscopy to investigate this phenomenon. Washed platelets were incubated with alpha-thrombin, adenosine diphosphate, or ionophore A23187 for increasing lengths of time. For alpha-thrombin, but not the other agonists, flow cytometry confirmed a dose- and time-dependent decrease in the binding of MoAbs specific for GP Ib alpha (AP-1, Bx-1), GP IX (FMC 25), or to the complex itself (SZ 1). Immunoglold staining performed using standard transmission or scanning electron microscopy high-lighted surface areas devoid of bound antibody. However, a quantitatively normal immunofluorescence was restored if paraformaldehyde-fixed, thrombin-stimulated platelets were permeabilized with Triton X-100 (Sigma Chemical Co, St Louis, MO) before MoAb addition, while immunogold staining was now seen to be concentrated within the interior of the platelet. Glutaraldehyde-fixed samples were then embedded in the resin Lowicryl K4M (Taab Laboratories Equipment Ltd, Aldermaston, England) and immunogold staining performed on thin sections using a polyclonal antibody to glycocalicin. An increased presence of GP Ib-IX complexes within surface-connected membrane systems of the thrombin-stimulated platelets was confirmed. Interestingly, GP Ib-IX movement was opposite to the thrombin-induced externalization of internal pools of GP IIb-IIIa complexes and of the alpha-granule membrane GP, GMP-140.
Assuntos
Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Trombina/farmacologia , Difosfato de Adenosina/farmacologia , Plaquetas/efeitos dos fármacos , Calcimicina/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Fixadores , Citometria de Fluxo , Imunofluorescência , Formaldeído , Humanos , Imuno-Histoquímica , Substâncias Macromoleculares , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , Octoxinol , Polietilenoglicóis , PolímerosRESUMO
We have used monoclonal antibodies AP-1 (anti-GP Ib alpha). AP-2 (anti-GP IIb-IIIa) and FMC 25 (anti-GP IX) in immunofluorescence and immunocytochemical studies on megakaryocytes (MK) isolated from a Bernard-Soulier syndrome (BSS) patient whose giant platelets were characteristically deficient in GP Ib-IX complexes. Electron microscopy showed that the patient's MK were similar in size to normal MK. However, a striking feature was the variable and intermittent nature of the demarcation membrane system which was often vacuolar in appearance. Permeabilized mature MK from normal individuals were strongly positive with AP-2, AP-1 and FMC 25. Those from the BSS patient were normal for AP-2, negative for AP-1 but weakly positive with FMC 25. Binding of the monoclonal antibodies to the patient's platelets was evaluated using flow cytometry. The results confirmed the absence of GP Ib alpha from the surface membranes, but showed the presence of small amounts of GP IX distributed throughout the platelet population. Our findings confirm that the membrane lesion in BSS is also to be found in MK and further show that the defect may affect differently individual constituents of the GP-Ib-IX complex.
Assuntos
Síndrome de Bernard-Soulier/patologia , Megacariócitos/ultraestrutura , Adulto , Síndrome de Bernard-Soulier/metabolismo , Plaquetas/química , Plaquetas/ultraestrutura , Imunofluorescência , Humanos , Masculino , Megacariócitos/química , Glicoproteínas de Membrana/análise , Microscopia EletrônicaRESUMO
Heparin-dependent thrombocytopenia (HDT) and associated thrombotic complications, are thought to be linked to the appearance of anti-platelet antibodies. Attempts were made to characterize the antibodies in the sera from 10 such patients. Western blotting against platelet antigens was inconclusive, often revealing multiple bands but with considerable variability from patient to patient. An often seen band of approximately 83 kDa was also given by some nonimmune sera and the antigen appeared to be predominantly intracellular in origin. Antibodies to major membrane glycoproteins were not readily apparent. This was confirmed by the MAIPA ("Monoclonal Antibody Immobilization of Platelet Antigens") test, performed to detect antibodies to GP Ib-IX and GP IIb-IIIa. Only one weak activity to GP Ib-IX and one weak activity to GP IIb-IIIa were detected. In contrast, an antibody to the PlA1 alloantigen was readily detected in the serum of an additional patient. The ability of HDT serum to induce the aggregation of control platelets was studied in detail. Aggregation was inhibited by EDU-3, a monoclonal antibody to GP IIb-IIIa complexes, and by the synthetic peptide RGDS, suggesting an involvement of the same pathway as used by physiologic agonists. However, in agreement with Chong et al (Thromb Res 55:291, 1989), we observed that aggregation was inhibited by rabbit IgG, suggesting that it was Fc-receptor mediated. Interestingly, 2E1, a monoclonal antibody to the Fc gamma RII receptor, induced platelet aggregation with a lag phase, a characteristic of HD antibodies. Nonetheless, for different donors, there was no correlation between the length of the lag phase induced by 2E1 and HD antibodies. Fc-receptor blockade should be considered as a means for diminishing the clinical complications of HDT.
Assuntos
Doenças Autoimunes/induzido quimicamente , Heparina/efeitos adversos , Receptores Fc/fisiologia , Trombocitopenia/induzido quimicamente , Adulto , Especificidade de Anticorpos , Antígenos de Plaquetas Humanas/imunologia , Autoanticorpos/imunologia , Feminino , Humanos , Técnicas de Imunoadsorção , Integrina beta3 , Masculino , Agregação Plaquetária , Glicoproteínas da Membrana de Plaquetas/imunologia , Trombastenia/complicações , Trombastenia/imunologiaRESUMO
The role of glycoprotein (GP) IIb-IIIa complexes and of adhesive proteins in mediating platelet aggregation is now well defined. However, less is known of the changes that occur once aggregation has begun. We report immunogold staining of thin sections of platelets or platelet aggregates, embedded in Lowicryl K4M, after the use of polyclonal antibodies to GP IIb or GP IIIa, fibrinogen (Fg), von Willebrand factor (vWF), and thrombospondin (TSP). Bound immunoglobulin G (IgG) was located by species-specific anti-IgG coupled to 5-nm gold particles and by electron microscopy. Initial experiments with platelet-rich plasma confirmed the feasibility of visualizing adhesive proteins between platelets in aggregates. Experiments then continued, using stirred suspensions of washed platelets incubated with alpha-thrombin. After 20 seconds, platelets were in contact without detectable release, although giant secretory vesicles containing adhesive proteins were seen. Internal pools of GP IIb-IIIa were progressively externalized within the aggregate. Secreted Fg was readily detected between platelets at 40 seconds. After 3 minutes, when most of the secretion had occurred, Fg had a patchwork-like distribution within the aggregate. After 6 minutes, zones with closely interspaced surface membranes, usually representing pseudopods, were dominant and Fg free. Results for vWF and TSP were similar to those for Fg. Nonetheless, GP IIb-IIIa complexes continued to be located between adjacent surface membranes throughout the aggregate. Thrombin-induced platelet aggregates were isolated, and sodium dodecyl sulfate-soluble extracts were obtained. Western blot experiments showed that, although fibrinopeptide A had been cleaved, degradation of adhesive proteins by platelet proteases had not occurred. These results emphasize that a platelet aggregate is a dynamic structure and suggest that not all surface-contact interactions are mediated by Fg or the other adhesive proteins tested in this study.
Assuntos
Plaquetas/ultraestrutura , Proteínas Sanguíneas/metabolismo , Adesividade Plaquetária , Agregação Plaquetária/efeitos dos fármacos , Glicoproteínas da Membrana de Plaquetas/metabolismo , Trombina/farmacologia , Difosfato de Adenosina/farmacologia , Plaquetas/metabolismo , Western Blotting , Grânulos Citoplasmáticos/metabolismo , Fibrinogênio/metabolismo , Fibrinopeptídeo A/metabolismo , Humanos , Imuno-Histoquímica , Microscopia Eletrônica , Trombospondinas , Fator de von Willebrand/metabolismoRESUMO
We recently reported that after activation of human platelets by thrombin, glycoprotein (GP) Ib-IX complexes are translocated to the surface-connected canalicular system (SCCS) (Blood 76:1503, 1990). As GPIb is a major receptor for von Willebrand factor (vWF) in platelet adhesion, we have now examined the consequences of thrombin activation on the organization of vWF bound to GPIb on the platelet surface. Studies were performed using monoclonal or polyclonal antibodies in either immunogold staining and electron microscopy (Au-EM) or in flow cytometry. When unstirred platelet-rich plasma was incubated with ristocetin, bound vWF was located by Au-EM as discrete masses regularly distributed over the cell surface. Platelets from a patient with Glanzmann's thrombasthenia, lacking GPIIb-IIIa complexes, gave a similar pattern, confirming that this represented binding to GPIb. That ristocetin was not precipitating vWF before their binding to the platelets was shown by the detection of similar masses on the surface of platelets of a patient with type IIB von Willebrand disease. Experiments were continued using washed normal platelets incubated in Tyrode-EDTA, the purpose of the EDTA being to limit the surface expression of endogenous vWF after platelet stimulation. Under these conditions, platelets were treated with ristocetin for 5 minutes at 37 degrees C in the presence of increasing amounts of purified vWF. This was followed by incubation with thrombin (0.5 U/mL) for periods of up to 10 minutes. Flow cytometry showed a time-dependent loss in the surface expression of vWF bound to GPIb and these changes were confirmed by Au-EM. In particular, immunogold staining performed on ultrathin sections showed that the bulk of the vWF was being cleared to internal membrane systems. Surface clearance of vWF during thrombin-induced platelet activation is a potential mechanism for regulating platelet adhesivity.
Assuntos
Plaquetas/fisiologia , Ativação Plaquetária , Glicoproteínas da Membrana de Plaquetas/metabolismo , Trombina/farmacologia , Fator de von Willebrand/metabolismo , Anticorpos , Plaquetas/efeitos dos fármacos , Plaquetas/ultraestrutura , Humanos , Técnicas In Vitro , Microscopia Eletrônica , Microscopia Imunoeletrônica , Glicoproteínas da Membrana de Plaquetas/efeitos dos fármacos , Ristocetina/farmacologiaRESUMO
Recent advances have resulted in the elucidation of the principal molecular pathways of platelet function. Parallel studies have led to the identification of glycoprotein antigens whose presence at the platelet surface indicates an activated state. Such markers include GMP-140 and other glycoproteins of intracellular membranes whose translocation requires secretion and fusion of granule membranes with those joined to the surface. Other markers include activation-dependent epitopes on the GP IIb-IIIa complex and adhesive proteins bound to the activated GP IIb-IIIa receptor. Such epitopes can be detected by specific monoclonal antibodies. Quantification of their binding by flow cytometry allows an estimation of epitope expression within the whole platelet population. Our studies are designed to answer the question of whether measuring these epitopes is useful for predicting thrombosis in patients with acute cardiovascular disease. For this, we have examined three states where platelet function may be modified and where the risk of thrombosis and/or bleeding is increased. These include (i) patients with severe burns, (ii) patients who have undergone coronary angioplasty, and (iii) patients receiving fibrinolytic therapy following myocardial infarction. Our results show that activated platelets can be detected in the circulation and that their level reflects the degree of the lesion. Nonetheless, we have as yet failed to show a direct correlation between their presence and a future pathological event.
Assuntos
Ativação Plaquetária , Trombose/sangue , Sequência de Aminoácidos , Angioplastia Coronária com Balão , Plaquetas/imunologia , Queimaduras/sangue , Humanos , Dados de Sequência Molecular , Terapia TrombolíticaRESUMO
In the Caucasian population, platelet incompatibility within the HPA-1 (Pl(A1/A2)) and HPA-5 (Br(a/b)) alloantigen systems are the two most likely causes of post-transfusion purpura (PTP) and neonatal alloimmune thrombocytopenia. However, the way in which HLA (class-II) antigens participate in alloantibody formation is unclear. The patient (M-J.G.) is a middle aged woman with two children who developed a severe PTP (< 2000 platelets/µ1) 8 days after receiving red cell concentrates during coronary bypass surgery. During treatment with intravenous gamma-globulin and corticosteroids, her platelet count peaked, fell again, and returned to normal over a period of several months. Western blotting and/or the monoclonal antibody specific-immobilization of platelet antigens (MAIPA) assay performed with serum prepared at the height of her initial thrombocytopenia revealed antibodies to both the Pl(A1) and the Br(a) alloantigens. This rare combination prompted us to study the expression of specific HLA class II antigens in the patient. HLA-DR and DQ typing was performed from genomic DNA by the recently developed polymerase chain reaction-restriction fragment length polymorphism procedure (PCR-RFLP). The patient was found to express the DRB1*1302/1303 and DRB3*0101/0301 alleles (serological specificities: HLA-DR6 and DR52a/c respectively). She also expressed the DQA1*0102/0501, DQB1*0601 and DQB1*0301 alleles. Thus, this case provides further evidence linking an immune response to Pl(A1) and Br(a) antigens with HLA-DR52a/c and DR6.
RESUMO
A young Italian man (A.P.) has a lifelong history of bleeding from gums and mucocutaneous tissue. Electron microscopy showed a wide diversity of platelet size including giant forms. In citrated platelet-rich plasma (PRP), platelet aggregation induced by adenosine diphosphate (ADP) and other agonists was much reduced. Both secretion and clot retraction were normal. The aggregation of washed platelets with ADP was improved but remained subnormal, as was aggregation with collagen and thrombin. Fibrinogen-binding was analyzed by flow cytometry using platelets in whole blood or PRP and was markedly decreased. Crossed immunoelectrophoresis of Triton X-100 extracts of (A.P.) platelets showed that GP IIb-IIIa levels were 40% to 50% of normal. Glycoprotein (GP) IIb and GP IIIa were of usual migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but their labeling was much reduced during lactoperoxidase-catalyzed iodination. Binding to (A.P.) platelets of four different 125I-labeled monoclonal antibodies to GP IIb-IIIa complexes was reduced to 12% to 20% of normal levels. However, when the patient's platelets were stimulated with alpha-thrombin, monoclonal antibody binding showed the same increase (approximately 20,000 sites) as normal platelets. Both flow cytometry and immunocytochemical studies showed that the distribution of residual surface GP IIb-IIIa within the total (A.P.) platelet population was heterogeneous and not related to platelet size. Staining of ultrathin sections confirmed the presence of an internal pool of GP IIb-IIIa. Monoclonal antibodies to other membrane glycoproteins bound normally to (A.P.) platelets. The patient has a selective deficiency of the surface pool of GP IIb-IIIa complexes that is manifested clinically by a mild Glanzmann's thrombasthenia-like syndrome.