RESUMEN
Coagulation factor XIII (FXIII) is a heterotetramer consisting of 2 catalytic A subunits (FXIII-A2) and 2 protective/inhibitory B subunits (FXIII-B2). FXIII-B, a mosaic protein consisting of 10 sushi domains, significantly prolongs the lifespan of catalytic subunits in the circulation and prevents their slow progressive activation in plasmatic conditions. In this study, the biochemistry of the interaction between the 2 FXIII subunits was investigated. Using a surface plasmon resonance technique and an enzyme-linked immunosorbent assay-type binding assay, the equilibrium dissociation constant (Kd) for the interaction was established in the range of 10(-10) M. Based on the measured Kd, it was calculated that in plasma approximately 1% of FXIII-A2 should be in free form. This value was confirmed experimentally by measuring FXIII-A2 in plasma samples immunodepleted of FXIII-A2B2. Free plasma FXIII-A2 is functionally active, and when activated by thrombin and Ca(2+), it can cross-link fibrin. In cerebrospinal fluid and tears with much lower FXIII subunit concentrations, >80% of FXIII-A2 existed in free form. A monoclonal anti-FXIII-B antibody that prevented the interaction between the 2 subunits reacted with the recombinant combined first and second sushi domains of FXIII-B, and its epitope was localized to the peptide spanning positions 96 to 103 in the second sushi domain.
Asunto(s)
Líquidos Corporales/química , Factor XIII/metabolismo , Factor XIIIa/metabolismo , Proteínas Recombinantes/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Formación de Anticuerpos , Reactivos de Enlaces Cruzados/farmacología , Ensayo de Inmunoadsorción Enzimática , Mapeo Epitopo , Factor XIII/inmunología , Factor XIIIa/inmunología , Fibrina/metabolismo , Humanos , Cinética , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Resonancia por Plasmón de SuperficieRESUMEN
Von Willebrand factor (VWF) is built up from a varying number of subunits, of which the larger molecules have higher haemostatic activity. Von Willebrand disease (VWD) and thrombotic thrombocytopenic purpura are the best known disorders with pathognomonic changes of the highly multimerised VWF forms. There is an established method to calculate the relative amount of large oligomers. Our aim is to quantify the degree of VWF multimerisation as well, to complete the densitometric analysis of VWF electrophoresis. After optimisation, we have defined this new parameter (M(MW)) as the molecular weight corresponding to the lower boundary of the largest 25% of VWF protein. M(MW) was significantly different (p < .0001) in platelet lysate, normal samples and VWD type 2 samples (10.4, 6.3, 2.1, respectively). There was strong correlation between the M(MW) and the amount of large multimers in normal samples (r(2) = 0.98) and in platelet lysate. However M(MW) was higher in platelet lysate, in which VWF is not cleaved by ADAMTS-13, than in healthy samples with the same amount of large multimers. Comparison of the new parameter and the collagen binding and ristocetin cofactor activity of VWF, showed that the functional tests are at least partially determined by the multimerisation; however, about 15% of VWD samples had normal activity to antigen ratios. The quantification of multimerisation aids the classification in these cases, especially at low antigen concentrations, and also might help in the detection of thrombotic conditions.
Asunto(s)
Densitometría/métodos , Factor de von Willebrand/química , Proteínas ADAM/metabolismo , Proteína ADAMTS13 , Tampones (Química) , Estudios de Casos y Controles , Colágeno/metabolismo , Densitometría/estadística & datos numéricos , Humanos , Técnicas In Vitro , Peso Molecular , Complejos Multiproteicos/química , Unión Proteica , Estructura Cuaternaria de Proteína , Enfermedades de von Willebrand/sangre , Factor de von Willebrand/análisis , Factor de von Willebrand/metabolismoRESUMEN
The present study describes severe multiplex cerebral ischaemic laesions in a male patient being diagnosed with polycythaemia vera (PV). In contrast to previous publications, unique platelet receptor pattern with normal platelet count was identified. Glycoprotein Ib receptor number on the surface of resting platelets was increased two-fold and almost three-fold in case of activated platelets compared to the controls. More over, in an in vitro study when whole blood was circulated both at venous and arterial shear conditions and shear rate was adjusted according to the blood viscosity, platelet aggregate/thrombus formation was characteristic on surfaces covered with purified von Willebrand factor while in case of controls the surface was covered with single platelets or platelet monolayer. Similar results with pathological findings have not been published in PV until now. Our result undersigns the necessity of antiplatelet therapy of PV patients, even at normal platelet count.
Asunto(s)
Isquemia Encefálica/sangre , Adhesividad Plaquetaria , Complejo GPIb-IX de Glicoproteína Plaquetaria/análisis , Policitemia Vera/complicaciones , Adulto , Células Cultivadas , Humanos , Masculino , Inhibidores de Agregación Plaquetaria/uso terapéutico , Recuento de Plaquetas , Pruebas de Función Plaquetaria , Policitemia Vera/tratamiento farmacológicoRESUMEN
BACKGROUND: Antibodies directed against Saccharomyces cerevisiae (ASCA), perinuclear components of neutrophils (pANCA), and porin protein C of Escherichia coli (anti-OmpC) are reported to be associated with disease phenotype and may be of diagnostic importance in inflammatory bowel disease (IBD). Since limited data are available from Eastern Europe, we assessed the above antibodies in Hungarian IBD patients. METHODS: In all, 653 well-characterized, unrelated consecutive IBD patients (Crohn's disease [CD]: 558, m/f: 263/295, duration: 8.1 +/- 10.7 years; ulcerative colitis [UC]: 95, m/f: 44/51, duration: 8.9 +/- 9.8 years) and 100 healthy subjects were investigated. Sera were assayed for anti-Omp and ASCA by enzyme-linked immunosorbent assay (ELISA) and ANCA by indirect immunofluorescence assay (IIF). TLR4 and NOD2/CARD15 variants were tested by polymerase chain reaction/restriction fragment length polymorphism (PCR-RFLP). Detailed clinical phenotypes were determined by reviewing the medical charts. RESULTS: Anti-Omp, ASCA, and atypical pANCA antibodies were present in 31.2%, 59.3%, and 13.8% of CD, 24.2%, 13.7%, and 48.5% of UC patients, and in 20%, 16%, and 5.6% of controls, respectively. ASCA and anti-Omp positivity were associated with increased risk for CD (odds ratio [OR](ASCA) = 7.65, 95% confidence interval [CI]: 4.37-13.4; OR(Omp) = 1.81, 95% CI: 1.08-3.05). In a logistic regression analysis, anti-Omp and ASCA were independently associated with ileal and noninflammatory disease, but not with a risk for surgery or response to steroids or infliximab. A serology dosage effect was also observed. ASCA and anti-Omp antibodies were associated with NOD2/CARD15, in addition to a gene dosage effect. No associations were found in UC. CONCLUSIONS: Serological markers were useful in the differentiation between CD and UC in an Eastern European IBD cohort. Reactivity to microbial components was associated with disease phenotype and NOD2/CARD15 genotype, further supporting the role of altered microbial sensing in the pathogenesis of CD.
Asunto(s)
Anticuerpos/sangre , Colitis Ulcerosa/inmunología , Enfermedad de Crohn/inmunología , Proteína Adaptadora de Señalización NOD2/genética , Adulto , Anticuerpos Anticitoplasma de Neutrófilos/inmunología , Colitis Ulcerosa/genética , Colitis Ulcerosa/cirugía , Enfermedad de Crohn/genética , Enfermedad de Crohn/cirugía , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/inmunología , Femenino , Fluoroinmunoensayo , Genotipo , Humanos , Hungría , Masculino , Mutación , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Porinas/inmunología , Saccharomyces cerevisiae/inmunologíaRESUMEN
To routinely test the formation of thrombi and the effect of drugs modifying it, proper test systems are needed. Their design should rely on the laws of rheology and the physiology of laminar flow. To best model physiological or pathological shear conditions, parallel/linear and rotational type flow chambers are developed. We have compared the initial phase of platelet thrombus formation in a parallel plate flow chamber (PPC) and a cone-and-plate chamber (CPC) under von Willebrand dependent shear conditions. Blood was allowed to flow through human collagen type III surfaces at a shear rate of 1000 s(-1) for 150 s. Thrombus deposition was characterized by surface coverage, average area and height of thrombi. VWF distribution within thrombi was analyzed with confocal laser scanning microscopy. Reduced surface-specific platelet adhesion and aggregation (surface coverage and average thrombus size) were observed in CPC along with a significant increase in single platelet disappearance from the circulating blood. Our data suggest that the higher rate of platelet consumption in this device, as opposed to PPC, is limiting the adhesion to the surface. Consequently, surface-specific processes and aggregation in the flowing blood are both assessed using CPC, while comprehensive evaluation of surface-specific processes is best achieved with PPC. Therefore, the choice of chamber type as a diagnostic tool is purpose-dependent.