RESUMEN
BACKGROUND: Insufficient control of von Willebrand factor (VWF) multimer size as a result of severely deficient ADAMTS-13 activity results in thrombotic thrombocytopenic purpura associated with microvascluar thrombosis and platelet consumption, features not seldom seen in severe sepsis and septic shock. METHODS: ADAMTS-13 activity and VWF parameters of 40 patients with severe sepsis or septic shock were compared with those of 40 healthy controls of the same age and gender and correlated with clinical findings and sepsis outcome. RESULTS: ADAMTS-13 activity was significantly lower in patients than in healthy controls [median 60% (range 27-160%) vs. 110% (range 63-200%); P < 0.001]. VWF parameters behaved reciprocally and both VWF ristocetin cofactor activity (RCo) and VWF antigen (VWF:Ag) were significantly (P < 0.001) higher in patients compared with controls. Neither ADAMTS-13 activity nor VWF parameters correlated with disease severity, organ dysfunction or outcome. However, a contribution of acute endothelial dysfunction to renal impairment in sepsis is suggested by the significantly higher VWF propeptide and soluble thrombomodulin levels in patients with increased creatinine values as well as by their strong positive correlations (creatinine and VWF propeptide r(s) = 0.484, P < 0.001; creatinine and soluble thrombomodulin r(s) = 0.596, P < 0.001). CONCLUSIONS: VWF parameters are reciprocally correlated with ADAMTS-13 activity in severe sepsis and septic shock but have no prognostic value regarding outcome.
Asunto(s)
Proteínas ADAM/metabolismo , Sepsis/diagnóstico , Choque Séptico/diagnóstico , Factor de von Willebrand/análisis , Proteína ADAMTS13 , Adulto , Anciano , Estudios de Casos y Controles , Creatinina/sangre , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Sepsis/sangre , Choque Séptico/sangre , Trombomodulina/sangreRESUMEN
BACKGROUND: After de novo synthesis in endothelial cells, the chemokine interleukin-8 (IL-8) is targeted to endothelial cell-specific storage vesicles, the Weibel-Palade bodies (WPBs), where it colocalizes with von Willebrand factor (VWF). OBJECTIVE: In this study we investigated a putative regulator function for VWF in the recruitment of IL-8 to WPBs. METHODS: We performed a quantitative analysis of the entry of IL-8 into the storage system of the endothelium using pulse-chase analysis and subcellular fractionation studies. RESULTS: Using pulse-chase analysis of IL-1beta-stimulated human umbilical vein endothelial cells, we found that a small part of de novo synthesized IL-8 was retained in endothelial cells after 4 h. In density gradients of endothelial cell homogenates nearly equimolar amounts of VWF and IL-8 were present in subcellular fractions that contained WPBs. Furthermore, we found that IL-8 binds to immobilized VWF under the slightly acidic conditions thought to prevail in the lumen of the late secretory pathway. CONCLUSIONS: These observations indicate that the sorting efficiency of IL-8 into the regulated secretory pathway of the endothelium is tightly controlled by the entry of VWF into WPBs.
Asunto(s)
Células Endoteliales/metabolismo , Interleucina-8/metabolismo , Cuerpos de Weibel-Palade/metabolismo , Factor de von Willebrand/metabolismo , Sitios de Unión , Células Cultivadas , Humanos , Interleucina-1beta/metabolismo , Cinética , Unión Proteica , Transporte de Proteínas , Fracciones Subcelulares/metabolismo , Trombina/metabolismo , Red trans-Golgi/metabolismoAsunto(s)
Proteínas ADAM/sangre , Análisis Químico de la Sangre/métodos , Hiperbilirrubinemia/sangre , Proteínas ADAM/deficiencia , Proteína ADAMTS13 , Humanos , Hiperbilirrubinemia/terapia , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Intercambio Plasmático , Púrpura Trombocitopénica Trombótica/sangre , Púrpura Trombocitopénica Trombótica/terapiaRESUMEN
BACKGROUND AND OBJECTIVES: Elevated levels of factor (F)VIII are associated with an increased risk of thrombosis. FVIII levels are determined mainly by von Willebrand factor (VWF). We have investigated the contribution of secretion and clearance rates to the elevated VWF antigen (VWF:Ag) and to the risk of thrombosis. VWF is secreted in equimolar amounts with its propeptide, which has a shorter half-life. VWF propeptide can be used as a measure of VWF secretion and allows estimation of the VWF half-life. METHODS AND RESULTS: We have measured VWF propeptide, VWF:Ag, FVIII:Ag and FVIII activity (FVIII:C) in the Leiden Thrombophilia Study. In controls, high VWF propeptide was associated with high VWF:Ag, FVIII:Ag and FVIII:C. In contrast to mature VWF:Ag, VWF propeptide was not influenced by blood groups. Using an ELISA-based assay we have shown that VWF propeptide lacks ABO antigens. Levels were higher in men and increased with age. A long VWF half-life was also associated with high VWF:Ag, FVIII:Ag and FVIII:C. The VWF half-life was influenced by blood group (10 h in O vs. 12 h in non-O individuals), but not by sex, and only slightly by age. VWF propeptide was higher in thrombosis patients than in controls. The VWF half-life was similar in patients and controls (11.4 and 11.1 h, respectively). CONCLUSIONS: Both secretion and clearance rates are important determinants of VWF and FVIII levels. However, mainly high VWF and FVIII levels caused by increased secretion seem to be associated with thrombosis. ABO blood group influences the clearance rates of VWF rather than VWF secretion rates.
Asunto(s)
Precursores de Proteínas/metabolismo , Trombofilia/metabolismo , Trombosis de la Vena/etiología , Trombosis de la Vena/metabolismo , Factor de von Willebrand/metabolismo , Sistema del Grupo Sanguíneo ABO , Adolescente , Adulto , Anciano , Antígenos/sangre , Antígenos/metabolismo , Biomarcadores/sangre , Biomarcadores/metabolismo , Estudios de Casos y Controles , Ensayo de Inmunoadsorción Enzimática , Estudios de Evaluación como Asunto , Factor VIII/metabolismo , Femenino , Semivida , Humanos , Masculino , Persona de Mediana Edad , Precursores de Proteínas/sangre , Valores de Referencia , Medición de Riesgo , Trombofilia/sangre , Trombofilia/complicaciones , Trombofilia/inmunología , Trombosis de la Vena/sangre , Trombosis de la Vena/inmunologíaRESUMEN
BACKGROUND: The cysteine-rich/spacer domains of ADAMTS13 contain a major binding site for antibodies in patients with acquired thrombotic thrombocytopenic purpura (TTP). OBJECTIVE: To study the heterogeneity of the antibody response towards these domains an immunoglobulin V-gene phage-display library was constructed to isolate monoclonal anti-ADAMTS13 antibodies from the immunoglobulin repertoire of a patient with acquired TTP. METHODS: Combined variable heavy chain (VH) and variable light chain (VL) segments, expressed as single-chain Fv fragments (scFv), were selected for binding to an ADAMTS13 fragment consisting of the disintegrin/thrombospondin type-1 repeat 1 (TSP1)/cysteine-rich/spacer domains. RESULTS: Seven different scFv antibody clones were identified that were assigned to four groups based on their homology to VH germline gene segments. Epitope-mapping revealed that scFv I-9 (VH1-69), I-26 (VH1-02), and I-41 (VH3-09) bind to an overlapping binding site in the ADAMTS13 spacer domain, whereas scFv I-16 (VH3-07) binds to the disintegrin/TSP1 domains. The affinity of scFv for the disintegrin/TSP1/cysteine-rich/spacer domain was determined by surface plasmon resonance analysis and the dissociation constants ranged from 3 to 254 nM. The scFv partially inhibited ADAMTS13 activity. However, full-length IgG prepared from the variable domains of scFv I-9 inhibited ADAMTS13 activity more profoundly. Plasma of six patients with acquired TTP competed for binding of scFv I-9 to ADAMTS13. CONCLUSION: Our data indicate that multiple B-cell clones producing antibodies directed against the spacer domain are present in the patient analyzed in this study. Our findings also suggest that antibodies with a similar epitope specificity as scFv I-9 are present in plasma of other patients with acquired TTP.
Asunto(s)
Proteínas ADAM/inmunología , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos/inmunología , Autoanticuerpos/inmunología , Linfocitos B/inmunología , Púrpura Trombocitopénica Trombótica/inmunología , Proteínas ADAM/genética , Proteína ADAMTS13 , Secuencias de Aminoácidos/genética , Secuencias de Aminoácidos/inmunología , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Especificidad de Anticuerpos/genética , Autoanticuerpos/química , Autoanticuerpos/genética , Clonación Molecular/métodos , Mapeo Epitopo/métodos , Epítopos/genética , Epítopos/inmunología , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/inmunología , Púrpura Trombocitopénica Trombótica/genética , Trombospondina 1/genética , Trombospondina 1/inmunologíaAsunto(s)
Factor VIII/fisiología , Factor de von Willebrand/fisiología , Animales , Coagulación Sanguínea/fisiología , Plaquetas/fisiología , Factor VIII/química , Factor VIII/historia , Historia del Siglo XX , Humanos , Agregación Plaquetaria , Conformación Proteica , Enfermedades de von Willebrand/fisiopatología , Factor de von Willebrand/química , Factor de von Willebrand/historiaRESUMEN
OBJECTIVE: To investigate the relation of endothelial cell activation with delayed cerebral ischaemia (DCI) and outcome after subarachnoid haemorrhage (SAH). METHODS: Concentrations of soluble (s) intercellular adhesion molecule-1, sE-selectin, sP-selectin, ED1-fibronectin, von Willebrand Factor (vWf), and vWf propeptide were measured within three days of SAH onset. The associations with poor outcome were investigated at three months in 106 patients. In 90 patients in whom the occurrence of cerebral ischaemia could be dated accurately, two analyses were undertaken: one for all ischaemic events (n = 32), including those related to treatment, and another for spontaneous DCI (n = 11). Concentrations of markers were dichotomised at their medians. The associations of endothelial cell activation markers with outcome were expressed as odds ratios (OR) from logistic regression and those with ischaemic events as hazard ratios (HR) derived from Cox regression. RESULTS: Early vWf concentrations were associated with poor outcome (crude OR = 4.6 (95% CI, 2.0 to 10.9; adjusted OR = 3.3 (1.1 to 9.8). Early levels of vWf were also positively related to occurrence of all ischaemic events (crude HR = 2.3 (1.1 to 4.9); adjusted HR = 1.8 (0.8 to 3.9) and with occurrence of spontaneous DCI (crude HR = 3.5 (0.9 to 13.1); adjusted HR = 2.2 (0.5 to 9.8). None of the other markers showed any associations. CONCLUSIONS: Concentrations of sICAM-1, sP-selectin, sE-selectin, and ED1-fibronectin do not predict the occurrence of DCI or outcome. The positive associations of raised early vWf concentrations with ischaemic events and poor outcome after SAH may reflect a predisposition to further ischaemic injury through formation of microthrombi in the cerebral circulation.
Asunto(s)
Aneurisma Intracraneal/complicaciones , Aneurisma Intracraneal/diagnóstico , Hemorragia Subaracnoidea/diagnóstico , Hemorragia Subaracnoidea/etiología , Adulto , Anciano , Biomarcadores/sangre , Encéfalo/diagnóstico por imagen , Encéfalo/patología , Movimiento Celular , Ectodisplasinas , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Ensayo de Inmunoadsorción Enzimática , Femenino , Fibronectinas/sangre , Humanos , Molécula 1 de Adhesión Intercelular/sangre , Aneurisma Intracraneal/sangre , Imagen por Resonancia Magnética , Masculino , Proteínas de la Membrana/sangre , Persona de Mediana Edad , Selectina-P/sangre , Hemorragia Subaracnoidea/sangre , Tomografía Computarizada por Rayos X , Factores de Necrosis Tumoral/sangre , Factor de von Willebrand/metabolismoRESUMEN
BACKGROUND: Protein S is a vitamin K-dependent protein with anticoagulant properties. It contains a so-called thrombin-sensitive region (TSR), which is susceptible to cleavage by coagulation factor Xa (FXa) and thrombin. Upon cleavage, the anticoagulant activity of protein S is abolished. OBJECTIVE: The aim of the present study was to determine whether protein S is cleaved within the TSR during activation of the coagulation system under near physiological conditions. RESULTS: In a reconstituted coagulation system containing apart from protein S only procoagulant constituents and synthetic phospholipid vesicles, protein S was cleaved at Arg60 by the FXa generated (3 mol min(-1) mol(-1) enzyme). FXa-catalyzed cleavage of protein S, however, was inhibited by factor Va and prothrombin by more than 70%. During clotting of recalcified citrated plasma in the presence of a synthetic lipid membrane, no FXa-catalyzed proteolysis of protein S was observed. Substituting platelets for phospholipid vesicles resulted both in the reconstituted system and in plasma in cleavage of the TSR. Cleavage was at Arg60 and was observed upon platelet activation, irrespective of the presence of FXa (13 pmol min(-1) 10(-8) platelets). No cleavage by thrombin was observed in either the reconstituted coagulation system or clotting plasma. CONCLUSION: These findings suggest that in vivo the anticoagulant activity of protein S is not down-regulated by FXa or thrombin during activation of coagulation. Our results rather suggest a role for a platelet protease in down-regulating the anticoagulant activity of protein S during the hemostatic response.
Asunto(s)
Factor Xa/metabolismo , Hemostasis , Proteína S/metabolismo , Trombina/metabolismo , Sitios de Unión , Coagulación Sanguínea , Plaquetas/citología , Plaquetas/fisiología , Células Cultivadas , Endotelio Vascular/citología , Humanos , Cinética , Liposomas/farmacología , Proteína S/fisiologíaRESUMEN
Vascular endothelial cells contain typical elongated vesicles, known as Weibel-Palade bodies. These organelles serve as a storage compartment for a variety of proteins that play a part in controlling vascular homeostasis, including von Willebrand factor, endothelin, P-selectin and interleukin-8. Upon activation of endothelial cells, Weibel-Palade bodies are translocated to the periphery of the cell and there fuse with the plasma membrane to release their contents into the blood circulation and subendothelial connective tissue. This process provides an adequate means by which endothelial cells can actively participate in controlling the arrest of bleeding upon vascular damage or modulate inflammatory reactions and other physiological and pathophysiological processes at the blood-tissue interface. Weibel-Palade bodies may also move to the nucleus of the cell, thus escaping secretion. This phenomenon may play a part in controlling stimulus-induced exocytosis.
Asunto(s)
Células Endoteliales/metabolismo , Endotelio Vascular/fisiología , Exocitosis/fisiología , Cuerpos de Weibel-Palade/metabolismo , Células Endoteliales/ultraestructura , Endotelio Vascular/metabolismo , Humanos , Factor de von Willebrand/metabolismoAsunto(s)
Plaquetas/fisiología , Factor V/genética , Trombastenia/complicaciones , Trombosis de la Vena/sangre , Trombosis de la Vena/genética , Humanos , Inmovilización/efectos adversos , Masculino , Persona de Mediana Edad , Mutación Puntual , Trombastenia/sangre , Trombastenia/genética , Trombosis de la Vena/etiologíaRESUMEN
von Willebrand factor is a multimeric plasma glycoprotein that is required for normal haemostasis. von Willebrand factor is synthesized by endothelial cells and megakaryocytes, and originates from its precursor pro-von Willebrand factor. The endoproteolytic processing of pro-von Willebrand factor results in mature von Willebrand factor and von Willebrand factor propeptide (also known as von Willebrand Ag II). In endothelial cells, the propeptide controls the polymerization and subsequent targeting of von Willebrand factor to the storage vesicles, the so-called Weibel-Palade bodies. Upon stimulation of the endothelial cells, the Weibel-Palade bodies are translocated to the plasma membrane of the cell, and mature von Willebrand factor and its propeptide are co-secreted. After release, these polypeptides have divergent fates and serve different biological functions. Mature von Willebrand factor both controls platelet adhesion and aggregation at sites of vascular injury and acts as a chaperone protein for coagulation factor VIII. The von Willebrand factor propeptide may serve a role in modulating inflammatory processes. This still growing body of information indicates that the biological function of the von Willebrand factor gene product is more diverse than was previously thought.
Asunto(s)
Factor de von Willebrand/genética , Animales , Endotelio Vascular/metabolismo , Hemostasis , Humanos , Megacariocitos/metabolismo , Transporte de Proteínas , Factor de von Willebrand/biosíntesis , Factor de von Willebrand/metabolismoRESUMEN
Previous studies have shown that factor VIII (FVIII) is expressed by multiple tissues. However, little is known about its cellular origin or its level of expression in different organs. In the present study, we examined FVIII gene expression in different tissues on a quantitative basis. Most of the tissues, especially liver and kidney, expressed high levels of FVIII mRNA compared to their level of expression of other hemostatic proteins, including von Willebrand factor (VWF). This was unexpected since FVIII is a trace protein. In situ hybridization analysis confirmed that liver and kidney were rich in FVIII mRNA. In the liver, a clear hybridization signal was detected in cells lining the sinusoids. FVIII mRNA analysis of purified liver cells confirmed the expression of FVIII mRNA by sinusoidal endothelial cells and Kupffer cells. Low but significant levels of FVIII mRNA were also detected in the hepatocytes. VWF mRNA was not detectable in these cells. Similarly, immunohistochemical staining of liver tissue revealed that FVIII protein is primarily associated with sinusoidal cells. VWF protein was predominantly located in the endothelium of larger vessels. In the kidney, FVIII synthesis was localized to the glomeruli and to tubular epithelial cells. Taken together, these results suggest that besides hepatocytes, non-parenchymal cells (e.g. sinusoidal endothelial cells) contribute to FVIII synthesis. VWF synthesis is primarily confined to extra-hepatic tissues.
Asunto(s)
Factor VIII/biosíntesis , Animales , Encéfalo/metabolismo , Endotelio Vascular/metabolismo , Factor VIII/genética , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Inmunoquímica , Hibridación in Situ , Riñón/citología , Riñón/metabolismo , Hígado/citología , Hígado/metabolismo , Pulmón/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Miocardio/metabolismo , Especificidad de Órganos , Reacción en Cadena de la Polimerasa , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Tromboplastina/biosíntesis , Tromboplastina/genética , Activador de Plasminógeno de Tipo Uroquinasa/biosíntesis , Activador de Plasminógeno de Tipo Uroquinasa/genética , Factor de von Willebrand/biosíntesis , Factor de von Willebrand/genéticaRESUMEN
Von Willebrand factor (VWF) is a multifunctional plasma protein that plays a prominent role in haemostasis. In endothelial cells, processing of its precursor pro-VWF results in the formation of two large polypeptides, mature VWF and a propeptide. These proteins are co-secreted on an equimolar basis but are cleared from the circulation at different rates. VWF levels are frequently elevated in response to vascular disorders. Similarly, propeptide levels are increased under these conditions, although primarily in fulminant vascular disease, such as thrombotic thrombocytopenic purpura and septicemia. In chronic vascular disease, e.g. diabetes or peripheral vascular disease, propeptide levels are much less elevated. The differential response of VWF and propeptide levels to vascular disease could provide a means to assess the extent and time course of endothelial cell activation. After secretion, the propeptide may play a role in modulating cellular adhesion processes. Thus, enhanced propeptide secretion seems not to be of merely diagnostic significance.
Asunto(s)
Precursores de Proteínas/fisiología , Enfermedades Vasculares/sangre , Factor de von Willebrand/fisiología , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Humanos , Precursores de Proteínas/sangre , Precursores de Proteínas/metabolismo , Factor de von Willebrand/metabolismoRESUMEN
Weibel-Palade bodies are endothelial cell-specific organelles, which contain von Willebrand factor (vWF), P-selectin, and several other proteins. Recently, we found that the small GTP-binding protein Ral is present in a subcellular fraction containing Weibel-Palade bodies. In the present study, we investigated whether Ral is involved in the regulated exocytosis of Weibel-Palade bodies. Activation of endothelial cells by thrombin resulted in transient cycling of Ral from its inactive GDP-bound to its active GTP-bound state, which coincided with release of vWF. Ral activation and exocytosis of Weibel-Palade bodies were inhibited by incubation with trifluoperazine, an inhibitor of calmodulin, before thrombin stimulation. Functional involvement of Ral in exocytosis was further investigated by the expression of constitutively active and dominant-negative Ral variants in primary endothelial cells. Introduction of active Ral G23V resulted in the disappearance of Weibel-Palade bodies from endothelial cells. In contrast, the expression of the dominant-negative Ral S28N did not affect the amount of Weibel-Palade bodies in transfected cells. These results indicate that Ral is involved in regulated exocytosis of Weibel-Palade bodies by endothelial cells.
Asunto(s)
Endotelio Vascular/metabolismo , Exocitosis , Cuerpos de Weibel-Palade/metabolismo , Proteínas de Unión al GTP ral/fisiología , Factor de von Willebrand/metabolismo , Calmodulina/fisiología , Células Cultivadas , Endotelio Vascular/efectos de los fármacos , Humanos , Mutación , Trombina/farmacología , Transfección , Proteínas de Unión al GTP rab3/genética , Proteínas de Unión al GTP rab3/metabolismo , Proteínas de Unión al GTP ral/genéticaRESUMEN
We designed a model system to study the role of von Willebrand factor (vWF) in the sorting of P-selectin and the biogenesis of Weibel-Palade body (WPB)-like organelles. For that purpose, a human epithelial cell line (T24) that synthesizes P-selectin mRNA, but which is devoid of vWF mRNA synthesis and storage organelles, was transfected with full-length vWF cDNA or a deletion mutant thereof. Stable transfectants of T24 with full-length vWF cDNA revealed the generation of WPB-like organelles as demonstrated by colocalization of vWF and P-selectin with double-labeling immunofluorescence. In contrast, T24 cells transfected with vWF delD'D3 cDNA, encoding a mutant that is unable to form vWF multimers, displayed only perinuclear vWF staining, whereas no indication was found for the presence of WPB-like organelles. The contents of the organelles in full-length vWF cDNA-transfected T24 cells were released on activation of the protein kinase C pathway, similar to the situation with genuine endothelial cells. The expression of vWF did not affect the biosynthesis of P-selectin, as deduced from the observation that untransfected and vWF cDNA-transfected T24 cells contained the same amount of P-selectin mRNA. We propose that the biosynthesis of multimeric vWF directs the generation of WPB-like organelles, as evidenced by the sequestering and anchoring of P-selectin into these storage granules.
Asunto(s)
Selectina-P/metabolismo , Cuerpos de Weibel-Palade/metabolismo , Factor de von Willebrand/metabolismo , Transporte Biológico/fisiología , ADN Complementario , Células Epiteliales/citología , Células Epiteliales/enzimología , Técnica del Anticuerpo Fluorescente , Expresión Génica/fisiología , Humanos , Selectina-P/genética , Proteína Quinasa C/metabolismo , ARN Mensajero/análisis , Transfección , Células Tumorales Cultivadas , Neoplasias de la Vejiga Urinaria , Factor de von Willebrand/genéticaRESUMEN
Patients with cross-reacting material (CRM)-reduced haemophilia A exhibit reduced levels of factor VIII antigen. In this study, we determined the molecular basis of the genetic defect in the factor VIII gene induced by either the Arg593-->Cys or the Asn618-->Ser missense mutation, identified in two CRM-reduced haemophilia A patients. We introduced either the Arg593-->Cys or the Asn618-->Ser mutation into a B-domain-deleted factor VIII cDNA and expressed the modified cDNAs in C127 cells. Reduced levels of factor VIII activity and factor VIII antigen in conditioned medium of transfected cells indicated that the secretion of both factor VIII variants was impaired. The ratio of factor VIII antigen present in cell extract to that in conditioned medium was 1.9 and 2.4 times higher for rFVIII-R593C and rFVIII-N618S, respectively, than for rFVIII. Metabolic labelling and immunoprecipitation of transfected cells revealed that rFVIII-R593C and rFVIII-N618S persisted somewhat longer inside the cell than factor rFVIII. Intracellular accumulation and subsequent degradation of factor VIII-R593C and factor VIII-N618S may explain the reduced levels of both factor VIII activity and antigen in plasma of mild haemophilia A patients with corresponding genetic defects.
Asunto(s)
Factor VIII/genética , Hemofilia A/genética , Intrones/genética , Mutación Missense/genética , Reacciones Cruzadas , Factor VIII/metabolismo , HumanosRESUMEN
The functional defect caused by substitution of Arg527 (--> Trp) and Arg531 (--> Gly, His) in factor VIII (FVIII), was explored by employing FVIII derived from patient plasma and recombinant FVIII variants. Mutation of these residues is associated with mild haemophilia A. For both FVIII-R527W and FVIII-R531H, activity was lower than antigen, indicating a functional defect for both variants. In contrast to FVIII-R527W, the amount of FVIII-R531H heterodimer present in plasma was reduced compared to heavy and light chain levels. Factor X (FX) activation experiments employing recombinant FVIII-R531G revealed that the activated FVIII-R531G heterotrimer was less stable than normal FVIIIa, apparently due to rapid dissociation of the A2 domain. These findings suggest that Arg531 is involved in maintaining the stability of both the heterodimer and the activated FVIII heterotrimer. Recombinant FVIII-R527W displayed reduced stimulation of FX activation, suggesting a defect in interaction with factor IXa (FIXa). The contribution of Arg527 in the interaction with FIXa was supported by the observation that FVIII-derived synthetic peptide Tyr511-Leu530 was able to inhibit FX activation and that this inhibition could be overcome by addition of increasing concentrations of FIXa. Furthermore, in the three-dimensional FVIII model residues Val517-Arg527 are located near the FIXa binding site Ser558-Gln565. Therefore we propose that Arg527 is part of an extended FIXa binding site, comprising residues Ser558-Gln565 and Val517-Arg527.
Asunto(s)
Sustitución de Aminoácidos/genética , Factor VIII/genética , Hemofilia A/genética , Factor X/genética , Factor X/metabolismo , Hemofilia A/metabolismo , Humanos , Mutación/genética , FenotipoRESUMEN
In endothelial cells von Willebrand factor (vWF) and P-selectin are stored in dense granules. so-called Weibel-Palade bodies. Upon stimulation of endothelial cells with a variety of agents including thrombin, these organelles fuse with the plasma membrane and release their content. Small GTP-binding proteins have been shown to control release from intracellular storage pools in a number of cells. In this study we have investigated whether small GTP-binding proteins are associated with Weibel-Palade bodies. We isolated Weibel-Palade bodies by centrifugation on two consecutive density gradients of Percoll. The dense fraction in which these subcellular organelles were highly enriched, was analysed by SDS-PAGE followed by GTP overlay. A distinct band with an apparent molecular weight of 28,000 was observed. Two-dimensional gel electrophoresis followed by GTP overlay revealed the presence of a single small GTP-binding protein with an isoelectric point of 7.1. A monoclonal antibody directed against RalA showed reactivity with the small GTP-binding protein present in subcellular fractions that contain Weibel-Palade bodies. The small GTPase RalA was previously identified on dense granules of platelets and on synaptic vesicles in nerve terminals. Our observations suggest that RalA serves a role in regulated exocytosis of Weibel-Palade bodies in endothelial cells.