RESUMEN
The platelet function analyser (PFA-100) is a biological tool designed to explore primary haemostasis. This system has thus been widely demonstrated as reliable in detecting von Willebrand factor (VWF) deficiency. However, most studies were based on patients benefitting from regular medical care and accurate diagnosis, and it would seem probable that the results were somewhat optimistic, and do not reflect its performances in 'real-world' situations. We have chosen to study the reliability of PFA-100 for screening VWF ristocetin cofactor (VWF:RCo) deficiency. We retrospectively analysed the results (n = 6431) of 4027 patients referred to our centre between October 1997 and June 2013 and in whom PFA-Epi, PFA-ADP, and VWF:RCo activity had been evaluated. We studied the influence of blood group on the results and the performances of each method in a subgroup of 213 patients with genetically confirmed von Willebrand disease. We have shown that the PFA-100 system, in our experience, constitutes an excellent screening test for detecting VWF:RCo deficiency, whatever the clinical situation, in 'real-world' conditions. The negative predictive value (NPV), the positive predictive value, the sensitivity and the specificity were respectively: 0.98, 0.51, 0.98 and 0.40. When values adjusted for blood group are used, NPV and sensitivity are inferior to those using normal values which have not been adjusted for blood group. We have shown the PFA-100 method to be more efficient in screening for VWF deficiency than the VWF:RCo technique.
Asunto(s)
Pruebas de Función Plaquetaria/instrumentación , Factor de von Willebrand/metabolismo , Sistema del Grupo Sanguíneo ABO/metabolismo , Adulto , Femenino , Humanos , Masculino , Valor Predictivo de las Pruebas , Enfermedades de von Willebrand/sangreRESUMEN
Type 2N von Willebrand's disease (VWD) is characterized by a factor VIII (FVIII) deficiency and a low FVIII/VWF ratio related to a markedly decreased affinity of von Willebrand factor (VWF) to FVIII. Type 2N VWD is diagnosed using assays allowing the measurement of plasma VWF capacity to bind FVIII (VWF:FVIIIB). These assays, crucial in order to distinguish type 2N VWD patients from mild haemophiliacs A and haemophilia A carriers, remain exclusively homemade and limited to laboratories possessing a high level of expertise in VWD. We evaluated the first commercial ELISA (Asserachrom® VWF:FVIIIB; Stago) comparated to a reference method in a multicentric study involving 205 subjects: 60 healthy volunteers, 37 haemophiliacs A, 17 haemophilia A carriers, 37 patients with type 2N VWD, 9 heterozygous carriers for a 2N mutation and 45 patients with miscellaneous other types of VWD (all previously characterized). A diluted plasma sample adjusted to 10 IU dL(-1) of VWF:Ag was incubated with a rabbit antihuman VWF polyclonal antibody. After removing the endogenous FVIII, recombinant FVIII (rFVIII) was added and bound rFVIII was quantified using a peroxydase-conjugated mouse antihuman FVIII monoclonal antibody. The intra-assay and inter-assay reproducibility was satisfactory. In all subgroups, both methods were well correlated. All type 2N VWD patients exhibited a markedly decreased VWF:FVIIIB (lower than 15%) and all heterozygous 2N carriers had a moderately decreased VWF:FVIIIB (between 30% and 65%). All controls (healthy subjects, haemophiliacs A and haemophilia A carriers) had a normal VWF:FVIIIB (higher than 80%) except one healthy volunteer and three haemophiliacs who exhibited a moderately decreased VWF:FVIIIB suggesting a heterozygous status for a 2N mutation. In conclusion, the Asserachrom® VWF:FVIIIB is easy to perform, standardized and accurate for type 2N VWD diagnosis with a 100% sensitivity and specificity.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Factor VIII/metabolismo , Enfermedad de von Willebrand Tipo 2/diagnóstico , Factor de von Willebrand/metabolismo , Hemofilia A/diagnóstico , Humanos , Mutación/genética , Juego de Reactivos para Diagnóstico , Valores de Referencia , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Enfermedad de von Willebrand Tipo 2/genética , Enfermedades de von Willebrand/diagnósticoRESUMEN
The D3 domain of von Willebrand factor (VWF) is involved in the multimerization process of the protein through the formation of disulfide bridges. We identified heterozygous substitutions, C1157F and C1234W, in the VWF D3 domain in two unrelated families with unclassified and type 2A von Willebrand disease, respectively. VWF was characterized by a low plasmatic level, an abnormal binding to platelet GPIb and a high capacity of secretion from endothelial cells following DDAVP infusion. Using site-directed mutagenesis and expression in mammalian cells, we have investigated the impact of these mutations upon the multimerization, secretion and storage of VWF. Using COS-7 cells both mutated recombinant VWF (rVWF) displayed only lower molecular weight multimers. Pulse-chase analysis and endoglycosidase H digestion experiments showed the intracellular retention of mutated rVWF in pre-Golgi compartments. Study of hybrid rVWF obtained with a constant amount of wild-type (WT) DNA and increasing proportions of mutated plasmids established that both substitutions reduced the release of WT VWF in a dose-dependent manner and failed to form high molecular weight multimers. Using transfected AtT-20 stable cell lines, we observed similar granular storage of the two mutants and WT rVWF. Our data suggest that cysteines 1157 and 1234 play a crucial role in the early step of the folding of the molecule required for a normal transport pathway, maturation and constitutive secretion. In contrast, their substitution does not prevent the storage and inducible secretion of VWF.
Asunto(s)
Mutación Missense , Factor de von Willebrand/genética , Adolescente , Adulto , Animales , Células COS , Chlorocebus aethiops , Cisteína , Dimerización , Células Endoteliales/metabolismo , Factor VIII/metabolismo , Salud de la Familia , Aparato de Golgi/metabolismo , Humanos , Persona de Mediana Edad , Peso Molecular , Pliegue de Proteína , Transducción Genética , Enfermedades de von Willebrand/genética , Factor de von Willebrand/química , Factor de von Willebrand/metabolismoRESUMEN
BACKGROUND: A quantitative description of bleeding symptoms in type 1 von Willebrand disease (VWD) has never been reported. OBJECTIVES: The aim was to quantitatively evaluate the severity of bleeding symptoms in type 1 VWD and its correlation with clinical and laboratory features. PATIENTS AND METHODS: Bleeding symptoms were retrospectively recorded in a European cohort of VWD type 1 families, and for each subject a quantitative bleeding score (BS) was obtained together with phenotypic tests. RESULTS: A total of 712 subjects belonging to 144 families and 195 controls were available for analysis. The BS was higher in index cases than in affected family members (BS 9 vs. 5, P < 0.0001) and in unaffected family members than in controls (BS 0 vs. -1, P < 0.0001). There was no effect of ABO blood group. BS showed a strong significant inverse relation with either von Willebrand ristocetin cofactor (VWF:RCo), von Willebrand antigen (VWF:Ag) or factor VIII procoagulant activity (FVIII:C) measured at time of enrollment, even after adjustment for age, sex and blood group (P < 0.001 for all the four upper quintiles of BS vs. the first quintile, for either VWF:RCo, VWF:Ag or FVIII:C). Higher BS was related with increasing likelihood of VWD, and a mucocutaneous BS (computed from spontaneous, mucocutaneous symptoms) was strongly associated with bleeding after surgery or tooth extraction. CONCLUSIONS: Quantitative analysis of bleeding symptoms is potentially useful for a more accurate diagnosis of type 1 VWD and to develop guidelines for its optimal treatment.
Asunto(s)
Hemorragia/diagnóstico , Enfermedades de von Willebrand/diagnóstico , Enfermedades de von Willebrand/genética , Sistema del Grupo Sanguíneo ABO , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Estudios de Cohortes , Europa (Continente) , Factor VIII/biosíntesis , Factor VIII/química , Salud de la Familia , Femenino , Humanos , Lactante , Masculino , Persona de Mediana Edad , Fenotipo , Estudios Retrospectivos , Ristocetina/química , Encuestas y Cuestionarios , Enfermedades de von Willebrand/sangre , Factor de von Willebrand/químicaRESUMEN
Recombinant factor VIIa (rFVIIa; NovoSeven, Novo Nordisk, Bagsvaerd, Denmark) appears effective and relatively safe for the treatment of bleeding and for surgical prophylaxis in patients with Glanzmann thrombasthenia as reported to the International Registry on rFVIIa and Congenital Platelet Disorders. One of the shortcomings of the Registry data is the heterogeneity of treatment protocol, including dosage, number of doses used, duration of treatment before declaration of failure, and mode of rFVIIa administration (bolus v continuous infusion). The data are not yet sufficient to define optimal regimens for various indications such as the type of bleeding or the type of procedures. The place of this drug compared to platelet transfusion in the overall management of patients with Glanzmann thrombasthenia will need to be determined in relationship to a number of challenges and unresolved issues in the clinical care of these patients. These issues include: how to improve local measures for patients with mucosal bleeds, optimal management of young women during menarche, optimal platelet transfusion regimens for various indications, the relationship between antiplatelet antibodies detected by monoclonal antibody-specific immobilization of platelet antigens (MAIPA) and effectiveness of platelet transfusion, whether there are other biological tests that may correlate with effectiveness of platelet transfusion, and management of pregnancy and delivery regarding antiplatelet immunization.
Asunto(s)
Factor VII/uso terapéutico , Proteínas Recombinantes/uso terapéutico , Trombastenia/tratamiento farmacológico , Coagulantes/uso terapéutico , Factor VIIa , Femenino , Humanos , Masculino , Transfusión de Plaquetas/efectos adversos , Embarazo , Complicaciones Hematológicas del Embarazo/tratamiento farmacológico , Complicaciones Hematológicas del Embarazo/terapia , Trombastenia/diagnóstico , Trombastenia/terapiaRESUMEN
Inherited factor (F)XI deficiency is a rare disorder in the general population, though it is commonly found in individuals of Ashkenazi Jewish ancestry. In particular, two mutations--a stop mutation (type II) and a missense mutation (type III)--which are responsible for FXI deficiency, predominate. The bleeding tendency associated with plasma FXI deficiency in patients is variable, with approximately 50% of patients exhibiting excessive post-traumatic or postsurgical bleeding. In this study, we identified the molecular basis of FXI deficiency in 10 patients belonging to six unrelated families of the Nantes area in France and one family of Lebanese origin. As in Ashkenazi Jewish or in French Basque patients, we have identified a new ancient mutation in exon 4 resulting in Q88X, specific to patients from Nantes, that can result in a severely truncated polypeptide. Homozygous Q88X was found in a severely affected patient with an inhibitor to FXI and in three other unrelated families, either as homozygous, heterozygous or compound heterozygous states. Other identified mutations are two nonsense mutations in the FXI gene, in exon 7 and 15, resulting in R210X and C581X, respectively, which were identified in three families. A novel insertion in exon 3 (nucleotide 137 + G), which causes a stop codon, was characterized. Finally, sequence analysis of all 15 exons of the FXI gene revealed three missense mutations resulting in G336R and G350A (exon 10) and T575M (exon 15). Two mutations (T575M and G350A) with discrepant antigen and functional values are particularly interesting because most of the described mutations are associated with the absence of secreted protein.
Asunto(s)
Codón sin Sentido , Deficiencia del Factor XI/genética , Factor XI/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Codón de Terminación/genética , Análisis Mutacional de ADN , Femenino , Efecto Fundador , Francia , Haplotipos , Heterocigoto , Homocigoto , Humanos , Masculino , Persona de Mediana Edad , Mutación Missense , LinajeRESUMEN
Type 2N von Willebrand disease encompasses all patients with factor VIII deficiency caused by a markedly decreased affinity of von Willebrand factor for factor VIII. It is recessively inherited and clinically similar to mild haemophilia. The differential biological diagnosis is of major importance for providing the optimal treatment and relevant genetic counselling. This accurate diagnosis is based on an evaluation of the factor VIII-binding capacity of plasma von Willebrand factor. Furthermore, molecular biology techniques allow the identification of missense mutations in the von Willebrand factor gene. All of these induce the substitution of amino acid residues located in the N terminal part of the mature von Willebrand factor molecule, which contains the factor VIII binding site. Most of them induce a classical type 2N von Willebrand disease phenotype with factor VIII deficiency but a normal level and multimeric pattern of von Willebrand factor.
Asunto(s)
Enfermedades de von Willebrand/fisiopatología , Técnicas de Laboratorio Clínico , Diagnóstico Diferencial , Humanos , Biología Molecular/métodos , Enfermedades de von Willebrand/clasificación , Enfermedades de von Willebrand/diagnóstico , Enfermedades de von Willebrand/genéticaRESUMEN
Type 2 von Willebrand disease causing defective von Willebrand factor-dependent platelet function comprises mainly subtypes 2A, 2B and 2M. The diagnosis of type 2 von Willebrand disease may be guided by the observation of a disproportionately low level of ristocetin cofactor activity or collagen-binding activity relative to the von Willebrand factor antigen level. The decreased platelet-dependent function is often associated with an absence of high molecular weight multimers (types 2A and 2B), but the high molecular weight multimers may also be present (type 2M and some type 2B), and supranormal multimers may exist (as in the Vicenza variant). Today, the identification of mutations in particular domains of the pre-provon Willebrand factor is helpful to classify these variants and to provide further insight into the structure-function relationship and the biosynthesis of von Willebrand factor. Thus, mutations in the D2 domain, involved in the multimerization process, are found in patients with type 2A, formerly named IIC von Willebrand disease. Mutations in the D3 domain characterize the Vicenza variant, or type IIE patients. Mutations in the A1 domain may modify the binding of von Willebrand factor multimers to platelets, either increasing (type 2B) or decreasing (types 2M and 2A/2M) the affinity of von Willebrand factor for platelets. In type 2A disease, molecular abnormalities identified in the A2 domain, which contains a specific proteolytic site, are associated with alterations in folding that impair the secretion of von Willebrand factor or increase its susceptibility to proteolysis. Finally, a mutation localized in the C terminus cysteine knot domain, which is crucial for the dimerization of von Willebrand factor subunit, has been identified in a rare subtype 2A, formerly named IID.
Asunto(s)
Plaquetas/fisiología , Enfermedades de von Willebrand/sangre , Factor de von Willebrand/fisiología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Exones , Humanos , Mutación , Enfermedades de von Willebrand/clasificación , Enfermedades de von Willebrand/diagnóstico , Factor de von Willebrand/genéticaRESUMEN
Type 2M von Willebrand disease (VWD) refers to variants with decreased platelet-dependent function that is not associated with the loss of high molecular weight (HMW) von Willebrand factor (VWF) multimers. This category includes the so-called "phenotype B" responsible for inexistent ristocetin-induced but normal botrocetin-induced binding of VWF to platelet glycoprotein lb. The missense mutation G1324S was identified in the first patient reported to display "phenotype B". We report here on the identification in four members of a French family of a missense mutation also affecting this glycine residue but changing it into an alanine residue. These individuals are heterozygous for this mutation and two of them display an additional quantitative VWF deficiency resulting from a stop codon at position 2470. After transient transfection in Cos-7 cells, the mutated recombinant protein harbouring the G1324A substitution was shown to exhibit normal multimers and inexistent ristocetin-induced but normal botrocetin-induced binding to GPIb, confirming the classification of this new mutation as a type 2M VWD mutation.
Asunto(s)
Sustitución de Aminoácidos , Mutación Missense , Mutación Puntual , Enfermedades de von Willebrand/genética , Factor de von Willebrand/genética , Adulto , Animales , Biopolímeros , Células COS , Chlorocebus aethiops , Codón/genética , Análisis Mutacional de ADN , Exones/genética , Femenino , Francia , Hemorragia/genética , Heterocigoto , Humanos , Masculino , Linaje , Fenotipo , Complejo GPIb-IX de Glicoproteína Plaquetaria/metabolismo , Glicoproteínas de Membrana Plaquetaria/metabolismo , Reacción en Cadena de la Polimerasa , Unión Proteica , Receptores de Superficie Celular/metabolismo , Proteínas Recombinantes de Fusión/genética , Ristocetina/farmacología , Transfección , Factor de von Willebrand/metabolismoRESUMEN
Aurin tricarboxylic acid (ATA) is known to inhibit ristocetin-induced platelet agglutination but not arachidonic acid-, epinephrine- or ADP-induced aggregation. Its capacity to abolish human von Willebrand factor (vWF)-platelet interactions was further investigated by measurement of platelet adhesion to collagen, platelet agglutination tests and binding studies. In flowing blood using parallel-plate perfusion chambers and human collagen, ATA inhibited platelet adhesion to completion in a dose-dependent manner only at the highest shear rate tested (2,600 s-1). It was without effect at 100 and 650 s-1. ATA completely abolished vWF-dependent platelet agglutination induced by ristocetin, botrocetin and asialo-vWF, respectively. 125I-vWF binding to ristocetin- and botrocetin-treated platelets, to heparin and to sulfatides as well as 125I-botrocetin binding to vWF was competitively inhibited by ATA. By contrast, binding of 125I-vWF to collagen was not affected. To further localize the domain of vWF interacting with ATA, experiments of inhibition of binding of selected 125I-monoclonal antibodies (MoAbs) to immobilized vWF by ATA were performed. Our data led to the conclusion that: 1) the interaction of ATA with vWF involves sequences of the A1 disulphide loop of vWF (residues 509-695) and close epitopes which interact with GPIb and 2) the inhibition of platelet adhesion by ATA occurs only at a high shear rate where vWF is known to play a key role. Thus ATA, which blocks the vWF/GPIb pathway by interfering with vWF and not with platelets, is a potential tool in preventing the early stages of thrombosis.
Asunto(s)
Ácido Aurintricarboxílico/farmacología , Colágeno/sangre , Disulfuros/sangre , Adhesividad Plaquetaria/efectos de los fármacos , Glicoproteínas de Membrana Plaquetaria/metabolismo , Factor de von Willebrand/efectos de los fármacos , Ácido Aurintricarboxílico/metabolismo , Sitios de Unión/fisiología , Unión Competitiva/fisiología , Venenos de Crotálidos/antagonistas & inhibidores , Pruebas de Inhibición de Hemaglutinación , Hemaglutininas/efectos de los fármacos , Humanos , Unión Proteica , Ristocetina/antagonistas & inhibidores , Estrés Mecánico , Factor de von Willebrand/metabolismoRESUMEN
We compared the effect of a synthetic dodecapeptide of residues 400-411 of the gamma chain of fibrinogen (gamma Fg 400-411) and of three synthetic peptides (15 to 18 aminoacids), of human von Willebrand Factor (vWF), containing the 1744-1747 Arg-Gly-Asp-Ser (RGDS) sequence, upon platelet adhesion to collagen in flowing blood. Both types of peptides are known to inhibit the binding of adhesive proteins to platelet membrane glycoprotein IIb/IIIa (GPIIb/IIIa). Collagen was coated onto plastic cover slips and exposed in parallel-plate perfusion chambers to reconstituted human blood at various shear rates for 5 min at 37 degrees C. At a shear rate of 2,600 s-1, RGDS peptides inhibited platelet adhesion to collagen in a dose-dependent manner and appeared to be more potent inhibitors than the gamma Fg 400-411 on a molar basis. No synergetic effect between RGDS and gamma Fg 400-411 peptides was observed. These results suggest that the RGDS peptides affect adhesion by inhibiting the GPIIb/IIIa-vWF interaction and confirm the involvement of this platelet receptor in vWF-mediated platelet adhesion to collagen at high shear rate.
Asunto(s)
Oligopéptidos/fisiología , Adhesividad Plaquetaria/fisiología , Glicoproteínas de Membrana Plaquetaria/fisiología , Factor de von Willebrand/química , Secuencia de Aminoácidos , Colágeno , Hemostasis/fisiología , Humanos , Datos de Secuencia Molecular , Perfusión , Recuento de Plaquetas , Estrés MecánicoRESUMEN
Inhibitors against von Willebrand factor (vWF) developed in two unrelated multitransfused patients (patients 1 and 2) with severe (type 3) von Willebrand disease (vWD) were analyzed. Both inhibitors were identified as antibodies of the IgG class by ELISA using immobilized purified vWF and either serum or purified Ig from the patients. Typing, mapping and functional studies of both antibodies revealed significantly distinct properties. Patient 1 antibody contained all subclasses of IgG (1, 2, 3 and 4) whereas antibody from patient 2 was a mixture of only IgG1 and 4. By ELISA using a series of immobilized purified proteolytic fragments of vWF, patient 1 antibody mainly bound to fragment SpIII and, to a lower extent, to fragments SpII and SpI; it poorly bound to P34 and the 39/34 kDa fragment. In contrast, patient 2 antibody only bound to fragments corresponding to the N-terminal portion of vWF but failed to bind to SpII. Functional studies were performed by testing the capacity of each antibody to inhibit vWF binding to its various ligands. Both antibodies blocked vWF binding to Factor VIII (FVIII), fibrillar type III collagen, bitiscetin and the subsequent induced binding to GPIb. Patient 1 antibody also blocked vWF binding to platelet GPIb when induced by ristocetin. However it failed to block vWF binding to GPIb when induced by botrocetin as well as the binding of botrocetin itself to vWF. Our data thus suggest that this inhibitor does not recognize the GPIb-binding site on vWF but the sites of vWF involved in its interaction with ristocetin. In contrast, we observed that patient 2 antibody blocked vWF binding to platelet GPIb induced by either agonist as well as vWF binding to botrocetin. Finally, the effect of the antibodies was tested on vWF binding to GPIIb/IIIa. As expected from the mapping experiments, only IgG from patient 1 blocked the interaction while IgG from patient 2 had no effect. In conclusion, we have shown that two multitransfused patients with type 3 vWD have developed alloantibodies with similar properties to those of polyclonal antibodies but with distinct effects on the functions of vWF.
Asunto(s)
Mapeo Epitopo , Isoanticuerpos/química , Enfermedades de von Willebrand/inmunología , Adulto , Sitios de Unión de Anticuerpos , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Niño , Colágeno/metabolismo , Venenos de Crotálidos/farmacología , Factor VIII/metabolismo , Femenino , Hemaglutininas/farmacología , Humanos , Inmunoglobulina G/efectos adversos , Inmunoglobulina G/sangre , Inmunoglobulina G/química , Concentración 50 Inhibidora , Radioisótopos de Yodo , Isoanticuerpos/efectos adversos , Isoanticuerpos/sangre , Masculino , Péptidos/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Proteínas Recombinantes/metabolismo , Ristocetina/farmacología , Venenos de Serpiente , Reacción a la Transfusión , Venenos de Víboras/farmacología , Factor de von Willebrand/antagonistas & inhibidores , Factor de von Willebrand/inmunología , Factor de von Willebrand/metabolismoRESUMEN
A protease present in plasma cleaves von Willebrand factor (vWF) at the peptide bond 842Tyr-843Met of the mature subunit. To quantify this vWF-cleaving protease activity in plasma we have developed a simple method based on the estimation by IRMA of the degradation of a constant amount of wild type recombinant vWF used as substrate, by serial dilutions of test plasma used as protease provider. vWFAg was estimated by two-site IRMA using as first coating antibody a monoclonal antibody (MoAb) whose epitope is localized on the C-terminal side of the cleavage site, and as second labeled antibody a pool of MoAbs specific for the N-terminal side. Because the proteolytic process leads to the progressive separation of the C- and N-terminal portions of the vWF subunit such an IRMA also shows a progressive apparent loss of vWFAg. In contrast, the levels of vWFAg estimated after proteolysis by regular IRMA remained essentially constant. Results obtained with this new method were compared with the analysis by SDS-agarose gel electrophoresis of the multimeric pattern of proteolyzed WT-rvWF and no significant difference was noted testing a series of 28 plasmas. As compared with normal pooled plasma, 14 normal individuals and 13 patients with various types of vWD had normal levels of protease activity (44-178%) by both methods. The validity of the method was confirmed by showing a lack of detectable protease activity in a patient with chronic relapsing thrombotic thrombocytopenic purpura. In conclusion our method appears as a useful tool for the quantification of the vWF-cleaving protease activity in plasma. Its sensitivity and specificity are similar to those of SDS-gel electrophoresis. However, this new IRMA has the major advantages of being much simpler and faster, and open to most research laboratories in the field.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Pruebas Enzimáticas Clínicas , Ensayo Inmunorradiométrico , Metaloendopeptidasas/sangre , Púrpura Trombocitopénica Trombótica/diagnóstico , Factor de von Willebrand/metabolismo , Proteínas ADAM , Proteína ADAMTS13 , Biopolímeros , Electroforesis en Gel de Poliacrilamida , Humanos , Metaloendopeptidasas/deficiencia , Metaloendopeptidasas/genética , Metaloendopeptidasas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Factor de von Willebrand/química , Factor de von Willebrand/inmunologíaRESUMEN
Type 2 vWD is defined by qualitative defects of vWF and is subdivided into four subtypes: 2N, 2B, 2A and 2M. The characterization of 150 unrelated French cases with type 2 vWD emphasizes the heterogeneity of this group. In 51 cases of type 2N vWD, new mutations were found not only in the D' domain (Cys25Tyr and Cys95Phe) but also in the D3 domain (Asp116Asn and Cys297Arg). In 42 cases of type 2B vWD, no new mutation was detected. In 45 cases with type 2A phenotype, three new candidate mutations were found in the A2 domain: Gln793Arg, Val841Phe and Leu876Pro. In addition, four new candidate mutations were detected in the A1 domain: Cys509Gly, Arg545His, Arg552Cys and Cys695Tyr. Finally, five new candidate mutations were identified in 12 patients with 2M (or unclassified) phenotype: Leu513Pro, Gly561A1a, Glu596Lys, Arg611Leu and IIe662Phe. For all candidate mutations, expression studies are in progress. This study of a large number of French variants of vWD brings further insight into the relationship between phenotype and genotype.
Asunto(s)
Enfermedades de von Willebrand/genética , Factor de von Willebrand/genética , Secuencia de Aminoácidos , Francia , Humanos , Datos de Secuencia Molecular , Mutación , Estructura Terciaria de ProteínaRESUMEN
The treatment of bleeds in Glanzmann's thrombasthenia is a challenging issue, especially when repeated platelet transfusions have induced anti-glycoprotein (GP) IIb-IIIa or anti-HLA allo-immunisation. In an attempt to find an alternative treatment regimen, we used recombinant factor VIIa (rFVIIa, NovoSeven, Novo Nordisk, Denmark) as first-line therapy in 3 patients with Glanzmann's thrombasthenia and anti-GPIIb-IIIa iso-antibodies who were scheduled for invasive procedures. The administration of an initial bolus dose of rFVIIa (70-110 microg/kg) was immediately followed by continuous infusion at the rate of 9-30 microg/kg/h for 3-15 days. The treatment resulted in an excellent clinical efficacy and tolerance in 2 cases. In the third patient, whereas efficacy was excellent at the surgical site, pharyngonasal bleeds of traumatic origin persisted for 10 days, and a severe thromboembolic complication occurred 5 days after discontinuation of rFVIIa. Complementary studies are needed for patients with congenital platelet disorders in order to evaluate the safety and the potential therapeutic place of rFVIIa treatment.
Asunto(s)
Factor VIIa/uso terapéutico , Trombastenia/tratamiento farmacológico , Adenocarcinoma/complicaciones , Adenocarcinoma/cirugía , Adulto , Anciano , Colecistectomía , Colectomía , Neoplasias del Colon/complicaciones , Neoplasias del Colon/cirugía , Femenino , Genes Recesivos , Humanos , Laparotomía , Masculino , Persona de Mediana Edad , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/inmunología , Proteínas Recombinantes/uso terapéutico , Trombastenia/complicacionesRESUMEN
As heparin-PF4 (H-PF4) complexes are the target for antibodies associated to heparin-induced thrombocytopenia (HIT), an ELISA has been developed and optimised for testing antibodies binding to H-PF4. This test was consistently negative in 50 healthy subjects (A492 < 0.3) and 35 patients with other causes of thrombocytopenia (A492 < 0.5). In contrast, 43 out of 44 HIT patients showed antibodies to H-PF4 (A492 = 1.70 +/- 0.81) including 5 patients with a negative platelet aggregation test. In one patient with HIT, antibodies to H-PF4 were already present at day 7, whereas platelet counts dropped < or = 100 x 10(9)/l only at days 11-12. Surprisingly, among 41 patients under heparin for > 7 days, 5 showed antibodies to H-PF4, without HIT. These findings underline the major interest of this ELISA for the early diagnosis of HIT. We also showed that LMWH as well as other sulphated polysaccharides can bind to HIT antibodies in the presence of PF4 and that their reactivity is dependent on the molecular weight and the sulphation grade. The mechanism for HIT involves platelet PF4 receptors which bind the macromolecular H-PF4 complexes formed in the presence of a well defined heparin/PF4 ratio.
Asunto(s)
Autoanticuerpos/inmunología , Heparina/efectos adversos , Factor Plaquetario 4/inmunología , Trombocitopenia/inducido químicamente , Adolescente , Adulto , Cromatografía de Afinidad , Ensayo de Inmunoadsorción Enzimática , Femenino , Heparina/química , Heparina/inmunología , Heparina/metabolismo , Humanos , Isotipos de Inmunoglobulinas/inmunología , Sustancias Macromoleculares , Masculino , Persona de Mediana Edad , Oligosacáridos/análisis , Agregación Plaquetaria , Factor Plaquetario 4/metabolismo , Pronóstico , Estudios Retrospectivos , Trombocitopenia/complicaciones , Trombocitopenia/inmunología , Trombosis/etiologíaRESUMEN
The incidence of factor VIII inhibitor was studied in a cohort of 56 previously untreated patients with severe hemophilia A (factor VIII below 1 U/dl). They received only one brand of highly purified factor VIII concentrate (HPSD-VIII) prepared by conventional chromatography with a solvent-detergent step for viral inactivation. Follow-up since the first infusion of HPSD-VIII was from 1 to 76 months (mean = 29) and cumulative exposure days (CED) from 1 to over 100 (median = 26). Five patients (9%) developed an inhibitor after 6 to 19 CED, only one being a high responder (2%), showing a low incidence of inhibitor compared with previous studies using high purity plasma-derived or recombinant products.
Asunto(s)
Factor VIII/antagonistas & inhibidores , Hemofilia A/tratamiento farmacológico , Preescolar , Factor VIII/administración & dosificación , Estudios de Seguimiento , Hemofilia A/sangre , Humanos , Lactante , Recién Nacido , Estudios Retrospectivos , Resultado del TratamientoRESUMEN
Fifty French previously untreated patients with severe hemophilia A (factor VIII < 1%), treated with only one brand of recombinant factor VIII (rFVIII), were evaluated for inhibitor development, assessment of risk factors and outcome of immune tolerance regimen. The median period on study was 32 months (range 9-74) since the first injection of rFVIII. Fourteen patients (28%) developed an inhibitor, four of whom (8%) with a high titer (> or = 10 BU). All inhibitor patients but one continued to receive rFVIII either for on-demand treatment or for immune tolerance regimen (ITR). Among these patients, inhibitor was transient in 2 (4%), became undetectable in 6 and was still present in 6. The prevalence of inhibitor was 12%. Presence of intron 22 inversion was found to be a risk factor for inhibitor development. Immune tolerance was difficult to achieve in our series despite a follow-up period of 16 to 30 months: immune tolerance was complete in only one out of the 3 patients undergoing low dose ITR and in one out of the 5 patients with high dose ITR.
Asunto(s)
Factor VIII/inmunología , Hemofilia A/inmunología , Tolerancia Inmunológica , Isoanticuerpos/biosíntesis , Niño , Preescolar , Inversión Cromosómica , Factor VIII/genética , Factor VIII/uso terapéutico , Estudios de Seguimiento , Francia , Hemofilia A/terapia , Humanos , Inmunización , Lactante , Intrones/genética , Isoanticuerpos/inmunología , Masculino , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/uso terapéutico , Factores de RiesgoRESUMEN
Von Willebrand factor (vWF) is an adhesive, multimeric glycoprotein present in plasma, platelets, and subendothelium, which has two main functions: (1) it serves as a carrier for factor VIII and (2) it plays a crucial role in platelet adhesion to subendothelium, acting as a "bridge" between platelet membrane glycoprotein (GP) Ib and GP IIb/IIIa and subendothelial components such as collagen and heparin. vWF is involved at high shear rates in the initial contact of platelets with the subendothelium, in their subsequent spreading, and in thrombus formation. The three pools of vWF (plasma, platelets, and subendothelium) are necessary for optimal adhesion. Specific fragments of vWF involved in binding to platelets, collagen, heparin, and factor VIII have been mapped by using a series of proteases and well-characterized monoclonal antibodies to distinct epitopes of vWF. Several groups, including ours, have identified at least eight functional domains on the 270-kd subunit that consists of 2,050 amino acids. The importance of the binding domains to GP Ib and to collagen is illustrated by the role of vWF fragment SpIII (amino acids 1 through 1,365) in promoting platelet adhesion to collagen. The role of the vWF-GP Ib axis and of the vWF-GP IIb/IIIa axis in platelet-vessel wall interactions has been demonstrated through the study of patients, monoclonal antibodies, recombinant fragments, and synthetic peptides. We have recently expressed fragments of vWF complementary DNA in Escherichia coli. One of these recombinant fragments, which spans amino acids 449 through 730, binds to platelets in the presence of ristocetin, to collagen, and to heparin and has the property of inhibiting ristocetin-induced platelet agglutination. The second fragment, which spans amino acids 914 through 1,364, binds to collagen.(ABSTRACT TRUNCATED AT 250 WORDS)