RESUMEN
BACKGROUND: Ultra-large von Willebrand factor and deficiency of its cleaving protease are important factors in the events leading to thrombotic microangiopathy; however, the mechanisms involved are only partly understood. Whereas pathological activation of the alternative complement pathway is linked to atypical hemolytic uremic syndrome, the role of complement activation in thrombotic thrombocytopenic purpura (TTP) is unknown. The aim of this study was to investigate whether signs of complement activation are characteristic of TTP. PATIENTS AND METHODS: Twenty-three patients with TTP (18 women, median age 38 years) and 17 healthy controls (13 women, median age 38 years) were included. Complement parameters (C3, Factors H, I, B and total alternative pathway activity) together with complement activation fragments (C3a) or complexes (C1rs-INH, C3bBbP, sC5b9) were measured by ELISA or RID. ADAMTS13 activity and anti-ADAMTS13 inhibitory antibodies were measured by the VWF-FRET73 assay. RESULTS: Increased levels of C3a, and SC5b9 were observed in TTP during acute episodes, as compared with healthy controls. Decreased complement C3 levels indicative of complement consumption occurred in 15% of acute TTP patients. Significant decrease of complement activation products C3a and SC5b9 was observed during plasma exchange (PEX). The sustained presence of anti-ADAMTS13 inhibitory antibodies in complete remission was associated with increased complement activation. CONCLUSION: These data document in an observational study the presence of complement activation in TTP. Further investigation is needed to determine its potential pathogenetic significance.
Asunto(s)
Activación de Complemento , Proteínas del Sistema Complemento/análisis , Púrpura Trombocitopénica Trombótica/inmunología , Proteínas ADAM/inmunología , Proteína ADAMTS13 , Adulto , Anticuerpos Neutralizantes/sangre , Autoanticuerpos/sangre , Biomarcadores/sangre , Estudios de Casos y Controles , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Hungría , Masculino , Persona de Mediana Edad , Intercambio Plasmático , Púrpura Trombocitopénica Trombótica/sangre , Púrpura Trombocitopénica Trombótica/terapia , RadioinmunoensayoRESUMEN
BACKGROUND: Type 3 von Willebrand disease (VWD) is an autosomal recessive bleeding disorder, characterized by virtually undetectable plasma von Willebrand factor (VWF) and consequently reduced plasma factor VIII levels. Genetic mutations responsible for type 3 VWD are very heterogeneous, scattered throughout the VWF gene and show high variability among different populations. METHODS: Twenty-five severe VWD patients were studied by direct sequencing of the 51 coding exons of the VWF gene. The total number of VWD type 3 families in Hungary is 24, of which 23 were investigated. RESULTS: Fifteen novel mutations were identified in 31 alleles, five being nonsense mutations (p.Q1238X, p.Q1898X, p.Q1931X, p.S2505X and p.S2568X), four small deletions and insertions resulting in frame shifts (c.1992insC, c.3622delT, c.5315insGA and c.7333delG), one a large partial deletion (delExon1-3) of the 5'-region, four candidate missense mutations (p.C35R, p.R81G, p.C295S, p.C623T) and one a candidate splice site mutation (c.1730-10C>A). Six previously described mutations were detected in 17 alleles, including the repeatedly found c.2435delC, p.R1659X and p.R1853X. Only one patient developed alloantibodies to VWF, carrying a homozygous c.3622delT. CONCLUSION: We report the genetic background of the entire Hungarian type 3 VWD population. A large novel deletion, most probably due to a founder effect, seems to be unique to Hungarian type 3 VWD patients with high allele frequency. In contrast to previous reports, none of the five patients homozygous for the large partial deletion developed inhibitors to VWF. This discrepancy raises the possibility of selection bias in some of the reports.
Asunto(s)
Enfermedad de von Willebrand Tipo 3/genética , Factor de von Willebrand/genética , Adolescente , Adulto , Niño , Femenino , Eliminación de Gen , Genotipo , Humanos , Hungría , Isoanticuerpos/química , Isoanticuerpos/genética , Masculino , Modelos Genéticos , Mutación , Mutación Missense , Sistema de Registros , Encuestas y CuestionariosAsunto(s)
Antineoplásicos/uso terapéutico , Trasplante de Células Madre Hematopoyéticas , Hemofilia A/complicaciones , Hepatitis B/complicaciones , Hepatitis C/complicaciones , Linfoma de Células del Manto/complicaciones , Linfoma de Células del Manto/terapia , Adulto , Enfermedad Crónica , Factor VIII/uso terapéutico , Hemofilia A/tratamiento farmacológico , Humanos , Masculino , Resultado del TratamientoRESUMEN
BACKGROUND: von Willebrand disease (VWD) Vicenza is characterized by low plasma von Willebrand factor (VWF) levels, the presence of ultra-large (UL) VWF multimers and less prominent satellite bands on multimer gels, and the heterozygous amino acid substitution R1205H in the VWF gene. The pathogenesis of VWD Vicenza has been elusive. Accelerated clearance is implicated as a cause of low VWF level. OBJECTIVES: We addressed the question, whether the presence of ultra-large multimers is a cause, or a result of accelerated VWF clearance, or whether it is an unrelated phenomenon. PATIENTS/METHODS: We studied the detailed phenotype of three Hungarian patients with VWD Vicenza, expressed the mutant VWF-R1205H in 293T cells and developed a mathematical model to simulate VWF synthesis and catabolism. RESULTS: We found that the half-life of VWF after DDAVP was approximately one-tenth of that after the administration of Haemate P, a source of exogenous wild-type (WT) VWF (0.81 + or - 0.2 vs. 7.25 + or - 2.38 h). An analysis of recombinant mutant VWF-R1205H showed that the biosynthesis and multimer structure of WT and mutant VWF were indistinguishable. A mathematical model of the complex interplay of VWF synthesis, clearance and cleavage showed that decreasing VWF half-life to one-tenth of normal reproduced all features of VWD Vicenza including low VWF level, ultra-large multimers and a decrease of satellite band intensity. CONCLUSION: We conclude that accelerated clearance alone may explain all features of VWD Vicenza.
Asunto(s)
Enfermedades de von Willebrand/metabolismo , Sustitución de Aminoácidos , Línea Celular , Electroforesis en Gel de Agar , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Masculino , Linaje , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Factor de von Willebrand/química , Factor de von Willebrand/metabolismoRESUMEN
In this paper we describe two pathways leading to platelet activation by crosslinking glycoprotein (GP) Ibalpha to the platelet Fc-receptor (FcgammaRII). First the monoclonal antibody (MoAb) 9C8, raised against human platelet GPIbalpha, dose-dependently induced platelet aggregation of citrate-anticoagulated platelet-rich plasma, an effect that can be inhibited by several activation inhibitors. The FcgammaRII-inhibitory MoAb IV.3 was able to prevent the aggregatory effects of MoAb 9C8, indicating that crosslinking of the antigen GPIbalpha to the FcgammaII-receptor is necessary for the activating effect. Secondly we observed a synergistic activating effect of two anti-von Willebrand factor (vWF) MoAbs IC1E7 and B724, both known to enhance vWF binding to GPIbalpha in the presence of shear or ristocetin. When these antibodies are added together to PRP, platelet aggregation is induced without further need for an additional modulator. This effect can be blocked by either MoAb IV.3 or an inhibitory anti-GPIb MoAb, indicating that again the platelet activation results from signaling through FcgammaRII crosslinked to vWF bound to GPIbalpha. In addition, both the anti-GPIb MoAb 9C8, or the two anti-vWF MoAbs 1C1E7 and B724 induce genuine platelet activation, as evidenced by the secretion of ATP and protein tyrosine phosphorylation. These findings with both anti-GPIb and anti-vWF MoAbs add further proof to recent reports demonstrating an interaction between the platelet receptors GPIb and FcgammaRII, suggesting a role for the FcgammaII-receptor in GPIb-related signaling.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Agregación Plaquetaria/efectos de los fármacos , Complejo GPIb-IX de Glicoproteína Plaquetaria/inmunología , Receptores de IgG/fisiología , Factor de von Willebrand/inmunología , Adenosina Trifosfato/metabolismo , Alprostadil/farmacología , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Aspirina/farmacología , Plaquetas/metabolismo , Humanos , Fragmentos Fab de Inmunoglobulinas/inmunología , Ratones , Ratones Endogámicos BALB C , Fosforilación , Fosfotirosina/análisis , Activación Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/fisiología , Inhibidores de Agregación Plaquetaria/farmacología , Procesamiento Proteico-Postraduccional , Receptores de IgG/efectos de los fármacos , Transducción de SeñalRESUMEN
Bernard Soulier Syndrome (BSS) is a rare inherited bleeding disorder caused by a defect in the glycoprotein (GP)Ib/IX/V complex. A patient with a bleeding problem was diagnosed as having BSS based on the prolonged bleeding time, the absence of ristocetin induced platelet aggregations, thrombocytopenia and the presence of giant platelets. Analysis of the platelets of the propositus, a 39-year-old Belgian female, by flow cytometry revealed a decreased expression of the GPIb/IX polypeptides. Western blotting confirmed these results and showed moreover that there was a decreased disulfide bridge formation between GPIb alpha and GPIb beta. After sequence analysis of the GPIb alpha, GPIb beta and GPIX genes, only a mutation in the GPIX gene at position 1826 (A-->G) was identified, changing Asn45-->Ser. Restriction analysis with Fnu4H1 demonstrated that the patient was homozygous for this mutation. As this Asn45-->Ser mutation in the GPIX gene was already found in four unrelated families, i.e. in a British, Austrian, Swedish and Finnish one, the occurrence of this mutation in a Belgian patient supports the hypothesis of Koskela et al. (1999) that the Asn45Ser mutation in GPIX appears to be an ancient mutation shared by northern and central European populations. Our present observation of a decreased disulfide bridge formation between GPIb alpha and GPIb beta shows that GPIX is not only needed for the correct assembly of the complex but might also be needed for the disulfide bridge formation between GPIb alpha and GPIb beta.
Asunto(s)
Sustitución de Aminoácidos , Síndrome de Bernard-Soulier/genética , Mutación Missense , Complejo GPIb-IX de Glicoproteína Plaquetaria/genética , Adenosina Difosfato/farmacología , Adulto , Bélgica , Síndrome de Bernard-Soulier/sangre , Síndrome de Bernard-Soulier/etnología , Western Blotting , Cistina/química , Análisis Mutacional de ADN , Etnicidad/genética , Europa (Continente)/epidemiología , Femenino , Frecuencia de los Genes , Humanos , Activación Plaquetaria/efectos de los fármacos , Complejo GPIb-IX de Glicoproteína Plaquetaria/química , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Conformación Proteica , Proteínas/farmacología , Receptores de TrombinaRESUMEN
Von Willebrand's disease (vWD) is the most common congenital haemorrhagic diathesis, characterized by the quantitative or qualitative disorder of von Willebrand factor (vWF). A number of methods have been used for the diagnosis of the disease, and the bleeding time determination is widely accepted as a screening test in spite of its low sensitivity. Our aim was to evaluate and compare the performance of two high shear systems (the O'Brien filter test and the PFA-100 device) in the screening and diagnosis of vWD. Thirty patients (n=13 type 1 with mild symptoms, n = 9 type 1 with severe symptoms, n = 2 type 2A, n = 3 type 2B and n = 3 type 3 vWD) and twenty controls were investigated. In mild vWD the platelet retention in the second phase of the filter test with citrated blood showed the highest sensitivity (91.6%). The sensitivity of the PFA-100 method with collagen-epinephrine cartridges in this group was 76.9%, while the bleeding time was prolonged only in 15.4% of the cases. In severe type 1, in type 2A and type 3 all functional tests reflected the bleeding tendency of the patients. In type 2B disease the bleeding time was prolonged only when the patient was thrombocytopenic, but both high shear systems revealed the disease independently of the presence of thrombocytopenia. The overall sensitivity of the bleeding time determination was 50% compared to the 80-90% sensitivity of the O'Brien filter test and the PFA-100 system. The sensitivity values of the filter test and the PFA-100 device with collagen-epinephrine cartridges were in the same range, but the collagen-ADP cartridges showed a lower (65.5%) sensitivity, though the results were specific and had high positive predictive value. We conclude that both high shear systems are suitable for the screening of vWD, and that they are superior to the traditional bleeding time determination in case of mild disease or type 2B vWD.
Asunto(s)
Pruebas de Coagulación Sanguínea/instrumentación , Filtración , Activación Plaquetaria , Pruebas de Función Plaquetaria/instrumentación , Enfermedades de von Willebrand/diagnóstico , Adenosina Difosfato/farmacología , Adolescente , Adulto , Tiempo de Sangría , Colágeno/farmacología , Epinefrina/farmacología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Activación Plaquetaria/efectos de los fármacos , Sensibilidad y Especificidad , Estrés Mecánico , Factor de von Willebrand/fisiologíaRESUMEN
In the differential diagnosis of primary and secondary thrombocytosis platelet function tests may play an important role. We examined the applicability of a platelet filter test (shear-dependent platelet aggregation) as a tool, to differentiate primary thrombocytosis (cases with myeloproliferative disorders) from secondary (reactive) thrombocytosis. The test was carried out in 53 patients suffering from myeloproliferative disorders associated with primary thrombocytosis and in 21 patients with other diseases complicated by secondary thrombocytosis. Using citrate as anticoagulant, the sensitivity of the O'Brien's test proved to be 77.1%, and its specificity 94.4%. Using heparin as anticoagulant the sensitivity and specificity of the test were found to be also reliably high. Based on these studies we suggest the use of the O'Brien's filterometer as a screening test in the differential diagnosis in patients with elevated platelet count. In the case of normal results, the causes of reactive thrombocytosis should be clarified first, while with abnormal results, haematological examination of the patients should be performed.
Asunto(s)
Pruebas de Función Plaquetaria/métodos , Trombocitosis/diagnóstico , Adenosina Difosfato/farmacología , Adulto , Anciano , Anciano de 80 o más Años , Anticoagulantes/farmacología , Tiempo de Sangría , Enfermedad Crónica , Ácido Cítrico/farmacología , Diagnóstico Diferencial , Epinefrina/farmacología , Reacciones Falso Positivas , Femenino , Filtración , Heparina/farmacología , Humanos , Masculino , Persona de Mediana Edad , Trastornos Mieloproliferativos/sangre , Trastornos Mieloproliferativos/diagnóstico , Activación Plaquetaria , Recuento de Plaquetas , Pruebas de Función Plaquetaria/instrumentación , Sensibilidad y Especificidad , Trombocitosis/sangre , Trombocitosis/etiología , Factor de von Willebrand/metabolismoRESUMEN
The ristocetin induced binding of vWF to GPIb, which is routinely tested in a platelet agglutination assay, can be reproducibly studied in an ELISA where plasma vWF binds to a captured rGPIb alpha-fragment (His1-Val289) in the presence of ristocetin. This binding is specific since the vWF-GPIb interaction could (i) be blocked by inhibitory anti-GPIb or anti-(vWF A1 domain) monoclonal antibodies (mAbs) and (ii) be enhanced by an anti-vWF mAb that also facilitates ristocetin induced platelet agglutination. Further studies were undertaken to determine whether the test could be used to differentiate vWF from patients with different types of von Willebrand's disease. The median vWF:RiCof activity in controls (n = 24) was 0.75 U/ml, in type 1 vWD patients (n = 17) 0.28 U/ml, in type 2A (n = 18) 0.055 U/ml, in type 2B (n = 4) 0.094 U/ml and in type 3 (n = 3) <0.0005 U/ml. Moreover, the values correlated well with those obtained from the vWF:RiCof-agglutination assay (r = 0.873). The vWF:RiCof-ELISA has several advantages: the use of a recombinant fragment instead of donor platelets results in a more reproducible test with a low inter- and intra-assay variability (<14% CV), the test can further be readily automated and for a single determination, only minimal amounts of patient plasma are required (8 microl).
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Ristocetina/análisis , Factor de von Willebrand/análisis , Humanos , Ristocetina/inmunología , Ristocetina/metabolismo , Sensibilidad y Especificidad , Factor de von Willebrand/inmunología , Factor de von Willebrand/metabolismoRESUMEN
Von Willebrand's disease (vWD) is the most common congenital haemorrhagic diathesis, characterized by the quantitative or qualitative disorder of von Willebrand factor (vWF). A number of methods have been used for the diagnosis of the disease, and the bleeding time determination is widely accepted as a screening test in spite of its low sensitivity. Our aim was to evaluate and compare the performance of two high shear systems (the O'Brien filter test and the PFA-100 device) in the screening and diagnosis of vWD. Thirty patients (n = 13 type 1 with mild symptoms, n = 9 type 1 with severe symptoms, n = 2 type 2A, n = 3 type 2B and n = 3 type 3 vWD) and twenty controls were investigated. In mild vWD the platelet retention in the second phase of the filter test with citrated blood showed the highest sensitivity (91.6%). The sensitivity of the PFA-100 method with collagen-epinephrine cartridges in this group was 76.9%, while the bleeding time was prolonged only in 15.4% of the cases. In severe type 1, in type 2A and type 3 all functional tests reflected the bleeding tendency of the patients. In type 2B disease the bleeding time was prolonged only when the patient was thrombocytopenic, but both high shear systems revealed the disease independently of the presence of thrombocytopenia. The overall sensitivity of the bleeding time determination was 50% compared to the 80-90% sensitivity of the O'Brien filter test and the PFA-100 system. The sensitivity values of the filter test and the PFA-100 device with collagen-epinephrine cartridges were in the same range, but the collagen-ADP cartridges showed a lower (65.5%) sensitivity, though the results were specific and had high positive predictive value. We conclude that both high shear systems are suitable for the screening of vWD, and that they are superior to the traditional bleeding time determination in case of mild disease or type 2B vWD.
Asunto(s)
Velocidad del Flujo Sanguíneo , Técnicas Hemostáticas , Tamizaje Masivo/métodos , Agregación Plaquetaria , Enfermedades de von Willebrand/sangre , Enfermedades de von Willebrand/diagnóstico , Adulto , Tiempo de Sangría , Estudios de Casos y Controles , Femenino , Humanos , Masculino , Persona de Mediana Edad , Sensibilidad y Especificidad , Trombocitopenia/sangre , Trombocitopenia/complicaciones , Enfermedades de von Willebrand/complicaciones , Enfermedades de von Willebrand/prevención & controlAsunto(s)
Trastornos de las Plaquetas Sanguíneas/diagnóstico , Pruebas de Función Plaquetaria/instrumentación , Trombastenia/diagnóstico , Enfermedades de von Willebrand/diagnóstico , Adenosina Difosfato , Adolescente , Albinismo/sangre , Tiempo de Sangría , Trastornos de las Plaquetas Sanguíneas/sangre , Niño , Colágeno , Epinefrina , Estudios de Evaluación como Asunto , Humanos , Masculino , Sensibilidad y Especificidad , Trombastenia/sangre , Enfermedades de von Willebrand/sangreAsunto(s)
Fibrinolíticos/uso terapéutico , Heparina de Bajo-Peso-Molecular/uso terapéutico , Complicaciones Cardiovasculares del Embarazo/tratamiento farmacológico , Trombofilia/tratamiento farmacológico , Adulto , Femenino , Fibrinolíticos/administración & dosificación , Fibrinolíticos/efectos adversos , Heparina de Bajo-Peso-Molecular/administración & dosificación , Heparina de Bajo-Peso-Molecular/efectos adversos , Humanos , Embarazo , Resultado del Embarazo , Trombofilia/genética , Resultado del TratamientoRESUMEN
We describe a patient with positive antinuclear antibodies, polyclonal gammopathy and high level of circulating immunocomplexes, resulting in vascular purpura. In addition, the patient had a slightly prolonged bleeding time and an isolated defect of ristocetin-induced platelet aggregation (RIPA) in platelet-rich plasma (PRP). The patient's plasma also inhibited RIPA in normal PRP and in normal platelet suspension. The activity and multimeric structure of plasmatic von Willebrand factor showed no alteration. We could demonstrate an autoantibody against platelet membrane glycoprotein (GP) Ib, using an ELISA-type assay. These data suggest an acquired Bernard-Soulier syndrome. We suggest that the patient had an immunocomplex-mediated leukocytoclastic vasculitis accompanied by production of antinuclear autoantibodies as well as the presence of an autoantibody against GPIb. The titer of the anti-GPIb antibody, however, was too low to induce significant platelet-type bleeding tendency, only laboratory alterations were found. Moreover, in a later stage of her disease, she developed a severe necrotizing vasculitis which was followed by a deep venous thrombosis.
Asunto(s)
Síndrome de Bernard-Soulier/complicaciones , Síndrome de Bernard-Soulier/inmunología , Anticuerpos Antinucleares/sangre , Anticuerpos Antinucleares/farmacología , Anticuerpos Monoclonales/farmacología , Antígenos/sangre , Antígenos/farmacología , Autoanticuerpos/sangre , Tiempo de Sangría , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Persona de Mediana Edad , Plasma/efectos de los fármacos , Plasma/inmunología , Agregación Plaquetaria/efectos de los fármacos , Pruebas de Función Plaquetaria/instrumentación , Complejo GPIb-IX de Glicoproteína Plaquetaria/inmunología , Poliarteritis Nudosa/inmunología , Ristocetina/farmacología , Trombosis de la Vena/inmunología , Factor de von Willebrand/inmunología , Factor de von Willebrand/farmacologíaRESUMEN
In the differential diagnosis of primary and secondary thrombocytosis, platelet function test can be used. We have examined the possible role of O'Brien's filter test in the differentiation of primary and secondary thrombocytosis in 53 patients with myeloproliferative diseases with primary thrombocytosis and in 21 patients with other disorders complicated by secondary thrombocytosis. By using heparin as an anticoagulant, the sensitivity of O'Brien's filter test proved to be 75%, and it's specificity was 85.7%. In blood samples anticoagulated with citrate, the sensitivity was 100% and specificity 83.3%. Based on these studies we suggest the use of O'Brien's filterometer as a screening test in the differential diagnosis in patients with elevated (> 400 x 10(9)/L) platelet count. In case of normal results, the causes of reactive thrombocytosis should be cleared first, while with pathologic results, haematological examination of the patients should be performed.
Asunto(s)
Hemofiltración/métodos , Trastornos Mieloproliferativos/sangre , Trombocitosis/diagnóstico , Enfermedad Crónica , Diagnóstico Diferencial , Humanos , Trastornos Mieloproliferativos/diagnóstico , Pruebas de Función PlaquetariaRESUMEN
Thromboembolic complications during pregnancy are the most common causes of maternal death. Here we report on thromboembolic prophylaxis of 60 pregnancies of 32 pregnant women with familial thrombophilia. Long-term Fraxiparine (Sanofi-Chinoin) as thromboprophylaxis was applied in 26 cases throughout pregnancy. UFH (Heparin-Ca inj.) was used in 11 cases, and there were 23 pregnancies without thromboembolic prophylaxis in our patient's case histories. Artificial abortions were not included in this paper. The ratio of successful pregnancies were: with Fraxiparine: 24/26 (92.3%), with UFH (Heparin-Ca): 8/11 (72.7%), without prophylaxis: 4/23 (17.4%). In the patient group treated with Fraxiparine there were no foetopathy, thrombocytopenia or bleeding complication. LMWH is recommended for pregnant women with familial thrombophilia. According to literature data and our own experiences the doses of LMWH in patients with familial thrombophilia, and -antiphospholipid syndrome, and -artificial heart value are suggested.