Identificación por nuestro grupo de una nueva mutación en el gen GP1BA (P.W246L) asociada al fenotipo PT-VWD. Sexta mutación reportada internacionalmente / Identification by our group of a novel mutation in the GP1BA gene (P.W246L) responsible for PT-VWD. The 6th mutation internationally reported

La enfermedad de von Willebrand tipo plaquetario (PT-VWD) y tipo 2B (2B-VWD) son trastornos hemorrágicos raros, caracterizados por agregación plaquetaria a bajas concentraciones de ristocetina (RIPA). El diagnóstico diferencial no es fácil y representa un desafío. Hasta el presente, sólo se habían reportado cinco mutaciones en el gen GP1BA relacionadas con este desorden. Describimos aquí la sexta mutación relacionada con PT-VWD, en un paciente con sintomatología hemorrágica severa, macro-trombocitopenia, leve agregación plaquetaria espontánea, RIPA positivo a 0,3 y 0,4 mg/mL, VWF:RCo/VWF: Ag<0,2 y estudios discriminatorios positivos para PT-VWD. VWFpp/VWF: Ag resultó normal a diferencia del 2B-VWD que en algunas oportunidades resulta afectado. El exón 28 del gen VWF del paciente y su madre no reveló mutaciones. Identificamos una sustitución G>T en el nucleótido 3805 en el gen GP1BA del paciente, resultando en un cambio de Trp a Leu en el residuo 246 (p.W246L), en la región de la GPIBa que une al VWF. Esta mutación no se identificó en su madre ni en 100 controles sanos. Es considerada como dañina por análisis in sílico. Consideramos que esta sustitución es responsable del fenotipo PT-VWD del paciente. Dada la ausencia de la misma en los 100 normales estudiados, no se considera un polimorfismo

Platelet-type von Willebrand disease (PT-VWD) and type 2B von Willebrand disease (2B-VWD) are rare bleeding disorders characterized by increased ristocetin-induced platelet aggregation (RIPA) at low concentrations. Diagnosis of either condition is not easy and the differential diagnosis is especially challenging. Five mutations in the GP1BA gene related to PT-VWD and near 50 patients are currently reported worldwide. We herein describe a patient with severe bleeding symptoms, macro thrombocytopenia, mild spontaneous platelet aggregation, positive RIPA at 0.3 and 0.4 mg/mL, VWF: RCo/VWF: Ag <0.2, normal VWFpp/VWF: Ag ratio, and RIPA mixing tests and cryoprecipitate challenge positive for PT-VWD. GP1BA gene was studied in the patient, his mother, and 100 healthy control subjects. We identified a substitution G>T at nucleotide 3805 in the patient's GP1BA gene, resulting in a Trp to Leu amino acid change at residue 246 (p.W246L), within the VWF binding region. This mutation was absent in his unaffected mother and also in the 100 controls, and was predicted as damaging by in silico analysis. The residue is located in a strongly conserved position in the phylogenetic tree. These findings argue in favor of considering this substitution does not represent a polymorphism, and is therefore responsible for the PT-VWD phenotype of the patient

Complicações do parto e resultados perinatais em gestantes portadoras da doença de von Willebrand / Delivery complications and perinatal results in pregnant women with von Willebrand disease

OBJETIVO: Analisar as complicações maternas associadas ao parto e puerpério, bem como os resultados perinatais, em gestantes portadoras da doença de von Willebrand. MÉTODOS: Foram analisados os prontuários de todas as gestantes com diagnóstico de doença de von Willebrand, cujo parto foi realizado nesta instituição no período compreendido entre março de 2001 e agosto de 2007. Foram investigadas características relativas à via de parto, incidência de complicações hemorrágicas no parto e no puerpério imediato, perfil dos exames laboratoriais maternos e resultados perinatais. As variáveis foram estudadas descritivamente, calculando-se freqüências absolutas e relativas, médias, medianas e desvios padrão. RESULTADOS: Foram revisadas 13 gestações em oito pacientes portadoras da doença. No período ocorreram 13.037 partos na instituição, perfazendo incidência de 0,1 por cento. Seis pacientes (75 por cento) apresentavam o tipo 1 da doença, e duas (25 por cento) o tipo 2. No terceiro trimestre, a média da atividade do fator VIIIc foi de 98,5 por cento. A cesárea foi realizada em nove casos (69 por cento), cuja anestesia foi do tipo raquidiana em sete casos. Como complicação do parto, um caso evoluiu com descolamento prematuro da placenta e foi realizada a cesárea. Um caso apresentou sangramento no primeiro pós-parto, necessitando reposição do fator VIII. Em dois casos foi realizada a reposição profilática do fator VIII antes da cesárea. A restrição do crescimento fetal ocorreu em cinco casos (38,5 por cento). O peso dos RNs apresentou média de 2676 gramas e um caso (7,8 por cento) apresentou Apgar de 1º minuto inferior a sete. CONCLUSÃO: O parto em gestantes com diagnóstico de doença de von Willebrand tem evolução favorável quando cuidados são tomados procurando oferecer assistência específica. O crescimento fetal deve ser monitorizado nessas gestantes.

OBJECTIVE: To study maternal complication associated to delivery and the puerperium period in pregnancies affected by von Willebrand's disease. METHODS: Chart data of all the pregnant women with diagnosis of von Willebrand disease were retrospectively reviewed. All cases with von Willebrand's disease that had given birth at this institution, between March 2001 and August 2007, were analyzed. The following variables were investigated: mode of delivery, hemorrhage complications during delivery and postpartum, maternal blood exams and perinatal results. Variables were studied descriptively, using absolute and relative frequencies, means, medians and standard deviations. RESULTS: 13 pregnancies of eight women with the disease were reviewed. During this sane period, there were 13,037 deliveries in the institution, resulting in an incidence of 0.1 percent. Six women (75 percent) were type 1 disease and, two (25 percent) were type 2. The last Factor VIIIc activity presented a mean value of 98.5 percent. A Cesarean section was performed in nine pregnancies, with epidural anesthesia in seven. Delivery complication occurred in two cases: one presented placental abruption and a Cesarean was performed. The other, presented postpartum hemorrhage in the first day and required reposition with factor VIII. Two cases received factor VIII before Cesarean section. Fetal growth restriction was detected in five pregnancies (38.5 percent). Mean birth weight was of 2676 grams and one case presented 1st minute Apgar score below seven. CONCLUSION: Delivery in patients with von Willebrand disease has a favorable evolution when specific assistance is provided. In these pregnancies,fetal growth should be monitored.

Enfermedad de Võn Willebrand y embarazo gemelar: a propósito de un caso / Von Willebrand disease and pregnancy twins: a purpose of a case

Durante el embarazo ocurren alteraciones importantes en varios órganos y sistemas particularmente el mecanismo hemostático expresado por episodios hemorrágicos trombóticos o ambos, con una marcada influencia en la morbimortalidad materna. Las mujeres con coagulopatías presentan durante el embarazo, parto y puerperio, un riesgo mayor de hemorragia, por lo que es necesario una adecuada evaluación y un manejo multidisciplinario del embarazo. La enfermedad Võn Willebrand es una coagulopatía poco común. Con el objetivo de revisar el manejo intraparto de pacientes con coagulapatías hereditarias. Se presenta el caso clínico de una mujer con embarazo gemelar portadora de Enfermedad de Võn Willebrand tipo 1, recomendando al final, un protocolo de estudio de las pacientes y sus hijos.

A Case of Type 2N von Willebrand Disease with Homozygous R816W Mutation of the VWF Gene in a Nepalese Woman / 대한진단검사의학회지

Type 2N von Willebrand disease (vWD) can be confused with hemophilia A due to decreased factor VIII levels and a bleeding tendency, and differential diagnosis is of importance for providing the optimal treatment and genetic counseling. For the accurate diagnosis of type 2N vWD, von Willebrand Factor (vWF) function tests, multimer assay and gene mutation analysis are needed. The patient was a 38-yr-old Nepalese woman with a history of bleeding manifestations from childhood, such as hemarthrosis, intramuscular hematoma, and menorrhagia. Family history revealed that her mother and elder brothers also had bleeding manifestations from childhood. When she had a laparotomy in 1991, she was diagnosed as hemophilia A with factor VIII level of 3.6% and was transfused with whole blood, factor VIII and cryoprecipitates. In June 2007, she was admitted to our hospital for further evaluation of bleeding tendency. Blood tests revealed normal CBC; bleeding time, 2 min; PT, 14.9 sec (11-14 sec); aPTT, 51.2 sec (24-38 sec); and factor VIII, 4.9% (50-150%). The prolonged aPTT was corrected by 1:1 mixing test to the levels of 106% and 84%, respectively, before and after 2 hr-incubation at 37degrees C. No abnormalities were found in the vWF antigen level (71.3%), ristocetin cofactor assay (130.4%), and multimer assay. Direct DNA sequencing of the VWF gene revealed homozygous missense mutation located in exon 19, c.2446C>T (p.Arg816Trp), confirming the diagnosis of type 2N vWD.

Combined occurrence of von Willebrand's disease and factor XIII deficiency: a case report.

We report the case of a three year old female child with combined occurrence of von Willebrand's disease and factor XIII deficiency, an extremely rare combination. The patient presented with prolonged bleeding following cuts and wounds. Clot solubility test using 5M urea was positive. Platelet aggregation using ristocetin was reduced, which corrected on adding normal plasma. Aggregation with other agonists was normal. We discuss the clinico- hematological profile of the case. Only one such case has been reported in literature in the past to the best of our knowledge.

Enfermedad de Von Willebrand y embarazo / Von Willerbrand's disease and pregnancy

Se presenta el caso de una paciente de 25 años quien sufre enfermedad de Von Willebrand en concomitancia con estado gravídico; se incluye una breve revisión de esta asociación expondiéndose seguimiento del embarazo y enfrentamiento al parto dando, en este último punto, especial importancia al uso de la desmopresina (DDAVP)

Dosagem da antitrombina III usando o substrato cromogênico Tos-Gly-Pro-Arg-NAN, em diversas situaçöes patológicas / Antithrombin III dosage using the chromogenic substrate Tos-Gly-Pro-Arg-NAN, in several pathological situations

O uso de método funcional para dosagem de antitrombina III (ATIII) é fundamental para o diagnóstico de deficiência deste inibidor da coagulaçao. OBJETIVO. Padronizar metodologia para dosagem da ATIII no plasma, utilizando-se microplacas, em diferentes situaçoes clínicas. MÉTODOS. A dosagem de ATIII foi feita utilizando-se o substrato cromogênico Tos-Gly-Pro-Arg-NAN, específico para trombina, e sintetizado no Departamento de Biofísica da Escola Paulista de Medicina. RESULTADOS. Dos 21 pacientes com trombose venosa (TV-P), 20 apresentaram valores superiores a 70 por cento (ll3 ñ 22 por cento), dos quais uma paciente de 22 anos apresentava deficiência congênita, com ATIII de 56 por cento e história de TVP recorrente, além de história familiar de TVP. O nível de ATIII em seis pacientes portadores de doença de von Willebrand foi normal (lO9 ñ 28 por cento), como esperado. Em 20 pacientes com insuficiência hepática foi observada reduçao importante do nível de ATIII (42 ñ 19 por cento), pois este inibidor é produzido no hepatócito, sendo bom parâmetro para avaliar a funçao hepática. Os três pacientes portadores de sepse com CIVD apresentaram níveis reduzidos de ATIII (45 + 5 por cento), que é consumida durante processo de ativaçao intravascular da coagulaçao. Os níveis de ATIII mostraram correlaçao significante com o TP e os níveis de fator V, ambos bons parâmetros para avaliaçao da funçao hepática e para monitorizaçao da CIVD. Houve correlaçao significante entre as dosagens realizadas com o substrato Tos-Gly-Pro-Arg-NAN, sintetizado na EPM, e o substrato S-2238 do laboratório Kabi. CONCLUSOES. A medida da ATIII usando substrato cromogênico Gly-Pro-Arg-NAN é de fácil execuçao e é sensível para o diagnóstico da deficiência deste inibidor em pacientes com insuficiência heopática, coagulaçao intravascular disseminada e trombofilia.

Thai national reference preparation in blood coagulation. IV. Home made reference plasma for von Willebrand's factor assay.

Two lots of home made reference plasma: FVIII R:Ag 30/8/88 and FVIII R:Ag 18/10/88 were prepared by lyophilization of pooled normal human plasma. Modification of J. Cejka's technique was used to determine FVIII R:Ag. This technique was tested for reliability i.e. precision, reproducibility and sensitivity. The concentration of FVIII R:Ag, determined by calibration against the 1st British Standard for FVIII R:Ag, human 66/355, which was established by National Institute for Biological Standard and Control (NIBS & C), London, the WHO International Laboratory for Biological Standard, were respectively 1.058 and 1.023 Ag units/ml for FVIII R:Ag 30/8/88 and FVIII R:Ag 18/10/88, respectively. The precision of the procedure and the accuracy of FVIII R:Ag concentration of both lots were verified by using them as standard curve to determine FVIII R:Ag in 4 unknown plasma samples, supplied by the UK Reference Laboratory for Anticoagulant Reagent & Control; WHO Collaborating Center for Quality Assessment, in Blood Coagulation Testing for International Quality Control Survey in Blood Coagulation. The results were very satisfactory. The coefficient of variation was between 2.22-5.47 per cent when compared with other 29 laboratories around the world. These home made reference preparation are stable at least up to 30 months at -70 degrees C, and can be applied for calibration of unknown sample instead of the 1st British Standard for FVIII R:Ag, human 66/355.

Response of patients with bleeding disorder to DDAVP administration.

DDAVP has been shown to provide hemostasis in patients with bleeding disorder. Thirty-one episodes of intravenous DDAVP administration (0.3-0.4 microgram/kg) in 22 patients with bleeding disorder were studied. There were 13 patients with hemophilia A, 1 with type I vWD and 8 with inherited and acquired platelet dysfunction. The age ranged from 2.3-26 yrs (mean +/- SD = 10 +/- 4.8). None of the 3 severe hemophilia A patients responded to the treatment. Two out of five episodes in 4 moderate hemophilia A patients responded clinically and had minute increments of F VIII:C. Ten out of eleven episodes (91%) in 6 mild hemophilia A patients had good responses. The dental procedures for these patients were successfully performed without blood component transfusion. The increments of F VIII:C ranged from 1.5-6.8 folds over the baseline levels (mean +/- SD = 2.5 +/- 1.4). In addition, two episodes of epistaxis in a vWD patient responded excellently and one dental procedure was successfully performed by giving DDAVP. The increments of F VIII:C and vWF:Ag ranged from 2.8-12.5 and 2.9-8 fold over the baseline levels respectively. The prolonged bleeding times were shorten to 6.5-7 minutes. Only three out of eight episodes in 8 inherited and acquired platelet dysfunction patients showed temporary responses. The bleeding time responses did not correlate with in vitro platelet aggregation.

Response of patients with hemophilia A and von Willebrand disease to desmopressin (DDAVP)

Factor VIII (FVIII) response to desmopressin (deamino-8-D-arginine vasopressin, abbreviated DDAVP) was studied in patients with mild Hemophilia A and von Willebrand Disease (vWD) who attend our Hemophilia Clinic. Thirty eight children and 9 adults had their F VIII components assayed 60 minutes after intravenous (IV) administration of DDAVP, 0.35 ug/kg. Among 27 hemophiliacs, F VIII coagulant activity (F VIII: C) increased from a mean of 16.8 to 59.2 u/dL; with an average 3.2-fold increase. In 20 vWD patients, the mean F VIII:C and von Willebrand Factor increased from a mean of 50.1 to 136%; and from 22.6 to 75.6%; with an average 3.0 and 5.7-fold increase, respectively. The overall F VIII:C response was good or excellent in 81.5% of the hemophiliaacs, and in 89.5% of the vWD patients tested. No significant side effects were observed. This study has demonstrated that IV DDAVP can cause an increase of F VIII:C and vWF to hemostatic levels, and thus it may be useful for the control of bleeding episodes in most of the patients tested in our clinic

Niveles de FvW:Ag en individuos de grupo sanguíneo 0 / FvW antigen serum levels in 0 blood group individuals

Dada la relación entre el grupo sanguíneo ABO y los niveles de FvW:Ag en 50 dadores de sangre tipo 0, mediante inmunoelectroforesis, con el objeto de obtener valores normales de referencia. El 22% de los dadores presentó niveles de FvW:Ag moderadamente más bajos que el intervalo normal de referencia (INR), 0,62-1,5 U/ml. Obtuvimos en el grupo dadores un promedio de 0.89 ñ 0,48 U/ml (2SD), y con ello un intervalo de 0,41-1,37 U/ml (IGD). Seleccionamos 30 pacientes grupo sanguíneo 0 con enfermedad de Von Willebrand tipo I (EvW), grupo vW, para conocer cómo se distribuían sus concentraciones de FvW:Ag en ambos intervalos. Obtuvimos un 86,6% (n =26) de pacientes con niveles de FvW:Ag dentro del IGD, en cambio sólo un 36,6% (n =11) de estos pacientes tenían sus concentraciones de FvW:Ag dentro del INR. Este hecho resultó significativo por la prueba chi cuadrado. Aunque estas observaciones fueron con un n relativamente pequeño, nos sugieren que a individuos con problemas hemorragíparos grupo sanguíneo 0, deberíamos estudiarlos con un control normal del mismo grupo; esto ayudaría sobre todo en aquellos casos donde se trata de diagnosticar formas leves (las más frecuentes) de la EvW por el laboratorio

Estudio de laboratorio del complejo F VIII plasmático en 181 pacientes con problemas hemorragíparos: IX región / Laboratory studies of plasma factor VIII complex in patients with haemorrhagic problems in Chile's IX region

El propósito del presente trabajo es proporcionar información sobre el comportamiento del F VIII plasmático en 181 pacientes con antecedentes hemorragíparos de la IX región, estudiados en nuestro laboratorio entre los años 1983 y 1987; además presentar la frecuencia de la enfermedad de Von Willebrand (EvW) en relación a las otras coagulopatías. Los paràmetros de laboratorio que analizamos fueron: T.S.Ivy, F VIII:C, FvW:Ag y F VIII:CoR. Obtuvimos un 72,4% de pacientes con alteraciones en los estudios. De este grupo, 84,7% correspondió a EvW, 12,2% a Hemofilias y 3,1% a disfunción plaquetaria mínima. Del grupo vW (n=111) el 46,8% correspondió a adultos y 53,2% a niños. Encontramos el T.S. prolongado 86,5% en adultos y 84,8% en niños; F VIII:C disminuido en adultos y niños 57,7% y 50,9%, respectivamente; FvW:Ag disminuido en ambos grupos 53,8 y 52,5%; F VIII:CoR disminuido 75% adultos y 71,2% niños. Aplicamos el coeficiente de Pearson revelando correlación significativa (p<0,01), en relación al F VIII:C/FvW:Ag y T.S./F VIII:CoR en adultos; hubo sólo correlación entre el estudio del F VIII:C/FvW:Ag en los niños. Concluimos que la EvW es la afección hereditaria de la hemostasis más frecuente, presentando gran variabilidad los resultados de los exámenes, por lo que es fundamental realizar el estudio del F VIII completo para lograr un diagnóstico inequívoco de laboratorio en este grupo de pacientes

Caracterizaçäo imunológica do fator agregador das plaquetas (PAF) no plasma circulante do boi / Immunological characterization of platelet aggregation factor (PAF) in bovine circulating plasma

A metodologia utilizada em experiências anteriores para a purificaçäo e caracterizaçäo do PAF bovino foi também empregada para o estudo da molécula do PAF no plasma circular do boi. Ao contário do que se esperava, a imunoeletroforese cruzada do plasma de boi em gel de agarose a 2% näo forneceu os resultados esperados. A utilizaçäo, poré, de gel de agarose a 1% e a 0,8% permitiu o paparecimento de vários picos de imunoprecipitado com identidade total, tendo o gel a 0,8% fornecido uma resoluçäo maior. A vaiaçäo da distância dos orifícios à linha demarcadora entre o gel superior e o gel inferior foi também estudada. Os melhores resultados foram obtidos quando os orifícios se situavam próximos da linha de separaçäo. Esses achados confirmam os resultados de experiências anteriores e sugerem que o PAF circula no plasma de boi como uma série de agregados de altos pesos moleculares

Determinaçäo do antígeno do fator VIII (VIIIR-Ag) por aglutinaçäo eritrocitária / DeteRmination of factoR VIII antigen (VIIIR-Ag) by eRythRocyte agglutination

Apresenta-se um método original, econômico, rápido e de fácil execuçäo para avaliaçäo semi-quantitativa do VIIIR Ag. Baseia-se em aglutinaçäo de eritrócitos sensibilizados com anticorpos específicos anti-antígeno do fator VIII. Com tal processo tornou-se fácil a diferenciaçäo diagnóstica entre hemofilia A e moléstia de von Willebrand e já permitiu algumas investigaçöes de pesquisa que confirmaram dados de literatura