Thrombotic microangiopathies: First report of 294 cases from a single institution experience in Argentina.

Introduction: Introduction: Thrombotic microangiopathies (TMAs) are rare disorders associated with fatal outcomes if left uncared for. However, healthcare problems in developing countries tend to limit medical assistance to patients. Methods: Methods: We prospectively studied an Argentine cohort of 294 consecutive patients from 2013 to 2016. Patients' subcategory classification relied on clinical symptoms and presence or absence of trigger events associated with TMA. Results: Main suspected disorders were the primary TMAs known as thrombotic thrombocytopenic purpura (TTP) (n = 72/294, 24%) and atypical haemolytic uraemic syndrome (aHUS) (n = 94/294, 32%). In acute phase, demographic parameters for acquired TTP (aTTP) (n = 28) and aHUS (n = 47) showed that both groups were characterised by a young median age (37 and 25 years, respectively) and female predominance (60% and 86%). Median of a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 activity was significantly lower in aTTP than in aHUS group (1.4% vs 83%) and was associated with a more severe thrombocytopenia (15 × 109 vs 53 × 109/L). Creatinine (Cr) and urea (Ur) were significantly increased in aHUS compared to aTTP subjects (Cr: 3.7 vs 0.7 mg/dL, Ur: 118 vs 33 mg/dL). Gastrointestinal and neurological symptoms were more frequent in aHUS and aTTP, respectively. Conclusion: The first description of a TMA cohort in Argentina revealed similar clinical presentations to those of other countries.

Phenotypic Parameters in Genotypically Selected Type 2B von Willebrand Disease Patients: A Large, Single-Center Experience Including a New Novel Mutation.

von Willebrand disease type 2B (VWD2B) expresses gain-of-function mutations that enhance binding of an individual's von Willebrand factor (VWF) to its platelet ligand, glycoprotein Ib (GPIb), and which are usually identified by increased ristocetin-induced platelet aggregation (RIPA). We describe here the phenotypic profile of 38 genotypically selected VWD2B-affected family members (AFMs) belonging to 19 unrelated families. Major bleeding was observed in 68.4% of AFMs (previous to their diagnosis and registered by lifetime interviews), with a total of 46 episodes (1.21/patient), and was found to be highly related to the individual bleeding score and presence of thrombocytopenia, but otherwise unrelated to other laboratory parameters. Excessive muco-cutaneous bleeding symptoms were often reported, the most frequent of which comprised menorrhagia, epistaxis, easy bruising, and bleeding after teeth extraction/in oral cavity. Eight unaffected family members were also studied. The prevalence of VWD2B within families was 0.826, and the penetrance of mutations was complete, making it mandatory to study entire family sets to complete diagnostic profiles. Seven heterozygous missense mutations were found, the most common being p.V1316M. In the p.R1308C group, 75% of the AFMs showed absence of RIPA at 0.5 mg/mL, 66.6% of whom had VWF:RCo < 10 IU/dL, and 50% of whom had VWF:CB < 10 IU/dL. In the p.S1310F group, none of the AFMs had VWF:RCo/VWF:Ag < 0.6 (RCo/Ag), but 100% had VWF:CB/VWF:Ag < 0.6/(CB/Ag). Patients with p.P1266L and p.R1304V were characterized as atypical VWD2B. Two de novo mutations were found in four AFMs belonging to two families. We also describe a novel mutation: p.Y1258C. Of our patients, 70.5% had O blood group. In conclusion, a normal RCo/Ag and a negative RIPA at 0.5 mg/mL do not necessarily rule out a diagnosis of VWD2B.

Factor von Willebrand y Enfermedad de von Willebrand: nuevos enfoques diagnósticos / Von Willebrand Factor and von Willebrand disease: new approaches to diagnosis / Fator Von Willebrand e doença de von Willebrand: novas abordagens diagnósticas

El factor von Willebrand (VWF) es una glicoproteína que se sintetiza en células endoteliales y en megacariocitos. Su vida media es de ~12 horas. Está formado por multímeros de diferentes pesos moleculares, pequeños, intermedios, grandes y extragrandes. La actividad funcional reside en los multímeros grandes, y los extragrandes son trombogénicos. Promueve la adhesión plaquetaria al subendotelio, la agregación plaquetaria y transporta al FVIII en plasma, protegiéndolo de su degradación por proteasas. La enfermedad de von Willebrand es el trastorno hemorrágico más frecuente; se describen deficiencias cuantitativas (parcial: VWD1; total: VWD3) o defectos cualitativos (VWD2A, VWD2M, VWD2B y VWD2N). La expresión clínica es variable (sangrado muco-cutáneo) y su herencia autosómica, dominante o recesiva, según las variantes. Los niveles del VWF dependen de factores genéticos y no genéticos que afectan el diagnóstico y la expresión clínica. Para llegar al diagnóstico se precisan varias pruebas, algunas inespecíficas. El laboratorio comienza con pruebas orientadoras, se continúa con pruebas confirmatorias, y posteriormente pruebas para definir la variante de VWD. El diagnóstico genotípico es fundamental para lograr el diagnóstico diferencial entre VWD2B vs. PT-VWD y VWD2N vs. Hemofilia A (leve-moderada), diferenciar VWD de AVWS y discriminar variantes VWD2.

Von Willebrand factor (VWF) is a glycoprotein with essential roles in both primary and secondary hemostasis, synthesized by endothelial cells and megakaryocytes. Its half-life is ~12 hours. VWF consists in multimers of different molecular weight: small, intermediate, large and ultra large. The functional activity resides in the large multimers; the ultra large are thrombogenic. VWF promotes platelet adhesion to subendothelium, platelet aggregation and binds FVIII, protecting it from proteolysis and preserving its hemostatic function. Von Willebrand disease is the most common bleeding disorder; qualitative defects (VWD2A, VWD2M, VWD2B and VWD2N) and quantitative deficiencies (VWD1 and VWD3) are described. The clinical expression is variable (mucocutaneous bleeding); VWF levels depend on genetic and non-genetic factors affecting diagnosis and clinical expression. The inheritance can be autosomal, dominant or recessive according to the variants. To reach diagnosis, several tests are required, being some of them unspecific. The laboratory testing begins with global tests, followed by confirmatory tests and further tests to define the variant of VWD. Genotypic studies are essential to achieve the differential diagnosis between VWD2B vs. PT-VWD, VWD2N vs. Hemophilia A (mild to moderate) and differentiate VWD from AVWS and discriminate VWD2 variants.

O fator de von Willebrand (vWF) é uma glicoproteína sintetizada em células endoteliais e em megacariócitos. Sua vida média é de ~12 horas. É constituído por multímeros de pesos moleculares diferentes, pequenos, intermediários, grandes e extragrandes. A atividade funcional reside nos multímeros grandes, sendo os extragrandes, trombogênicos. Promove adesão das plaquetas ao subendotélio, a agregação plaquetária e transporta o FVIII em plasma, protegendo-o de sua degradação. A doença de von Willebrand é o distúrbio hemorrágico mais frequente; são descritas deficiências quantitativas (parcial: VWD1; total: VWD3) ou defeitos qualitativos (VWD2A, VWD2M, VWD2B e VWD2N). A expressão clínica é variável, (sangramento mucocutâneo), e sua herança autossômica dominante ou recessiva de acordo com as variantes. Os níveis de vWF dependem de fatores genéticos e não-genéticos que afetam o diagnóstico e a expressão clínica. Para fazer o diagnóstico, vários testes são necessários, alguns inespecíficos. O laboratório começa com testes orientadores, continua com testes de confirmação e, mais tarde, com testes para definir a variante de VWD. O diagnóstico genotípico é essencial para alcançar o diagnóstico diferencial entre VWD2B vs. PT-VWD e VWD2N vs. Hemofilia A (leve a moderada), diferenciar VWD de AVWS, discriminar variantes VWD2.

VWF and ADAMTS13 behavior in estradiol-treated HUVEC.

OBJECTIVES: In this study, the role of 17ß-estradiol (E2) in the regulation of von Willebrand factor (VWF) and ADAMTS13 synthesis, storage, and secretion was investigated in cultured human umbilical vein endothelial cells (HUVEC). METHODS: HUVEC were grown to 80-90% confluence and replaced with fresh medium containing E2 (1 nm) or vehicle for 24 h, after which the supernatant medium and cell lysates were collected to measure VWF and ADAMTS13. VWF was evaluated by VWF:Ag and multimeric analysis. ADAMTS13 was evaluated by SDS-PAGE. VWF and ADAMTS13 mRNA were quantified by real-time PCR after E2 or vehicle exposure for 18 h. A functional effect of ADAMTS13 on HUVEC VWF protein synthesis was further evaluated using a short hairpin RNA (shRNA) to knockdown the expression of endogenous ADAMTS13. RESULTS: E2 did not increase the release or intracellular VWF levels in HUVEC. However, E2 increased the production of intracellular ADAMTS13, although there was no evidence of significant effects of their release into culture medium. Incubation of HUVEC with E2 resulted in a significantly increased expression of VWF and ADAMTS13 mRNA. ADAMTS13 gene inactivation upregulates release and intracellular VWF levels in E2-treated HUVEC. CONCLUSION: The results demonstrated that E2 may play a role in the regulation of VWF and ADAMTS13 gene expression and in its production in human endothelial cells. The mechanism of the protective effects of E2 on the cardiovascular system could be explained by the intracellular regulation of VWF produced by ADAMTS13.

Hemorragias mayores asociadas a cirugía en pacientes con enfermedad de Von Willebrand leve / Major haemorrhages associated with surgery in patients with mild von Willebrand disease

Introducción: Los pacientes con enfermedad de von Willebrand frecuentemente sangran frente a desafíos hemostáticos. Objetivos: Nuestro propósito fue identificar marcadores predictivos de hemorragia mayor en cirugías en pacientes con las variantes tipo 1 y posible tipo 1 de la enfermedad. Material y Métodos: Se registraron las hemorragias mayores en cirugías anteriores al diagnóstico y los parámetros de laboratorio en 311 pacientes. Éstos se agruparon de acuerdo con la ausencia (grupo A) o presencia (grupo B) de hemorragia mayor en cirugías. Resultados: Presentaron hemorragia mayor el 26 por ciento de los pacientes y 17,5 por ciento de las cirugías. No hubo diferencias en el porcentaje de pacientes tipo 1 (32,6 por ciento) y posible tipo 1 (24,8 por ciento) que tuvieron hemorragia mayor. Tampoco se observaron diferencias en la prevalencia del grupo sanguíneo O, edad, género, historia familiar y niveles de FVIII y VW entre los grupos A y B. La hemorragia post exodoncia fue el antecedente clínico más frecuente (P<0,000; RR=2,11; IC 95 por ciento = 1,3-3,5) y podría definir riesgo de hemorragias mayores. El bleeding score y el número de sitios de sangrado no resultaron predictivos de hemorragias mayores. Las cesáreas (24,6 por ciento) y adenoamigdalectomías (22,3 por ciento) fueron las cirugías con mayor frecuencia de hemorragias mayores. Conclusión: Los pacientes con VWD tipo 1 y posible tipo 1 mostraron similar incidencia de hemorragia mayor en cirugías. Los niveles de FVIII y VWF, el tiempo de sangría, historia familiar y grupo sanguíneo no resultaron efectivos como marcadores predictivos de hemorragia mayor. Sin embargo, el antecedente de sangrado post exodoncia y el tipo de cirugía a llevar a cabo (cesáreas y adenoamigdalectomías) en pacientes con enfermedad de von Willebrand tipo 1 y posible tipo 1 parecen importantes como marcadores de riesgo.

Introduction: Patients with von Willebrand disease frequently bleed under haemostatic challenges. Objectives: The aim of our study was to identify predictive markers of perioperative major haemorrhage in type 1 and possible type 1 patients von Willebrand disease. Material and Methods: We recorded perioperative bleeding complications previous to diagnosis and laboratory parameters in 311 patients. They were grouped according to the absence (group A) or presence (group B) of perioperative major haemorrhage. Results: Twenty-six per cent of patients and 17.5 per cent of surgical procedures presented major haemorrhage. There was no difference neither between percentages of type 1 (32.6 per cent) and possible type 1 patients (24.8 per cent) who had major haemorrhage nor in the prevalence of O blood group, age, gender, family history and levels of FVIII and VWF, between group A and B. A history of bleeding after tooth extraction was the most frequent clinical feature (P<0.000; RR=2.11; CI 95 per cent = 1.3-3.5) observed in patients with major haemorrhage, and could defi ne risk factor. The bleeding score and the number of bleeding sites were not predictors of major haemorrhages. Caesarean sections (24.6 per cent) and adeno-tonsillectomies (22.3 per cent) showed the highest frequency of major haemorrhage. Conclusion: Type 1 and possible type 1 VWD patients showed similar incidence of perioperative major haemorrhage. The levels of FVIII and VWF, the bleeding time, blood group and family history did not prove to be effective as predictive markers of major haemorrhage. However, the personal history of bleeding after tooth extraction and the type of surgery (caesarean section and adeno-tonsillectomies) in patients with either type 1 or possible type 1 von Willebrand disease shown to be important in determining risk.

Effect of estradiol, progesterone and testosterone on apoptosis- and proliferation-induced MAPK signaling in human umbilical vein endothelial cells.

Sex hormones induce death or cell proliferation in various cell lines and in primary cultures. However, the signal transduction pathways involved in the regulation of proliferation and apoptosis in endothelial cells have not been fully elucidated. Here, we report that progesterone and testosterone induce apoptosis in HUVECs in a p38- and JNK-dependent manner, and that estradiol promotes proliferation via the activation of ERK2. We showed that, at physiological doses, progesterone and testosterone promoted p38, but not JNK, phosphorylation. Hormone inhibitors, on the other hand, prevented p38 phosphorylation. When supraphysiological doses were applied, both p38 and JNK were phosphorylated, causing apoptotic cell death. The addition of hormone inhibitors at an appropriate concentration did not prevent cell death or the phosphorylation of p38 and JNK. Estradiol, at physiological doses, promoted an increase in ERK2 phosphorylation that was blocked by fulvestrant. At physiological and supraphysiological doses, it promoted a proliferative effect. In conclusion, these findings suggest that JNK has an important pro-apoptotic function following progesterone and testosterone treatment in human endothelial cells, and that ERK2 has a proliferative effect following estradiol treatment.

Major haemorrhage related to surgery in patients with type 1 and possible type 1 von Willebrand disease.

Patients with von Willebrand disease (VWD) frequently bleed under a challenge. The aim of our study was to identify predictive markers of perioperative major haemorrhage in type 1 (VWF:RCo = 15-30 IU dl(-1)) and possible type 1 (VWF:RCo = 31-49 IU dl(-1)) VWD patients. We recorded perioperative bleeding complications previous to diagnosis and laboratory parameters in 311 patients with 498 surgical procedures. The patients were grouped according to the absence (A) or presence (B) of perioperative major haemorrhages. Eighty-one patients (26%) and 87 surgical procedures (17.5%) presented major haemorrhages associated with surgeries. There was no difference between the percentage of type 1 and possible type 1 VWD patients who had major haemorrhages (32.6% and 24.8% respectively; p = ns). No difference in the prevalence of O blood group, age, gender, positive family history and laboratory test results (FVIII and VWF) was observed, independent of the haemorrhagic tendency. Bleeding after tooth extraction was the most frequent clinical feature observed in patients with perioperative major haemorrhages. The bleeding score and the number of bleeding sites (> or = 3) were not predictors of major haemorrhage associated with surgery. Caesarean section and adenotonsillectomy showed the highest frequency of major haemorrhages (24.6% and 22.3%, respectively). In conclusion, type 1 and possible type 1 VWD patients showed similar incidence of perioperative major haemorrhages. Laboratory tests and positive family history did not prove to be effective at predicting major haemorrhages in patients that had either type 1 or possible type 1 VWD. The history of bleeding after tooth extraction could define risk factors of major haemorrhage.