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

Pilot study of homocysteine and cysteine in patients with thrombosis in different vascular sites. Epidemiology and response to folate.

Hyperhomocysteinemia is a risk factor for arterial and venous thrombosis. However, lowering homocysteine (Hcy) with vitamins not only failed to improve outcomes but also may lead to recurrent events. Our objectives were to evaluate Hcy and cysteine (Cys) levels in patients with thrombosis in different vascular sites, and their response to folate. One hundred and sixty four consecutive patients with thrombosis (42.1% arterial (AT), 36% venous (VT), 4.9% both venous and arterial thrombosis (AVT) and 17% unusual site (UST)) were included. Hcy and Cys were highest in patients with AVT and UST (p=0.0006). Ninety-three patients were treated, 70% were followed-up. Hcy levels normalized after therapy in all patients. Cys levels tended to vary after therapy according to the site of thrombosis. We observed a significant correlation between folate and Hcy (r: 0.48; p=0.005) among homozygous for MTHFR. A significant inverse relation was observed between Hcy and folate among homozygous and heterozygous (r: 0.462, p=0.007 and r: 0.267; p=0.04, respectively). No correlation was observed between folate and Cys. In conclusion, our observations suggest that Hcy and Cys might be implicated in thrombosis in different vascular sites, and respond differently to folate.

Assessment of platelet activation in myeloproliferative disorders with complementary techniques.

Bleeding and thrombosis in myeloproliferative disorders (MPD) are common events, sometimes both are present in the same patient during the course of the disease. Platelet activation in patients with MPD is often suggested. The present study analyses the presence of circulating activated platelets, using simultaneously flow cytometry and aggregometric studies in MPD. We studied 28 patients: 13 with polycythaemia vera, seven with essential thrombocythaemia, and eight chronic myeloid leukaemia. We performed functional tests, aggregation and adenosine triphosphate (ATP) release and flow cytometric assays (mepacrine staining and platelet activation markers CD62, CD63 and fibrinogen binding (B-FG)). Twenty-one MPD samples (75%) had reduced aggregation and ATP release. Acquired delta-SPD was detected in 11 of 28 MPD patients (39%), and we found no association between reduced mepacrine labelling and abnormal ATP release. High levels of activation markers were obtained: CD62 in 19 of 28 patients (68%), CD63 in 13 of 28 patients (46%) and B-FG in 19 of 28 patients (68%). The most prevalent abnormality was a reduced aggregation and ATP release. The lack of association between ATP release and mepacrine labelling suggests that other mechanisms, besides the deficit of intraplatelet ATP/adenosine diphosphate, might occur. High levels of activation markers were also observed. We conclude that both tests are complementary and necessary to understand the functional status of platelets in MPD.