thrombosis from a_prothrombin_mutation_[autosaved]
TRANSCRIPT
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THROMBOSIS FROM A PROTHROMBIN MUTATIONCONVEYING ANTITHROMBIN RESISTANCE
Yuhri Miyawaki, M.Sc., Atsuo Suzuki, M.Sc., Junko Fujita, B.Sc., Asuka Maki, B.Sc.. All e. t.c
.
Presentado por:Juliana Sandoval y Sandra Quiroz
RestrepoEstudiantes III semestre
Facultad de medicina UPB
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INTRODUCTION
• This work is about the finding of a mutation on the gene which codifies the coagulation factor Prothrombine in patients with inherited thrombosis, such mutation leads to a resistance to antithrombine.
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THROMBOSIS
• Thrombosis is defined as the formation of a clot which appears after a injury produced to the endothelium of a vein or a artery. This occlusion can lead to cell or tissue death and represents a bigger risk if they start forming in brain and heart.
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PROTHROMBIN
• The prothrombine also called Factor II is the precursor of thrombin (which is the active form of the Factor II) in the coagulation cascade; it is also part of the Vitamin K-Dependent factors and its synthesized in the liver for approximated 72 hours period of life.
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ANTITHROMBIN
Is located in the first chromosome 1q23-q25.1 locus.
It is a glycoprotein, that have an important role in the control of blood clotting and subsequent
clot dissolution
Antithrombin III is the most representative.
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CORRELACION
PROTHROBIN
ANTITROMBIN
Hemostasis
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OBJECTIVE GENERAL
• TO DETERMINATE THE THROMBOSIS FROM A PROTHROMBIN MUTATION CONVEYING ANTITHROMBIN
RESISTANCE.
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MATERIALES Y METODOS
PCR- RFLP
ELISA
Dot: Blot
DNAc
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MATERIALES Y METODOS
PCR• DNA plantilla, primers, DNTps, pstI, buffers, DNA polimerasa
PCR-
RFLP
• Electroforesis, gel de agarosa , enzima de restricción.
ELISA
• Reacción entre antígenos y anticuerpos, requiere un paso de separacion
Dot-
blot• DNAc
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CASO CLINICO
• Paciente de 17 años de edad, japonesa.• El origen de su familia es Yukuhashi en la parte norte de
las islas de Kyushu.
• Antecedente personales: Presenta su primer episodio de trombosis venosa profunda a los 11 años
• Tratamiento: Desde el inicio de la enfermedad hasta la actualidad manejada con warfarina.
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CASO CLINICO
• Antecedentes familiares:
nueve de los miembros de su familia habían presentado uno o más episodios de trombosis venosa profunda, de los cuales:
Dos familiares tenia un embolismo pulmonar y otros tres murieron a
causa de la trombosis.
Cinco miembros de la familia, incluido el caso índice, habían tenido una trombosis de menores
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CASO CLINICO
Panel A shows the family pedigree. The proband (IV-1) is indicated by an arrow. Solid symbols represent affected family members, open symbols unaffected family members, and slashed
symbols deceased family members. Circles represent female family members, and squares male family members.
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MATERIALES Y METODOS
ANALISIS DE DNA
Con el PCR se amplían todos los fragmentos de los 14 exones.
Para detectar el sitio exacto de la mutación se utilizo la PCR - RFLP
Se utilizaron 2 cebadores.
Se introdujo en un sitio PstI para la amplificación de un alelo mutante.
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MATERIALES Y METODOS
RECOMBINATES DE PROTROMBINA:
una sonda DNAc , para obtener un vector de protrombina mutante (PCR)
(hibridación), en células renales; con el fin de determinar cual tenia mayor expresión por la protrombina
Dot- blot
ELISA
5′-TGTAGAAGCCATATTTCCCcTgC-3′ 5′-TTTCCCATCCaGGTCACAGCCTTCA-3
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MATERIALES Y METODOS
LAS PRUEBAS FUNCIONALES DE PROTROMBINAS RECOMBINANTES
Se realizaron tres pruebas de actividad de protrombina:
• Una etapa de coagulación• Coagulación en 2 etapas• 1 ensayo cromogénico que utiliza s2238 ( sustrato de trombina colorea la
escisión)
Para examinar las funciones de la protrombina recombinante en el plasma
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MATERIALES Y METODOS
FORMACIÓN DEL COMPLEJO TROMBINA- ANTITROMBINA :
• Protrombina mutada• Protrombina nueva complejo TAT se utilizaron 2 pruebas :
Con heparina y sin heparina en rangos diferentes de tiempo y detenidas con PPACK
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MATERIALES Y METODOS
PRUEBA DE GENERACION DE TROMBINA:
NUEVA
MUTANTE
MUTANTEHETEROCI
GOTICA
Mezcla1: Plasma deficiente en protrombina ,con protrombinas recombinadas (100%)
Mezcla2:antitrombina eficiente en plasma con antitrombina humana (50%)
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RESULTADOS
ANÁLISIS DEL DNA
Heterocigótica La misma mutación fue
detectada en su madre y en otros tres miembros de la familia
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RESULTADOS
ANÁLISIS DEL DNA
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RESULTADOS
• PROTROMBINAS RECOMBINANTES
Se utilizó ELISA para medir los niveles de protrombina en el medio de cultivo después de la absorción de sulfato de bario
Se encontraron que ambos, el tipo salvaje y la protrombina mutante fueron absorbidos completamente.
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RESULTADOS
• PRUEBAS FUNCIONALES DE PROTROMBINAS RECOMBINANTES
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RESULTADOS• FORMACION DEL COMPLEJO TROMBINA-
ANTITROMBINA
• Sin heparina : nueva incrementa de forma tiempo dependiente. Y la protrombina mutante , no fue legible durante los primeros 30 minutos.
• Con heparina: ambos incrementan , pero la protrombina mutante se ve mas afectada.
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RESULTADOS
PRUEBA DE GENERACIÓN DE TROMBINA
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DISCUSION
Akhavan S, Mannucci P, Lak M, et al.
AGREE “Prothrombin deficiency is an autosomal recessive disorder associated with a
moderately severe bleeding tendency”
Poort SR, Rosendaal FR, Reitsma PH,Bertina RM
AGREE A common genetic variationin the 3′-untranslated region
of the prothrombingene is associated with
elevatedplasma prothrombin levels
and an increasein venous thrombosis
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DISCUSIONRosendaal FR. AGREE knowledge about the
aetiology of venous thrombosis has advanced
with the discovery of several ctors that contribute to the
incidence of thrombosis, particularly the role of
coagulation abnormalities
De Stefano V, Finazzi G, Mannucci P.
AGREE Patients with hereditary thrombophilia often present with unusual
clinical episodes of venous thrombosis at a
young age and recurrencein atypical vessels, often with a family history of
the condition
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CONCLUSION• It was achieved to demonstrate that the replacement of arginine with
leucine, which is the major cause of mutation, explains the resistance of prothrombin.
• Thrombosis is more lethal in men than in women, because the affectation over coronary vessels.
• A thrombin-generation assay revealed that the peak activity of the mutant prothrombin was fairly low.
• Young women with a predisposition to thrombophilia are exposed to risk factors like planning anticonceptive, by favoring the condition of venous insufficiencies.
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