seminario biologia mol

RECOMBINANT CLOTTING FACTOR VIII CONCENTRATES: HETEROGENEITY AND HIGH-PURITYEVALUATION

Presented by:

Luis Miguel Ruiz Velasquez

3rd Semester

Medicine Student

Lello Zolla

INTRODUCTION

The coagulation cascade, is a very complex step-by-step process that occurs in the body when a blood vessel is injured

Several special proteins known as coagulation factors are activated one after the other in a “cascade” effect. The end result is a blood clot that creates a barrier over the injury site, protecting it until it heals

This process also involves a feedback system that regulates clot formation in the body so that clots are removed when the injury site is healed.

http://www.medicinapreventiva.com.ve/laboratorio/imagenes/coagul.jpg

There are several proteins that play special roles in coagulation cascade like: factor XII, XI, IX, VIII, X, V, II, and fibrinogen as well as prekallikrein (PK) and high molecular weight kininogen (HK).They encompasses the intrinsec and extrinsec pathway

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Human clotting factor VIII (FVIII) is a non-enzymatic plasma glycoprotein, an obligated step in the intrinsic pathway of the blood coagulation cascade.

FVIII circulates in plasma and associates with von Willebrand factor in a noncovalent complex, and its compose by three different domains (A,B,C)

hemophilia A is caused by a reduction in the amount or activity of factor VIII.

The lack of this factor of the coagulation cascade results in the formation of fibrin deficient clots which makes coagulation much more prolonged, and the clot more unstable.

Hemophilia A is inherited as an X-linked recessive trait, and thus occurs in males and in homozygous females.

http://www.plataformasinc.es/-cascada-de-coagulacion/6989-1-esl--Factor-VIIIa-de-la-cascada-de-coagulacion.jpg

When a bledding procces occurs, the coagulation cascade actives and several factor like FVIII triger the blood clot synthesis, the deficience of FVIII is called hemophilia A.

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OBJECTIVE

The objective of this study was to compare the heterogeneity and the high purity of both rFVIII preparations before and after thrombin digestion

MATERIALES Y METODOS

SDS PAGESodium dodecyl sulfato polyacrylamida gel electrophoresis

Electroforesis en gel de poliacrilamida con dodecil sulfato El principio: migración de solutos o partículas cargadas en

medio liquido bajo la influencia de un campo eléctrico.

Poliacrilamida involucra geles que varían en composición y tamaño del poro.

Dodecil sulfato es un detergente que desnaturaliza las proteínas eliminando su estructura secundaria y terciaria sin cambiar enlaces sulfuro y confiere una carga negativa .Se usa para analizar proteínas

2DE ELECTROFORESIS EN DOS DIMENSIONES

Analiza proteínas

Se separan por dos propiedades. Se inicia con electroforesis en una dimensión. Pero después se separa la segunda molécula por una segunda características en una dirección a noventa grados de la primera

La posibilidad de que dos moléculas sean iguales es poco probable , por esto, se separan con efectividad en dos dimensiones.

http://www.proteomefactory.com/images/2demousebrain.jpg

DIGESTION EN GEL Es parte de la preparación de la muestra

para la identificación de proteínas por espectrometría de masa. Corta enzimáticamente la proteína en péptidos

Etapas: Decoloración. reducción y alquilación de cisteínas Clivaje proteolítico Extracción de péptidos

SECUENCIACION

Usa segmentos del DNA original cortados con enzimas de restricción

Marca el DNA radiactivamente o químicamente con compuestos fluorescentes

Usa la electroforesis para separar fragmentos

RESULTADOS

Figure 1. (A) 1-D SDS-PAGE of three different rFVIII preparations. Forty microgram of each rFVIII was separated on 5–16% w/v

•Las bandas 2-3-4-11-12, corresponden a factor VIII recombinante truncado

•Demostrado por secuenciación

•Se observan bandas al mismo nivel por la proteólisis desencadenada por la trombina

La electroforesis en dos dimensiones revelo la presencia de haptoglobina, proapolipoproteina, glutatión transferasa y B lactamasa

ReferenceAgree or disagree

What he or she say?

DISCUSSION

ReferenceAgree or disagree

What he or she say?

Jankowski et al

In the case of Helixate NexGensand Advates, B domain was removed from full-lengthprotein

Hansenet al.

this cleavage on theprecursor could be mediated by cell surface bound proteases

Reference Agree or disagree

What he or she say?

Basilico et al

The low number of peptides identified in biotechnologicalpreparations could be due to

the presence of albumin.

agree

CONCLUSIONS

The 2DE is better than SDS PAGE , in the searching of purity grade.

The use of rfactor VIII does not rule out the presence of contaminants

They should expose about the repercutions of the impure rFVIII

The thrombin clivage remove the different bands in the SDS PAGE of FVIII inactive pro-cofactor