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Expression analysis of missense pros1 mutations identified in protein s deficient patients

Objectif



Research objectives and content
Protein S (PS) is a vitamin K-dependent plasma glycoprotein that inhibits blood coagulation by acting as the cofactor of activated protein C in the inactivation of the procoagulant factors Va and VIIIa. In plasma, around a 60% of PS forms a noncovalent complex with the Beta-chain of the complement C4b-binding protein and only the remaining 40% of PS circulates as free protein and is active as a cofactor of APC. Inherited PS deficiency is an autosomal dominant disease associated with an increased risk for the development of venous thrombotic events, which confirms the physiological role of PS as an antithrombotic protein. The human PS gene (PROS1) spans about 80kb of genomic DNA on the centromeric region of chromosome 3 and it contains 15 exons which are transcribed in a 3.5 kb mRNA. Direct analysis of PROS1 in PS deficient families has revealed the presence of several different missense mutations which are associated with one of two different abnormal PS phenotypes: type I (reduced plasma concentration of PS antigen) or type III (reduced plasma concentrations of the free and active form of PS). Many of these mutations have been identified by our group in Spanish PS deficient families. The reason why such protein S amino acids substitutions result in the PS deficient phenotype is as yet unknown and although molecular modelling studies may predict the role of these and other amino acids in the structure and function of PS, the definite proof of this role will come from expression studies of their respective protein S mutants. The main objective of the proposed research project is to perform the expression studies required to analyse the molecular fate of several PROS1 missense mutations that have been identified in Spanish patients with thrombosis associated with type I or type III PS deficiency. With this purpose an expression vector containing the normal or the mutated PROS1 cDNAs will be constructed, transfected into a mammalian cell line that allows for a correct post-translational processing of protein S and their expression products will be analysed after culture of the transfected cells.
Training content (objective, benefit and expected impact)
Training in the methodologies required for recombinant gene expression studies will be obtained. Particularly, this will be: vector construction, mutagenesis of cloned cDNAs, cell transfection, cell culture and analysis of the expression products. Knowledge and experience in these methodologies will be of great benefit for future mutant analysis, either in the PS gene or in other genes associated with thromboembolic or other inherited diseases.
Links with industry / industrial relevance (22)
The results of these studies could be of interest in the pharmaceutical industry for the production of recombinant protein S molecules with therapeutical properties in the treatment of venous thrombotic diseases. The availability of recombinant PS mutants can also be of use to determine the sensibility of PS assays for common PS missense mutantions. In particular, this can be applied to analyse the sensibility for these mutants PS molecules of the new free PS antigen assay that has been recently described and is being developed in Dr. Dahlbdck laboratory.

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Coordinateur

LUND UNIVERSITY
Contribution de l’UE
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Adresse
Wallenberg laboratoriet, University Hospital
221 85 LUND
Suède

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