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The carboxyl-terminal region of coagulation factors: role in biosynthesis and function of FVII and FX

Branchini, Alessio (2011) The carboxyl-terminal region of coagulation factors: role in biosynthesis and function of FVII and FX. PhD Thesis , Università degli Studi di Ferrara.

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    -BACKGROUND- Factor VII (FVII), factor IX (FIX), factor X (FX) and protein C (PC), belonging to the family of coagulation vitamin K-dependent serine proteases, share high sequence and structural homology at the gene and protein level. However, their carboxyl-terminal region displays striking differences in length and aminoacid composition. While this region of FIX and PC has been demonstrated to be essential for efficient biosynthesis and secretion, little is shown for FVII and FX. -AIMS- The main aim was to investigate the carboxyl-terminal region of FVII and FX as determinant of biosynthesis/secretion and/or function. In the study we took advantage of the characterization of i) a natural variant of FVII characterized by a nonsense mutation (R402X) leading to a slightly truncated protein (-4 residues), and ii) natural anti-FVII inhibitory antibodies developed in a patient with an altered carboxyl-terminal region. The study was approached both by assays in patient’s plasma and by expression of the recombinant FVII variants in eukaryotic cells. To address the issue of the role of the carboxyl-terminal region of FX, a panel of progressively truncated FX variants has been expressed and characterized. -MAIN RESULTS AND CONCLUSIONS - The main results of the studies can be summarized as follows. i) Demonstration that the truncated FVII-402X molecule, albeit poorly secreted, possesses an increased specific activity, which explains the association of the R402X nonsense mutation with an asymptomatic phenotype; ii) identification of an anti-FVII inhibitory antibody in a patient homozygous for a frequent FVII frameshift mutation (11125delC) and data supporting the FVII carboxyl-terminal region as the main epitope of this antibody; iii) demonstration that the carboxyl-terminal region of FVII is essential for efficient biosynthesis and secretion, and the presence of an inverse relationship between the extent of the deletion of the carboxy-terminus and secretion levels. The deleted variants however possess a normal specific activity, thus not supporting a functional role for the very last residues of FVII; iv) demonstration that efficient secretion of FX, at variance from its highly homologous FVII, FIX and PC proteins, is not affected by short deletions (up to 21 residues) of the carboxyl-terminal region, which seems to have a functional role. Taken together these data indicate a differential role of the carboxyl-terminal region of FVII and FX, which might have contributed to divergence and evolution of these serine proteases from the common ancestor enzyme. -METHODOLOGICAL APPROACHES- Production and expression of recombinant proteins, ELISA-based and functional assays have been exploited in this study to investigate the expression and the activity of different natural or recombinant variants of coagulation FVII and FX.

    Item Type:Thesis (PhD Thesis)
    Date:22 February 2011
    Tutor:Bernardi, Francesco - Pinotti, Mirko
    Coordinator:Bernardi, Francesco
    Institution:Università degli Studi di Ferrara
    Divisions:Dipartimento > Biochimica e biologia molecolare
    Subjects:Area 05 - Scienze biologiche > BIO/10 Biochimica
    Uncontrolled Keywords:carbossi-terminale, coagulazione, FVII, FX, carboxyl-terminal, coagulation
    Deposited on:13 Apr 2012 15:02


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