Multicatalytic Serine Proteases in Blood Coagulation



    To identify the catalytic site of factor IXs site directed mutageneses will be used in combination with mass spectrometry. Recombinant fIX will be produced using an E.coli expression system followed by in vitro refolding of the protein. The enzymatic activity of different fIX variants will be investigated in a chromogenic substrate assay. Finally, crystallization and structure determination of fIXs will be performed using robot-supported crystallization screening with the vapor diffusion or batch method. X-ray diffraction data will be collected and processed using standard software packages.

    Blood coagulation factor IX plays a critical role in the initiation, maintenance and regulation
    of the blood coagulation cascade. Factor IX shares with related coagulation factors a common
    domain architecture including a trypsin-like serine protease domain. Analogously, factor
    IX is synthesised as an inactive single chain zymogen form; this pro-form becomes proteolytically
    activated by members of the coagulation cascade. Upon the correct cleavage, the
    newly generated N-terminus (V16) forms a salt bridge with D194, whereby the formation of
    an active site with trypsin-like substrate specificity is induced. Unexpectedly, we have discovered
    an alternative proteolytic activity with chymotryptic specificity that results from noncanonical
    proteolytic activation of the zymogen factor IX. This alternative active site is different
    and independent from the classical active site (S195). The aim of this project is to exactly define the
    catalytic mechanism of the novel activity (factor IXs) including the identification of the catalytic
    residues, to elucidate its mechanism of activation, and to determine the crystal structure
    of factor IXs. The results will provide a rational for a more complete understanding of the
    physiology and pathophysiology of the haemophilic coagulation factor IX.
    Tatsächlicher Beginn/ -es Ende1/01/1031/12/11

    Systematik der Wissenschaftszweige 2002

    • 1411 Molekularbiologie