Viribus unitis - the molecular synergism between coagulation factors VIII and IX

Abstract

The complex formed by the serine protease activated factor IX (FIXa) and its cofactor activated factor VIII (FVIIIa) plays a central role in the coagulation cascade. Detailed structural insight of FVIII and FIX driving complex formation and action are still lacking. In this thesis, with footprinting approaches coupled to mass spectrometry we aimed to gain novel insights into the molecular mechanism at the basis of the FIXa-FVIIIa complex. The main findings of this work include the identification of five surface-exposed hydrophobic residues unique to FVIII C1 domain contributing to von Willebrand Factor and/or FIXa interactions. Through Hydrogen-Deuterium eXchange Mass Spectrometry (HDX) we dove deeper into the FVIIIa life-cycle discovering a novel FIXa interactive region (L631-Y636). With focus on FIXa, HDX was employed to unravel molecular rearrangements in response to cofactor interaction. This led to the identification of allosteric changes occurring in FIXa and of a basic patch (Exosite II) directly implied in the interaction with FVIIIa. We finally investigated the main changes at the basis of the conversion of FIX into FIXa, we found that no major differences could be detected between FIX and FIXa, implying that FIXa is largely zymogen-like. Moreover, HDX-MS coupled to covalent footprinting by Tandem-Mass-Tags labelling highlighted the importance of charged residues of the 220-loop. Taken together our findings provide novel insight into the necessary and important steps needed to sustain FIXa-FVIIIa complex formation and functioning in the coagulation cascade. Our discoveries also aim to assist the development of therapeutics for the treatment of hemostatic disorders.