Structure and function of coagulation factors

Principal investigators: Sander Meijer PhD and prof Koen Mertens PhD

The coagulation cascade comprises several serine proteases that act in combination with a non-enzymatic co-factor on phospholipid-containing membranes. Over the past decade we have been focusing on the mechanism by which activated factor IX assembles with its co-factor, factor VIII. These proteins are indispensable for proper functioning of the coagulation cascade as a functional absence of factor VIII (FVIII) and factor IX is associated with the bleeding disorders hemophilia A and hemophilia B. FVIII is composed of a series of repeated domains which appear in the order A1-a1-A2-a2-B-a3-A3-C1-C2. The A-domains of FVIII mediate the binding to activated factor IX and factor X, whereas the C2 domain has been implicated in binding to phospholipids. After activation, the A2 domain rapidly dissociates from activated factor VIII (FVIIIa) resulting in a dampening of the activity of the activated factor X-generating complex. The amino acid residues that affect A2 domain dissociation are therefore critical for the FVIII co-factor function. We have now employed chemical footprinting in conjunction with mass spectrometry to identify lysine residues that contribute to the stability of activated FVIII. We hypothesized that lysine residues, which are buried in FVIII and surface-exposed in dissociated activated FVIII (dis-FVIIIa), may contribute to interdomain interactions (Figure 1). Mass spectrometry analysis revealed that residues K1967 and K1968 of region T1964-Y1971 are buried in FVIII and are exposed to the surface in dis-FVIIIa. This result, combined with the observation that the FVIII variant K1967I is associated with hemophilia A, suggests that these residues contribute to the stability of activated FVIII. Kinetic analysis revealed that the FVIII variants K1967A and K1967I exhibit an almost normal co-factor activity. However, these variants also showed an increased loss in co-factor activity over time compared with that of FVIII wild type (WT). Remarkably, the co-factor activity of a K1968A variant was enhanced and sustained for a prolonged time relative to that of FVIII WT. Surface plasmon resonance analysis demonstrated that A2 domain dissociation from activated FVIII was reduced for K1968A and enhanced for K1967A. In conclusion, mass spectrometry analysis combined with site-directed mutagenesis studies revealed that the lysine couple K1967-K1968 within region T1964-Y1971 has an opposite contribution to the stability of FVIIIa.

  Surface-exposed residues (in red) involved in retaining the A2 domain in activated FVIII

Key publication

Last edited on: 18 February 2016