Evaluation of a potential redox switch in blood coagulation tenase

Abstract

Blood coagulation factor IXa (FIXa) activates factor X that leads to thrombin and fibrin formation and a stable thrombus. FIXa catalytic efficiency is markedly enhanced when bound to the co-factor, factor VIII (FVIII), and a negatively charged phospholipid surface in the tenase complex. Small redox active peptides and protein oxidoreductases have been shown previously to have some FIXa co-factor activity and thiol modifying agents have been reported to influence FVIII activity. These observations suggested that FIXa might contain an allosteric disulfide that is regulated by FVIII. This idea was tested by measuring the influence of FVIII on the redox state of FIXa disulfide bonds and the effect of plasma oxidoreductases on FIXa activity. The redox state of nine of the 11 disulfide bonds in FIXa was measured using differential cysteine labelling and mass spectrometry and all were oxidized in the protein, and this did not change upon binding of the enzyme to FVIII. All eight disulfide bonds in FVIII were also predominantly oxidized and this did not appreciably change upon FIXa binding. In addition, relevant protein reductants in the circulation inhibited rather than activated FIXa activity. In conclusion, we found no evidence that the co-factor function of FVIII involves a change in the redox state of one or more FIXa disulfide bonds. Austen, D.E. (1970). Thiol groups in the blood clotting action of factor VIII. Br J Haematol 19, 477-484. Bayele, H.K., Murdock, P.J., and Pasi, K.J. (2010). Residual factor VIII-like cofactor activity of thioredoxin and related oxidoreductases. Biochim Biophys Acta 1800, 398-404.

A proximity-based in silico approach to identify redox-labile disulfide bonds: The example of FVIII

“…Early work [ 26 ] also highlighted a decrease in FVIII cofactor activity (FVIII:C) as well as a dissociation from its carrier protein von Willebrand Factor (VWF), another redox-labile protein that increases FVIII half-life in the circulation by ~6-fold [ 65 ]. In contrast to this, infusion of N -acetylcysteine (NAC) was associated with a short-lived rise in measurable FVIII activity, although this increase in FVIII activity was only observed in patients with an adverse reaction to the NAC infusion and a resultant rise in VWF release from the endothelium [ 66 , 67 ]. There is also contradictory evidence in the literature regarding the effect of reduction on FIXa and the tenase complex (FIXa/FXa), whose formation is catalysed by FVIII.…”

“…There is also contradictory evidence in the literature regarding the effect of reduction on FIXa and the tenase complex (FIXa/FXa), whose formation is catalysed by FVIII. A more recent study investigating allosteric disulfide bond regulation of FIXa showed there was no change in FVIII or FIXa disulfide bond status when complexed, but did show a decrease in the low inherent activity of the tenase (FIXa/FX) complex when thioredoxin or PDI were present [ 67 ], again suggesting that reduction of FVIII supresses FVIII activity.…”

SAXS analysis of the intrinsic tenase complex bound to a lipid nanodisc highlights intermolecular contacts between factors VIIIa/IXa

“…[29][30][31][32][33][34][35] Calculating an ab initio molecular envelope of the Xase:ND complex allowed us to computationally dock fVIIIa to fIXa (Figure 4F) and investigate the feasibility of previously proposed protein-protein interactions as well as speculate on novel points of contact (Figure 5). Formation of the Xase complex is not predicted to alter the redox states of the disulfide bridges in fVIIIa or fIXa, 87 limiting any conformational rearrangements to flexible linkers between domains.…”

SAXS analysis of intrinsic tenase complex bound to lipid nanodisc highlights intermolecular contacts between factors VIIIa/IXa

“…[27][28][29][30][31][32][33] Calculating an ab initio molecular envelope of the Xase:ND complex allowed us to computationally dock fVIIIa to fIXa (Figure 3F) and investigate the feasibility of previously proposed protein-protein interactions as well as speculate on novel points of contact (Figure 4). Formation of the Xase complex is not predicted to alter the redox states of the disulfide bridges in fVIIIa or fIXa, 78 limiting any conformational rearrangements to flexible linkers between domains.…”