Contribution of factor VIII light-chain residues 2007–2016 to an activated protein C-interactive site

Summary

Although factor (F) VIIIa is inactivated by activated protein C (APC) through cleavages in the FVIII heavy chain-derived A1 (Arg³³⁶) and A2 subunits (Arg⁵⁶²), the FVIII light chain (LC) contributes to catalysis by binding the enzyme. ELISA-based binding assays showed that FVIII and FVIII LC bound to immobilised active site-modified APC (DEGRAPC) (apparent K _d ~270 nM and 1.0 μM, respectively). Fluid-phase binding studies using fluorescence indicated an estimated K _d of ~590 nM for acrylodan-labelled LC binding to DEGR-APC. Furthermore, FVIII LC effectively competed with FVIIIa in blocking APC-catalysed cleavage at Arg³³⁶ (K _i = 709 nM). A binding site previously identified near the C-terminal end of the A3 domain (residues 2007–2016) of FVIII LC was subjected to Ala-scanning mutagenesis. FXa generation assays and western and dot blotting were employed to assess the contribution of these residues to FVIIIa interactions with APC. Virtually all variants tested showed reductions in the rates of APC-catalysed inactivation of the cofactor and cleavage at the primary inactivation site (Arg³³⁶), with maximal reductions in inactivation rates (~3-fold relative to WT) and cleavage rates (~3 to ~9-fold relative to WT) observed for the Met2010Ala, Ser2011Ala, and Leu2013Ala variants. Titration of FVIIIa substrate concentration monitoring cleavage by a dot blot assay indicated that these variants also showed ~3-fold increases relative to WT while a double mutant (Met2010Ala/Ser2011Ala) showed a >4-fold increase in K _m. These results show a contribution of a number of residues within the 2007–2016 sequence, and in particular residues Met2010, Ser2011, and Leu2013 to an APC-interactive site.

Keywords

Factor VIIIa - activated protein C - factor VIII mutants - proteolysis

• References

• 1 Mann KG, Nesheim ME, Church WR. et al. Surface-dependent reactions of the vitamin K-dependent enzyme complexes. Blood 1990; 76: 1-16.
• 2 Vehar GA, Keyt B, Eaton D. et al. Structure of human factor VIII. Nature 1984; 312: 337-342.
• 3 Andersson LO, Forsman N, Huang K. et al. Isolation and characterisation of human factor VIII: molecular forms in commercial factor VIII concentrate, cryoprecipitate, and plasma. Proc Natl Acad Sci USA 1986; 83: 2979-2983.
• 4 Fay PJ, Anderson MT, Chavin SI. et al. The size of human factor VIII heterodimers and the effects produced by thrombin. Biochim Biophys Acta 1986; 871: 268-278.
• 5 Eaton D, Rodriguez H, Vehar GA. Proteolytic processing of human factor VIII. Correlation of specific cleavages by thrombin, factor Xa, and activated protein C with activation and inactivation of factor VIII coagulant activity. Biochemistry 1986; 25: 505-512.
• 6 Fay PJ, Mastri M, Koszelak ME. et al. Cleavage of factor VIII heavy chain is required for the functional interaction of a2 subunit with factor IXA. J Biol Chem 2001; 276: 12434-12439.
• 7 Lollar P. The association of factor VIII with von Willebrand factor. Mayo Clinic proceedings Mayo Clinic 1991; 66: 524-534.
• 8 Regan LM, Fay PJ. Cleavage of factor VIII light chain is required for maximal generation of factor VIIIa activity. J Biol Chem 1995; 270: 8546-8552.
• 9 Fay PJ. Activation of factor VIII and mechanisms of cofactor action. Blood Rev 2004; 18: 1-15.
• 10 Fay PJ, Beattie TL, Regan LM. et al. Model for the factor VIIIa-dependent decay of the intrinsic factor Xase. Role of subunit dissociation and factor IXa-catalysed proteolysis. J Biol Chem 1996; 271: 6027-6032.
• 11 Fay PJ, Smudzin TM, Walker FJ. Activated protein C-catalysed inactivation of human factor VIII and factor VIIIa. Identification of cleavage sites and correlation of proteolysis with cofactor activity. J Biol Chem 1991; 266: 20139-20145.
• 12 Plantier JL, Rolli V, Ducasse C. et al. Activated factor X cleaves factor VIII at arginine 562, limiting its cofactor efficiency. J Thromb Haemost 2010; 08: 286-293.
• 13 Walker FJ, Fay PJ. Regulation of blood coagulation by the protein C system. FASEB J 1992; 06: 2561-2567.
• 14 Eaton DL, Wood WI, Eaton D. et al. Construction and characterisation of an active factor VIII variant lacking the central one-third of the molecule. Biochemistry 1986; 25: 8343-8347.
• 15 Manithody C, Fay PJ, Rezaie AR. Exosite-dependent regulation of factor VIIIa by activated protein C. Blood 2003; 101: 4802-4807.
• 16 Krishnaswamy S, Williams EB, Mann KG. The binding of activated protein C to factors V and Va. J Biol Chem 1986; 261: 9684-9693.
• 17 Fay PJ, Walker FJ. Inactivation of human factor VIII by activated protein C: evidence that the factor VIII light chain contains the activated protein C binding site. Biochim Biophys Acta 1989; 994: 142-148.
• 18 Walker FJ, Scandella D, Fay PJ. Identification of the binding site for activated protein C on the light chain of factors V and VIII. J Biol Chem 1990; 265: 1484-1489.
• 19 Nogami K, Shima M, Nishiya K. et al. A novel mechanism of factor VIII protection by von Willebrand factor from activated protein C-catalysed inactivation. Blood 2002; 99: 3993-3998.
• 20 O’Brien LM, Huggins CF, Fay PJ. Interacting regions in the A1 and A2 subunits of factor VIIIa identified by zero-length cross-linking. Blood 1997; 90: 3943-3950.
• 21 Foster PA, Fulcher CA, Houghten RA. et al. Localisation of the binding regions of a murine monoclonal anti-factor VIII antibody and a human anti-factor VIII alloantibody, both of which inhibit factor VIII procoagulant activity, to amino acid residues threonine351-serine365 of the factor VIII heavy chain. J Clin Invest 1988; 82: 123-128.
• 22 Mimms LT, Zampighi G, Nozaki Y. et al. Phospholipid vesicle formation and transmembrane protein incorporation using octyl glucoside. Biochemistry 1981; 20: 833-840.
• 23 Takeyama M, Wakabayashi H, Fay PJ. Factor VIII light chain contains a binding site for factor X that contributes to the catalytic efficiency of factor Xase. Biochemistry 2012; 51: 820-828.
• 24 Wakabayashi H, Koszelak ME, Mastri M. et al. Metal ion-independent association of factor VIII subunits and the roles of calcium and copper ions for cofactor activity and inter-subunit affinity. Biochemistry 2001; 40: 10293-10300.
• 25 Lollar P, Fay PJ, Fass DN. Factor VIII and factor VIIIa. Methods Enzymol 1993; 222: 128-143.
• 26 Varfaj F, Wakabayashi H, Fay PJ. Residues surrounding Arg336 and Arg562 contribute to the disparate rates of proteolysis of factor VIIIa catalysed by activated protein C. J Biol Chem 2007; 282: 20264-20272.
• 27 Varfaj F, Neuberg J, Jenkins PV. et al. Role of P1 residues Arg336 and Arg562 in the activated-Protein-C-catalysed inactivation of Factor VIIIa. Biochem J 2006; 396: 355-362.
• 28 Ngo JC, Huang M, Roth DA. et al. Crystal structure of human factor VIII: implications for the formation of the factor IXa-factor VIIIa complex. Structure 2008; 16: 597-606.
• 29 Shen BW, Spiegel PC, Chang CH. et al. The tertiary structure and domain organisation of coagulation factor VIII. Blood 2008; 111: 1240-1247.
• 30 Mather T, Oganessyan V, Hof P. et al. The 2.8 A crystal structure of Gla-domainless activated protein C. EMBO J 1996; 15: 6822-6831.
• 31 Wakabayashi H, Griffiths AE, Fay PJ. Factor VIII lacking the C2 domain retains cofactor activity in vitro. J Biol Chem 2010; 285: 25176-25184.
• 32 Yegneswaran S, Wood GM, Esmon CT. et al. Protein S alters the active site location of activated protein C above the membrane surface. A fluorescence resonance energy transfer study of topography. J Biol Chem 1997; 272: 25013-25021.
• 33 McMullen BA, Fujikawa K, Davie EW. et al. Locations of disulfide bonds and free cysteines in the heavy and light chains of recombinant human factor VIII (antihemophilic factor A). Protein science 1995; 04: 740-746.
• 34 O’Brien LM, Mastri M, Fay PJ. Regulation of factor VIIIa by human activated protein C and protein S: inactivation of cofactor in the intrinsic factor Xase. Blood 2000; 95: 1714-1720.