Hydrogen–Deuterium Exchange Mass Spectrometry Identifies Activated Factor IX-Induced molecular Changes in Activated Factor VIII

Abstract

Hydrogen–deuterium exchange mass spectrometry (HDX-MS) was employed to gain insight into the changes in factor VIII (FVIII) that occur upon its activation and assembly with activated factor IX (FIXa) on phospholipid membranes. HDX-MS analysis of thrombin-activated FVIII (FVIIIa) revealed a marked increase in deuterium incorporation of amino acid residues along the A1–A2 and A2–A3 interface. Rapid dissociation of the A2 domain from FVIIIa can explain this observation. In the presence of FIXa, enhanced deuterium incorporation at the interface of FVIIIa was similar to that of FVIII. This is compatible with the previous finding that FIXa contributes to A2 domain retention in FVIIIa. A2 domain region Leu631-Tyr637, which is not part of the interface between the A domains, also showed a marked increase in deuterium incorporation in FVIIIa compared with FVIII. Deuterium uptake of this region was decreased in the presence of FIXa beyond that observed in FVIII. This implies that FIXa alters the conformation or directly interacts with this region in FVIIIa. Replacement of Val634 in FVIII by alanine using site-directed mutagenesis almost completely impaired the ability of the activated cofactor to enhance the activity of FIXa. Surface plasmon resonance analysis revealed that the rates of A2 domain dissociation from FVIIIa and FVIIIa-Val634Ala were indistinguishable. HDX-MS analysis showed, however, that FIXa was unable to retain the A2 domain in FVIIIa-Val634Ala. The combined results of this study suggest that the local structure of Leu631-Tyr637 is altered by FIXa and that this region contributes to the cofactor function of FVIII.

Keywords

hydrogen–deuterium exchange - factor VIII - factor IXa - A2 domain retention - interactive regions

Authors' Contributions

J.v.G., N.F., M.B.-S, and M.A.P. performed the experiments; C.v.d.Z. provided technical assistance; J.v.G., N.F., M.v.d.B., and A.B.M. designed the experiments; E.E. assisted with setting up of HDX-MS experiments; J.v.G., N.F., M.A.P., and M.v.d.B. analyzed the results; N.F. and J.v.G. made the figures; and J.v.G., N.F., M.v.d.B., and A.B.M. wrote the article.

^* Both authors contributed equally to this work.

Publication History

Received: 24 June 2020

Accepted: 21 October 2020

Article published online:
10 December 2020

• References

• 1 Fay PJ. Activation of factor VIII and mechanisms of cofactor action. Blood Rev 2004; 18 (01) 1-15
• 2 Mannucci PM, Tuddenham EG. The hemophilias--from royal genes to gene therapy. N Engl J Med 2001; 344 (23) 1773-1779
• 3 Vehar GA, Keyt B, Eaton D. et al. Structure of human factor VIII. Nature 1984; 312 (5992): 337-342
• 4 Lenting PJ, van Mourik JA, Mertens K. The life cycle of coagulation factor VIII in view of its structure and function. Blood 1998; 92 (11) 3983-3996
• 5 Kamikubo Y, Mendolicchio GL, Zampolli A. et al. Selective factor VIII activation by the tissue factor-factor VIIa-factor Xa complex. Blood 2017; 130 (14) 1661-1670
• 6 Fay PJ. Factor VIII structure and function. Int J Hematol 2006; 83 (02) 103-108
• 7 van Dieijen G, Tans G, Rosing J, Hemker HC. The role of phospholipid and factor VIIIa in the activation of bovine factor X. J Biol Chem 1981; 256 (07) 3433-3442
• 8 Fay PJ. Subunit structure of thrombin-activated human factor VIIIa. Biochim Biophys Acta 1988; 952 (02) 181-190
• 9 Lollar P, Parker CG. pH-dependent denaturation of thrombin-activated porcine factor VIII. J Biol Chem 1990; 265 (03) 1688-1692
• 10 Lollar P, Parker ET. Structural basis for the decreased procoagulant activity of human factor VIII compared to the porcine homolog. J Biol Chem 1991; 266 (19) 12481-12486
• 11 Ngo JC, Huang M, Roth DA, Furie BC, Furie B. Crystal structure of human factor VIII: implications for the formation of the factor IXa-factor VIIIa complex. Structure 2008; 16 (04) 597-606
• 12 Stoilova-McPhie S, Villoutreix BO, Mertens K, Kemball-Cook G, Holzenburg A. 3-Dimensional structure of membrane-bound coagulation factor VIII: modeling of the factor VIII heterodimer within a 3-dimensional density map derived by electron crystallography. Blood 2002; 99 (04) 1215-1223
• 13 Shen BW, Spiegel PC, Chang CH. et al. The tertiary structure and domain organization of coagulation factor VIII. Blood 2008; 111 (03) 1240-1247
• 14 Meems H, Meijer AB, Cullinan DB, Mertens K, Gilbert GE. Factor VIII C1 domain residues Lys 2092 and Phe 2093 contribute to membrane binding and cofactor activity. Blood 2009; 114 (18) 3938-3946
• 15 Gilbert GE, Kaufman RJ, Arena AA, Miao H, Pipe SW. Four hydrophobic amino acids of the factor VIII C2 domain are constituents of both the membrane-binding and von Willebrand factor-binding motifs. J Biol Chem 2002; 277 (08) 6374-6381
• 16 Bajaj SP, Schmidt AE, Mathur A. et al. Factor IXa:factor VIIIa interaction. helix 330-338 of factor ixa interacts with residues 558-565 and spatially adjacent regions of the a2 subunit of factor VIIIa. J Biol Chem 2001; 276 (19) 16302-16309
• 17 Fay PJ, Beattie T, Huggins CF, Regan LM. Factor VIIIa A2 subunit residues 558-565 represent a factor IXa interactive site. J Biol Chem 1994; 269 (32) 20522-20527
• 18 Pantazatos D, Gessner CR, Woods Jr VL, Gilbert GE. Changes in the factor VIII C2 domain upon membrane binding determined by hydrogen-deuterium exchange MS. Biochem J 2014; 461 (03) 443-451
• 19 Bloem E, van den Biggelaar M, Wroblewska A. et al. Factor VIII C1 domain spikes 2092-2093 and 2158-2159 comprise regions that modulate cofactor function and cellular uptake. J Biol Chem 2013; 288 (41) 29670-29679
• 20 Przeradzka MA, van Galen J, Ebberink EHTM. et al. D' domain region Arg782-Cys799 of von Willebrand factor contributes to factor VIII binding. Haematologica 2020; 105 (06) 1695-1703
• 21 van den Biggelaar M, Madsen JJ, Faber JH. et al. Factor VIII interacts with the endocytic receptor low-density lipoprotein receptor-related protein 1 via an extended surface comprising “hot-spot” lysine residues. J Biol Chem 2015; 290 (27) 16463-16476
• 22 Song H, Olsen OH, Persson E, Rand KD. Sites involved in intra- and interdomain allostery associated with the activation of factor VIIa pinpointed by hydrogen-deuterium exchange and electron transfer dissociation mass spectrometry. J Biol Chem 2014; 289 (51) 35388-35396
• 23 Hendrix H, Lindhout T, Mertens K, Engels W, Hemker HC. Activation of human prothrombin by stoichiometric levels of staphylocoagulase. J Biol Chem 1983; 258 (06) 3637-3644
• 24 Mertens K, Bertina RM. Activation of human coagulation factor VIII by activated factor X, the common product of the intrinsic and the extrinsic pathway of blood coagulation. Thromb Haemost 1982; 47 (02) 96-100
• 25 Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72: 248-254
• 26 Lenting PJ, ter Maat H, Clijsters PP, Donath MJ, van Mourik JA, Mertens K. Cleavage at arginine 145 in human blood coagulation factor IX converts the zymogen into a factor VIII binding enzyme. J Biol Chem 1995; 270 (25) 14884-14890
• 27 Foster PA, Fulcher CA, Marti T, Titani K, Zimmerman TS. A major factor VIII binding domain resides within the amino-terminal 272 amino acid residues of von Willebrand factor. J Biol Chem 1987; 262 (18) 8443-8446
• 28 Meems H, van den Biggelaar M, Rondaij M, van der Zwaan C, Mertens K, Meijer AB. C1 domain residues Lys 2092 and Phe 2093 are of major importance for the endocytic uptake of coagulation factor VIII. Int J Biochem Cell Biol 2011; 43 (08) 1114-1121
• 29 van den Biggelaar M, Bierings R, Storm G, Voorberg J, Mertens K. Requirements for cellular co-trafficking of factor VIII and von Willebrand factor to Weibel-Palade bodies. J Thromb Haemost 2007; 5 (11) 2235-2242
• 30 Rouser G, Siakotos AN, Fleischer S. Quantitative analysis of phospholipids by thin-layer chromatography and phosphorus analysis of spots. Lipids 1966; 1 (01) 85-86
• 31 Bloem E, Meems H, van den Biggelaar M, Mertens K, Meijer AB. A3 domain region 1803-1818 contributes to the stability of activated factor VIII and includes a binding site for activated factor IX. J Biol Chem 2013; 288 (36) 26105-26111
• 32 McGinniss MJ, Kazazian Jr HH, Hoyer LW, Bi L, Inaba H, Antonarakis SE. Spectrum of mutations in CRM-positive and CRM-reduced hemophilia A. Genomics 1993; 15 (02) 392-398
• 33 Ahmed RPH, Ivaskevicius V, Kannan M, Seifried E, Oldenburg J, Saxena R. Identification of 32 novel mutations in the factor VIII gene in Indian patients with hemophilia A. Haematologica 2005; 90 (02) 283-284
• 34 Bloem E, Meems H, van den Biggelaar M, van der Zwaan C, Mertens K, Meijer AB. Mass spectrometry-assisted study reveals that lysine residues 1967 and 1968 have opposite contribution to stability of activated factor VIII. J Biol Chem 2012; 287 (08) 5775-5783
• 35 Fay PJ, Jenkins PV. Mutating factor VIII: lessons from structure to function. Blood Rev 2005; 19 (01) 15-27
• 36 Mertens K, Celie PH, Kolkman JA, Lenting PJ. Factor VIII-factor IX interactions: molecular sites involved in enzyme-cofactor complex assembly. Thromb Haemost 1999; 82 (02) 209-217
• 37 Healey JF, Lubin IM, Nakai H. et al. Residues 484-508 contain a major determinant of the inhibitory epitope in the A2 domain of human factor VIII. J Biol Chem 1995; 270 (24) 14505-14509
• 38 Jenkins PV, Dill JL, Zhou Q, Fay PJ. Clustered basic residues within segment 484-510 of the factor VIIIa A2 subunit contribute to the catalytic efficiency for factor Xa generation. J Thromb Haemost 2004; 2 (03) 452-458
• 39 Soeda T, Nogami K, Nishiya K. et al. The factor VIIIa C2 domain (residues 2228-2240) interacts with the factor IXa Gla domain in the factor Xase complex. J Biol Chem 2009; 284 (06) 3379-3388
• 40 Bovenschen N, Boertjes RC, van Stempvoort G. et al. Low density lipoprotein receptor-related protein and factor IXa share structural requirements for binding to the A3 domain of coagulation factor VIII. J Biol Chem 2003; 278 (11) 9370-9377