Acidic Region Residues 1680–1684 in the A3 Domain of Factor VIII Contain a Thrombin-Interactive Site Responsible for Proteolytic Cleavage at Arg¹⁶⁸⁹

Abstract

Factor VIII (FVIII) is activated by thrombin-catalyzed cleavage at Arg³⁷², Arg⁷⁴⁰, and Arg¹⁶⁸⁹. Our previous studies suggested that thrombin interacted with the FVIII C2 domain specific for cleavage at Arg¹⁶⁸⁹. An alternative report demonstrated, however, that a recombinant (r)FVIII mutant lacking the C2 domain retained >50% cofactor activity, indicating the presence of other thrombin-interactive site(s) associated with cleavage at Arg¹⁶⁸⁹. We have focused, therefore, on the A3 acidic region of FVIII, similar to the hirugen sequence specific for thrombin interaction (54–65 residues). Two synthetic peptides, spanning residues 1659–1669 with sulfated Tyr¹⁶⁶⁴ and residues 1675–1685 with sulfated Try¹⁶⁸⁰, inhibited thrombin-catalyzed FVIII activation and cleavage at Arg¹⁶⁸⁹. Treatment with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide to cross-link thrombin with either peptide showed possible contributions of both 1664–1666 and 1683–1684 residues for thrombin interaction. Thrombin-catalyzed activation and cleavage at Arg¹⁶⁸⁹ in the alanine-substituted rFVIII mutants within 1663–1666 residues were similar to those of wild type (WT). Similar studies of 1680–1684 residues, however, demonstrated that activation and cleavage by thrombin of the FVIII mutant with Y1680A or D1683A/E1684A, in particular, were severely or moderately reduced to 20 to 30% or 60 to 70% of WT, respectively. Surface plasmon resonance-based analysis revealed that thrombin interacted with both Y1680A and D1683A/E1684A mutants with approximately sixfold weaker affinities of WT. Cleavage at Arg¹⁶⁸⁹ in the isolated light-chain fragments from both mutants was similarly depressed, independently of the heavy-chain subunit. In conclusion, the 1680–1684 residues containing sulfated Tyr¹⁶⁸⁰ in the A3 acidic region also contribute to a thrombin-interactive site responsible for FVIII activation through cleavage at Arg¹⁶⁸⁹.

Keywords

factor VIII - thrombin - A3 domain - recombinant - proteolysis

Note

An account of this work was presented at the ASH 2019 annual meeting and exposition, Orland, Florida, United States, December 9, 2019.

Authors' Contributions

Y.N. performed the experiments, analyzed the data, made the figures, and wrote the paper; H.M. performed the experiments; K.N. designed the research, interpreted the data, wrote the paper, edit the manuscript, and approved the final version to be published.

Publication History

Received: 27 August 2020

Accepted: 02 January 2021

Article published online:
16 February 2021

• References

• 1 Mann KG, Nesheim ME, Church WR, Haley P, Krishnaswamy S. Surface-dependent reactions of the vitamin K-dependent enzyme complexes. Blood 1990; 76 (01) 1-16
• 2 Wood WI, Capon DJ, Simonsen CC. et al. Expression of active human factor VIII from recombinant DNA clones. Nature 1984; 312 (5992): 330-337
• 3 Vehar GA, Keyt B, Eaton D. et al. Structure of human factor VIII. Nature 1984; 312 (5992): 337-342
• 4 Fay PJ, Anderson MT, Chavin SI, Marder VJ. The size of human factor VIII heterodimers and the effects produced by thrombin. Biochim Biophys Acta 1986; 871 (03) 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 (02) 505-512
• 6 Fay PJ, Mastri M, Koszelak ME, Wakabayashi H. Cleavage of factor VIII heavy chain is required for the functional interaction of a2 subunit with factor IXA. J Biol Chem 2001; 276 (15) 12434-12439
• 7 Donath MS, Lenting PJ, van Mourik JA, Mertens K. The role of cleavage of the light chain at positions Arg1689 or Arg1721 in subunit interaction and activation of human blood coagulation factor VIII. J Biol Chem 1995; 270 (08) 3648-3655
• 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 (15) 8546-8552
• 9 Fay PJ. Activation of factor VIII and mechanisms of cofactor action. Blood Rev 2004; 18 (01) 1-15
• 10 Binnie CG, Lord ST. The fibrinogen sequences that interact with thrombin. Blood 1993; 81 (12) 3186-3192
• 11 Rydel TJ, Ravichandran KG, Tulinsky A. et al. The structure of a complex of recombinant hirudin and human alpha-thrombin. Science 1990; 249 (4966): 277-280
• 12 Stone SR, Braun PJ, Hofsteenge J. Identification of regions of alpha-thrombin involved in its interaction with hirudin. Biochemistry 1987; 26 (15) 4617-4624
• 13 Sheehan JP, Sadler JE. Molecular mapping of the heparin-binding exosite of thrombin. Proc Natl Acad Sci U S A 1994; 91 (12) 5518-5522
• 14 Sheehan JP, Wu Q, Tollefsen DM, Sadler JE. Mutagenesis of thrombin selectively modulates inhibition by serpins heparin cofactor II and antithrombin III. Interaction with the anion-binding exosite determines heparin cofactor II specificity. J Biol Chem 1993; 268 (05) 3639-3645
• 15 Esmon CT, Lollar P. Involvement of thrombin anion-binding exosites 1 and 2 in the activation of factor V and factor VIII. J Biol Chem 1996; 271 (23) 13882-13887
• 16 Myles T, Yun TH, Hall SW, Leung LL. An extensive interaction interface between thrombin and factor V is required for factor V activation. J Biol Chem 2001; 276 (27) 25143-25149
• 17 Myles T, Yun TH, Leung LL. Structural requirements for the activation of human factor VIII by thrombin. Blood 2002; 100 (08) 2820-2826
• 18 Nogami K, Shima M, Hosokawa K. et al. Factor VIII C2 domain contains the thrombin-binding site responsible for thrombin-catalyzed cleavage at Arg1689. J Biol Chem 2000; 275 (33) 25774-25780
• 19 Nogami K, Zhou Q, Myles T, Leung LL, Wakabayashi H, Fay PJ. Exosite-interactive regions in the A1 and A2 domains of factor VIII facilitate thrombin-catalyzed cleavage of heavy chain. J Biol Chem 2005; 280 (18) 18476-18487
• 20 Nogami K, Saenko EL, Takeyama M, Giddings JC, Yoshioka A, Shima M. Identification of a thrombin-interactive site within the FVIII A2 domain that is responsible for the cleavage at Arg372. Br J Haematol 2008; 140 (04) 433-443
• 21 Minami H, Nogami K, Yada K, Shima M. Identification Of The thrombin-binding site on factor VIII regulating Arg³⁷² cleavage in the factor VIII heavy chain. Blood 2013; 122 (21) 3570
• 22 Wakabayashi H, Griffiths AE, Fay PJ. Factor VIII lacking the C2 domain retains cofactor activity in vitro. J Biol Chem 2010; 285 (33) 25176-25184
• 23 Michnick DA, Pittman DD, Wise RJ, Kaufman RJ. Identification of individual tyrosine sulfation sites within factor VIII required for optimal activity and efficient thrombin cleavage. J Biol Chem 1994; 269 (31) 20095-20102
• 24 Shima M, Scandella D, Yoshioka A. et al. A factor VIII neutralizing monoclonal antibody and a human inhibitor alloantibody recognizing epitopes in the C2 domain inhibit factor VIII binding to von Willebrand factor and to phosphatidylserine. Thromb Haemost 1993; 69 (03) 240-246
• 25 Nogami K, Ogiwara K, Matsumoto T, Nishiya K, Takeyama M, Shima M. Mechanisms of human neutrophil elastase-catalysed inactivation of factor VIII(a). Thromb Haemost 2011; 105 (06) 968-980
• 26 Foster PA, Fulcher CA, Houghten RA, de Graaf Mahoney S, Zimmerman TS. Localization 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 (01) 123-128
• 27 Okuda M, Yamamoto Y. Usefulness of synthetic phospholipid in measurement of activated partial thromboplastin time: a new preparation procedure to reduce batch difference. Clin Lab Haematol 2004; 26 (03) 215-223
• 28 Nogami K, Zhou Q, Wakabayashi H, Fay PJ. Thrombin-catalyzed activation of factor VIII with His substituted for Arg372 at the P1 site. Blood 2005; 105 (11) 4362-4368
• 29 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 (03) 820-828
• 30 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
• 31 Takeyama M, Nogami K, Matsumoto T, Noguchi-Sasaki M, Kitazawa T, Shima M. An anti-factor IXa/factor X bispecific antibody, emicizumab, improves ex vivo coagulant potentials in plasma from patients with acquired hemophilia A. J Thromb Haemost 2020; 18 (04) 825-833
• 32 Saenko EL, Scandella D. The acidic region of the factor VIII light chain and the C2 domain together form the high affinity binding site for von willebrand factor. J Biol Chem 1997; 272 (29) 18007-18014
• 33 Leyte A, van Schijndel HB, Niehrs C. et al. Sulfation of Tyr1680 of human blood coagulation factor VIII is essential for the interaction of factor VIII with von Willebrand factor. J Biol Chem 1991; 266 (02) 740-746
• 34 Newell JL, Fay PJ. Acidic residues C-terminal to the A2 domain facilitate thrombin-catalyzed activation of factor VIII. Biochemistry 2008; 47 (33) 8786-8795
• 35 Bihoreau N, Paolantonacci P, Bardelle C. et al. Structural and functional characterization of Factor VIII-delta II, a new recombinant Factor VIII lacking most of the B-domain. Biochem J 1991; 277 (Pt 1): 23-31
• 36 Donath MJ, de Laaf RT, Biessels PT. et al. Characterization of des-(741-1668)-factor VIII, a single-chain factor VIII variant with a fusion site susceptible to proteolysis by thrombin and factor Xa. Biochem J 1995; 312 (Pt 1): 49-55
• 37 Brown CM, Ray M, Eroy-Reveles AA, Egea P, Tajon C, Craik CS. Peptide length and leaving-group sterics influence potency of peptide phosphonate protease inhibitors. Chem Biol 2011; 18 (01) 48-57
• 38 Pipe SW, Kaufman RJ. Characterization of a genetically engineered inactivation-resistant coagulation factor VIIIa. Proc Natl Acad Sci U S A 1997; 94 (22) 11851-11856
• 39 Higuchi M, Wong C, Kochhan L. et al. Characterization of mutations in the factor VIII gene by direct sequencing of amplified genomic DNA. Genomics 1990; 6 (01) 65-71
• 40 Chiu PL, Bou-Assaf GM, Chhabra ES. et al. Mapping the interaction between factor VIII and von Willebrand factor by electron microscopy and mass spectrometry. Blood 2015; 126 (08) 935-938
• 41 Hill-Eubanks DC, Lollar P. von Willebrand factor is a cofactor for thrombin-catalyzed cleavage of the factor VIII light chain. J Biol Chem 1990; 265 (29) 17854-17858