Characterisation of an antibody specific for coagulation factor VIII that enhances factor VIII activity

 

 Buy Article Permissions and Reprints

Summary

Many reports have identified factor (F)VIII inhibitory antibodies with epitopes located in all subunits of the FVIII molecule. Antibodies that promote FVIII activity do not appear to have been reported. We characterised, for the first time, a unique anti-FVIII monoclonal antibody, mAb216, that enhanced FVIII coagulant activity. The mAb216 shortened the activated partial thromboplastin time and specifically increased FVIII activity by ~1.5-fold dose-dependently. FXa generation and thrombin generation were similarly increased by ~1.4- and ~2.5-fold, respectively. An A2 epitope, not overlapping the common A2 epitope, was identified and the antibody was shown to enhance thrombin (and FXa)-catalysed activation of FVIII by modestly accelerating cleavage at Arg³⁷². The presence of mAb216 mediated an ~1.5-fold decrease in Km for the FVIII-thrombin interaction. Enhanced FVIII activity was evident to an equal degree, even the presence of anti-FVIII neutralising antibodies with epitopes in each subunit. In addition, mAb216 depressed the rates of heat-denatured loss of FVIII activity and FVIIIa decay by 2 to ~2.5-fold. We have developed an anti-A2, FVIII mAb216 that augmented procoagulant activity. This enhancing effect could be attributed to an increase in thrombin-induced activation of FVIII, mediated by cleavage at Arg³⁷² and a tighter interaction of thrombin with the A2 domain. The findings may cast new light on new principles for improving the treatment of haemophilia A patients.

• 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.
  PubMedGoogle Scholar
• 2 Wood WI, Capon DJ, Simonsen CC. et al. Expression of active human factor VIII from recombinant DNA clones. Nature 1984; 312: 330-337.
  CrossrefPubMedGoogle Scholar
• 3 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 coagulation activity. Biochemistry 1986; 25: 505-512.
  CrossrefPubMedGoogle Scholar
• 4 Fay PJ. Activation of factor VIII and mechanisms of cofactor action. Blood Rev 2004; 18: 1-15.
  CrossrefPubMedGoogle Scholar
• 5 Shima M. Characterization of factor VIII inhibitors. Int J Hematol 2006; 83: 109-118.
  CrossrefPubMedGoogle Scholar
• 6 Saenko EL, Shima M, Rajalakshmi KJ. et al. A role for the C2 domain of factor VIII in binding to von Willebrand factor. J Biol Chem 1994; 269: 11601-11605.
  PubMedGoogle Scholar
• 7 Scandella D, Gilbert GE, Shima M. et al. Some factor VIII inhibitor antibodies recognize a common epitope corresponding to C2 domain amino acids 2248 through 2312, which overlap a phospholipid-binding site. Blood 1995; 86: 1811-1819.
  PubMedGoogle Scholar
• 8 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: 25774-25780.
  CrossrefPubMedGoogle Scholar
• 9 Nogami K, Shima M, Hosokawa K. et al. Role of factor VIII C2 domain in factor VIII binding to factor Xa. J Biol Chem 1999; 274: 31000-31007.
  CrossrefPubMedGoogle Scholar
• 10 Fay PJ, Scandella D. Human inhibitor antibodies specific for the factor VIII A2 domain disrupt the interaction between the subunit and factor IXa. J Biol Chem 1999; 274: 29826-29830.
  CrossrefPubMedGoogle Scholar
• 11 Zhong D, Saenko EL, Shima M. et al. Some human inhibitor antibodies interfere with factor VIII binding to factor IX. Blood 1998; 92: 136-142.
  PubMedGoogle Scholar
• 12 Nogami K, Lapan KA, Zhou Q. et al. Identification of a factor Xa-interactive site within residues 337–372 of the factor VIII heavy chain. J Biol Chem 2004; 279: 15763-15771.
  CrossrefPubMedGoogle Scholar
• 13 Mimms LT, Zampighi G, Nozaki Y. et al. Phospholipid vesicle formation and transmembrane protein incorporation using octyl glucoside. Biochemistry 1981; 20: 833-840.
  CrossrefPubMedGoogle Scholar
• 14 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: 240-246.
  Article in Thieme ConnectPubMedGoogle Scholar
• 15 Matsumoto T, Shima M, Takeyama M. et al. The measurement of low levels of factor VIII or factor IX in hemophilia A and hemophilia B plasma by clot waveform analysis and thrombin generation assay. J Thromb Haemost 2006; 04: 377-384.
  CrossrefPubMedGoogle Scholar
• 16 Lollar P, Fay PJ, Fass DN. Factor VIII and factor VIIIa. Methods Enzymol 1993; 222: 128-143.
  PubMedGoogle Scholar
• 17 Hemker HC, Giesen P, Al Dieri R. et al. Calibrated automated thrombin generation measurement in clotting plasma. Pathophysiol Haemost Thromb 2003; 33: 4-15.
  CrossrefPubMedGoogle Scholar
• 18 Dargaud Y, Béguin S, Lienhart A. et al. Evaluation of thrombin generating capacity in plasma from patients with haemophilia A and B. Thromb Haemost 2005; 93: 475-480.
  Article in Thieme ConnectPubMedGoogle Scholar
• 19 Wakabayashi H, Varfaj F, Deangelis J. et al. Generation of enhanced stability factor VIII variants by replacement of charged residues at the A2 domain interface. Blood 2008; 112: 2761-2769.
  CrossrefPubMedGoogle Scholar
• 20 Ansong C, Fay PJ. Factor VIII A3 domain residues 1954–1961 represent an A1 domain-interactive site. Biochemistry 2005; 44: 8850-8857.
  CrossrefPubMedGoogle Scholar
• 21 Fay PJ, Haidaris PJ, Smudzin TM. Human factor VIIIa subunit structure. Reconstitution of factor VIIIa from the isolated A1/A3-C1-C2 dimer and A2 subunit. J Biol Chem 1991; 266: 8957-8962.
  PubMedGoogle Scholar
• 22 Blanco AN, Peirano AA, Grosso SH. et al. An ELISA system to detect anti-factor VIII antibodies without interference by lupus anticoagulants. Preliminary data in hemophilia A patients. Haematologica 2000; 85: 1045-1050.
  PubMedGoogle Scholar
• 23 Lacroix-Desmazes S, Moreau A. Sooryanarayana. et al. Catalytic activity of antibodies against factor VIII in patients with hemophilia A. Nat Med 1999; 05: 1044-1047.
  CrossrefPubMedGoogle Scholar
• 24 Kerschbaumer RJ, Riedrich K, Kral M. et al. An antibody specific for coagulation factor IX enhances the activity of the intrinsic factor X-activating complex. J Biol Chem 2004; 279: 40445-40450.
  CrossrefPubMedGoogle Scholar
• 25 Nogami K, Saenko EL, Takeyama M. et al. Identification of a thrombin-interactive site within the FVIII A2 domain that is responsible for the cleavage at Arg372. Br J Haematol 2008; 140: 433-443.
  CrossrefPubMedGoogle Scholar
• 26 Nogami K, Zhou Q, Myles T. et al. Exosite-interactive regions in the A1 and A2 do-mains of factor VIII facilitate thrombin-catalyzed cleavage of heavy chain. J Biol Chem 2005; 280: 18476-18487.
  CrossrefPubMedGoogle Scholar