title: von Willebrand factor contributes to longer half-life of PEGylated factor VIII in vivo.
abstract: PEGylation of B-domain deleted factor VIII (PEG-FVIII-BDD) prolongs the half-life of the molecule by approximately twofold in animals (Mei et al., Blood 2010; 116: 270). To investigate the role of von Willebrand factor (vWF) in the catabolism of PEG-FVIII-BDD in vivo, a FVIII-BDD mutant (F8V), which is incapable of binding vWF, was generated by deleting the vWF-binding region in the a3 domain of FVIIII-BDD. F8V was expressed, purified and PEGylated by site-specific conjugation. The biochemical and biological properties of F8V and PEGylated F8V (PEG-F8V) were evaluated in vitro and in vivo. The specific activity of purified F8V by a chromogenic assay was similar to FVIII-BDD and PEGylation had minimal impact on the specific activity of F8V in this assay. Analysis by Biacore indicated that both F8V and PEG-F8V display greatly reduced vWF binding in vitro. Pharmacokinetic studies in FVIII knockout (HaemA) mice showed that the terminal half-life (T1/2 ) of F8V was dramatically reduced relative to FVIII-BDD (0.6 h vs. 6.03 h). PEGylation of F8V promoted a significant increase in T1/2 , although PEGylation did not fully compensate for the loss in vWF binding. PEG-F8V showed a shorter T1/2 than PEG-FVIII-BDD both in HaemA mice (7.7 h vs. 14.3 h) and in Sprague-Dawley male rats (2.0 ± 0.3 h vs. 6.0 ± 0.5 h). These data demonstrated that vWF contributes to the longer T1/2 of PEG-FVIII-BDD. Furthermore, this suggests that the clearance of the FVIII:vWF complex, through vWF receptors, is not the sole factor which places an upper limit on the duration of PEG-FVIII circulation in plasma.
authors.affiliation: Biologics Research, Bayer HealthCare Pharmaceuticals, San Francisco, CA 94158, USA. liang.tang@bayer.com
authors.forename: L
authors.initials: L
authors.lastname: Tang
authors.forename: L
authors.initials: L
authors.lastname: Leong
authors.forename: D
authors.initials: D
authors.lastname: Sim
authors.forename: E
authors.initials: E
authors.lastname: Ho
authors.forename: J-M
authors.initials: JM
authors.lastname: Gu
authors.forename: D
authors.initials: D
authors.lastname: Schneider
authors.forename: R I
authors.initials: RI
authors.lastname: Feldman
authors.forename: F
authors.initials: F
authors.lastname: Monteclaro
authors.forename: H
authors.initials: H
authors.lastname: Jiang
authors.forename: J E
authors.initials: JE
authors.lastname: Murphy
mesh_terms: D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D005169:Factor VIII / Q000302:isolation & purification / Q000378:metabolism* / Q000493:pharmacokinetics; D006207:Half-Life; D006801:Humans; D051379:Mice; D011092:Polyethylene Glycols / Q000378:metabolism* / Q000493:pharmacokinetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D017207:Rats, Sprague-Dawley; D017384:Sequence Deletion; D013379:Substrate Specificity; D014841:von Willebrand Factor / Q000378:metabolism*
chemical_list: D014841:von Willebrand Factor; D011092:Polyethylene Glycols; D005169:Factor VIII