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von W illebrand factor contributes to longer half‐life of PEGylated factor VIII in vivo
Author(s) -
Tang L.,
Leong L.,
Sim D.,
Ho E.,
Gu J.M.,
Schneider D.,
Feldman R. I.,
Monteclaro F.,
Jiang H.,
Murphy J. E.
Publication year - 2013
Publication title -
haemophilia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.213
H-Index - 92
eISSN - 1365-2516
pISSN - 1351-8216
DOI - 10.1111/hae.12116
Subject(s) - pegylation , in vivo , von willebrand factor , peg ratio , chemistry , von willebrand disease , recombinant dna , in vitro , microbiology and biotechnology , pharmacology , biochemistry , polyethylene glycol , medicine , immunology , platelet , biology , finance , gene , economics
Summary 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 (v WF ) 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 (T 1/2 ) of F8V was dramatically reduced relative to FVIII‐ BDD (0.6 h vs. 6.03 h). PEGylation of F8V promoted a significant increase in T 1/2 , although PEGylation did not fully compensate for the loss in vWF binding. PEG ‐F8V showed a shorter T 1/2 than PEG ‐FVIII‐ BDD both in HaemA mice (7.7 h vs. 14.3 h) and in S prague‐ D awley male rats (2.0 ± 0.3 h vs. 6.0 ± 0.5 h). These data demonstrated that vWF contributes to the longer T 1/2 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.