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Biological activity of recombinant factor VIII variants lacking the central B‐domain and the heavy‐chain sequence Lys 713 ‐Arg 740 : discordant in vitro and in vivo activity
Author(s) -
Mertens K.,
Donath M. J. S. H.,
Leen R. W. van,
KeyzerNellen M. J. M. de,
Verbeet M. Ph.,
Bos J. M. Klaasse,
Leyte A.,
Mourik J. A. van
Publication year - 1993
Publication title -
british journal of haematology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.907
H-Index - 186
eISSN - 1365-2141
pISSN - 0007-1048
DOI - 10.1111/j.1365-2141.1993.tb08656.x
Subject(s) - factor ixa , factor x , factor ix , in vitro , recombinant dna , von willebrand factor , in vivo , microbiology and biotechnology , factor vii , chemistry , coagulation , biological activity , tissue factor , factor v , cofactor , mutant , biochemistry , biology , thrombin , medicine , platelet , immunology , genetics , enzyme , gene , thrombosis
Summary. Recombinant factor VIII variants with overlapping deletions spanning the region Lys 713 ‐Ile 1668 have been expressed in mammalian cells, and analysed for biological activity both in vitro and in vivo . Two distinct assay systems were used to measure the activity in vitro . The one‐stage coagulation assay served to assess factor VIII procoagulant activity while a spectrophotometric assay was used for the quantification of factor VIII cofactor activity in factor IXa‐dependent factor X activation. Deletion of the entire B‐domain (Ser 741 ‐Arg 1648 ) resulted in a protein with similar procoagulant and cofactor activity. In contrast, factor VIII‐del(713–163 7), which has a deletion that also comprises the heavy‐chain sequence Lys 713 ‐Arg 740 , had lost factor VIII procoagulant activity while factor VIII cofactor activity was retained. This functional inconsistency was further addressed by comparing purified factor VIII‐del(713–1637) with factor VIII‐del(868–1562). a mutant with normal in vitro activity. Kinetic studies of factor Xa formation revealed that higher concentrations of thrombin were required to develop the cofactor activity from factor VIII‐del(713–1637) than needed for factor VIII‐del(868–1562) or plasma factor VIII. The physiological significance of this finding was assessed in dogs with haemophilia A. Both deletion mutants were similar to plasma factor VIII with regard to binding to von Willebrand factor and half‐life and recovery. Employing the cuticle bleeding time model, factor VIII‐del(868–1562) was found to be indistinguishable from plasma factor VIII, whereas factor VIII‐del(713–1637) was less effective. The increased throm‐bin‐resistance of factor VIII‐del(713–1637) thus limits both procoagulant activity and haemostatic efficacy in cuticle bleeding. These observations suggest that the heavy‐chain sequence Lys 713 ‐Arg 740 , although dispensable for factor VIII cofactor function per se , is involved in the proteolytic activation of factor VIII both in vitro and in vivo .