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Biological activity of recombinant factor VIII variants lacking the central B-domain and the heavy-chain sequence Lys713-Arg740: discordant in vitro and in vivo activity.

Abstract：

:Recombinant factor VIII variants with overlapping deletions spanning the region Lys713-Ile1668 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 (Ser741-Arg1648) resulted in a protein with similar procoagulant and cofactor activity. In contrast, factor VIII-del(713-1637), which has a deletion that also comprises the heavy-chain sequence Lys713-Arg740, 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 thrombin-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 Lys713-Arg740, although dispensable for factor VIII cofactor function per se, is involved in the proteolytic activation of factor VIII both in vitro and in vivo.

journal_name

Br J Haematol

journal_title

British journal of haematology

authors

Mertens K,Donath MJ,van Leen RW,de Keyzer-Nellen MJ,Verbeet MP,Klaasse Bos JM,Leyte A,van Mourik JA

doi

10.1111/j.1365-2141.1993.tb08656.x

subject

Has Abstract

pub_date

1993-09-01 00:00:00

pages

133-42

issue

eissn

0007-1048

issn

1365-2141

journal_volume

pub_type

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Biological activity of recombinant factor VIII variants lacking the central B-domain and the heavy-chain sequence Lys713-Arg740: discordant in vitro and in vivo activity.