Biochemical and functional characterization of recombinant von Willebrand factor produced on a large scale

Abstract

Recombinant von Willebrand factor (r-vWF) was produced in serum-free medium on a large scale in recombinant Chinese hamster ovary cells and was purified from fermentation supernatant by a combination of anion exchange chromatography and heparin affinity chromatography. Heparin affinity chromatography yielded r-vWF polymers of different degrees of multimerization. r-vWF was analysed by qualitative and quantitative functional analysis. We could show that while binding of r-vWF to platelets did not depend on multimerization of the molecule, ristocetin-induced platelet aggregation, binding to collagen and binding to heparin correlated directly with the extent of multimerization. Binding of recombinant coagulation factor VIII (r-FVIII) to r-vWF was studied by real-time biospecific interaction analysis and surface plasmon technology. The data indicated that binding of r-FVIII did not depend on r-vWF multimerization. Real-time biospecific interaction analysis suggested a potential stoichiometry of 2 to 2.5 r-vWF subunits per r-FVIII molecule. Kinetic analysis of the r-vWF-r-FVIII interaction gave a binding rate constant of 3 x 10(6) M-1 s-1 and an association constant of 2.5 x 10(9) M-1. Reaction of r-vWF with carbohydrate-specific lectins demonstrated that r-vWF contained a high proportion of N-glycans composed of mannose, galactose, glucose, N-acetylglucosamine and terminal sialic acid. Carbohydrate moities were covalently bound to the protein structure and were quantitatively removed from r-vWF only after protein denaturation. The results demonstrated that r-vWF produced on large scale under serum-free culture conditions exhibited qualitative and quantitative functional properties comparable to human plasma-derived vWF.

A novel von Willebrand factor binding protein expressed by Staphylococcus aureus a aThe GenBank accession number for the sequence reported in this paper is AY032850.

“…Recombinant vWf was a kind gift from Professor F. Dorner, Vienna, Austria. Recombinant vWf has been shown to mediate platelet aggregation and to promote binding of collagen and coagulation factor VIII with activity comparable to human-plasma-derived vWf (Fischer et al, 1997). Goat anti-vWf antibodies were purchased from Kordia ; antibodies against recombinant vWbp (see below) developed in chicken were obtained from Immunsystem AB ; mouse anti-E-tag antibodies and horseradish peroxidase (HRP)-labelled anti-mouse antibodies were from Amersham Biosciences.…”

Structure and function of von Willebrand factor

“…1d) Biological functions VWF performs a large number of functions in the human body [14][15][16]71]; binding to factor VIII and platelet surface glycoprotein is prominent among them [6,72,73]. The binding of VWF to platelets is regulated by its initial interaction with connective tissue and also by shear stress in flowing blood.…”

Binding of von Willebrand factor by coagulase-negative staphylococci

“…rvWF is composed of mature subunits [13]. Fischer et al showed that rvWF produced on a large scale under serum-free culture conditions exhibits all the qualitative and quantitative functional properties which allow it to mediate platelet aggregation, promote collagen binding and binding of coagulation factor VIII with activity comparable to human plasma-derived vWF [14]. Herrmann et al also observed similar adhesion promotion for S. aureus when recombinant vWF was used [12].…”