The chromogenic assay used to monitor the potency of factor VIII does not consistently give the same result as a plasma substrate-based test. The standard measurement of factor VIII coagulant activity at the FDA employs the plasma substrate assay, while the European Pharmacopoeia mandates a chromogenic test. Discrepancies between these two tests affect product fill and patient care. To understand and resolve the disparities between the tests, we are evaluating molecular parameters that influence the assays. Strategy: 1) Examine the contact residues for factor VIII on the N-terminal domain of vWF, and assess the role they play in binding and releasing FVIII from vWF. 2) Study the kinetic interaction of commercial factor VIII products with von Willebrand factor (vWF), factors IXa and X, thrombin, and phospholipid. Surface plasmon resonance (SPR), measured by BIA core, is employed to assess whether specific vWF peptides affect different phases of interaction between vWF and FVIII products. The method is also used to investigate the interaction of commercial factor VIII products, with other coagulation proteins, in the presence or absence of vWF. Preliminary findings: 1) An Escherichia coli expression system was selected to be evaluated for expression of the vWF gene. This system offered the potential of easy manipulation and high protein yield. High-level expression of soluble recombinant vWF (rvWF) proteins were obtained from the pET E. coli system. Two major polypeptide fragments of rvWF, 32 Kd and 49 Kd, were isolated and purified. Kinetic studies of these fragments with factor VIII are on-going. 2) Several synthetic peptides contained within the N-terminal domain of vWF were generated to probe the precise contact regions of vWF with factor VIII. The interaction of a commercial recombinant factor VIII (rFVIII) (Recombinate) with immobilized vWF was inhibited by vWF N-terminal domain residues 14-29, suggesting that this region of vWF may be important in binding factor VIII. In contrast, the interaction of Recombinate with vWF was not inhibited by vWF residues 66-82. Under study is whether these peptides can inhibit vWF interaction with other factor VIII products, e.g. B-domainless FVIII and plasma-derived FVIII. 3) The kinetic binding properties of three commercial rFVIII products to vWF was examined. Two of the rFVIII products contained B domain sequences; the third had none. rFVIII that lacked the B domain bound significantly differently from the other products. The dissociation constant (KD) of the B-domainless product was 2.25x10-10 M, compared to 9.0x10-10 M, and 9.26x10-10 M for the other two. Thus, not all rFVIII products have the same kinetic interactions with vWF, and interactions with vWF may be modulated by the B domain of FVIII. We are currently investigating whether the B-domain can modulate the susceptibility of FVIII to thrombin cleavage.
