Engineering FVIIa Variants with Enhanced Activity

The formation of a complex between factor VIIa (FVIIa) and tissue factor (TF) triggers the blood coagulation cascade in response to a vascular injury. TF has the dual biological function of localizing FVIIa to the site of injury and converting the latent enzyme into an efficient catalyst of factor VII, IX, and X activation. The hemostatic effect of recombinant FVIIa in hemophilia patients, where FVIIa is present at supraphysiological concentrations, is however largely mediated by factor X activation on the surface of activated platelets to which FVIIa binds with relatively low affinity. In this setting, it would be pharmacodynamically advantageous to dispose of FVIIa variants with enhanced intrinsic activity. Moreover, such variants might shed light on how TF induces maturation of the active conformation of FVIIa. Intuitively, more active variants of FVIIa could be designed by exploiting structural differences between FVIIa before and after binding to TF. To get the most out of this approach, the structures of free, uninhibited FVIIa and TF-bound FVIIa should be available for comparison. This is because an active-site inhibitor shifts FVIIa into an active conformation and locks it there, and conformational transitions in the same direction normally induced by TF binding may already have occurred upon inhibitor incorporation or be obscured or even prevented. Unfortunately, crystallization attempts with FVIIa without an active-site inhibitor have not been fruitful, neither with nor without TF. Nevertheless, a few structural clues have been identified, one of which was an attempted mimick by mutagenesis that led to the discovery of the modestly superactive L305V-FVIIa (1). The design and generation of more active FVIIa variants by exploiting sequence differences between FVIIa and homologous enzymes, whose active sites expedite catalytic events at a higher rate, has been more successful. The most striking outcome of this particular endeavour is V158D/E296V/M298Q-FVIIa (FVIIa_DVQ) (2, 3).In FVIIa_DVQ, three amino acid residues in FVIIa have been replaced by those occupying the corresponding positions in thrombin and factor IXa. Interestingly, the conformational changes induced by the three replacements have been shown to be a subset of those brought about by TF. It is fair to state that FVIIa_DVQ is a conformational intermediate between free and TF-bound FVIIa and that the molecule strives to imitate the latter. FVIIa_DVQ is dramatically more efficient in cleaving substrates in the absence of TF compared with FVIIa, whereas the specific activities of the two forms of FVIIa bound to TF are comparable. This has also been demonstrated on cellular representations of the two conditions, namely platelets and LPS-stimulated monocytes, respectively. The high activity of FVIIa_DVQ has also manifested itself as an increased potency and more rapid effect in murine bleeding models compared with FVIIa and in vitro normalization of human hemophilic blood clotting. An indication of an improved efficacy rate of FVIIa_DVQ compared with the efficient drug recombinant FVIIa was observed in a clinical trial (4).

1. Persson E, Bak H., Olsen O.H. Substitution of valine for leucine 305 in factor VIIa increases the intrinsic enzymatic activity. J Biol Chem.2001;276(31):29195-29199.

2. Persson E, Kjalke M., Olsen O.H. Rational design of coagulation factor VIIa variants with substantially increased intrinsic activity. Proc Natl Acad Sci USA.2001;98(24):13583-13588.

3. Persson E., Olsen O.H., Bjørn S.E., Ezban M. Vatreptacog alfa from conception to clinical proof of concept. Semin Thromb Hemost. 2012;38(3):274-281.

4. De Paula E.V., Kavakli K., Mahlangu J., et al. Recombinant factor VIIa analog (vatreptacog alfa [activated]) for treatment of joint bleeds in hemophilia patients with inhibitors: a randomized controlled trial. J Thromb Haemost. 2012;10(1):81-89.