Intravascular inhibition of factor VIIa and the analogue NN1731 by antithrombin

N1731 is a recombinant activated factor VII (rFVIIa) analogue with increased intrinsic activity. This also applies to its reactivity towards antithrombin (AT), the role of which was investigated in a pharmacokinetic (PK) study. NN1731 or rFVIIa was administered to normal and haemophilia A dogs and elimination was measured by FVIIa clot activity, FVIIa- and FVIIa-AT antigen. In vitro AT complex formation was studied in canine plasma spiked with NN1731 or rFVIIa. Based on FVIIa antigen concentrations, PK profiles in normal and haemophilia A dogs were similar for NN1731 and rFVIIa with antigen half lives, t½ ≈ 1·8 h. In contrast, PK profiles based on activity measurements were distinctly different. NN1731 induced a strong, short lasting (t½ ≈ 0·5 h) pro-coagulant response, whereas rFVIIa induced a lower, longer lasting (t½ ≈ 1·1 h) response. Western Blot and FVIIa-AT antigen analysis demonstrated in vivo AT complex formation that accounted for these divergences. AT complex formation with FVIIa or NN1731 in vitro in canine plasma was considerably slower than the in vivo reaction. The results suggest that in vivo inhibition by AT contributes significantly to define drug duration in haemophilia treatment with rFVIIa and in particular with the NN1731 analogue

resonance energy transfer and quenching.

Effects on the conformation of FVIIa by sT

A loop of coagulation factor VIIa influencing macromolecular substrate specificity.

AbstractCoagulation factor VIIa (FVIIa) belongs to a family of proteases being part of the stepwise, self-amplifying blood coagulation cascade. To investigate the impact of the mutation Met298{156}Lys in FVIIa, we replaced the Gly283{140}–Met298{156} loop with the corresponding loop of factor Xa. The resulting variant exhibited increased intrinsic activity, concurrent with maturation of the active site, a less accessible N-terminus, and, interestingly, an altered macromolecular substrate specificity reflected in an increased ability to cleave factor IX (FIX) and a decreased rate of FX activation compared to that of wild-type FVIIa. In complex with tissue factor, activation of FIX, but not of FX, returned to normal. Deconvolution of the loop graft in order to identify important side chain substitutions resulted in the mutant Val158{21}Asp/Leu287{144}Thr/Ala294{152}Ser/Glu296{154} Ile/Met298{156}Lys-FVIIa with almost the same activity and specificity profile. We conclude that a lysine residue in position 298{156} of FVIIa requires a hydrophilic environment to be fully accommodated. This position appears critical for substrate specificity among the proteases of the blood coagulation cascade due to its prominent position in the macromolecular exosite and possibly via its interaction with the corresponding position in the substrate (i.e. FIX or FX)