The short half-life of coagulation factor VIIa (FVIIa) in circulation is the result of elimination through multiple pathways of which inactivation by the plasma inhibitor antithrombin (AT) accounts for as much as 65% of the total clearance in humans. Remarkably, the rate of inhibition in vivo is about 30 times greater than the uncatalyzed rate of inhibition in vitro suggesting the presence of rate enhancing components in vivo (Agerso et al. (2011) J Thromb Haemost, 9:333-338). Prime candidates include endogenous heparin-like glycosaminoglycans (GAGs) potentiating the reactivity of antithrombin, or tissue factor (TF) which upon binding to FVIIa increases its susceptibility to inhibition. In the present study site-directed mutagenesis of FVIIa was undertaken to identify variants with altered AT reactivity in order to explore the relationship between the reactivity of FVIIa with AT in vitro and in vivo as well as the nature of endogenous rate enhancing components. The pharmacokinetic properties of FVIIa variants were determined in Sprague Dawley rats as this model recapitulates the aspects of AT-mediated FVIIa clearance observed in humans and allows for interaction of human FVIIa with endogenous rat TF. Similar to the human situation, inactivation of wild-type FVIIa in rat is evident as an accumulation of circulating FVIIa-AT complexes and a progressive divergence of the pharmacokinetic profiles representing FVIIa clot activity and total FVIIa antigen. Initially, the ability to modulate the in vivo complex formation with AT was investigated using two FVIIa variants exhibiting enhanced (>200%) or reduced ( Interestingly, among the generated FVIIa variants with altered AT reactivity, two subsets were identified that displayed differential in vitro reactivity with AT depending on the type cofactor present. Accordingly, one group exhibited a greater susceptibility to inhibition relative to wild-type FVIIa in the presence of heparin but not in the presence of TF, while the other group demonstrated the opposite behavior. Endowed with the ability to report on the cofactor identity from the rate of inhibition relative to wild-type FVIIa, variants from each group were tested for their tendency to accumulate as complexes with AT following intravenous administration to rats. Supporting a contribution from endogenous GAGs to the in vivo inactivation of FVIIa, the measured in vivo peak levels of accumulated FVIIa-AT complexes were found to directly correlate with the in vitro rate constants determined for the variants in the presence of heparin, but not when the cofactor was TF or the combination of TF and heparin. Altogether, these results 1) demonstrate a direct relationship between the in vitro reactivity of FVIIa with AT in the presence of heparin and the clearance of FVIIa through this pathway in vivo, and 2) identify heparin-like GAGs as the likely rate enhancing component of FVIIa inhibition in vivo. Disclosures Ostergaard:Novo Nordisk A/S: Employment. Hansen:Novo Nordisk A/S: Employment. Pelzer:Novo Nordisk A/S: Employment. Agerso:Novo Nordisk A/S: Employment. Pedersen:Novo Nordisk A/S: Employment. Glue:Novo Nordisk A/S: Employment. Johnsen:Novo Nordisk A/S: Employment. Andersen:Novo Nordisk A/S: Employment. Bjelke:Novo Nordisk A/S: Employment. Breinholt:Novo Nordisk A/S: Employment. Stennicke:Novo Nordisk A/S: Employment. Gandhi:Novo Nordisk A/S: Employment. Olsen:Novo Nordisk A/S: Employment. Hermit:Novo Nordisk A/S: Employment.