Else Marie Nicolaisen

Generation of Gla-domainless FVIIa by cathepsin G-mediated cleavage.

Cougulation factor VII contains ten λ‐carboxyglutamic acid residues in the N‐terminal region (Gla‐domain) which are essential for the hemostatic function of FVII. The present study shows that granulocyte cathepsin G degrades the Gla‐domain of FVIIa in vitro. Characterization of the truncated FVIIa by SDS‐PAGE and N‐terminal amino acid sequence analysis revealed that cleavage had occurred between Tyr‐44 and Ser‐45 and that further cleavage was only obtained on extensive cathepsin G exposure. Cleavage of vitamin K‐dependent coagulation factors by cathepsin G may play a role in vivo, and it offers a convenient way of obtaining proteins deprived of their Gla‐domain for functional and structural studies.

FVIIa derivatives obtained by autolytic and controlled cathepsin G mediated cleavage.

The heavy chain of coagulation factor VII contains a serine esterase entity. A partial cleavage in the heavy chain occurs during purification and activation of the single‐chain zymogen, presumably as a result of autolysis. Neutrophil cathepsin G initially generates a Gla‐domainless FVIIa without coagulant activity. However, on extended exposure cleavage also occurs in the heavy chain, resulting in a complete loss of enzyme activity. Four cleavage sites on the heavy chain, two susceptible to trypsin‐like autolysis and two susceptible to chymotrypsin‐like cathepsin G‐mediated catalysis have been identified. The hydrolysis of peptide bonds in the heavy chain might contribute to regulation of the coagulation process in vivo.

Investigating clearance mechanisms for recombinant activated factor VII in a perfused liver model

Clearance mechanisms for recombinant activated human FVII (rFVIIa; NovoSeven), a heterogeneously glycosylated protein, have yet to be fully elucidated, but may involve the liver. The effects of the gamma-carboxy glutamic acid (Gla) domain and the sialic acid content of the protein on rFVIIa clearance were investigated following intravenous administration of rFVIIa lacking the Gla domain, des(1-44) rFVIIa and asialo-rFVIIa in pharmacokinetic (PK) studies and perfused rat livers. PK parameters for both rFVIIa and des(1-44) rFVIIa had similar biphasic clearance profiles, as well as half-lives ([t(1/2)]=80 and 88 minutes, respectively), while asialo-rFVIIa was cleared quickly (t(1/2)=21 minutes) with a linear clearance profile. Perfused liver studies with all proteins (10 nM) mirrored the trends in profiles observed in the PK study. rFVIIa and des(1-44) rFVIIa were cleared to a similar extent, 41% and 35%, respectively, after 1 h, whereas plasma-derived FVII from humans (which has a higher sialylation content than rFVIIa) was cleared to a lesser extent (21%). Asialo-rFVIIa, on the other hand, was almost totally cleared and when an excess of asialo-orosomucoid was added to the perfusate, its clearance was significantly reduced (by 34%) and also for rFVIIa, albeit to a lesser extent (by 14%). Together these data suggest that carbohydrate receptor(s) (e.g. the asialoglycoprotein receptor, ASGPR) play a role in asialo-rFVIIa and rFVIIa clearance. In vivo and liver clearance data correlated well showing similar trends and indicated that rFVIIa clearance is not affected by the Gla domain, but rather by a subpopulation of N-glycosylated structures on rFVIIa.

Evaluation of the Metal Binding Sites in a Recombinant Coagulation Factor Viii Identifies Two Sites with Unique Metal Binding Properties.

Abstract Coagulation factor VIII is a glycosylated, non-covalent heterodimer consisting of a heavy chain (A1-A2-B domains) and a light chain (A3-C1-C2 domains). The association of the chains, and the stability and function of the dimer depend on the presence of metal ions. We applied X-ray fluorescence, X-ray crystallographic structure determination with anomalous signals at different wavelengths, and colorimetric measurements to evaluate the metal binding sites in a recombinant factor VIII molecule, turoctocog alfa. We identified a metal binding site in domain A3 dominated by Cu+ binding and a site in domain A1 dominated by Zn2+ binding.