Incorporation of an Active Site Inhibitor in Factor VIIa Alters the Affinity for Tissue Factor

doi:10.1074/jbc.272.18.11863

Incorporation of an Active Site Inhibitor in Factor VIIa Alters the Affinity for Tissue Factor*

From Vessel Wall Biology, Health Care Discovery, Novo Nordisk A/S, Niels Steensens Vej 1, DK-2820 Gentofte, Denmark and the^§Department of Biochemistry, University of Texas Health Center, Tyler, Texas 75710

Abstract

Recent studies showed that the administration of active site-inhibited factor VIIa blocked factor VIIa/tissue factor-induced fibrin and thrombus formation in ex vivo andin vivo model systems. These studies suggest that inactivated factor VIIa competes efficiently with plasma factor VII(a) for a limited number of tissue factor sites. In the present study, we compared the interactions of factor VIIa and active site-inhibited factor VIIa with tissue factor. Competition studies of factor VIIa and active site-inhibited factor VIIa in a factor X activation assay showed that the affinity of the latter for relipidated tissue factor was 5-fold higher than that of factor VIIa. Radioligand binding studies with a human bladder carcinoma cell line (J82) and surface plasmon resonance studies using soluble tissue factor demonstrated a faster association and a slower dissociation for the active site-inhibited factor VIIa. Studies of equilibrium binding to cell surface tissue factor showed that the affinity of active site-inhibited VIIa was 5-fold higher than that of factor VIIa to non-functional tissue factor sites, whereas both inactivated factor VIIa and factor VIIa bound to functional tissue factor sites with the same high affinity. Comparison of the CD spectra of factor VIIa and active site-inactivated factor VIIa revealed structural differences in the protease domain. The potential physiological implications of these findings are discussed.

Footnotes

↵* This work was supported in part by Grant HL-42813 from NHLBI, National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ To whom correspondence should be addressed: Vessel Wall Biology-Biochemistry, Novo Nordisk A/S, Hagedornsvej 1, DK-2820 Gentofte, Denmark. Tel.: 45-44-43-81-72; Fax: 45-44-43-81-10; E-mail:bbsn{at}novo.dk.
↵¶ Recipient of Research Career Development Award KO4-02594 from NHLBI, National Institutes of Health, during this investigation.
↵1 The abbreviations used are: FVIIa, activated coagulation factor VII; FFR-FVIIa,d-Phe-l-Phe-l-Arg methylene FVIIa; des-(1–44), lacking the N-terminal 44 amino acids; des-(1–44)-FVIIa, FVIIa lacking the N-terminal 44 amino acids; des-(1–44)-FFR-FVIIa, FFR-FVIIa lacking the N-terminal 44 amino acids; TF, tissue factor; sTF, soluble extracellular portion of tissue factor (residues 1–219); FX, coagulation factor X; FXa, activated FX; dansyl, 5-dimethylaminonaphthalene-1-sulfonyl.
- Received October 11, 1996.
- Revision received December 27, 1996.