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Volume 272, Number 18, Issue of May 2, 1997 pp. 12209-12214
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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Characterization of Placental Bikunin, a Novel Human Serine Protease Inhibitor

(Received for publication, December 11, 1996)

Katherine A. Delaria , Daniel K. Muller ^§ , Christopher W. Marlor ^§ , James E. Brown ^¶ , Rathindra C. Das , Steven O. Roczniak and Paul P. Tamburini

From  Preclinical Research, Biotechnology Unit, ^§ Institute for Bone and Joint Disease and Cancer, and  Institute for Research Technologies, Pharmaceuticals Division, Bayer Corporation, West Haven, Connecticut 06516 and ^¶ Process Science, Biotechnology Unit, Berkeley, California 94710

We reported previously the cloning of a novel human serine protease inhibitor containing two Kunitz-like domains, designated as placental bikunin, and the subsequent purification of a natural counterpart from human placental tissue (Marlor, C. W., Delaria, K. A., Davis, G., Muller, D. K., Greve, J. M., and Tamburini, P. P. (1997) J. Biol. Chem. 272, 12202-12208). In this report, the 170 residue extracellular domain of placental bikunin (placental bikunin_(1-170)) was expressed in baculovirus-infected Sf9 cells using its putative signal peptide. The resulting 21.3-kDa protein accumulated in the medium with the signal peptide removed and could be highly purified by sequential kallikrein-Sepharose and C₁₈ reverse-phase chromatography. To provide insights as to the potential in vivo functions of this protein, we performed an extensive investigation of the inhibitory properties of recombinant placental bikunin_(1-170) and both of its synthetically prepared Kunitz domains. All three proteins inhibited a number of serine proteases involved in the intrinsic pathway of blood coagulation and fibrinolysis. Placental bikunin_(1-170) formed inhibitor-protease complexes with a 1:2 stoichiometry and strongly inhibited human plasmin (K_i = 0.1 nM), human tissue kallikrein (K_i = 0.1 nM), human plasma kallikrein (K_i = 0.3 nM) and human factor XIa (K_i = 6 nM). Conversely, this protein was a weaker inhibitor of factor VIIa-tissue factor (K_i = 1.6 µM), factor IXa (K_i = 206 nM), factor Xa (K_i = 364 nM), and factor XIIa (K_i = 430 nM). This specificity profile was to a large extent mimicked, albeit with reduced potency, by the individual Kunitz domains. As predicted from this in vitro specificity profile, recombinant placental bikunin_(1-170) prolonged the clotting time in an activated partial thromboplastin time assay.

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