Characterization of placental bikunin, a novel human serine protease inhibitor

Abstract

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 vive 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.