CHARACTERIZATION OF PLACENTAL BIKUNIN, A NOVEL HUMAN SERINE-PROTEASE INHIBITOR

We reported previously the cloning of a novel human serine protease in hibitor containing two Kunitz-like domains, designated as placental bi kunin, and the subsequent purification of a natural counterpart from h uman 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 bacul ovirus-infected Sf9 cells using its putative signal peptide. The resul ting 21.3-kDa protein accumulated in the medium with the signal peptid e removed and could be highly purified by sequential kallikrein-Sephar ose and C-18 reverse-phase chromatography. To provide insights as to t he potential in vivo functions of this protein, we performed an extens ive investigation of the inhibitory properties of recombinant placenta l bikunin((1-170)) and both of its synthetically prepared Kunitz domai ns. All three proteins inhibited a number of serine proteases involved in the intrinsic pathway of blood coagulation and fibrinolysis. Place ntal bikunin((1-170)) formed inhibitor-protease complexes with a 1:2 s toichiometry and strongly inhibited human plasmin (K-i = 0.1 nM), huma n 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 mu M), facto r 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, alb eit with reduced potency, by the individual Kunitz domains. As predict ed from this in vitro specificity profile, recombinant placental bikun in((1-170)) prolonged the clotting time in an activated partial thromb oplastin time assay.