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 C 18 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 M), 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.Blood clotting, resulting either from the extrinsic pathway following tissue injury or the intrinsic pathway following contact activation, involves tightly regulated proteolytic cascades (1). The intrinsic pathway is initiated by activation of factor XII either through proteolysis or contact with negatively charged surfaces. Activated factor XIIa, in turn, converts plasma prekallikrein to kallikrein, which can then activate additional factor XII. Factor XIIa activates factor XI, which, in turn, activates factor IX. Activated factor IX forms a complex with factor VIIIa, phospholipid, and calcium, which converts factor X to factor Xa. Factor X is also activated by the factor VIIatissue factor complex operating within the extrinsic pathway. Thrombin generation by factor Xa in complex with factor Va leads ultimately to the formation of the fibrin clot. Thrombus formation is also regulated by the fibrinolytic system whereby plasmin, formed from plasminogen by the action of kallikrein, tissue plasminogen activator (tPA), 1 or urokinase, breaks down both fibrinogen and fibrin (2).Protease inhibitors play critical roles in the regulation of the coagulation and fibrinolytic systems. Tissue factor pathway i...

Activation of Hepatocyte Growth Factor (HGF) by Endogenous HGF Activator Is Required for Metanephric Kidney Morphogenesis in Vitro

“…This observation suggests that the ureteric bud creates a local gradient of HGF activity and Met signaling. In preliminary experiments, we have identified both HGF activator inhibitor-1 and -2 (HAI-1 and HAI-2) in the ureteric bud (37)(38)(39).…”

Pro-urokinase-type Plasminogen Activator Is a Substrate for Hepsin

“…At various time points, 50-l aliquots were removed and added to 150 l of 667 nM HAI-2 (final concentration in 250 l was 400 nM) in Hepes buffer to stop further pro-uPA cleavage. At the concentration used, HAI-2 specifically inhibits plasmin (29), HGFA (30), hepsin, and matriptase but not the newly generated uPA (Table 1). Our results on HAI-2 inhibition of plasmin and HGFA (data not shown) were consistent with the published data (29,30).…”

Sequence and Conformational Specificity in Substrate Recognition

“…It potently inhibits many serine proteases with subnanomolar affinities (66) and is believed to be a physiological inhibitor of hepatocyte growth factor (HGF) activator (67) as well as hepsin (68) and matriptase (69,70), two type II transmembrane serine proteases that, like HGF activator, can convert latent pro-hepatocyte growth factor/scatter factor (pro-HGF/SF) into the two-chain active signaling heterodimer. Additionally, HAI2 is a physiological inhibitor of prostasin, a glycosylphosphatidylinositol-anchored protease involved in regulation of matriptase activation and shedding (71,72).…”

“…HAI2, also known as placental bikunin, is another compound inhibitor composed of two Kunitz domains (66). It potently inhibits many serine proteases with subnanomolar affinities (66) and is believed to be a physiological inhibitor of hepatocyte growth factor (HGF) activator (67) as well as hepsin (68) and matriptase (69,70), two type II transmembrane serine proteases that, like HGF activator, can convert latent pro-hepatocyte growth factor/scatter factor (pro-HGF/SF) into the two-chain active signaling heterodimer.…”