Characterization of placental bikunin, a novel human serine protease inhibitor.
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 (Ki = 0.1 nM), human tissue kallikrein (Ki = 0.1 nM), human plasma kallikrein (Ki = 0.3 nM) and human factor XIa (Ki = 6 nM). Conversely, this protein was a weaker inhibitor of factor VIIa-tissue factor (Ki = 1.6 microM), factor IXa (Ki = 206 nM), factor Xa (Ki = 364 nM), and factor XIIa (Ki = 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.
Delaria
K A
KA
Preclinical Research, Biotechnology Unit, Bayer Corporation, West Haven, Connecticut 06516, USA.
Muller
D K
DK
Marlor
C W
CW
Brown
J E
JE
Das
R C
RC
Roczniak
S O
SO
Tamburini
P P
PP
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001779:Blood Coagulation Factors / Q000037:antagonists & inhibitors*; D002460:Cell Line; D002846:Chromatography, Affinity; D010450:Endopeptidases / Q000378:metabolism*; D005260:Female; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D008562:Membrane Glycoproteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010314:Partial Thromboplastin Time; D010446:Peptide Fragments / Q000737:chemistry; D010920:Placenta / Q000378:metabolism*; D011247:Pregnancy; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D018411:Spodoptera; D014162:Transfection; D014360:Trypsin Inhibitor, Kunitz Soybean; D014361:Trypsin Inhibitors / Q000737:chemistry
D001779:Blood Coagulation Factors; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D011994:Recombinant Proteins; C109148:SPINT2 protein, human; D014361:Trypsin Inhibitors; D014360:Trypsin Inhibitor, Kunitz Soybean; D010450:Endopeptidases