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 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 vitrospecificity profile, recombinant placental bikunin(1–170)prolonged the clotting time in an activated partial thromboplastin time assay. 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 vitrospecificity 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 (1Mann K.G. Lorand L. Methods Enzymol. 1993; 222: 1-10Google Scholar). 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 VIIa-tissue 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), 1The abbreviations used are: tPA, tissue plasminogen activator; TFPI, tissue factor pathway inhibitor; Suc, succinyl; AMC, 7-amido-4-methylcoumarin; Boc,t-butoxycarbonyl; Bz, benzoyl; pNa,p-nitroanilide; Tricine,N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; Fmoc,N-(9-fluorenyl)methoxycarbonyl; HPLC, high performance liquid chromatography; APTT, activated partial thromboplastin time. or urokinase, breaks down both fibrinogen and fibrin (2Marder V.J. Frances C.W. Doolittle R.F. Colman R.W. Hirsh J. Marder V.J. Salzman E.W. Hemostasis and Thrombosis: Basic Principles and Clinical Practice. Lippincott, Philadelphia1982: 145-163Google Scholar). Protease inhibitors play critical roles in the regulation of the coagulation and fibrinolytic systems. Tissue factor pathway inhibitor (TFPI), a multivalent Kunitz-type inhibitor, is a major regulator of the extrinsic pathway through factor Xa-dependent inhibition of factor VIIa-tissue factor (3Davie E.W. Fujikawa K. Kisiel W. Biochemistry. 1991; 30: 10363-10370Google Scholar). C1 inhibitor is thought to be the major physiological inhibitor of the intrinsic pathway enzymes plasma kallikrein and factor XII (4Travis J. Salvesen G.S. Annu. Rev. Biochem. 1983; 52: 655-709Google Scholar). More recently, TFPI-2, a serine protease inhibitor structurally related to TFPI, has been proposed to be an important physiological inhibitor of enzymes involved in both coagulation and fibrinolysis (5Petersen L.C. Sprecher C.A. Foster D.C. Blumberg H. Hamamoto T. Kisiel W. Biochemistry. 1996; 35: 266-272Google Scholar). In a previous study, we identified and cloned a human cDNA encoding a novel protein containing two Kunitz-like domains which we termed placental bikunin (6Marlor C.W. Delaria K.A. Davis G. Muller D.K. Greve J.M. Tamburini P.P. J. Biol. Chem. 1997; 272: 12202-12208Google Scholar). In this study a soluble recombinant fragment of this protein, placental bikunin(1–170), 2Residues of the placental bikunin sequence and fragments thereof are numbered as defined in Ref. 6Marlor C.W. Delaria K.A. Davis G. Muller D.K. Greve J.M. Tamburini P.P. J. Biol. Chem. 1997; 272: 12202-12208Google Scholar according to their position within the open reading frame for full-length mature placental bikunin. In this scheme, placental bikunin(1–170) refers to a protein containing the NH2-terminal 170 amino acids of the mature protein produced by removal of the signal peptide. The NH2-terminal(7–64) domain and COOH-terminal(102–159) domain refer to individual functional Kunitz domains normally present within residues 7–64 and 102–159, respectively, of mature placental bikunin. was expressed and found to be a potent serine protease inhibitor. Using the recombinant protein and both of its synthetic NH2- and COOH-terminal Kunitz domains, we dissect the specificity of the protein more comprehensively. In doing so, we demonstrate that placental bikunin is a potent inhibitor of plasma and tissue kallikreins, plasmin and factor XIa. Bovine chymotrypsin, bovine trypsin, and tPA (single chain form from human melanoma cell culture), urokinase, Suc-A-A-P-F-AMC, Boc-L-G-R-AMC, Suc-A-A-A-AMC, Boc-Q-G-R-AMC, Bz-P-F-R-pNa, and P-F-R-AMC were from Sigma. Neutrophil elastase was from Athens Research and Technology, Inc. (Athens, GA). Human plasmin, factor VIIa, tissue factor (lipidated), and (CH3SO2-D-cyclohexylpyrosyl)-G-R-pNa (Spectrozyme tPA substrate) were from American Diagnostica, Inc. (Greenwich, CT). Human plasma kallikrein and human factors IXa, X, Xa, XIa, and XIIa were from Enzyme Research Laboratories (South Lafayette, IN). Tos-G-P-K-AMC, Suc-A-A-P-R-pNa, and Boc-E(OBzl)-A-R-AMC, were from Bachem Bioscience (King of Prussia, PA). H-D-I-P-R-pNa was from Chromogenix AB. Bovine pancreatic kallikrein, human tissue kallikrein, and recombinant aprotinin were kindly provided by Bayer AG (Wuppertal, Germany). A cDNA fragment encoding an NH2-terminal 170-amino acid fragment of placental bikunin was expressed in Sf9 cells (7Luckow V.A. Summers M.D. Virology. 1989; 170: 31-39Google Scholar) as follows. Placental bikunin cDNA obtained by polymerase chain reaction and contained within the pCRII vector as described previously (6Marlor C.W. Delaria K.A. Davis G. Muller D.K. Greve J.M. Tamburini P.P. J. Biol. Chem. 1997; 272: 12202-12208Google Scholar) was liberated by digestion with HindIII and XbaI. This fragment was gel purified and then cloned into the M13mp19 vector (New England Biolabs, Beverly, MA).In vitro mutagenesis (8Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 488-492Google Scholar) was used to generate aPstI site between the upstream XbaI site and the sequence encoding the ATG start site at the 5′ end of the cDNA insert. The oligonucleotide used for the mutagenesis had the sequence CGC GTC TCG GCT GAC CTG GCC CTG CAG ATG GCG CAC GTG TGC GGG. A stop codon (TAG) and BglII/XmaI site was similarly engineered at the 3′ end of the cDNA using the oligonucleotide CTG CCC CTT GGC TCA AAG TAG GAA GAT CTT CCC CCC GGG GGG GTG GTT CTG GCG GGG CTG. The stop codon was in-frame with the sequence encoding placental bikunin and caused termination immediately following the lysine at amino acid residue 170 of the mature protein sequence. The mutated cDNA thus encoded a truncated placental bikunin protein containing both Kunitz domains but which was devoid of the putative transmembrane domain. The product from digestion with PstI andBglII was isolated and cloned into the BacPac8 vector (CLONTECH) for expression of placental bikunin(1–170). The expression of placental bikunin(1–170) by Sf9 insect cells was optimal at a multiplicity of infection of 1 when the medium was harvested at 72 h post-infection. Sf9 cell culture supernatant (2–3 liters) was harvested by centrifugation (1,500 × g for 30 min), adjusted to pH 8.0 by the addition of 1 m Tris-HCl (pH 8.0) to a final concentration of 50 mm, then applied at 2.0-ml min−1 to a 5-ml column comprised of bovine pancreatic kallikrein (70 mg) which had been immobilized onto CNBr-activated Sepharose 4B (Pharmacia Biotech Inc.) according to the manufacturer's instructions. After loading, the column was washed with 0.1m Tris-HCl (pH 8.0) containing 0.1 m NaCl until the A 280 nm of the wash could no longer be detected. The column was washed further with 0.1 m Tris-HCl (pH 8.0) containing 0.5 m NaCl and then eluted with 0.2m acetic acid (pH 2.0). Fractions (2.0 ml) were collected in tubes containing 0.25 of 1 m Tris-HCl (pH and for the to bovine as described (6Marlor C.W. Delaria K.A. Davis G. Muller D.K. Greve J.M. Tamburini P.P. J. Biol. Chem. 1997; 272: 12202-12208Google Scholar). were adjusted to pH with acid and to a C18 reverse-phase column × with acid containing at a of 1 The placental bikunin(1–170) was eluted with a of in acid 50 Fractions containing inhibitory were in (pH 0.1 m and at until The highly purified protein an nm of × to amino acids 7–64 and of mature placental bikunin (6Marlor C.W. Delaria K.A. Davis G. Muller D.K. Greve J.M. Tamburini P.P. J. Biol. Chem. 1997; 272: 12202-12208Google Scholar) were an peptide using was a with an of The chain used were as for and for and for and for In was when and were performed in and for h at The peptide was at in a for and washed were purified by a C18 column × 30 using a of either in acid (placental or a of in acid (placental The purified synthetic to placental and placental were to the functional NH2-terminal(7–64) and COOH-terminal(102–159) Kunitz domains using an of the of W. J. Chem. 1991; Scholar) as follows. A containing in 0.1 m Tris-HCl (pH was to a of reduced peptide and for the NH2-terminal(7–64) and COOH-terminal(102–159) in 0.1m Tris-HCl (pH containing m to a final concentration of 0.3 peptide in 0.1 m Tris-HCl (pH and 1 The containing either domain were at for h and then with 50 (pH 8.0) containing 0.1 m was isolated by immobilized bovine pancreatic kallikrein described for the purification of recombinant placental bikunin(1–170). Fractions containing inhibitory were adjusted to pH with then applied to a C18 reverse-phase column × The NH2-terminal(7–64) Kunitz domain was purified using a of in acid at a of The COOH-terminal(102–159) Kunitz domain was purified using a of in were in and at until To the stoichiometry with trypsin, the protease nm) was with recombinant placental bikunin nm) or aprotinin nm) in of 50 (pH 0.1 m for 30 at by the addition of final which formation of was at To the stoichiometry with plasma kallikrein, nm protease was with recombinant placental bikunin nm) in of 50 (pH 8.0) containing 0.1 m NaCl and After 30 at was final and at protease was and obtained from of inhibitor to (K were as described previously (6Marlor C.W. Delaria K.A. Davis G. Muller D.K. Greve J.M. Tamburini P.P. J. Biol. Chem. 1997; 272: 12202-12208Google Scholar) using for inhibitors C. 1989; Scholar) of and for to Kunitz domains and placental bikunin(1–170), (1Mann K.G. Lorand L. Methods Enzymol. 1993; 222: 1-10Google Scholar) site of trypsin, plasma and tissue kallikreins, and plasmin were by as described T. Methods Enzymol. Scholar). The concentration of bovine was by with as described J. Biol. Chem. Scholar). The of the NH2-terminal(7–64) and COOH-terminal Kunitz and aprotinin were by with and by amino acid The concentration of placental bikunin(1–170) was by amino acid The of factors VIIa, IXa, X, Xa, XIa, and XIIa, tissue urokinase, and tPA were the manufacturer's of protease with inhibitor for at in of the were initiated with to the following bovine pancreatic kallikrein with = and P-F-R-AMC (K m = human tissue kallikrein, 50 Tris-HCl (pH 50 and with = nm and P-F-R-AMC (K m = human 0.1 (pH and with = nm and (K m = human factor Xa, Tris-HCl (pH 0.1 m bovine with = and (K m = and human factor XIa A with 1 bovine with = 0.1 (K m = 50 The following were performed following of protease with inhibitor for 30 at with = 50 and 30 (K m = with = 50 and (K m = human plasmin, 50 Tris-HCl (pH 0.1 m and with = 50 and (K m = human plasma kallikrein with = nm and P-F-R-AMC (K m = and factor XIIa with = and (K m = Factor VIIa-tissue factor inhibition was by nm factor VIIa, with inhibitor in of A for 30 at were initiated with 1 final and the A nm was Factor IXa inhibition was as described (5Petersen L.C. Sprecher C.A. Foster D.C. Blumberg H. Hamamoto T. Kisiel W. Biochemistry. 1996; 35: 266-272Google Scholar). factor IXa nm) was for at with inhibitor in 50 Tris-HCl (pH containing 0.1 = and Factor X nm) was then and the for 30 at was then and factor Xa To the inhibition of tPA, tPA was with inhibitor for h at in Tris-HCl (pH containing NaCl and were initiated with to tPA nm), inhibitor μm), in Tris-HCl (pH containing and The of formed for h at was from the in the A of was a = = nm) the of the was at nm a were from of inhibitor which were by by U. using the following 1 i and are the in the or of a inhibitor concentration of and the concentration of i were obtained by for the of using the following i = i for inhibition by placental bikunin(1–170) were an stoichiometry of 1:2 and that the two Kunitz inhibitory domains are and The of recombinant placental bikunin(1–170) and aprotinin the was as follows. in Tris-HCl (pH containing NaCl and was ml) to a within a The was to with a activation and were in the addition of 0.1 of plasma the and concentration of were to clotting, which was digestion was performed as follows. placental bikunin(1–170) in was to a in of Tris-HCl containing 6 m and 1 and for h in the at the addition of final an additional the was applied to a reaction containing C18 eluted in then to by The was with of acid with for to in 0.1 of then to NH2-terminal NH2-terminal sequence was performed as described Methods Enzymol. but with (6Marlor C.W. Delaria K.A. Davis G. Muller D.K. Greve J.M. Tamburini P.P. J. Biol. Chem. 1997; 272: 12202-12208Google Scholar). acid was performed as described previously P.P. J. J. J. A. D. H. Biochem. Scholar). gel was performed with according to the manufacturer's and protein was with using was performed a of with the following 50 30 were a washed with and eluted at with acetic To the protease inhibitory of placental bikunin, a cDNA encoding the extracellular domain of 170 amino acids of placental bikunin its natural signal peptide (6Marlor C.W. Delaria K.A. Davis G. Muller D.K. Greve J.M. Tamburini P.P. J. Biol. Chem. 1997; 272: 12202-12208Google Scholar) was prepared by mutagenesis and expressed in the The protein was purified from the cell culture supernatant by sequential kallikrein-Sepharose and C18 reverse-phase of was the in the of the protein was to be as a of the inhibitory of the culture supernatant was to placental bikunin(1–170). This is the that the of the inhibitory present in the supernatant to the kallikrein-Sepharose the placental bikunin(1–170) the column of the column and of of medium between and 1 of placental bikunin(1–170) amino acid of recombinant placental bikunin(1–170) from Sf9 of is the of inhibitor to by with the A of is the of inhibitor to by with the in a The final was highly as by gel and a of with the of placental bikunin(1–170). NH2-terminal sequence of the protein the for as obtained for the natural protein (6Marlor C.W. Delaria K.A. Davis G. Muller D.K. Greve J.M. Tamburini P.P. J. Biol. Chem. 1997; 272: 12202-12208Google Scholar). The sequence at residue and with the sequence the amino acid was to the of placental bikunin(1–170). placental bikunin(1–170) was then and then purification of the resulting peptide for the sequence for of the from the digestion of placental of fragments resulting from the digestion of purified placental bikunin(1–170) with amino acid bikunin residue was performed according to amino acid in a was performed according to of of either bovine or human plasma kallikrein with purified placental that of inhibitor can with two of protease the of with aprotinin a stoichiometry of in the inhibitor complex To to dissect the of the individual domains within this bikunin, synthetic were to the NH2-terminal(7–64) and Kunitz domains of placental bikunin. reduced = and COOH-terminal(102–159) domain = were of which had the amino acid of using as the by purification a purified NH2-terminal(7–64) of the reduced and domains of the reduced which of and The of peptide 1 that had in in the formation of three from the present within of the reduced A of serine proteases was used to the specificity and of recombinant placental bikunin(1–170) and its individual Kunitz domains a Kunitz inhibitor of defined was in for placental bikunin(1–170) was a potent inhibitor of proteases and also of potential to its putative physiological this protein was a potent inhibitor of human plasma and tissue kallikreins, human plasmin and factor XIa. The of recombinant placental plasmin, plasma kallikrein, and factor XIa were and respectively, with placental bikunin(1–170) was also a more potent inhibitor aprotinin enzymes of the intrinsic pathway of factors IXa, Xa, and XIIa, the i were in the of for the of placental bikunin(1–170) its individual Kunitz domains, and aprotinin with serine Kunitz Kunitz i for placental bikunin(1–170) were by to the protein from bovine from bovine from human from bovine from human 0.5 from human 0.25 from human no inhibition at the inhibitor concentration in no inhibition at the inhibitor concentration in from human no inhibition at the inhibitor concentration in from human from human from human no inhibition at the inhibitor concentration in (K i are in are the from i for placental bikunin(1–170) were by to the protein from bovine from human no inhibition at the inhibitor concentration in in a (K i are in are the from that recombinant placental bikunin(1–170) be a more inhibitor of the intrinsic coagulation pathway To this placental bikunin(1–170) and aprotinin were for their to the APTT, a of the intrinsic pathway of As predicted from the in vitro specificity recombinant placental bikunin(1–170) was more potent aprotinin in the To a of the clotting at aprotinin but recombinant placental bikunin(1–170) was placental bikunin(1–170) was a inhibitor of the factor VIIa-tissue factor complex and factor Xa, that be a inhibitor of the extrinsic pathway of This was by the that placental bikunin(1–170) had no the time when at to placental bikunin(1–170) urokinase, tPA, or elastase at to 1 The individual Kunitz domains within placental bikunin were functional protease inhibitors and the domain was potent tissue kallikrein and factor XIa. factor XIIa was the NH2-terminal domain more potent the COOH-terminal domain. the specificity of the COOH-terminal domain more that of recombinant placental bikunin(1–170) that the of the COOH-terminal protease was In this we the expression and functional of placental bikunin. the natural protein was in (6Marlor C.W. Delaria K.A. Davis G. Muller D.K. Greve J.M. Tamburini P.P. J. Biol. Chem. 1997; 272: 12202-12208Google Scholar) the functional were performed with a recombinant form of the A cDNA sequence encoding a placental bikunin fragment containing both Kunitz domains but truncated immediately to the putative transmembrane (6Marlor C.W. Delaria K.A. Davis G. Muller D.K. Greve J.M. Tamburini P.P. J. Biol. Chem. 1997; 272: 12202-12208Google Scholar) was expressed in Sf9 This was to that expression of the full-length protein its as a soluble The putative signal peptide encoded within the cloned cDNA (6Marlor C.W. Delaria K.A. Davis G. Muller D.K. Greve J.M. Tamburini P.P. J. Biol. Chem. 1997; 272: 12202-12208Google Scholar) was as by the Sf9 system as by its from the NH2-terminal sequence of the purified with the recombinant protein, the individual Kunitz domains were prepared synthetically and as by in with formation of the number of placental bikunin(1–170) and the synthetic NH2-terminal(7–64) and COOH-terminal(102–159) Kunitz domains were potent serine protease protein was a potent inhibitor of trypsin, chymotrypsin, plasmin, factor XIa, and tissue and plasma kallikrein, a inhibitor of factors IXa, Xa, and with placental bikunin(1–170) that both Kunitz domains were functional and could the formation of complexes with The that placental bikunin(1–170) was functional to the that the encoded by the full-length cDNA as a and in the for protease placental bikunin(1–170) inhibited two kallikrein and factor XIa, of the contact was an inhibitor of factor VIIa-tissue factor This that is involved in the regulation of the extrinsic coagulation with placental bikunin(1–170) was potent in the a of the of coagulation by the intrinsic but the a of the of coagulation by the extrinsic pathway. placental bikunin an in vitro profile that be of a physiological regulator of both contact activation and the intrinsic pathway of the the play a upstream in fibrinolysis through activation of plasmin, which in in fibrinolysis. The high of placental the and plasmin that this inhibitor could to fibrin The that the full-length protein with is with the that to the of contact activation and fibrinolysis of at tissue surfaces. In placental bikunin(1–170) i that were with the individual domains. been obtained with the recombinant protein containing three of the Kunitz domain had from a to for the factor factor complex of the individual domains L.C. K. J. Biochem. 1996; Scholar). has been for the of with its and that the tissue complex more when to complex with factor Xa Methods Enzymol. 1993; 222: Scholar). a this is with a inhibition factor Xa to form the extrinsic pathway The that placental bikunin the intrinsic pathway through a further We that the NH2- and COOH-terminal Kunitz domains a number of the COOH-terminal Kunitz domain is more potent factor XIa. the COOH-terminal domain of placental bikunin factor XIa in which in the subsequent formation of a complex through of the NH2-terminal domain with factor XIIa or This as a inhibition for the extrinsic pathway factor XIa is to form the pathway is the the in factor XIa between the synthetic NH2- and COOH-terminal Kunitz domains could the that the putative residue within the NH2-terminal domain of placental bikunin (6Marlor C.W. Delaria K.A. Davis G. Muller D.K. Greve J.M. Tamburini P.P. J. Biol. Chem. 1997; 272: 12202-12208Google Scholar) a in the specificity of this domain. of fibrinolysis and coagulation been in open aprotinin W. A. G. 1989; and are to from the inhibition of kallikrein, plasmin, and the intrinsic pathway of coagulation H. G. 1983; Scholar). placental bikunin(1–170) is more potent aprotinin as an inhibitor of plasma kallikrein, and the intrinsic pathway of further of the of this novel human bikunin in this is In placental bikunin also be for for which aprotinin has been to be potential is the of the of cells into tissue G. J. Scholar). In placental bikunin be for the of of to or injury T. A. 1993; A. C. A. W. J. a that from the of the T. A. 1993; A. C. A. W. J. J. H. S. J. J. Scholar) through the of serine proteases as kallikrein R.W. J. Scholar). We the following of the at the Bayer Research for important acid and and