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Mapping the Interaction between High Molecular Mass Kininogen and the Urokinase Plasminogen Activator Receptor

2004; Elsevier BV; Volume: 279; Issue: 16 Linguagem: Inglês

10.1074/jbc.m313850200

1083-351X

Fakhri Mahdi, Zia Shariat‐Madar, Alice A. Kuo, Maria E. Carinato, Douglas B. Cines, Alvin H. Schmaier,

Blood Coagulation and Thrombosis Mechanisms

The urokinase plasminogen activator receptor (uPAR) is a multifunctional, GPI-linked receptor that modulates cell adhesion/migration and fibrinolysis. We mapped the interaction sites between soluble uPAR (suPAR) and high molecular mass kininogen (HK). Binding of biotin-HK to suPAR was inhibited by HK, 56HKa, and 46HKa with an IC50 of 60, 110, and 8 nm, respectively. We identified two suPAR-binding sites, a higher affinity site in the light chain of HK and 46HKa (His477-Gly496) and a lower affinity site within the heavy chain (Cys333-Lys345). HK predominantly bound to suPAR fragments containing domains 2 and 3 (S-D2D3). Binding of HK to domain 1 (S-D1) was also detected, and the addition of S-D1 to S-D2D3 completely inhibited biotin-HK or -46HKa binding to suPAR. Using sequential and overlapping 20-amino acid peptides prepared from suPAR, two regions for HK binding were identified. One on the carboxyl-terminal end of D2 (Leu166-Thr195) blocked HK binding to suPAR and to human umbilical vein endothelial cells (HUVEC). This site overlapped with the urokinase-binding region, and urokinase inhibited the binding of HK to suPAR. A second region on the amino-terminal portion of D3 (Gln215-Asn255) also blocked HK binding to HUVEC. Peptides that blocked HK binding to uPAR also inhibited prekallikrein activation on HUVEC. Therefore, HK interacts with suPAR at several sites. HK binds to uPAR as part of its interaction with its multiprotein receptor complex on HUVEC, and the biological functions that depend upon this binding are modulated by urokinase. The urokinase plasminogen activator receptor (uPAR) is a multifunctional, GPI-linked receptor that modulates cell adhesion/migration and fibrinolysis. We mapped the interaction sites between soluble uPAR (suPAR) and high molecular mass kininogen (HK). Binding of biotin-HK to suPAR was inhibited by HK, 56HKa, and 46HKa with an IC50 of 60, 110, and 8 nm, respectively. We identified two suPAR-binding sites, a higher affinity site in the light chain of HK and 46HKa (His477-Gly496) and a lower affinity site within the heavy chain (Cys333-Lys345). HK predominantly bound to suPAR fragments containing domains 2 and 3 (S-D2D3). Binding of HK to domain 1 (S-D1) was also detected, and the addition of S-D1 to S-D2D3 completely inhibited biotin-HK or -46HKa binding to suPAR. Using sequential and overlapping 20-amino acid peptides prepared from suPAR, two regions for HK binding were identified. One on the carboxyl-terminal end of D2 (Leu166-Thr195) blocked HK binding to suPAR and to human umbilical vein endothelial cells (HUVEC). This site overlapped with the urokinase-binding region, and urokinase inhibited the binding of HK to suPAR. A second region on the amino-terminal portion of D3 (Gln215-Asn255) also blocked HK binding to HUVEC. Peptides that blocked HK binding to uPAR also inhibited prekallikrein activation on HUVEC. Therefore, HK interacts with suPAR at several sites. HK binds to uPAR as part of its interaction with its multiprotein receptor complex on HUVEC, and the biological functions that depend upon this binding are modulated by urokinase. Recent investigations indicate that high molecular mass kininogen (HK) 1The abbreviations used are: HK, high molecular mass kininogen; uPAR, urokinase plasminogen activator receptor; PK, prekallikrein; suPAR, soluble urokinase plasminogen activator receptor; scuPA, single chain urokinase plasminogen activator; HUVEC, human umbilical vein endothelial cell(s). 1The abbreviations used are: HK, high molecular mass kininogen; uPAR, urokinase plasminogen activator receptor; PK, prekallikrein; suPAR, soluble urokinase plasminogen activator receptor; scuPA, single chain urokinase plasminogen activator; HUVEC, human umbilical vein endothelial cell(s). binds to endothelial cell membranes through an interaction with a multiprotein receptor complex comprising at least cytokeratin 1, gC1qR, and the urokinase plasminogen activator receptor (uPAR) (1Hasan A.A.K. Zisman T. Schmaier A.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3615-3620Crossref PubMed Scopus (162) Google Scholar, 2Shariat-Madar Z. Mahdi F. Schmaier A.H. J. Biol. Chem. 1999; 274: 7137-7145Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 3Herwald H. Dedio J. Kellner R. Loos M. Muller-Esterl W. J. Biol. Chem. 1996; 271: 13040-13047Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 4Joseph K. Grebrehiwet B. Peerschke E.I.B. Reid K.B.M. Kaplan A.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8552-8557Crossref PubMed Scopus (214) Google Scholar, 5Colman R.W. Pixley R.A. Najamunnisa S. Yan W. Wang J. Mazar A. McCrae K.R. J. Clin. Invest. 1997; 100: 1481-1487Crossref PubMed Scopus (206) Google Scholar). The three proteins co-localize on the endothelial cell membrane (6Mahdi F. Shariat-Madar Z. Figueroa C.D. Schmaier A.H. Blood. 2001; 97: 2342-2350Crossref PubMed Scopus (109) Google Scholar). The same three proteins form a receptor complex for factor XII (7Mahdi F. Shariat-Madar Z. Figueroa C.D. Schmaier A.H. Blood. 2002; 99: 3585-3596Crossref PubMed Scopus (143) Google Scholar), but binding of factor XII in vivo is likely limited both by the low plasma concentration of free Zn2+, which is below the requirement for FXII binding and by the much higher plasma concentration of HK (7Mahdi F. Shariat-Madar Z. Figueroa C.D. Schmaier A.H. Blood. 2002; 99: 3585-3596Crossref PubMed Scopus (143) Google Scholar). Binding of HK to this multiprotein receptor complex predominates, localizing prekallikrein (PK) to the cell surface. The plasma concentration of PK and the ambient free Zn2+ concentration in plasma also prevent FXI from binding to HK under conditions where platelets or other cells are not activated (8Mahdi F. Shariat-Madar Z. Schmaier A.H. J. Biol. Chem. 2003; 278: 43983-43990Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). PK bound to HK on endothelial cells is proteolyzed by membrane-expressed prolylcarboxypeptidase to form plasma kallikrein (9Shariat-Madar Z. Mahdi F. Schmaier A.H. J. Biol. Chem. 2002; 277: 17962-17969Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 10Moreira C.R. Schmaier A.H. Mahdi F. da Motta G. Nader H.B. Shariat-Madar Z. FEBS Lett. 2002; 523: 167-170Crossref PubMed Scopus (54) Google Scholar). This multiprotein receptor complex thereby regulates the assembly and activation of the plasma kallikrein/kinin system. The requirements for HK binding to each component of this receptor complex and the effects of other biologically relevant ligands, e.g. urokinase, on this binding have not been well delineated. It is known that both the heavy and light chains of HK interact with a region of cytokeratin 1 coded by exon 1 (2Shariat-Madar Z. Mahdi F. Schmaier A.H. J. Biol. Chem. 1999; 274: 7137-7145Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Antibody to this region completely inhibits HK binding to cytokeratin 1 as well as to the receptor complex (6Mahdi F. Shariat-Madar Z. Figueroa C.D. Schmaier A.H. Blood. 2001; 97: 2342-2350Crossref PubMed Scopus (109) Google Scholar). Likewise, some antibodies to gC1qR and uPAR completely block HK binding to the proteins individually as well as when they are part of the complex expressed on endothelial cells (6Mahdi F. Shariat-Madar Z. Figueroa C.D. Schmaier A.H. Blood. 2001; 97: 2342-2350Crossref PubMed Scopus (109) Google Scholar, 8Mahdi F. Shariat-Madar Z. Schmaier A.H. J. Biol. Chem. 2003; 278: 43983-43990Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). However, it is unclear whether each component of the cellular complex recognizes discrete or overlapping portions of HK and whether each molecule of HK binds to more than one component of the complex at the same time. To begin to address these issues, we sought to identify the regions in HK and uPAR required for binding. The results indicate the existence of multiple potential sites of interaction between this ligand and receptor and provide insight into the assembly of HK on its multireceptor complex and the influence of urokinase on this interaction. Proteins and Materials—Single-chain and two-chain HK (specific activity of 13-17 units/mg and 17 units/ml, respectively) in 4 mm sodium acetate-HCl and 0.15 m NaCl, pH 5.3, plasma kallikrein, and PK (specific activity of 22-27 units/mg) in 4 mm sodium acetate-HCl and 0.15 m NaCl, pH 5.3, were purchased from Enzyme Research Laboratories, Inc. (South Bend, IN). Carboxymethylated papain was prepared as previously reported (11Meloni F.J. Schmaier A.H. J. Biol. Chem. 1991; 266: 6786-6794Abstract Full Text PDF PubMed Google Scholar). 56-kDa (56HKa) and 46-kDa (46HKa) kallikrein-cleaved HK was prepared by dialyzing 1 mg of single-chain HK into HEPES carbonate buffer (137 mm NaCl, 3 mm KCl, 12 mm NaHCO3, 14.7 mm HEPES, 5.5 mm dextrose containing 2 mm CaCl2, and 1 mm MgCl2) pH 7.4. After dialysis, plasma kallikrein in a molar ratio of 1:200 to HK was added (12Meloni F.J. Gustafson E.J. Schmaier A.H. Blood. 1992; 79: 1233-1244Crossref PubMed Google Scholar). The mixture was incubated at 37 °C for various amounts of time, and the reaction was stopped by adding SDS-PAGE sample buffer. Aliquots from each time point were analyzed by SDS-PAGE. Once the optimal time of incubation to generate 56- and 46-kDa HK was determined, larger amounts were incubated at the appropriate molar ratios, and the reactions were stopped by adding 1 mm diisopropyl fluorophosphate. The cleaved HK then was dialyzed against HEPES carbonate buffer, pH 7.4, to remove the diisopropyl fluorophosphate. The integrity of the cleaved HK was analyzed on a 8% SDS-PAGE, and the protein concentration was estimated using the Bio-Rad protein assay. A biotinylation kit and ImmunoPure streptavidin horseradish peroxidase dihydrochloride (3,3′,5,5′-tetramethylbenzidine dihydrochloride) were supplied by Pierce. Prestained low molecular mass standard, nitrocellulose, and polyacrylamide were purchased from Bio-Rad. SDS gel electrophoresis samples were stained with Coomassie Brilliant Blue R-250 (Bio-Rad). cDNA encoding full-length uPAR was generously provided by Dr. F. Blasi (Copenhagen, Denmark). Peptides and Antibodies—The peptides corresponding to domains 3-5 of HK (see Table I) were synthesized at the Protein and Carbohydrate Structure Facility of the University of Michigan (Ann Arbor, MI) as previously reported (13Herwald H. Hasan A.A.K. Godovac-Zimmermann J. Schmaier A.H. Muller-Esterl W. J. Biol. Chem. 1995; 270: 14634-14642Abstract Full Text Full Text PDF PubMed Google Scholar, 14Hasan A.A.K. Cines D.B. Zhang J. Schmaier A.H. J. Biol. Chem. 1994; 269: 31822-31830Abstract Full Text PDF PubMed Google Scholar, 15Hasan A.A.K. Cines D.B. Herwald H. Schmaier A.H. Muller-Esterl W. J. Biol. Chem. 1995; 270: 19256-19261Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Sequential and overlapping peptides corresponding to each domain of suPAR were synthesized at Multiple Peptide Systems (San Diego, CA) (see Table II). All of the peptides from suPAR are numbered based upon the full-length sequence including the 22-amino acid signal peptide of uPAR. The peptides used were colorless, odorless, and >95% pure as determined by reverse phase high pressure liquid chromatography and mass spectrometry. A monoclonal antibody against uPAR (3B10FC) was generously provided by Dr. Robert F. Todd III from the University of Michigan (Ann Arbor, MI) (16Mizukami I.F. Garni-Wagner B.A. DeAngelo M. Flint A. Lawrence D.A. Cohen R.L. Todd III, R.F. Clin. Immunol. Immunopathol. 1994; 71: 96-104Crossref PubMed Scopus (30) Google Scholar). Monoclonal anti-uPAR antibodies E180 and E33 were the kind gifts of Dr. A. Mazar (Attenuon, San Diego, CA).Table IKininogen peptides and their influence to inhibit HK/46HKa binding to suparPeptideaEach peptide is named for the first three letters of the sequence using the single-letter amino acid code followed the number of amino acids in the sequence.SequencebEach letter represents the single-letter amino acid code.PositioncThe numbers represent the amino acids position of the mature HK without its signal sequence (14, 15).HK domainIC50dThe values given represent the micromolar IC50 values of biotin-HK or biotin-46HKa binding to suPAR by each of the peptides.Biotin-HKBiotin-46HKaμmKIC11KICVGCPRDIP244-2543No inhibitionNo inhibitionNAT26NATFYFKIDNVKKARVQVVAGKKYFI276-30132020LDC27LDCNAEVYVVPWEKKIYPTVNCOPLGM331-35732030CNA13CNAEVYVVPWEKK333-34531510GKE19GKEQGHTRRHDWGHEKQRK402-4205300HNL21HNLGHGHKHERDQGHGHQRGH421-44152010GHG19GHGLGHGHEQQHGLGHGHK440-458530HVL24HVLDHGHKHKHGHGHGKHKNKGKK471-49450.70.5HKH20HKHGHGHKHKNKGKKNGKH479-4985220a Each peptide is named for the first three letters of the sequence using the single-letter amino acid code followed the number of amino acids in the sequence.b Each letter represents the single-letter amino acid code.c The numbers represent the amino acids position of the mature HK without its signal sequence (14Hasan A.A.K. Cines D.B. Zhang J. Schmaier A.H. J. Biol. Chem. 1994; 269: 31822-31830Abstract Full Text PDF PubMed Google Scholar, 15Hasan A.A.K. Cines D.B. Herwald H. Schmaier A.H. Muller-Esterl W. J. Biol. Chem. 1995; 270: 19256-19261Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar).d The values given represent the micromolar IC50 values of biotin-HK or biotin-46HKa binding to suPAR by each of the peptides. Open table in a new tab Table IISequential and overlapping synthetic peptides from uPAR and their influence on biotin-HK binding to suPAR or endothelial cellsPeptideaEach peptide is named for the first three letters of the sequence using the single-letter amino acid code followed the number of amino acids in the sequence.SequencebEach letter represents the single-letter amino acid code.PositioncThe numbers represent the amino acids position of the sequence of full-length urokinase plasminogen activator receptor including the signal sequence.uPAR domainIC50dThe values represent the micromolar IC50 values of biotin-HK binding to suPAR or endothelial cells (HUVEC) by each of the peptides prepared from the sequence of uPAR.suPARHUVECμmDLC20DLCNQGNSGRAVTYSRSRYI96-115120ECI20ECISCGSSDMSCERGRHQSL116-1352>300>300QCR20QCRSPEEQCLDVVTHWIQEG136-1552100>300EEG20EEGRPKDDRHLRGCGYLPGC156-1752100>300LRG20LRGCGYLPGCPGSNGFHNND166-18520.97YLP20YLPGCPGSNGFHNNDTFHFL171-19022025PGS20PGSNGFHNNDTFHFLKCCNT176-19521.840FHN20FHNNDTFHFLKCCNTTKCNE181-200214TKC19TKCNEGPILELENLPQNGR196-2142>300>300QCY21QCYSCKGNSTHGCSSEETFLI215-235312DCR20DCRGPMNQCLVATGTHEPKN236-255322TAS20TASMCQHAHLGDAFSMNHID265-2843>300VSC20VSCCTKSGCNHPDLDVQYRS285-3043>300a Each peptide is named for the first three letters of the sequence using the single-letter amino acid code followed the number of amino acids in the sequence.b Each letter represents the single-letter amino acid code.c The numbers represent the amino acids position of the sequence of full-length urokinase plasminogen activator receptor including the signal sequence.d The values represent the micromolar IC50 values of biotin-HK binding to suPAR or endothelial cells (HUVEC) by each of the peptides prepared from the sequence of uPAR. Open table in a new tab Preparation of Wild Type and Mutant suPAR—Soluble urokinase plasminogen activator receptor (suPAR), isolated domains of suPAR (1Hasan A.A.K. Zisman T. Schmaier A.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3615-3620Crossref PubMed Scopus (162) Google Scholar, 2Shariat-Madar Z. Mahdi F. Schmaier A.H. J. Biol. Chem. 1999; 274: 7137-7145Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 3Herwald H. Dedio J. Kellner R. Loos M. Muller-Esterl W. J. Biol. Chem. 1996; 271: 13040-13047Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar), a fragment containing recombinant soluble suPAR domains 1 and 2 and 3 (S-D1, S-D2D3) and scuPA were prepared and expressed using the Drosophila Expression System (Invitrogen, Carlsbad, CA) according to manufacturer's instructions, as described previously (17Bdeir K. Kuo A. Mazar A. Sachais B.S. Xiao W. Gawlak S. Harris S. Higazi A.A. Cines D.B. J. Biol. Chem. 2000; 275: 28532-28538Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 18Bdeir K. Kuo A. Sachais B.S. Rux A.H. Bdeir Y. Mazar A. Higazi A.A.R. Cines D.B. Blood. 2003; 102: 3600-3608Crossref PubMed Scopus (56) Google Scholar). Isolated domains 1,2, and 3 of suPAR were prepared from wild type protein by sequential digestion with chymotrypsin and pepsin as previously reported (19Higazi A.A. Mazar A. Wang J. Quan N. Griffin R. Reilly R. Henkin J. Cines D.B. J. Biol. Chem. 1997; 272: 5348-5353Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Mutagenesis of suPAR in pMT/Bip/V5 (Invitrogen) was performed with the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) (17Bdeir K. Kuo A. Mazar A. Sachais B.S. Xiao W. Gawlak S. Harris S. Higazi A.A. Cines D.B. J. Biol. Chem. 2000; 275: 28532-28538Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). The suPAR domain 2 and 3 mutants are shown in Table III. Wild type and variant suPARs were purified from the media using a monoclonal (E180 or E33) anti-uPAR antibody affinity column. Wild type scuPA was purified from the media using a monoclonal anti-uPA antibody column and was analyzed by SDS-PAGE and Western blot (17Bdeir K. Kuo A. Mazar A. Sachais B.S. Xiao W. Gawlak S. Harris S. Higazi A.A. Cines D.B. J. Biol. Chem. 2000; 275: 28532-28538Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). All suPAR mutants are numbered based upon the full-length sequence of suPAR including the signal peptide.Table IIIsuPAR mutantsMutantSequenceaEach peptide is named for the first three letters of the sequence using the single-letter amino acid code followed the number of amino acids in the sequence. Each letter represents the single-letter amino acid code. The numbers represent the amino acid positions of the mature uPAR that includes the amino acids of the signal sequence.Domain 2Wild type174PGSNGFHNNDTFHPLKCCNT195H182A,N184A176PGSNGFANADTFHPLKCCNT195H182A,D185A176PGSNGFANNATFHPLKCCNT195N184Q174PGSNGFHNQDTFHPLKCCNT195Domain 3Wild type215QCYSCKGNSTHGCSSEETFLI235N222Q215QCYSCKGQSTHGCSSEETFLI235E230A,E231A215QCYSCKGNSTHGCSSAATFLI235a Each peptide is named for the first three letters of the sequence using the single-letter amino acid code followed the number of amino acids in the sequence. Each letter represents the single-letter amino acid code. The numbers represent the amino acid positions of the mature uPAR that includes the amino acids of the signal sequence. Open table in a new tab Gel Electrophoresis and Immunoblot Analysis—The proteins were separated using 8-15% acrylamide SDS-PAGE and then transferred to nitrocellulose membranes at 100 volts for 1 h. The electroblots then were incubated in blocking buffer (5% (w/v) dry milk, 0.05% Tween 20, 0.15 m NaCl, and 20 mm Tris-HCl, pH 7.4) for 1 h (20Schmaier A.H. Farber A. Schein R. Sprung C. J. Lab. Clin. Med. 1988; 112: 182-192PubMed Google Scholar). The membranes were then incubated with monoclonal antibody to uPAR diluted at 1:100 for 1 h. The membranes were washed and incubated with horseradish peroxidase-conjugated sheep anti-mouse (1:2000) for 1 h, and antibody binding was detected with an ECL system from Amersham Biosciences. All of the steps were carried out at room temperature. Endothelial Cell Culture—Human umbilical vein endothelial cells (HUVEC), endothelial cell growth medium, trypsin-EDTA, and trypsin neutralizing solutions were purchased from Clonetics (San Diego, CA). The cells were cultured according to the manufacturer's recommendations. The cells between the first and fifth passages were subcultured onto fibronectin-coated, 96-well plates 24 h prior to the start of the experiment (14Hasan A.A.K. Cines D.B. Zhang J. Schmaier A.H. J. Biol. Chem. 1994; 269: 31822-31830Abstract Full Text PDF PubMed Google Scholar). Cell viability was determined using trypan blue exclusion. The cell numbers were determined by direct counting on a hemocytometer. Biotinylation and Iodination—Five mg of HK in 200 μl was dialyzed against 0.01 m sodium phosphate, 0.15 m NaCl, pH 7.4 (14Hasan A.A.K. Cines D.B. Zhang J. Schmaier A.H. J. Biol. Chem. 1994; 269: 31822-31830Abstract Full Text PDF PubMed Google Scholar). A 5-fold molar excess of sulfo-NHS-LC-Biotin was added. After incubation for 2 h on ice, the sample was loaded onto 10-ml Econo-Pac 10 DG column (Bio-Rad). Biotinylated HK (biotin-HK) was monitored by absorbance at 280 nm using an extinction coefficient of 7.0 for HK and a protein assay (Bio-Rad). Biotin-HK had a specific activity of 13 units/mg. 46HKa was biotinylated in the same way. Peptides PGS20, HKH20, CNA13, QCY21, and DCR20 were synthesized with biotinylated labels at Multiple Peptide Systems. suPAR and HK were radiolabeled with Na125I using Iodogen precoated tubes (Pierce) at a ratio of 100 μCi of 125I/100 μg of protein to avoid oxidative injury (17Bdeir K. Kuo A. Mazar A. Sachais B.S. Xiao W. Gawlak S. Harris S. Higazi A.A. Cines D.B. J. Biol. Chem. 2000; 275: 28532-28538Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Free 125I was removed by gel filtration using Sephadex G25 (PD-10; Amersham Biosciences). Biotin-Protein or -Peptide Binding Studies—Biotinylated proteins or peptides in the absence or presence of competing proteins or peptides in HEPES carbonate buffer, pH 7.4 (8Mahdi F. Shariat-Madar Z. Schmaier A.H. J. Biol. Chem. 2003; 278: 43983-43990Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar), were added to microtiter plates coated with monolayers of HUVEC or with other proteins or peptides (1 μg/ml) coated by an overnight incubation at 4 °C in 0.1 m sodium carbonate, pH 9.6. After blocking the wells with 1% gelatin, the cuvettes were incubated with biotinylated HK, 46HKa, or peptides for 1 h at 37 °C. The wells were washed three times with HEPES carbonate buffer, pH 7.4. The bound biotinylated proteins or peptides were measured using ImmunoPure streptavidin horseradish peroxidase conjugate (Pierce) and peroxidase-specific fast reacting substrate, 3,3′,5,5′-tetramethylbenzidine dihydrochloride (Pierce) by measuring the absorbance of the reaction mixture at 450 nm using a microplate autoreader EL 311 (Bio-Tek Instrument, Winooski, VT), as previously described (14Hasan A.A.K. Cines D.B. Zhang J. Schmaier A.H. J. Biol. Chem. 1994; 269: 31822-31830Abstract Full Text PDF PubMed Google Scholar, 21Hasan A.A.K. Cines D.B. Ngaiza J.R. Jaffe E.A. Schmaier A.H. Blood. 1995; 85: 3134-3143Crossref PubMed Google Scholar). Binding of Iodinated HK to suPAR—suPAR was immobilized onto Immulon 4HB plates (Thermo Labs Systems, Franklin, MA) in 0.01 m sodium phosphate, 0.15 m NaCl, pH 7.4 overnight at 4 °C at a concentration of 1 μg/ml. Binding of 125I-HK to suPAR was measured in the presence of varying concentrations of unlabeled HK in HEPES carbonate buffer. 125I-suPAR binding to Chinese hamster ovary cells expressing scuPA was performed as previously reported (17Bdeir K. Kuo A. Mazar A. Sachais B.S. Xiao W. Gawlak S. Harris S. Higazi A.A. Cines D.B. J. Biol. Chem. 2000; 275: 28532-28538Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Prekallikrein Activation on Endothelial Cells—The ability of peptides from suPAR to block PK activation on endothelial cells was determined. HUVEC were grown as monolayers in microtiter plate wells. HK and PK (20 nm each) were then added to the wells in the absence or presence of 100 μm peptide DLC20, LRG20, YLP20, PGS20, FHN20, TKC19, QCY21, or DCR20 from domains 1-3 of suPAR (see Table II) in HEPES carbonate buffer, pH 7.4, for 1 h at 37 °C. After washing three times with HEPES carbonate buffer, pH 7.4, 0.4 mm H-D-Pro-Phe-Arg-pNA was added, and hydrolysis of the substrate was measured over the next 1 h at 405 nm (22Motta G. Rojkjaer R. Hasan A.A.K. Cines D.B. Schmaier A.H. Blood. 1988; 91: 516-528Crossref Google Scholar). Characterization of HK Species—One μg of HK, 46HKa, and 56HKa was analyzed using 8% SDS-PAGE under reduced and nonreduced conditions. Under reduced conditions, HK, 56HKa, and 46HKa migrated predominantly at 120 kDa, at 64 and 56 kDa, and at 64 and 46 kDa, respectively (Fig. 1A). These results indicate that 56HKa and 46HKa retained their heavy chains and their respective light chains. Under nonreduced conditions, HK, 56HKa and 46HKa migrated as single bands at 120, 110, and 108 kDa, respectively (Fig. 1B), consistent with previous reports of a change in the molecular mass of plasma HK when it is activated by plasma kallikrein (20Schmaier A.H. Farber A. Schein R. Sprung C. J. Lab. Clin. Med. 1988; 112: 182-192PubMed Google Scholar). Binding of Biotin-HK to HUVEC and suPAR—We then determined whether these forms of HK differed in their affinity for HUVEC or for suPAR. Initial investigations were with HUVEC. Native HK, 56HKa, and 46HKa blocked the binding of biotinylated HK to HUVEC with IC50 values of 115, 115, and 50 nm, respectively (Fig. 2A). Native HK, 56HKa, and 46HKa blocked biotin-HK binding to suPAR with IC50 values of 60, 110, and 8 nm, respectively (Fig. 2B). These data indicate that 46HKa is a more potent inhibitor of the binding of native HK to HUVEC and suPAR than are the less activated forms of HK. This outcome was consistent with previously reported information that kallikrein-cleaved HK bound more avidly to uPAR than did intact HK (5Colman R.W. Pixley R.A. Najamunnisa S. Yan W. Wang J. Mazar A. McCrae K.R. J. Clin. Invest. 1997; 100: 1481-1487Crossref PubMed Scopus (206) Google Scholar). The data also demonstrated that native HK also bound to suPAR, albeit with lower affinity than its cleaved counterparts. This information suggested that cleavage of HK enhanced its binding to suPAR. This increase in the affinity of binding for 46HKa was less pronounced on HUVEC, which express additional binding sites for the kininogens. Binding Sites for suPAR within Domain 5 of HK—It has been shown in previous studies that HK bound to cells through determinants within both its light and heavy chains (13Herwald H. Hasan A.A.K. Godovac-Zimmermann J. Schmaier A.H. Muller-Esterl W. J. Biol. Chem. 1995; 270: 14634-14642Abstract Full Text Full Text PDF PubMed Google Scholar, 15Hasan A.A.K. Cines D.B. Herwald H. Schmaier A.H. Muller-Esterl W. J. Biol. Chem. 1995; 270: 19256-19261Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Therefore, we next sought to identify the region(s) in domain 5 of HK that bound to suPAR and inhibited the binding of native HK and 46HKa (13Herwald H. Hasan A.A.K. Godovac-Zimmermann J. Schmaier A.H. Muller-Esterl W. J. Biol. Chem. 1995; 270: 14634-14642Abstract Full Text Full Text PDF PubMed Google Scholar, 14Hasan A.A.K. Cines D.B. Zhang J. Schmaier A.H. J. Biol. Chem. 1994; 269: 31822-31830Abstract Full Text PDF PubMed Google Scholar, 15Hasan A.A.K. Cines D.B. Herwald H. Schmaier A.H. Muller-Esterl W. J. Biol. Chem. 1995; 270: 19256-19261Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Binding of biotin-HK to suPAR was blocked by peptides GKE19, HNL21, GHG19, HKH20, and HVL24 with IC50 values of 300, 20, 30, 2, and 0.7 μm, respectively (Table I). HKH20 was a weaker inhibitor of biotin-46HKa binding (Table I). Peptides HKH20, HNL21, and HVL24 inhibited biotin-46HKa binding to suPAR with IC50 values of 20, 10, and 0.5 μm, respectively (Table I). These data indicate that the HVL24 region corresponding to amino acids 471-494 of domain 5 of HK has the highest affinity for suPAR, although other portions of domain 5 also had the capacity to bind. Binding Sites for suPAR within Domain 3 of HK—A similar investigation was performed with peptides within the cell-binding region in domain 3 of HK (13Herwald H. Hasan A.A.K. Godovac-Zimmermann J. Schmaier A.H. Muller-Esterl W. J. Biol. Chem. 1995; 270: 14634-14642Abstract Full Text Full Text PDF PubMed Google Scholar) (Table I). Peptides NAT26, LDC27, and CNA13 inhibited biotin-HK binding to suPAR with IC50 values of 20, 20, and 15 μm, respectively (Table I), whereas peptide KIC11 from the amino-terminal end of this domain was inactive (Table I). The protein CM-papain, a domain 3-binding protein, inhibited the binding of biotin-HK to suPAR with an IC50 values of 1.5 μm (data not shown). NAT12, LDC27, and CNA13 inhibited biotin-46HKa binding to suPAR with IC50 values of 20, 30, and 10 μm, respectively (Table I). CM-papain was an equipotent inhibitor of biotin-46HKa and biotin-HK binding (IC50 = 1.5 μm) (data not shown). Taken together, these data suggested that HK bound to suPAR through regions found on both its heavy and light chains, although the binding of suPAR to domain 5 peptides was at least 10-fold more avid (compare the IC50 value of HVL24 with CNA13). This conclusion was supported by the finding that biotin-HKH20 and biotin-CNA13 bound specifically to suPAR (Fig. 3). Binding Sites for HK in suPAR—We next investigated the binding sites in suPAR for HK. As a first step, the epitope recognized by a monoclonal anti-uPAR antibody (3B10FC) that blocked HK binding to cultured endothelial cells (6Mahdi F. Shariat-Madar Z. Figueroa C.D. Schmaier A.H. Blood. 2001; 97: 2342-2350Crossref PubMed Scopus (109) Google Scholar, 16Mizukami I.F. Garni-Wagner B.A. DeAngelo M. Flint A. Lawrence D.A. Cohen R.L. Todd III, R.F. Clin. Immunol. Immunopathol. 1994; 71: 96-104Crossref PubMed Scopus (30) Google Scholar) was partially mapped. On immunoblot, 3B10FC bound to suPAR domain 2 and to fragments containing domains 2 + 3 (D2D3 and S-D2D3); 3B10FC bound weakly to D3, but not to D1, under the same experimental conditions (Fig. 4). These data are consis