©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mapping the Cell Binding Site on High Molecular Weight Kininogen Domain 5 (*)

(Received for publication, February 9, 1995; and in revised form, June 9, 1995)

Ahmed A. K. Hasan (1) Douglas B. Cines (2) Heiko Herwald (3) Alvin H. Schmaier (1)(§) Werner Müller-Esterl (3)

From the  (1)Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan 48109-0724, the (2)Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, and the (3)Institute of Physiological Chemistry and Pathobiochemistry, Johannes Gutenberg University, Mainz, Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Investigations mapped the region(s) on the light chain of high molecular weight kininogen (HK) that participates in cell binding. Sequential and overlapping peptides of domain 5 (D5(H)) were synthesized to determine its cell binding site(s). Three peptides from non-overlapping regions on D5(H) were found to inhibit biotin-HK binding to endothelial cells. Peptides GKE19 and HNL21 weakly inhibited biotin-HK binding with IC of 792 and 215 µM, respectively. Peptide HKH20 inhibited biotin-HK binding with an IC of 0.2 µM. Two peptides, GGH18 and HVL24, which overlapped HKH20, also inhibited biotin-HK binding to endothelial cells with IC values of 108 and 0.8 µM, respectively. Biotinylated HKH20 directly bound to endothelial cells. HK and HKH20 bound at or near the same site on endothelial cells because HK inhibited biotin-HKH20 binding (IC = 0.2 µM). A polyclonal anti-HKH20 antibody also blocked biotin-HK binding. Peptides HKH20 and HVL24 and anti-HKH20 antibody also inhibited the procoagulant activity of plasma HK. These data indicated that the cell and artificial surface binding sites on D5(H) overlap. The orientation of HK on endothelial cells may be critical for the assembly and activation of contact system enzymes and the expression of kininogen's anti-thrombin activity.


INTRODUCTION

Plasma high molecular weight kininogen (HK) (^1)is a multifunctional protein. It is a parent protein of bradykinin and serves as a cofactor for factor XI and prekallikrein assembly on biologic membranes(1, 2, 3, 4) . The docking of HK to platelet and endothelial cell membranes requires its binding by regions on both its heavy and light chains(5, 6, 7, 8) . Further investigations have shown that kininogen domain 3 (D3) contains the heavy chain cell binding site(s), while the carboxyl-terminal portion of bradykinin and the amino-terminal region of the common portion of kininogen light chain (domain 4 (D4)) subsumes another cell membrane interaction site(9, 10) .

It is of interest that HK and its related protein, low molecular weight kininogen (LK), have multiple touchdown sites for cell binding. Presumably, these binding sites orient the proteins for their biologic activities. In particular, the delivery of bradykinin to its receptors on endothelial cells is important to stimulate prostacyclin synthesis, superoxide formation, nitric oxide formation, tissue plasminogen activator secretion, and smooth muscle hyperpolarization factor liberation(11, 12, 13, 14, 15) . Further, placement of kininogens on platelets and endothelial cells blocks alpha-thrombin from binding and activating these cells(5, 9, 16, 17) . These activities contribute to the constitutive anticoagulant nature of cell membranes in the intravascular compartment. In a previous study, we demonstrated that monoclonal antibody HKL12 prolonged the activated partial thromboplastin time of normal plasma and blocked the procoagulant activity of HK(18) . The epitope of this antibody was mapped to the center portion of HK domain 5 (D5(H)). This antibody also inhibited the binding of radiolabeled HK to M protein on the surface of Streptococcus pyogenes bacteria(19) . Since D5(H) is known to be the artificial surface binding region(20, 21, 22) , finding that an antibody that inhibits its procoagulant activity also blocks HK binding to bacteria suggested that D5(H) also may contain the HK cell binding site. Using synthetic peptides, we have defined the specific sequences on D5(H) that participate in HK binding to endothelial cells.


EXPERIMENTAL PROCEDURES

Proteins

HK and LK were purified from plasma simultaneously using sequential carboxymethyl-papain-Sepharose and reactive blue-Sepharose affinity chromatography(10, 17) . Briefly, diisopropylfluorophosphate-treated fresh frozen plasma (100 ml) was thawed at 37 °C, and benzamidine-HCl (10 mM), polybrene (40 µg/ml), EDTA (2 mM), phenylmethylsulfonyl fluoride (PMSF) (0.2 mM), soybean trypsin inhibitor (0.2 mg/ml), aprotinin (100 units/ml), and NaCl (2 M) were added(23) . The plasma was applied to a carboxymethyl-papain-Sepharose 4B column (2.5 20 cm) that had been equilibrated with a mixture of 2 M NaCl, 1 mM benzamidine-HCl, 40 µg/ml polybrene, 0.2 mM PMSF, and 0.02% (w/v) NaN(3) in 50 mM phosphate buffer, pH 7.5(24) . After the column was washed with equilibration buffer, HK and LK were eluted in a single peak by the addition of a solution containing 2 mM EDTA and 0.02% (w/v) NaN(3) in 50 mM phosphate buffer, pH 11.5. 5-ml fractions were collected into tubes containing 0.25 ml of 4 mM PMSF in 1 M sodium acetate, pH 4.2, to give a final pH of 6.0. The fractions containing HK and LK were then applied to a reactive blue-Sepharose column (Sigma) equilibrated with 0.01 M sodium acetate, pH 6.8. Bound LK and HK were eluted using the same buffer containing 0.3 and 1 M NaCl, respectively. HK (120 kDa) and LK (66 kDa) migrated as single bands on reduced SDS-polyacrylamide gel electrophoresis (PAGE). HK reacted with monoclonal antibodies to its heavy and light chains by enzyme-linked immunosorbent assay and Western blotting, while LK was recognized only by antibodies directed to the common heavy chain but not to the HK light chain. Purified HK retained its procoagulant activity and had a specific activity of 12-20 units/mg (see below).

Peptides

Peptides of D5(H) were synthesized both in the Proteinchemisches Zentrallabor of the Johannes Gutenberg University (Mainz, Germany) and in the Protein and Carbohydrate Structure Facility of the University of Michigan (Ann Arbor, MI). The peptides were made by solid-phase synthesis using FMOC (N-(9-fluorenyl)methoxycarbonyl) moiety chemistry. All peptides were purified by preparative reverse-phase high performance liquid chromatography. The purified peptides were characterized by analytical high performance liquid chromatography, amino acid analysis, and mass spectrometry. All synthesized peptides had greater than 99% correlation between their formula weight and their molecular weight determined by mass spectrometry. On reverse-phase high performance liquid chromatography, geq91.3% of each synthesized peptide was within a peak of its expected molecular weight. Each peptide was colorless, odorless, and soluble in water. In this manuscript, each peptide is named using the one-letter code of the first three amino acid residues followed by the number of amino acids in the peptide.

Antibodies

Monoclonal antibodies HKL12, which is directed to D5(H), and HKL16, which is directed to HK domain 6 (D6(H)), were purified from ascites by protein A affinity chromatography(18) . The purified antibodies were quantified by absorbance at 280 nm using an extinction coefficient (E) of 14 or by the dye-binding assay of Bradford(25) . The F(ab`)(2) fragment of monoclonal antibody HKL12 was prepared by pepsin cleavage (1:50 pepsin to antibody ratio (wt:wt)) in 0.1 M sodium acetate, pH 4.5, for 24 h. The reaction was stopped by raising the pH to 8.0 with 2.5 M Tris. The F(ab`)(2) fragments were separated from their Fc portions by affinity chromatography using protein A(26) . A polyclonal rabbit antibody to peptide HKH20 was prepared by covalently coupling peptide HKH20 to a carrier protein, key hole limpet hemocyanin, using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimidebulletHCl (27) with minor modifications(28) . The resultant conjugates were used to immunize rabbits by standard procedures(29) . Antisera to peptide conjugates were tested by an indirect enzyme-linked immunosorbent assay using free peptides (1 µg/ml) or HK (1 µg/ml) as the coating antigen. Immunoselection of specific antibodies was performed by affinity chromatography on peptides covalently coupled to Affi-Gel 10 (4 mg of peptide/ml of gel). The immunoselected antibodies appeared as pure immunoglobulin heavy and light chains on SDS-PAGE under reducing conditions.

Biotinylation of HK and Peptide HKH20

2 mg of lyophilized HK was mixed in 1 ml of 0.01 M sodium phosphate, pH 7.2, containing 0.25 mM NHS-LC-biotin (Pierce) for 30 min at room temperature. The reaction mixture was added to a 10-ml desalting column (Econo-Pac 10DG, Bio-Rad), preequilibrated with a buffer containing 0.1 M sodium phosphate, 0.15 M NaCl, and 0.1 mM phenylmethylsulfonyl fluoride, pH 7.2. The protein concentration in each fraction was determined from the absorbance at 280 nm using an extinction coefficient of 7.0 for HK (24) . Incorporation of biotin into HK was determined by the 2-(4`-hydroxyazo-benzene) benzoic acid assay(30) , performed according to the manufacturer's instructions (Pierce). Each molecule of HK was labeled with two to three molecules of biotin (biotin-HK) without loss of either its procoagulant or antigenic properties. Biotinyl-glycyl-HKHGHGHGKHKNKGKKNGKH (biotin-HKH20) was synthesized by the Protein and Carbohydrate Structure Facility at the University of Michigan (Ann Arbor, MI) and was purified and analyzed as described above. The peptide was odorless, colorless, and water soluble. Each molecule of peptide contained one molecule of biotin at its amino terminus.

Functional and Immunochemical Assays

HK procoagulant activity was measured using a one-stage, kaolin-activated coagulant assay with a commercial-activated partial thromboplastin time reagent (Organon Technika, Durham NC) (23) with total kininogen-deficient plasma as substrate. This plasma was donated to this laboratory by the late Mayme Williams (Philadelphia, PA). HK and biotin-HK samples were compared to a daily standard curve using pooled normal human plasma diluted 1/10 to 1/1,000 with 0.01 M Tris, 0.15 M NaCl at pH 7.4. 1 unit of activity was defined as that amount of HK procoagulant activity in 1 ml of pooled normal plasma. In certain experiments, investigations were performed to determine if synthetic peptides of D5(H) or D6(H) interfered with the procoagulant activity of HK in normal plasma. In these examinations, the peptides were added to pooled normal plasma (George King, Inc., Overland Park, KS, lot 313D) at three final concentrations (500, 250, and 100 µM), and the residual procoagulant activity using an activated partial thromboplastin time was measured. In other experiments, purified anti-HKH20 antibody or rabbit IgG were incubated with pooled normal human plasma in 1.25- to 18-fold molar excess antibody to plasma HK concentration (670 nM), and the residual plasma HK procoagulant activity was measured. The concentrations of purified HK and biotin-HK were determined by radial immunodiffusion(23, 31) , by their absorbance at 280 nm using an extinction coefficient of 7.0 for HK(24) , and by the Bradford method (25) with identical results.

Endothelial Cells

Cultures of human umbilical vein endothelial cells (HUVEC) were established as previously described (32, 33, 34, 35) . HUVEC were passaged two to four times in medium 199 (Life Technologies, Inc.) supplemented with 10% heat-inactivated fetal calf serum (Flow Laboratories, McLean, VA), penicillin-streptomycin (Life Technologies, Inc.), and endothelial cell growth factor (34) and grown to confluence on fibronectin-coated 96-well microtiter plates (Nunclon, product of Nunc Intermed, Denmark and procured from Thomas Scientific, Swedsboro, NJ). HUVEC were always used within 24 h of becoming confluent.

Binding of Biotin-HK and Biotin-HKH20 to HUVEC

All binding experiments were performed at 37 °C. HUVEC grown to confluence on 96-well microtiter plates (4 10^4 cells/well) were washed five times in HEPES-Tyrode's binding buffer. The cells were incubated with 20 nM biotin-HK in HEPES-Tyrode's buffer containing 50 µM Zn at 37 °C for various times up to 3 h to achieve equilibrium. Nonspecific binding, unless stated otherwise, was determined by measuring binding in the presence of 50-fold molar excess unlabeled HK or in the absence of 50 µM Zn with equivalent results (10) . Specific binding was determined by subtracting nonspecific binding from total binding. To determine whether a D5(H) synthetic peptide could bind directly to endothelial cells, increasing concentrations of biotin-HKH20 were incubated with HUVEC in the absence or presence of 100-fold molar excess unlabeled peptide in HEPES-Tyrode's buffer containing 50 µM of Zn at 37 °C for 60 min. To exclude proteolytic degradation of the peptides during the time of the binding experiments, simultaneous experiments were performed under the above conditions except that 0.1 mM captopril, 0.1 mM bacitracin, 0.01 mM phosphoramidon, and 0.2 mM PMSF were added. The stability of some of the peptides also was examined by SDS-PAGE. Biotin-HKH20 and biotin-HNL21 were incubated for 3 h at 37 °C over endothelial cells and then electrophoresed on a 20% SDS-PAGE. After transfer to nitrocellulose, they were compared for their structural integrity with the same peptides that were not incubated over the cells.

Cell-associated biotin-HK or biotin-HKH20 was measured using ImmunoPure streptavidin horseradish peroxidase conjugate (Pierce) and peroxidase-specific fast-reacting substrate, 3,3`,5,5`,tetramethylbenzidine dihydrochloride (turbo-TMB, Pierce). To do this, the cells were washed three times with HEPES-Tyrode's buffer containing 50 µM Zn and incubated with 100 µl of streptavidin-horseradish peroxidase conjugate (1:500) in binding buffer containing 50 µM Zn for an additional hour at room temperature. The cells were then quickly washed five times with 0.01 M sodium phosphate, 0.15 M NaCl, pH 7.4, and the substrate, turbo-TMB (100 µl), was added for 5 min at room temperature. The color reaction was stopped by adding 1 M phosphoric acid (100 µl), and bound biotinylated protein was quantified by measuring the absorbance of the reaction mixture at 450 nm using a Microplate auto reader EL 311 (Bio-Tek Instrument, Winooski, VT).


RESULTS

The Influence of Monoclonal Antibodies to D5 on HK Binding to Endothelial Cells

Investigations were performed with monoclonal antibodies to D5(H) and D6(H) to determine their influence on the binding of biotin-HK to endothelial cells. Monoclonal antibody HKL12 to D5(H)(18) , both as whole immunoglobulin and as a F(ab`)(2) fragment, inhibited biotin-HK from binding to endothelial cells (data not shown). Monoclonal antibody HKL12 at 400 nM inhibited 50% of binding of biotin-HK (20 nM). HKL16, a monoclonal antibody directed to the prekallikrein and factor XI binding site on D6(H) had little influence on biotin-HK binding to endothelial cells (data not shown). The finding that HKL12 partially inhibited the binding of HK to endothelial cells suggested that a cellular binding site for HK was on D5(H) similar to the HK binding site for M protein(19) . However, since the degree of the inhibition was incomplete, we surmised that this antibody and its F(ab`)(2) fragment bound to a site adjacent to the actual D5(H) cell binding site.

Synthetic Peptides of D5

With the knowledge that HKL12 partially inhibited biotin-HK binding to endothelial cells and that this antibody blocked D5(H) from binding to the surface of a bacterium(19) , a series of sequential and overlapping peptides to D5(H) were synthesized (Table 1). Peptides VSP21, GKE19, HNL21, GHG19, FKL20, and HKH20 sequentially spanned the entire amino acid sequence of D5(H) (Fig. 1, top). Peptides GGH18, HVL24, HKH10, GHG10, and KNK10 are peptides with overlapping sequences of HKH20 (Fig. 1, bottom).




Figure 1: Diagram of sequential and overlapping synthetic peptides of D5(H). The top of the figure shows the entire domain structure of HK. The middleportion contained within the dottedlines shows the sequence of peptides that span D5(H)(18) . The numbersabove and below the verticallines represent the amino acid location on the HK protein. The full sequence of each of the peptides is given in Table 1. The bottom of the figure shows peptides with overlapping sequences that span peptide HKH20.



Inhibition of Biotin-HK Binding to Endothelial Cells by Synthetic Peptides of D5

In our initial investigations, we sought to determine which of these peptides within D5(H) inhibited biotin-HK binding to endothelial cells and their relative potency (Fig. 2). Starting at the amino-terminal portion of D5(H), synthetic peptide VSP21 did not interfere with biotin-HK binding (Fig. 2B). Synthetic peptides GKE19 and HNL21 partially inhibited binding with an IC of 792 ± 116 and 215 ± 3 µM, respectively (Fig. 2A). Peptides GHG19 and FKL20 from the middle portion of D5(H) had no influence on biotin-HK binding to endothelial cells (Fig. 2, A and B). In contrast, peptide HKH20 from the carboxyl-terminal portion of D5(H) was a potent inhibitor of biotin-HK binding with an IC of 0.23 ± 0.02 µM (Fig. 2B). Overlapping peptides (GGH18, HVL24) of the HKH20 sequence were then synthesized to analyze this region in greater detail (Fig. 1, Table 1). Peptide GGH18 consisted of 10 additional residues on the amino terminus and lacked 12 carboxyl-terminal residues of HKH20. Peptide HVL24 had 8 additional residues at the amino terminus and was 4 residues shorter at the carboxyl terminus than HKH20. Both GGH18 and HVL24 inhibited biotin-HK binding with an IC of 108 ± 6 and 0.8 ± 0.09 µM, respectively. These data indicated that there were two candidate regions on D5(H), one of low affinity in the amino-terminal area and another of much higher affinity in the carboxyl-terminal portion, that can participate in HK cell binding. Since the apparent IC for the synthetic peptides from these two regions differed by 10^3, further investigations concentrated on HKH20, a peptide that bound to HUVEC with higher affinity.


Figure 2: Inhibition of biotin-HK binding to endothelial cells by synthetic peptides of D5(H). Biotin-HK (20 nM) in HEPES-Tyrode's buffer containing 50 µM Zn was incubated with endothelial cells in the absence or presence of increasing concentrations (0.1 µM to 1 mM) of various peptides within D5(H). The sequence of the peptides used in these experiments is given in Table 1. PanelA: GKE19, ; HNL21, bullet; GGH18, ; GHG19, ▴. PanelB: FKL20, ; HKH20, bullet; HVL24, ; VSP21, ▴. The percent bound biotin-HK shown was determined by comparing the binding of biotinylated ligand in the presence and absence of each peptide. The data shown are the mean ± S.E. of three independent experiments.



Binding of Biotin-HKH20 to Endothelial Cells

We then investigated if biotin-HKH20 would directly bind to endothelial cells (Fig. 3). Biotin-HKH20 specifically bound to endothelial cells when added at concentrations geq30 nM. At each concentration of added biotin-HKH20 (30 nM to 10 µM), a 100-fold molar excess of unlabeled HKH20 blocked binding (Fig. 3). Investigations were next performed to determine the specificity of biotin-HKH20 binding to endothelial cells (Fig. 4). HK was able to prevent biotin-HKH20 from binding to endothelial cells with an IC of 0.2 µM (Fig. 4A). These data indicated that HK and biotin-HKH20 bound at or near the same site on endothelial cells. The binding of biotin-HKH20 was highly specific. Peptides GHG19 from D5(H) and LDC27 from D3 (Table 1) both at 1 mM had no influence on biotin-HKH20 binding (Fig. 4A). Peptide GGH18, which overlapped the sequence of HKH20, inhibited biotin-HKH20 binding 40% at 100 µM (Fig. 4A). Further, peptides HKH20 and HVL24, the latter of which overlaps the amino-terminal portion of HKH20 by 16 residues, inhibited biotin-HKH20 binding equally with an IC of 1.0 µM (Fig. 4B). These studies showed that biotin-HKH20 was specifically displaced from the endothelial cell membrane only by peptides with overlapping sequences. Both HKH20 and HVL24 were stable for the duration of incubation with endothelial cells. When these peptides were incubated with endothelial cells in the presence of 0.1 mM captopril, 0.1 mM bacitracin, 0.01 mM phosphoramidon, and 0.2 mM PMSF, the IC values of these peptides on biotin-HKH20 binding were identical to that reported above for incubations performed in the absence of inhibitors (data not shown). Peptide stability during the incubation period with endothelial cells was examined further. When biotin-HKH20 was incubated with HUVEC for up to 3 h at 37 °C, there was no change in the apparent molecular mass of the incubated versus unincubated peptides as seen on electroblotted nitrocellulose of a 20% SDS-PAGE of the peptides (data not shown).


Figure 3: Direct binding of biotin-HKH20 to endothelial cells. Increasing concentrations of biotin HKH20 (1 nM to 10 µM) in HEPES-Tyrode's buffer containing 50 µM Zn was incubated with endothelial cells in the absence () or presence (bullet) of 100-fold molar excess unlabeled HKH20. The sequence of HKH20 is given in Table 1. The data presented are the mean ± S.E. of three experiments.




Figure 4: Inhibition of biotin-HKH20 binding to endothelial cells. PanelA, biotin-HKH20 (500 nM) in HEPES-Tyrode's buffer containing 50 µM Zn was incubated with endothelial cells in the presence of 0.1 nM to 3 µM HK () or GGH18 (bullet), LDC27 (), or GHG19 (▴). The percent bound biotin-HKH20 shown was determined by comparing the binding of biotin-HKH20 in the presence of HK or peptide competitors to that seen in the absence of the competitors. The data shown are the mean ± S.E. of three independent experiments for HK and single, representative experiments for each of the peptides. PanelB, biotin-HKH20 (500 nM) in HEPES-Tyrode's buffer containing 50 µM Zn was incubated with endothelial cells in the presence of increasing concentrations of HKH20 () or HVL24 (bullet). The percent bound biotin-HKH20 shown was determined by comparing the binding of biotin-HKH20 in the presence and absence of each peptide. The data shown are the mean ± S.E. for three experiments. The absence of error bars seen for some of the points indicates that the errors were very small.



Studies were performed to map further the cell binding region of HKH20 (Fig. 5). KNK10, which consists of the carboxyl-terminal 10 amino acids of HKH20, did not inhibit biotin-HKH20 from binding to endothelial cells. At 700 µM, GHG10, which consists of the middle 10 amino acids of HKH20, inhibited the binding of biotin-HKH20 by 50%. HKH10, which consists of the amino-terminal 10 amino acids of HKH20, inhibited biotin-HKH20 binding with an IC of 80 µM. Thus, these first 10 amino acids of HKH20 were 80-fold less potent inhibitors than HKH20 itself. Again, the presence of protease inhibitors (captopril, bacitracin, phosphoramidon, and PMSF) all at the same concentrations used above did not enhance inhibition of biotin-HKH20 binding by these peptides (data not shown). These data indicated that under the conditions of the binding experiments, these small peptides were stable as well.


Figure 5: Mapping of the HKH20 endothelial cell binding region. Biotin-HKH20 (500 nM) in HEPES-Tyrode's buffer containing 50 µM Zn was incubated with endothelial cells in the presence of 100 nM to 1 mM HKH20 (), HKH10 (bullet), KNK10 (), or GHG10 (▴). The percent bound biotin-HKH20 shown was determined by comparing the binding of biotin-HKH20 in the presence and absence of each competitor. The data shown are the mean ± S.E. of three experiments. The absence of error bars seen for some of the points indicates that the errors were very small.



Effect of Anti-HKH20 Peptide Antibody on HK Binding and Procoagulant Activity

Since peptide HKH20 had the highest inhibitory capacity, a rabbit anti-HKH20 antibody was produced. The anti-HKH20 antibody, unlike preimmune rabbit IgG, inhibited biotin-HK binding in a concentration-dependent fashion with an IC of 30 nM, i.e. a 3-fold molar excess antibody to biotin-HK (Fig. 6A). Since D5(H) contains the surface binding region for HK procoagulant activity(20, 21, 22) , we determined if the anti-HKH20 antibody also would inhibit HK procoagulant activity (Fig. 6B). Equimolar concentrations of preimmune rabbit IgG to anti-HKH20 antibody to plasma HK (670 nM) inhibited the procoagulant activity of HK <5% (data not shown). In contrast, anti-HKH20 antibody reduced plasma HK procoagulant activity in concentrations >3.6 µM (i.e. 5-fold molar excess above that of plasma HK). These data suggested that the binding site that comprised the HKH20 sequence in D5(H) was critical for the expression of HK procoagulant activity on artificial surfaces.


Figure 6: The influence of anti-HKH20 antibody on HK binding and procoagulant activity. PanelA, influence of polyclonal anti-HKH20 antibody from rabbit on biotin-HK binding to endothelial cells. Biotin-HK (10 nM) in HEPES-Tyrode's buffer containing 50 µM Zn was incubated with endothelial cells in the presence of 1-1000 nM anti-HKH20 antibody () or preimmune rabbit IgG (bullet). The percent of bound biotin-HK in the presence of each immunoglobulin is shown. The data shown are the mean ± S.E. of three independent experiments. PanelB, influence of anti-HKH20 antibody on plasma HK procoagulant activity. Normal human plasma, which contains HK at a concentration of 670 nM, was incubated 2 h at 37 °C with 1.25-18-fold molar excess (0.835-12 µM) preimmune IgG or purified anti-HKH20 antibody. One part of the antibody-treated normal plasma was incubated with 1 part of activated partial thromboplastin reagent, 1 part kaolin (10 mg/ml), and 1 part total kininogen-deficient plasma for 5 min followed by the addition of 1 part 30 mM calcium chloride. The time to clot formation was measured. The residual procoagulant activity in the antibody-treated plasma in each sample was measured by comparing the time to clot formation in the same assay to that of a 1/10 to 1/1000 dilution of normal human plasma. After the influence of an equal molar excess of preimmune rabbit IgG was subtracted, the fold reduction of plasma HK procoagulant activity was compared with the concentration of purified anti-HKH20 antibody incubated with plasma HK. The graph presented represent the mean ± S.E. of three experiments for 0.835-3.6 µM anti-HKH20 IgG and the means of triplicate determinations on the same day for 8.4-12 µM anti-HKH20 IgG.



Influence of D5 Peptides on HK Procoagulant Activity

Investigations were next performed to determine if the synthetic peptides themselves, which inhibited HK binding to endothelial cells, also interfered with HK procoagulant activity. As previously reported, peptide SDD31 of D6(H) blocked the procoagulant activity of plasma HK presumably by blocking prekallikrein and factor XI binding and activation on the artificial surfaces (36, 37, 38) . Both HKH20 and HVL24 at 500 µM also produced a greater than 2-fold inhibition in HK-mediated procoagulant activity in normal plasma, similar to SDD31 (Fig. 7). This inhibition of the coagulation time was concentration dependent over a range of 100-500 µM (data not shown). It was also specific to this region on D5(H). No other peptide that comprised any other portion of D5(H) inhibited HK procoagulant activity. The anticoagulant activity of HKH20 and HVL24 must have been dependent in part upon the size and affinity of the peptides because HKH10 at 500 µM did not block the procoagulant activity of HK in plasma (data not shown).


Figure 7: The influence of D5(H) and D6(H) peptides on HK procoagulant activity. Normal human plasma was incubated 5 min at room temperature with 500 µM GKE19, HNL21, GHG19, FKL20, GGH18, HVL24, HKH20, or SDD31. One part of peptide-treated plasma was incubated with 1 part of activated partial thromboplastin reagent and 1 part kaolin for 5 min followed by the addition of 1 part 30 mM calcium chloride. The time to clot formation was measured. The bargraph indicates the fold prolongation of the activated partial thromboplastin time of the peptide-treated plasma versus an untreated plasma. A value of 1 indicates no prolongation. The values presented are the mean ± S.E. of three experiments.




DISCUSSION

This investigation identifies the endothelial cell binding regions on D5(H). The present investigations map non-overlapping areas of D5(H), which have cell binding properties. Two tandem sequences, GKE19 and HNL21, on the amino-terminal portion of D5(H) have relatively low affinity for cells (IC = 792 and 215 µM, respectively). A second sequence, HKH20, which is on the carboxyl-terminal portion of D5(H), directly binds to endothelial cells and inhibits biotin-HK binding with an IC of 0.23 µM. Since the IC for inhibition of biotin HK binding by GKE19 and HNL21 is 3443- and 935-fold higher, respectively, than HKH20, we believe that HKH20 represents the major cell attachment site of the HK light chain. Whether the HKH20 region in native HK binds with higher affinity than the GKE19 and/or the HNL21 regions is not known. Alternatively, peptides GKE19 and HNL21 could bind to HUVEC because they mimic the sequence or amino acid composition of HKH20. A pentapeptide HGHKH present in GGH18 and HVL24 also is present in HNL21. A tetrapeptide GHGH present in GGH18, HVL24, and HKH20 is seen twice in HNL21. It is of interest that monoclonal antibody HKL12 and its F(ab`)(2), whose target epitope is located on peptide GHG19(18) , partially inhibited HK binding to cells even though it was not directed to a cell binding region itself. These data are consistent with either a steric effect of the antibodies or a change in the conformation of the cell binding site(s) of HK as result of the antibody binding to an adjacent region. This latter phenomena would be similar to what we previously reported about a monoclonal antibody directed to the heavy chain of the kininogens(39) .

The finding that anti-HKH20 antibody and synthetic peptides HKH20 and HVL24 also inhibited the procoagulant activity of plasma HK are consistent with the notion that this region of D5(H) is the HK artificial surface binding site. The procoagulant activity of HK consists of two activities: the ability to bind to artificial surfaces and the ability to approximate prekallikrein and factor XI to the surface(20, 21, 22, 36, 37, 38) . The finding that the peptide SDD31 blocked the procoagulant activity is consistent with inhibition of prekallikrein and factor XI binding to HK, a function of D6(H)(36, 37, 38) . The additional finding that HKH20 and HVL24 reduce the procoagulant activity of HK indicates that these D5(H) peptides must be interfering with the binding of HK to artificial, negatively charged surfaces(20, 21, 22) . Although we have not investigated the direct binding of HKH20 and HVL24 to artificial surfaces, the data presented are consistent with the notion that these D5(H) peptides compete with the HK light chain for kaolin binding. This idea is further supported by the recent finding that HK specifically binds to a bacterial surface protein by the HKH20 sequence on D5(H)(19) . A segment of the histidine-glycine-rich region on D5(H), which overlaps peptide HNL21, has been implicated in binding to artificial surfaces(21, 22) . Our finding that HNL21 is a relatively weak inhibitor of cell binding and does not interfere with the procoagulant activity of HK suggests that HNL21 may be of minor importance for the docking of HK to ``natural'' surfaces such as endothelial cell membranes. Overall, our data suggest that the major binding region(s) on D5(H) for biologic surfaces overlaps with binding segments for artificial surfaces such as kaolin and dextran sulfate. This finding indicates that efforts by many laboratories to characterize HK interaction with artificial surfaces pointed to the region on D5(H) that interacts with biologic membranes.

It is quite striking that HK has been characterized to have multiple linear amino acid sequences that participate in cell binding. Peptide LDC27 on D3(9, 40) , peptide GFSPFRSSRIG on D4(10) , and peptide HKH20 on D5(H) are separated regions on the HK protein molecule that inhibit HK binding to endothelial cells to varying degrees. HKH20 on D5(H) is a more potent peptide inhibitor of HK binding to endothelial cells (IC = 0.23 µM) than LDC27 on D3 (IC = 60 µM) (40) . Peptide GFSPFRSSRIG on D4 is the weakest inhibitor (IC = 1 mM), although its presence in HK is essential for maximal binding of HK to endothelium(10, 17) . The significance of these multiple and discrete binding regions for HK is not known. The data are consistent with a model in which these domains form a discontinuous interface with a complementary docking site(s), i.e. putative receptor(s). Placement of kininogen on cells contributes to the anticoagulant nature of the intravascular compartment by selectively inhibiting alpha-thrombin's activation of platelets and endothelial cells(5, 6, 9, 17) . The full biologic significance of kininogens interacting with their cell binding site(s) will not be fully appreciated until the kininogen receptor(s) is characterized.


FOOTNOTES

*
This work was supported in part by Grant HL35553 (to A. H. S.), Grants HL40387 and HL50790 (to D. B. C.), and Deutsche Forschungsgemeinschaft (MU 598/5-1), the Volkswagen Stiftung (I-69637), and Fonds der Chemischen Industrie (163323) (to W. M.-E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Division of Hematology and Oncology, Dept. of Internal Medicine, University of Michigan, 102 Observatory St., Ann Arbor, MI 48109-0724. Tel.: 313-747-3124; Fax: 313-764-2566.

(^1)
The abbreviations used are: HK, high molecular weight kininogen; D3, domain 3 of kininogens; D4, domain 4 of kininogens; D5(H) and D6(H), domains 5 and 6, respectively, of HK; LK, low molecular weight kininogen; PMSF, phenylmethylsulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis; HUVEC, human umbilical vein endothelial cells; biotin-HK, biotinylated HK; Biotin-HKH20, biotinyl-glycyl-HKHGHGHGKHKNKGKKNGKH.


ACKNOWLEDGEMENTS

We express appreciation to Dr. Weersak Nawarawong for assistance in the performance of some of the preliminary experiments in this investigation and to Dr. Armin Maidhof for help with the antibody production.


REFERENCES

  1. Roche e Silva, M., Beraldo, W. T., and Rosenfeld, G. (1949) Am. J. Physiol. 156,261-273
  2. Berrettini, M., Schleef, R. R., Heeb, M. J., Hopmeier, P., and Griffin, J. H. (1992) J. Biol. Chem. 267,19833-19839 [Abstract/Free Full Text]
  3. Gurewich, V., Johnstone, M., Loza, J-P., and Pannell, R. (1993) FEBS Lett. 318,317-321 [CrossRef][Medline] [Order article via Infotrieve]
  4. Loza, J-P., Gurewich, V., Johnstone, M., and Pannell, R. (1994) Thromb. Haemostasis 71,347-352 [Medline] [Order article via Infotrieve]
  5. Meloni, F. J., and Schmaier, A. H. (1991) J. Biol. Chem. 266,6786-6794 [Abstract/Free Full Text]
  6. Meloni, F. J., Gustafson, E. J., and Schmaier, A. H. (1992) Blood 79,1233-1244 [Abstract]
  7. Zini, J. M., Schmaier, A. H., and Cines, D. B. (1993) Blood 81,2936-2946 [Abstract]
  8. Reddigari, S. R., Kuna, P., Miragliotta, G., Shibayama, Y., Nishikawa, K., and Kaplan, A. P. (1993) Blood 81,1306-1311 [Abstract]
  9. Jiang, Y., Müller-Esterl, W., and Schmaier, A. H. (1992) J. Biol. Chem. 267,3712-3717 [Abstract/Free Full Text]
  10. Hasan, A. A. K., Cines, D. B., Zhang, J., and Schmaier, A. H. (1994) J. Biol. Chem. 269,31822-31830 [Abstract/Free Full Text]
  11. Hong, S. L. (1980) Thromb. Res. 18,787-796 [Medline] [Order article via Infotrieve]
  12. Holland, J. A., Pritchard, K. A., Pappolla, M. A., Wolin, M. S., Rogers, N. J., and Stemerman, M. B. (1990) J. Cell. Physiol. 143,21-25 [Medline] [Order article via Infotrieve]
  13. Palmer, R. M. J., Ferrige, A. G., and Moncada, S. (1987) Nature 327,524-526 [CrossRef][Medline] [Order article via Infotrieve]
  14. Smith, D., Gilbert, M., and Owen, W. G. (1985) Blood 66,835-839 [Abstract]
  15. Nakashima, M., Mombouli, J-V., Taylor, A. A., and Vanhoutte, P. M. (1993) J. Clin. Invest. 92,2867-2871 [Medline] [Order article via Infotrieve]
  16. Puri, R. N., Zhou, F., Hu, C-J., Colman, R. F., and Colman, R. W. (1991) Blood 77,500-507 [Abstract]
  17. Hasan, A. A. K., Cines, D. B., Ngaiza, J. R., Jaffe, E. A., and Schmaier, A. H. (1995) Blood, 85,3134-3143 [Abstract/Free Full Text]
  18. Kaufmann, J., Haasemann, M., Modrow, S., and Müller-Esterl, W. (1993) J. Biol. Chem. 268,9079-9091 [Abstract/Free Full Text]
  19. Ben Nasr, A., Herwald, H., Müller-Esterl, W., and Björck, L. (1994) Biochem. J. 305,173-180
  20. Schmaier, A. H., Schutsky, D., Farber, A., Silver, L. D., Bradford, H. N., and Colman, R. W. (1987) J. Biol. Chem. 262,1405-1411 [Abstract/Free Full Text]
  21. De La Cadena, R. A., and Colman, R. W. (1992) Protein Sci. 1,151-160 [Abstract/Free Full Text]
  22. Kunapuli, S., De La Cadena, R. A., and Colman, R. W. (1993) J. Biol. Chem. 268,2486-2492 [Abstract/Free Full Text]
  23. Schmaier, A. H., and Colman, R. W. (1989) Methods Enzymol. 169,276-296 [Medline] [Order article via Infotrieve]
  24. Johnson, D. A., Salvesen, G., Brown, M. A., and Barrett, A. J. (1987) Thromb. Res. 48,187-193 [Medline] [Order article via Infotrieve]
  25. Bradford, M. M. (1976) Anal. Biochem. 72,248-254 [CrossRef][Medline] [Order article via Infotrieve]
  26. Schmaier, A. H., Bradford, H. N., Lundberg, D., Farber, A., and Colman, R. W. (1990) Blood 75,1273-1281 [Abstract]
  27. Goodfriend, T. L., Levine, L., and Fasman, G. (1964) Science 144,1344-1345
  28. Haasemann, M., Buschko, J., Faussner, A., Roscher, A. A., Hoebeke, J., Burch, R. M., and Müller-Esterl, W. (1991) J. Immunol. 147,3882-3892 [Abstract/Free Full Text]
  29. Müller-Esterl, W., Johnson, D., Salvesen, G., and Barrett, A. A. (1988) Methods Enzymol. 163,240-256 [Medline] [Order article via Infotrieve]
  30. Green, N. M. (1965) Biochem. J. 94,23-24
  31. Schmaier, A. H., Silver, L. D., Adams, A. L., Fischer, G. L., Munoz, P. G., Vroman, L., and Colman, R. W. (1984) Thromb. Res. 33,51-67 [Medline] [Order article via Infotrieve]
  32. Jaffe, E. A., Nachman, R. L., Becker, C. G., and Minick, C. R. (1973) J. Clin. Invest. 52,2745-2756 [Medline] [Order article via Infotrieve]
  33. Cines, D. B., Lyss, A. P., Beeber, M., Bina, M., and DeHoratius, R. J. (1984) J. Clin. Invest. 73,611-625 [Medline] [Order article via Infotrieve]
  34. Maciag, T., Cerundolo, J., Ilsley, S., Kelley, P. R., and Forand, R. (1979) Proc. Natl. Acad. Sci. U. S. A. 76,5674-5678 [Abstract]
  35. Schmaier, A. H., Kuo, A., Lundberg, D., Murray, S., and Cines, D. B. (1988) J. Biol. Chem. 263,16327-16333 [Abstract/Free Full Text]
  36. Tait, J. F., and Fujikawa, K. (1986) J. Biol. Chem. 261,15396-15401 [Abstract/Free Full Text]
  37. Tait, J. F., and Fujikawa, K. (1987) J. Biol. Chem. 262,11651-11657 [Abstract/Free Full Text]
  38. Vogel, R., Kaufmann, J., Chung, D. W., Kellermann, J., and Müller-Esterl, W. (1990) J. Biol. Chem. 265,12494-12502 [Abstract/Free Full Text]
  39. Jiang, Y. P., Nawarawong, W., Meloni, F. J., and Schmaier, A. H. (1992) Thromb. Haemostasis 68,143-148 [Medline] [Order article via Infotrieve]
  40. Herwald, H., Hasan, A. A. K., Godovac-Zimmermann, J., Schmaier, A. H., and Müller-Esterl, W. (1995) J. Biol. Chem. 270,14634-14642 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.