(Received for publication, February 9, 1995; and in revised form, June 9, 1995)
From the
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) were
synthesized to determine its cell binding site(s). Three peptides from
non-overlapping regions on D5
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
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.
Plasma high molecular weight kininogen (HK) ()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 -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
). This
antibody also inhibited the binding of radiolabeled HK to M protein on
the surface of Streptococcus pyogenes bacteria(19) .
Since D5
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
also may contain the HK cell
binding site. Using synthetic peptides, we have defined the specific
sequences on D5
that participate in HK binding to
endothelial 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).
Figure 1:
Diagram of sequential and overlapping
synthetic peptides of D5. 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
(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.
Figure 2:
Inhibition of biotin-HK binding to
endothelial cells by synthetic peptides of D5. 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
. The sequence of the peptides used in these experiments
is given in Table 1. PanelA: GKE19,
;
HNL21,
; GGH18,
; GHG19, ▴. PanelB: FKL20,
; HKH20,
; 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.
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 (
) 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
(
), 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 (
). 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 (
), 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.
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 (
). 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.
Figure 7:
The influence of D5 and
D6
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.
This investigation identifies the endothelial cell binding
regions on D5. The present investigations map
non-overlapping areas of D5
, which have cell binding
properties. Two tandem sequences, GKE19 and HNL21, on the
amino-terminal portion of D5
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
, 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`)
, 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 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
(36, 37, 38) . The additional
finding that HKH20 and HVL24 reduce the procoagulant activity of HK
indicates that these D5
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
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
(19) . A segment of the histidine-glycine-rich
region on D5
, 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
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
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 are separated regions on the HK
protein molecule that inhibit HK binding to endothelial cells to
varying degrees. HKH20 on D5
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
-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.