From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
Human High Molecular Weight Kininogen Binds To Human Umbilical Vein
Endothelial Cells Via Its Heavy and Light Chains
By Sesha R. Reddigari, Piotr Kuna, Gina Miragliotta, Yoji Shibayama, Katsumi Nishikawa, and Allen P. Kaplan
High molecular weight kininogen (HK) is a multifunctional
plasma glycoprotein that occupies a critical position in
pathways that link inflammation and coagulation. Excision
of the vasoactive peptide bradykinin by plasma kallikrein
results in kinin-free HK that consists of a 65-Kd N-terminal
heavy chain (HK-HC) linked t o the C-terminal45-Kd light
chain (HK-LC) by a disulfide bridge. HK-HC is an inhibitor
of SH-proteases and HK-LC contains the binding sites
for coagulation cofactors prekallikrein and factor XI. HK
has previously been shown t o bind specifically to human umbilical vein endothelial cells (HUVEC) in a zinc2+dependent manner by a single class of high-affinity binding
sites. We have further characterizedthat interaction in order
to determine the cell-binding regions of HK. Competition
binding experiments have indicated that either HK-LC or
HK-HC was able t o inhibit the binding of labeled HK with
of 77 nmol/L and 89
a 50% inhibitory concentration
nmol/L, respectively. Cleaved two-chain HK (HKa) had an
IC50 of 73 nmol/L, whereas uncleaved HK had an ICsoof
335 nmol/L. Direct binding experiments have indicated that
HUVEC bind both purified ['251]HK-HC and [1251]HK-LCin a
zinc*+-dependentmanner and that HK-LC did not displace
bound HK-HC. The light chain of low molecular weight kininogen or prekallikrein-binding region of HK did not inhibit
the binding of HK t o HUVEC. Our results, therefore, indicate
that (1) HK is capable of binding to endothelial cells via
both heavy and light chain moieties, (2) HKa has a higher
affinity to HUVEC, and (3)purified heavy and light chains
are capableof directly binding t o HUVEC. The data are consistent with the presence of a single high-affinity site for
HK on endothelial cells within which are subsites that bind
to heavy and light chains.
8 1993 by The American Society of Hematology.
H
that contains identical heavy chain and Bk sequences but a
different light chain sequence.'* The heavy chain of LK is
an inhibitor of thiol proteases, but the function of LKs light
chain is still unknown.
Owing to its multifunctional nature, interaction of HK
with vascular endothelium may be of pathophysiologic
significance. Although it has been reported previously that
HK and LK bind to endothelial cell^,'^-'^ platelet^,'^.'^ and
neutrophils," the mechanism of interaction has remained
unclear. We have previously presented preliminary evidence suggesting that HK binds to HUVEC via its heavy
chain (HK-HC) region." Recently, Jiang et alZ0have shown
that the domain D3 of HK-HC, which is one of the three
cystatinlike regions of HK-HC, was capable of inhibiting
binding of HK to HUVEC. Direct binding of HK-HC or
domain D3 to HUVEC was not demonstrated in these
studies. We now present evidence that both HK-HC and
HK-LC bind to HUVEC in a zinc2+-dependent manner
with very similar affinities and address possible mechanisms of binding.
IGH MOLECULAR weight kininogen (HK) is a 1 15Kd plasma protein occupying a critical position in the
pathways that link hemostasis and the inflammatory response.' Its plasma concentration ranges from 55 to 90 pg/
mL2-4and it circulates as a bimolecular complex with prekallikrein and as a bimolecular or trimolecular complex with
factor XI5 (one or two molecules of HK to one molecule of
factor XI). Surface-mediated activation of the contact system
in plasma leads to activation of factor XI1 and prekallikrein
that in turn leads to cleavage of HK and release of bradykinin
(Bk), a vasoactive peptide that has been shown to promote
venular dilation, vascular permeability, pain, hypotension,
and
Excision of Bk converts HK into a two-chain molecule in
which the two chains are linked by a disulfide bridge. The
65-Kd region N-terminal to Bk's position is known as the
heavy chain (HK-HC) and the 49- to 56-Kd region C-terminal
to Bk is known as the light chain (HK-LC).9 The latter is
transient and undergoes further conversion by plasma kallikrein to a relatively more stable 45- to 49-Kd HK-LC."
HK-HC is responsible for the calpainlike thiol protease inhibitory activity of HK." The HK-LC, on the other hand,
contains the domains responsible for binding to ( I ) negatively
charged surfaces that activate the contact system and ( 2 )
prekallikrein and factor XI.' Plasma also contains a 68-Kd
glycoprotein known as low molecular weight kininogen (LK)
From the Division of Allergy, Rheumatology and Clinical Immunology, Department of Medicine, State University of New York, Stony
Brook, NY; and the Division of Biochemistry, Institute of Bioactive
Science, Nippon Zoki Phurmacwtical Company, Hyogo, Japan.
Submitted August 24, 1992; accepted October 29, 1992.
Address reprint requests to Sesha R. Reddiguri, Division of Allergy,
Rheumatology and Clinicul Immunology, Department of Medicine,
State University o f New York, Stony Brook. N Y , I 1794.
The publication costs of this article were defiayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section I734 solely to
indicate this fact.
0 I993 by The American Society of Hematology.
0006-4971/93/8105-0031$3.00/0
1306
MATERIALS AND METHODS
Endothelial cell culture. Endothelial cells were isolated from human umbilical veins according to the method of Jaffe et aI2' and
cultured as described previo~sly.'~
Proteins. HK, HK-LC, and HK-HC were prepared from fresh
normal human plasma (NHP) according to the method of Keribiriou
and Griffin? HK-HC was further purified by passage through an
affinity column containing the anti-HK-LC monoclonal antibody
No. I 15-2 1'' covalently coupled to agarose. The HK-LC-dependent
clotting activity of purified HK-HC was <0.003 U/mg. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) has
shown that the proteins were pure and that there was no cross-contamination between HK-HC and HK-LC preparations (Fig I). The
coagulant activities of HK were determined according to Proctor and
Rapaportz3and were found to be 12 U/mg. The light chain of low
molecular weight kininogen (residues 402 to 427) and the prekalliwere synthesized
krein-binding region (residues 565 to 595) of HK22,24
by the Center for Analysis and Synthesis of Macromolecules, State
University of New York, Stony Brook.
Blood, Vol81, No 5 (March 1). 1993: pp 1306-131 1
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
INTERACTION OF KININOGEN WITH HUVEC
A 1
2
, .,
3
-4-5----=-.
1307
B 1
STD
2
STD
----=-P-
ko
- 94
kD
- 68
-94
68
-
- 43
-
- 30
-43
-30
Fig 1. SDS-PAGE of HK, HKa, HK-LC, and HK-HC. Purified HK and its derivatives were subjected to electrophoresison 10%polyacrylamide
gels and stained with Coomassie blue R-250.(A) HK (reduced, lane 11, HKa (unreduced, lane 2) HKa (reduced. lane 3).(B) HK-LC (reduced,
lane 1). HK-HC (reduced, lane 2). Molecular weight standards are shown on t h e right of each panel.
Protein concentrations were determined according to the method
of Bradford*' using human IgG as the reference protein. SDS-PAGE
was performed according to Laemmli.26
Radiolaheling cfprordns. HK, HK-HC, and HK-LC were iodinated with ["SI]NaI by lodobeads (Pierce Chemical CO, Rockford,
IL) in 0.01 mol/L sodium acetate. 0. I5 mol/L NaCI, pH 5.5. according
to manufacturer's recommendations. Following iodination, unreacted
['251]Nalwas separated by desalting. Specific labeling, determined by
trichloroacetic acid (TCA) precipitation following addition of bovine
serum albumin (BSA) as carrier protein. ranged from 1.7 X IOR to
2.6 X IO9 cpm/mg protein. LC thus labeled did not lose clotting
activity. Radioactivity incorporation was >95%.
HK used in some experiments was labeled with tritium by periodate
oxidation followed by reduction with ('HlNaBH, according to the
method described by Van Lenten and Ashwell for labeling casein."
Briefly, HK in 0. I mol/L sodium acetate, 0. I5 mol/L sodium chloride,
pH 5.6, was made 5 mmol/L in sodium periodate. After incubation
at 0°C for I5 minutes. the oxidation reaction was stopped by addition
of excess ethylene glycol and the solution was desalted on a column
equilibrated with 0.01 mol/L phosphate buffer containing 0.1 5 mol/
L NaCI. pH 7.5. One milliliter of ['HJNaBH, (50 Ci/mmol) in 0.01
mol/L NaOH was added to unlabeled oxidized HK, mixed for 30
minutes at 25°C before 0.4 mg of unlabeled NaBH, was added, and
the incubation continued for another 30 minutes. Labeled HK then
subjected to desalting on a Biogel P6 (Bio-Rad Laboratories, Richmond, CA) in I O mmol/L sodium acetate, 0. I5 mol/L NaCI, pH 5.5
and stored in aliquots at -70°C until use. Specific radioactivity, as
determined by TCA precipitation, was 0.4 X IO9 dpm/mg protein.
['HIHK retained its clotting activity of 12 U/mg and was pure as
checked by SDS-PAGE followed by autoradiography. Radioactivity
incorporation was 97%.
Binding of H K IO HUVEC. All binding studies were performed
at the third cell passage at 4°C to minimize ligand internalization.
HUVEC were washed four times over a 30-minute interval with
PMSF-treated preincubation buffer ( I37 mmol/L NaCI. 4 mmol/L
KCI, I I mmol/L glucose, I O mmol/L HEPES, I mmol/L CaCI,.
and 0.5 mg/mL BSA) and then reincubated with the same buffer
containing 50 pmol/L ZnC12(binding buffer) for 20 minutes. HUVEC
were then incubated with 100 p L of binding buffer containing labeled
HK, HK-HC, or HK-LC in the presence or absence of agonists as
indicated in the figure legends. Following incubation, the unbound
material was aspirated and the cells washed four times with a total
of 0.8 mL of binding buffer and the bound radioactivity was determined following solubilization of cells with I % SDS in which the
monolayer dissolved almost immediately. The number of cells per
well as counted after each experiment averaged 2.2 X IO4 cells per
well. The cell viability was greater than 95% as tested by trypan blue
exclusion before and after the binding experiments. Each measurement was in triplicate and the experiments were repeated two times.
In experiments designed to determine zinc2+dependency, zinc chloride was omitted from the preincubation step.
RESULTS
Binding of HK to HUVEC and inhibition by e.~ce.wtinlabeled IIK. HK-LC. and HK-HC. HUVEC were incubated
with 1 pg/mL (8.7 nmol/L) [-'H]HK in the presence and absence of 100 pg/mL unlabeled HK and the extent of binding
was followed as a function of time. As shown in Fig 2, [-'H]HK
was observed to bind to HUVEC in a time-dependent manner, reachinga plateau of2.5 pmol/106 HUVEC by 120 minutes. In the presence of excess unlabeled HK, this binding
was inhibited significantly to approximately I .2 pmol/106
HUVEC, confirming specific binding of HK to HUVEC. To
identify the cell-binding regions on kininogen molecule, we
incubated HUVEC with 8.7 nmol/L [-'H]HK in the presence
of 100-fold excess of either purified unlabeled HK-HC or
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
REDDlGARl ET AL
1308
z
a
n
1
-5E2
0
0
20
40
60
80
100
120
I
I
I
I
I
3
log agonist (nM)
2
5
4
140
Time (mln)
Fig 2. HK-HC and KK-LC inhibit the binding of ['HIHK to HUVEC.
Confluent HUVEC in a 96-well cell culture plate were incubated
with 8.7 nmol/L ['HIHK at 4°C in binding buffer (see Materials and
Methods section) in the absence ( 0 )or presence of 100-fold molar
HK-HC
),
(U), or HK-LC (A)and
excess of unlabeled agonists: HK (I
bound HK determined as a function of time. Each point represents
mean (+SDI of six separate incubations.
100
0
n
80
Y
r
60{
--t Light chain of LK
Y
--t Prekallikrein binding domain
HK-LC. Both chains substantially inhibited the binding of
HK (Fig 2), suggesting that HK binds to HUVEC via site(s)
on its HC and LC regions.
Next, we examined whether the binding of [3H]HK can
be reversed by these factors. HUVEC were incubated with
8.7 nmol/L [3H]HK as described before and at the 30-minute
time point 1 00-fold excess of unlabeled HK, HK-HC, or HKLC was added and bound [3H]HK was determined at 60 and
120 minutes. All three competitors completely reversed the
specific binding of HK to HUVEC (Fig 3), again suggesting
1
08
oc
0.6
0
n
2o
of HK
-F
I
o ~ " ' ~ " ' l " ' ~ ' ' ' l ' ' ' 1
0
200
400
600
800
1000
[Competitor] nM
Fig 4. Competitive inhibition of ['Z61]HK binding to HUVEC. (A)
Determination of ICsoconcentrations. HUVEC were incubated with
8.7nm0l/L['~~1]HKinthepresenceofO.69.8,140,279,419,559,
and 698 nmol/L cold HK (U); 0,21.75,43.5,65.25,87,174.435,
and 652.5 nmol/L HK-HC (A);0, 21.75. 43.5, 65.25, 87, 130.5,
174,435, and 652.5 nmol/L HK-LC (+) or 0.56.5,113,226,452,
678,904, and 1.130 nmol/Ltwo-chain HK (Y) for 2 hours and bound
ligand was determined. Data were processed by the computer program ALLFIT as described in the Materials and Methods section.
Data points represent mean of three separate incubations. (B) Light
chain of LK and prekallikrein binding region of HK do not inhibit the
binding of [1z51]HKto HUVEC. HUVEC were incubated with 0. 87,
435, and 870 nmol/L of LC-LK or prekallikrein-binding region of HK
for 2 hours and bound HK determined. Data represent mean of six
determinations (fSD).
0.4
v)
e
n
0.2
0
0
20
40
60
80
100
120
140
Time (min)
Fig 3. HUVEC-bound HK is reversed by HK-HC and HK-LC. HUVEC were incubated with 8.7 nmol/L ['HIHK and bound ligand was
determined as described before ( 0 ) .To replicate sets of wells 100,
(I
or )
HK-LC
, (A)were added
fold excess of unlabeled HK (01HK-HC
at the 30-minute time point and bound HK determined at 60 and
120 minutes. Nonspecific binding in the absence of zinc chloride
was subtracted from total bound cpm. Each point is mean of six
separate incubations.
that HUVEC possess binding site(s) that are specific to both
HK-HC and HK-LC.
Next, the inhibition by these factors was quantitated in a
competitive inhibition experiment. HUVEC were incubated
with 8.7 nmol/L ['251]HKin the presence of 0 to 870 nmol/
L unlabeled HK, HK-HC, HK-LC, and two-chain HK under
equilibrium conditions and bound HK was determined (Fig
4A). The data were analyzed using the computer program
ALLFIT, which uses the standard four-parameter logistic
function to calculate the 50% inhibition concentrations
(Laboratory of Theoretical and Physical Biology, NIH, Bethesda, MD). Results indicated that HK, HK-LC, HK-HC,
and two-chain HK inhibited the binding of labeled HK with
ICso of 335 nmol/L (standard error ? 11.5%), 77 nmol/L
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
INTERACTION OF KININOGEN WITH HUVEC
1309
B 1 2 .-t-+ Zinc
a
-
:
.+-
Zinc
10
0
0
c
-
8
z
0
p
6
0
?
*"
4
a
m
0
E 2
I
,
20
40
I
0
.
.
.
I
.
.
.
l
.
.
.
I
.
I
60
Time
80
.
.
I
.
.
.
I
100
120
140
0
20
60
40
(min)
80
Time
100
120
140
(min)
Fig 5. Binding of HK-LC and HK-HC to HUVEC is zinc dependent. HUVEC were incubated with 8.7 nmol/L HK-LC (A) or HK-HC (B) in
the absence (m) or presence ( 0 )of 50 pmol/Lzinc chloride and bound ligand determined as a function of time. Data represent mean of three
separate determinations.
(2I3.6%), 89 nmol/L (2I 1.7%).and 73 nmol/L (+ 18%).respectively, suggesting that HK-HC, HK-LC, and HKa inhibit
the binding of labeled HK with very similar affinities. These
ICs0 numbers were then used to calculate the apparent Ki
values using the equation K, = 8/3(11 - TI) as described by
Muller,'K where K, = affinity constant, I, = ICsaof the agonist,
and TIis the concentration of labeled ligand. Results indicated
that although native HK inhibited the binding of labeled HK
with an apparent Ki of 122 nmol/L, HK-HC. HK-LC, and
the two-chain HK inhibited the same with comparable apparent Ki values of 25 nmol/L, 30 nmol/L, and 20 nmol/L,
respectively. This approach to the calculation of Ki from ICs0
has been previously applied by Meloni et al.29who have validated this method by showing that Ki values obtained thus
were not significantly different from those obtained by other
concentration-dependent binding/inhibition experiments.
Next, we assessed whether the synthetically prepared light
chain moiety (residues 402 to 427) of LK and the prekallikrein-binding region of HK (residues 565 to 595 of HK) influenced the binding oflabeled HK. HUVEC were incubated
with 8.7 nmol/L ["'IIHK in the presence 0, 87. 435, and
870 nmol/L of each peptide, and after 2 hours bound HK
was determined as described before. Neither peptide had any
effect on the binding of HK to HUVEC (Fig 49).
Direct and :in$' dependent binding o f l f K - L C and HKH C to H U V J X . Because the results of experiments described above indicated the presence of binding site(s) for
both chains of HK, we next examined whether purified HKLC and HK-HC can directly bind HUVEC. HUVEC were
incubated with 8.7 nmol/L ["SI]HK-HC (Fig 5A) or 8.7
nmol/L [12'I]HK-LC(Fig 5B) in the same binding buffer (used
for HK binding) and the time course of its binding followed
as described before. Both chains were able to bind HUVEC
in a time-dependent manner. However, when zinc2+ was
omitted from the binding buffer, the binding was drastically
decreased, suggesting that the binding is dependent upon the
presence of zinc ions.
Binding qf NK-HC is not significantlv inhibited by HKLC. We next assessed whether addition of increasing concentrations of unlabeled HK-LC would inhibit the binding
of labeled HK-HC. HUVEC were incubated with 8.9 nmol/
L purified labeled ['251]HK-HCin the presence ofO, 87,217.5,
435, and 870 nmol/L of unlabeled HK-LC for 90 minutes
and bound ligand was determined. Results, shown in Fig 6,
indicated that even at 870 nmol/L of HK-LC, greater than
80% of HK-HC was still bound to HUVEC. Since specific
binding of labeled HK was inhibited well below 870 nmol/
L of the competitor (Fig 4A), the data shown in Fig 6 indicate
that the binding of HK-HC is not inhibited by HK-LC.
DISCUSSION
Human HK has been demonstrated to bind to cultured
HUVEC specifically with a B,, of 0.5-3.0 X IO6 molecules
0 nM
87 nM
217 5 nM
435 nM
870 nM
[H K- LC]
Fig 6. HK-LC does not inhibit the bindingof HK-HC. H W E C were
incubated with indicated amounts of unlabeled HK-LC for 2 hours
as described in the Materials and Methods section and bound ligand
determined. Bars represent mean (iSD) of four separate determinations.
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
REDDIGARI ET AL
1310
of HK per cell in a zinc*+-dependent manner and a kd of 35
to 53 n m ~ l / L . ~The
~ . 'binding
~
was suggested to occur via a
single class of high-affinity binding sites. We have recently
demonstrated the in situ binding of HK to umbilical vein
end0the1ium.l~We now extend these studies to determine
the cell-binding regions of HK. Our data show that purified
unlabeled HK-HC and HK-LC, in addition to native HK,
reversibly inhibited the binding of labeled HK to HUVEC
(Figs 2, 3). Control experiments have indicated that the light
chain of LK did not inhibit the binding of HK (Fig 4B),
which points to the specificity of the binding reaction. These
results infer that both chains of HK are capable of binding
to HUVEC, and this conclusion was corroborated by direct
binding experiments where [1251]HK-LC
or [1251]HK-HC
were
shown to specifically bind to HUVEC in a zinc2+-dependent
manner (Fig 5). Comparison of IC50values or apparent K,s
(25 nmol/L for HK-HC and 30 nmol/L for HK-LC) suggested
that both chains compete with HK for binding to HUVEC
with equal affinity. The closeness of these numbers may explain why the binding of HK to HUVEC appeared to occur
via a single class of binding sites.13.14.19
However, because the
presence of a single class of sites was deduced from binding
experiments in which the maximum labeled ligand concentration was only 80 nmol/L, we performed saturation binding
experiments with 8 to 700 nmol/L HK to see whether higher
ligand concentrations would yield data that might fit a two
site model. The results (not shown), which yielded a kd of
1 I5 nmol/L, did not fit a two-site model.
Preliminary data of Zini et a13' have indicated the presence
of two binding sites for HK on HUVEC following stimulation
with Bk and phorbol myristic acetate (PMA). These workers
have reported that preincubation of HUVEC with Bk for 3
to 4 hours upregulated the binding of HK to HUVEC via
the heavy chain moiety. Preincubation with PMA resulted
in the expression of a second binding site on HUVEC for
HK but not LK. Because HK and LK have identical heavy
chain moiety but different light chains, these data infer that
the second binding site involves the light chain of HK. Neither
direct binding of the light chain nor inhibition of HK binding
by the light chain were reported in that abstract. Our data,
on the other hand, demonstrate that HK-LC and HK-HC
bind to unstimulated HUVEC directly and inhibit the binding
of labeled HK. Studies of H K s interaction with plateletsI7
have similarly demonstrated binding sites on both HK-HC
and HK-LC with apparent K, values of 30 and 11 nmol/L,
respectively, which are comparable to our data with HUVEC.
It can be speculated that HUVEC possess two binding sites
with similar affinities for HK because the two chains with
very dissimilar primary structures are capable of binding to
the cells independently. However, because (1) the specific
binding of intact HK to HUVEC was completely abolished
by either chain and (2) the binding of HK-HC was not affected
by 100-fold excess of purified HK-LC (Fig 6), it is likely that
a single cell surface receptor with two interacting sites is involved in the binding. Similar conclusions were drawn with
respect to the interaction of HK with platelets in the study
by Meloni et al," where purified LK completely inhibited
the binding of labeled HK to platelets, but HK-LC failed to
inhibit the binding of labeled LK. Because LK and HK have
identical heavy chains but unrelated light chains, these authors
have suggested that platelets possess a single receptor (with
contiguous sites for binding each of the two chains) to which
a molecule of native HK binds in a less than optimal manner.
Based on our present data, a similar mechanism can be applied to endothelial cells also. The apparent K, of 122 nmol/
L for HK compared with 25 to 30 nmol/L for the individual
chains may corroborate this theory. If a molecule of native
HK binds less than optimally, it is possible that cleavage by
kallikrein relieves some of the rigidity imparting more flexibility to the resulting two-chain molecule or exposing the
binding site(s) completely so that it can interact more readily
with the receptor via either chain. The lower apparent K, of
20 nmol/L for two-chain HK compared with 122 nmol/L
for the intact molecule supports this likelihood. However,
further studies are needed to clarify this issue.
Kininogens are the only source of the proinflammatory
peptide Bk and, given the potential proinflammatory nature
of vascular endothelial cells,31interaction of HK and LK
with HUVEC may have physiologic and pathologic significance. It is not known whether the other components of the
Bk-forming pathway, factor XI1 and prekallikrein, interact
directly with the endothelium. Because the prekallikrein
binding region of HK did not interfere with the binding of
HK to the cells (Fig 4B), it can be concluded that this portion
of HK is not involved in binding to the cells. This leaves
open the possibility that prekallikrein may attach to the cells
via HK. The presence of contact system components on the
endothelial cell surface might facilitate generation of Bk on
the cell surface upon activation of the contact system by inflammatory agents such as e n d o t ~ x i n .Vascular
~ ~ , ~ ~ endothelial cells are also known to express activators of factor XII,34
and certain bacterial enzymes are capable of activating factor
XI1 and/or ~ r e k a l l i k r e i n .Factor
~ ~ . ~ ~XIIa formed as a result
of contact activation can convert prekallikrein to kallikrein
that in turn can digest cell-bound HK to liberate Bk. We
have recently shown that plasma kallikrein can recognize
and liberate Bk from HUVEC-bound HK in a time-dependent fashion. Although the aforementioned activators can
generate Bk in the fluid phase, Bk is likely to be inactivated
by kininases present in blood very quickly because Bk has a
half-life of only 15 to 20 seconds in blood of most species.37
Bk generated on a cell surface may interact more efficiently
with its cell surface receptors before being either degraded
by plasma kininases and/or swept away from the site of injury
by blood flow, thus constituting a mechanism for bradykinininduced endothelial cell activation, plasma and inflammatory
cell extravasation, and hypotension. We believe this analysis
of the kinin-forming cascade has particular relevance for inflammatory reactions mediated by Bk.
REFERENCES
1. Kaplan AP, Silverberg M: The coagulation-kinin pathway of
human plasma. Blood 70:1, 1987
2. Adam A, Albert A, Calay G, Closset J, Damas J, Franchimont
P Human kininogens of low and high molecular mass: Quantification
by radioimmunoassay and determination of reference values. Clin
Chem 31:423, 1985
3. Liimmle B, Zuraw BL, Heeb MJ, Schwarz HP, Berrettini M,
Curd JG, Griffin JH: Detection and quantitation of cleaved and un-
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
INTERACTION OF KININOGEN WITH HUVEC
cleaved high molecular weight kininogen in plasma by ligand blotting
with radiolabeled plasma kallikrein or factor XI. Thromb Haemost
59:151, 1988
4. Reddigari SR, Kaplan A P Quantification of human high molecular weight kininogen by immunoblotting with a monoclonal antilight chain antibody. J Immunol Methods 19, 1989
5. Mandle RJ, Colman RW, Kaplan A P Identification of prekallikrein and high molecular weight kininogen as a complex in plasma.
Proc Natl Acad Sci USA 73:4179, 1976
6. Erdos EG: Kininases, in Erdos EG (eds): Handbook of Experimental Pharmacology. Heidelberg, Springer, 1979, p 427
7. Marceau F, Lussier A, Regoli D, Girond J: Pharmacology of
kinins: Their relevance to tissue injury and inflammation. Gen Pharmacol 14:209, 1983
8. Armstrong D: Pain. Handbook Exp Pharmacol 25:434, 1970
9. Keribiriou DM, Griffin JH: Human high molecular weight kininogen: Studies of structure-function relationships and of proteolysis
of the molecule occuring during contact activation of plasma. J Biol
Chem 245:12020, 1979
IO. Reddigari SR, Kaplan AP: Cleavage of high molecular weight
kininogen by purified kallikreins and upon contact activation of
plasma. Blood 71:1334, 1988
1 1. Higashiyama S, Ohkubo I, Ishiguro H, Kunimatsu M, Sawaki
K, Sasaki M: Human high molecular weight kininogen as a thiol
protease inhibitor: Presence of the entire inhibition capacity in the
native form of heavy chain. Biochemistry 25:1669, 1986
12. Kitamura N, Kitagawa H, Fukushima D, Takagaki Y, Miyata
T, Nakanishi S Structural organization of the human kininogen gene
and a model for its evolution. J Biol Chem 260:8610, 1985
13. Schmaier AH, Kuo A, Lundberg D, Murray S, Cines DB: The
expression of high molecular weight kininogen on human umbilical
vein endothelial cells. J Biol Chem 31: 16327, 1988
14. van Iwaarden F, de Groot PG, Bouma BN: The binding of
high molecular weight kininogen to cultured human endothelial cells.
J Biol Chem 263:4698, 1988
15. Nishikawa K, Shibayama Y, Kuna P, Calcaterra E, Kaplan
AP, Reddigari S R Generation of vasoactive peptide bradykinin from
human umbilical vein endothelium-bound high molecular weight
kininogen by plasma kallikrein. Blood 80:1988, 1992
16. Gustafson EJ, Schutsky D, Knight LC, Schmaier AH: High
molecular weight kininogen binds to unstimulated platelets. J Clin
Invest 78:310, 1986
17. Meloni FJ, Gustafson EJ, Schmaier AH: High molecular weight
kininogen binds to platelets by its heavy and light chains and when
bound has altered susceptibility to kallikrein cleavage. Blood 79: 1233,
1992
18. Gustafson EJ, Schmaier AH, Wachtfogel YT, Kaufman N,
Kucich U, Colman RW: Human neutrophils contain and bind high
molecular weight kininogen. J Clin Invest 84:28, 1989
19. Kuna P, Kaplan AP, Reddigari SR: Human high molecular
weight kininogen binds to human umbilical cord endothelial cells
through its heavy chain. Blood 76:426, 1990
131 1
20. Jiang Y, Muller-Ester1 W, Schmaier AH: Domain 3 of kininogens contains a cell-binding site and a site that modifies thrombin
activation of platelets. J Biol Chem 267:3712, 1992
2 I . Jaffe EA: Culture of human endothelial cells. Transplant Proc
12:49, 1980
22. Reddigari SR, Kaplan A P Monoclonal antibody to human
high molecular weight kininogen recognizes its prekallikrein binding
site and inhibits its coagulant activity. Blood 74:695, 1989
23. Proctor PR, Rapaport SI: The partial thromboplastin time
with kaolin: A simple screening test for first stage plasma clotting
deficiencies. Am J Clin Pathol 35:212, 1961
24. Tait J, Fujikawa K Identification of the binding site plasma
prekallikrein in human high molecular weight kininogen. J Biol Chem
261:15396, 1986
25. Bradford MM: A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of
protein-dye binding. Anal Biochem 72:248, 1976
26. Laemmli U K Cleavage of structural proteins during the assembly of the head of bactereophage T4. Nature 227:680, I970
27. Lenten LV, Ashwell G Studies on the chemical and enzymatic
modification of glycoproteins. J Biol Chem 246:1889, 1971
28. Muller R: Determination of affinity and specificity of antihapten antibodies by competitive radioimmunoassay. Methods Enzymol 92:589, 1983
29. Meloni FJ, Schmaier AH: Low molecular weight kininogen
binds to platelets to modulate thrombin-induced platelet activation.
J Biol Chem 266:6786, 1991
30. Zini JM, Schmaier AH, Cines D: Bradykinin stimulates the
binding of its precursors, high and low molecular weight kininogens,
to human endothelial cells. Blood 78:75, 1991
31. Cotran RS: New roles for the endothelium in inflammation
and immunity. Am J Pathol 129:407, 1987
32. Pettinger WA, Young R Endotoxin-induced kinin (bradykinin) formation: Activation of Hageman factor and plasma kallikrein
in human plasma. Life Sci 9:313, 1970
33. Roeise 0, Bouma BN, Stadaas JO, Aasen AO: Dose dependence of endotoxin-induced activation of the plasma contact system:
An in vitro study. Circ. Shock 26:419, 1988
34. Wiggins RC, Loskutoff DJ, Cochrane CG, Griffin JH, Edgington TS: Activation of rabbit Hageman factor by homogenates
of cultured rabbit endothelial cells. J Clin Invest 65:197,
1980
35. Yamamoto T, Shibuya Y, Nishino N, Okabe H, Kambara T:
Activation of Hageman factor by Pseudomonas aeruginosa elastase
in vitro. Biochim Biophys Acta 1038:231, 1990
36. Molla A, Yamamoto T, Akaike T, Miyoshi S, Maeda H:
Activation of Hageman factor and prekallikrein and generation
of kinin by various microbial proteinases. J Biol Chem 264: 10589,
1989
37. Stewart JM: Chemistry and biologic activity of peptides related
to bradykinin, in Erdos EG (eds): Bradykinin, Kallidin and Kallikrein.
New York, Springer-Verlag, 1980, p 227
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1993 81: 1306-1311
Human high molecular weight kininogen binds to human umbilical
vein endothelial cells via its heavy and light chains
SR Reddigari, P Kuna, G Miragliotta, Y Shibayama, K Nishikawa and AP Kaplan
Updated information and services can be found at:
http://www.bloodjournal.org/content/81/5/1306.full.html
Articles on similar topics can be found in the following Blood collections
Information about reproducing this article in parts or in its entirety may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests
Information about ordering reprints may be found online at:
http://www.bloodjournal.org/site/misc/rights.xhtml#reprints
Information about subscriptions and ASH membership may be found online at:
http://www.bloodjournal.org/site/subscriptions/index.xhtml
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American
Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Copyright 2011 by The American Society of Hematology; all rights reserved.