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Regulatory Control of the Terminal Complement Proteins at the Surface of Human
Endothelial Cells: Neutralization of a C5b-9 Inhibitor by Antibody to CD59
By Karen K. Hamilton, Zhao Ji, Scott Rollins, Betty H. Stewart, and Peter J. Sims
Functionally inhibitory antibody to the plasma membrane
complement inhibitor CD59 has been used to investigate
control of the terminal complement proteinsat the endothelial cell surface. Antibodies against purified human erythrocyte CD59 (polyclonal anti-CD59 and monoclonal antibodies [MoAbs] 1F1 and 1F5) were found to bind specifically to
monolayers of cultured human umbilical vein endothelial
cells, and by Western blotting to recognize an 18- to 21-Kd
endothelial protein. When bound to the endothelial monolayer, anti-CD59 (immunoglobulin G or Fab fragment)
potentiated membrane pore formation induced upon C9
binding to C5b-8, and augmented the C5b-9-induced cellular responses, including stimulated secretion of von Willebrand factor and expression of catalytic surface for the
prothrombinase enzyme complex. Although potentiating
endothelial responses to the terminal complement proteins, anti-CD59 had no effect on the response of these
cells to stimulation by histamine. Taken together, these
data suggest that human endothelial cells express the
CD59 cell surface inhibitor of the terminal complement
proteins, which serves to protect these cells from poreforming and cell-stimulatory effects of the C5b-9 complex.
These data also suggest that the inactivation or deletion of
this cell surface regulatory molecule would increase the
likelihood for procoagulant changes in endothelium exposed to complement activation in plasma.
0 1990 by The American Society of Hematology.
T
sponses, including vesiculation and endocytosis, which serve
to remove these plasma membrane-inserted proteins from the
cell surface.4-” Recently, a membrane protein that inhibits
C5b-9-mediated hemolysis has been identified in human
erythrocyte^.'^*'^ This 18- to 21-Kd protein has been sequenced and found to be identical to leukocyte CD59, a
glycosyl-phosphatidylinositol-linked membrane protein distributed on lymphocytes, neutrophils, and monocyte^.'^.'^
Although CD59 has not been detected in human platelets,
antibody raised against this protein has been shown to
augment C9 activation by membrane C5b-8, and to potentiate CSb-9-induced platelet activation responses.15 These
data imply the presence of a C5b-9 inhibitor on the surface of
these cells that shares epitopes with CD59. W e now report
evidence for a C5b-9-inhibitory protein on the surface of
human endothelial cells that is antigenically related or
identical to CD59, and that serves to protect these cells from
stimulatory effects arising through complement activation.
HE TERMINAL complement proteins C5b-9 are established as mediators of immune destruction of microorganisms and transplanted heterologous tissue. In addition,
when deposited on homologous blood and vascular cell
membranes, these proteins have been shown to induce the
nonlytic activation of a variety of cell responses, through
pathways that appear to involve influx of extracellular
calcium and activation of intracellular protein kinase C
(PKC).1-4For example, human endothelial cells exposed to
the human C5b-9 proteins exhibit an increase in cytosolic
calcium concentration, which is accompanied by induced
secretion of their intracellular storage granules containing
von Willebrand factor ( v W F ) . ~In addition to inducing
secretory fusion of the Weibel-Palade bodies with the plasma
membrane, exposure to C5b-9 induces vesiculation of the
endothelial cell surface, exposing binding sites for coagulation factor Va, and thereby increasing membrane-catalyzed
prothrombinase activity of this normally nonthrombogenic
ce11.4
Cellular resistance to the effects of the C5b-9 proteins can
occur through a variety of mechanisms, including membrane
and fluid phase inhibitors that block their assembly and
membrane insertion, as well as compensatory cellular re~~
~
From the Cardiovascular Biology Research Program, Oklahoma
Medical Research Foundation: the Departments of Microbiology
and Immunology and Medicine. Oklahoma University Health
Sciences Center: and the St Francis of Tulsa Medical Research
Institute. Oklahoma City, OK.
Submitted April 4.1990; accepted August 17. 1990.
Supported by Grant Nos. H W l 7 4 9 , HL36061, and HL36946
from the Heart, Lung and Blood Institute. National Institutes of
Health. P.J.S. is an Established Investigator of the American Heart
Association.
Address reprint requests to Peter J. Sims. MD, PhD, Cardiovascular Biology Research Program, Oklahoma Medical Research
Foundation, 825 NE 13th Si. Oklahoma City. OK 73104.
The publication costs of this article were defrayed 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 I990 by The American Society of Hematology.
0006-4971/90/7612-0004$3.00/0
2572
EXPERIMENTAL PROCEDURES
Endothelial cell culture. As described previously,’ human umbilical vein endothelial cells (HUVE) were harvested using 0.1%
collagenase, and grown in Medium 199 containing 20% fetal bovine
serum (FBS), heparin, endothelial growth supplement, and penicillin and streptomycin until confluent. Cells were subcultured using
trypsin after reaching confluence. First passage cells were grown in
48-well (1 cm’) or 96-well (0.3 cmZ)tissue culture plates and used 2
to 5 days after subculturing.
Preparation of rabbit anti-endothelial cell antibody. Rabbits
were immunized with partially purified human endothelial cell
plasma membranes. The details of membrane preparation, immunization, and immunoglobulin G (IgG) purification have been reported
previously.’
Preparation of serum deficient in the complement component C8.
Human serum was depleted of C8 by absorption against rabbit
antibody to C8 coupled to agarose, as described previously.’
Purification of human complement proteins C8 and C9. Human
C8 and C9 were purified as reported previously.’ These proteins were
iodinated to specific activities of 2,501 cpm/ng (C8) and 1,454
cpm/ng (C9) using immobilized lactoperoxidase and glucose oxidase (Enzymobead Radioiodination Reagent; BioRad, Richmond,
CA).
Purification of an 18- to 21-Kd inhibitor of C5b-9 from human
erythrocyte membranes. Isolation of the 18- to 21-Kd inhibitor
Blood, Vol76, No 12 (December 15). 1990: pp 2572-2577
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2573
ENDOTHELIAL CELL COMPLEMENT C5B-9 INHIBITOR
from erythrocyte membranes (P18), preparation of rabbit antibody
to the erythrocyte inhibitor (anti-P18), and preparation of Fab
fragments of anti-P18 IgG have been reported previously in
The full-length sequence of the 18- to 21-Kd erythrocyte inhibitor
has been reported and has established its identity with the leukocyte
antigen CD59.I3 Therefore, the antibody against the erythrocyte
inhibitor, previously referred to as anti-P18, will be designated
anti-CD59 henceforth. Monoclonal antibodies (MoAbs) 1F1 and
1F5, reactive with CD59, were provided by Drs Motowo Tomita
(Showa University, Tokyo, Japan) and Hidechika Okada (Nagoya
City University School of Medicine, Nagoya, Japan).
Radioiodination of antibodies. MoAbs 1F1 and 1F5 were
radiolabeled with IODO-GEN (Pierce Chemical, Rockford, IL).
Specific activities were 6,221 cpm/ng (1F1) and 4,856 cpm/ng
( I F5).
Binding of radiolabeled antibodies to CD59 to cultured human
endothelial cells. Except where specified, all cell experiments were
performed using Hanks balanced salt solution, modified to contain
10 mmol/L HEPES and 1% bovine serum albumin (Hanks-HEPESBSA). Cells were washed free of serum-containing medium and
fixed 10 minutes with 1% paraformaldehyde in phosphate-buffered
saline, pH 7.4. Cells were washed three times to remove fixative, and
incubated with '251-anti-CD59,'251-lFl,or '251-1F5in the presence
and absence of a 20-fold excess of the unlabeled antibody for 30
minutes at room temperature. Cells were then washed rapidly five
times with 1 mL vol of Hanks-HEPES-BSA chilled to 4"C,
solubilized in 4% sodium dodecyl sulfate (SDS), and radioactivity
measured.
Immunoblotting of endothelial proteins with antibody to CDS9.
Second-passage HUVE were grown to confluence (75 cm'). After
washing free of medium, cells were removed by scraping. The
washed cells were pelleted, denatured at 100°C in 10% SDS and 20
mmol/L N-ethylmaleimide (without reduction), and subjected to
15% polyacrylamide gel electrophoresis under nonreducing conditions. After transfer to nitrocellulose and blocking with 10%nonfat
dry milk, blots were incubated overnight with either polyclonal
anti-CD59 (10 pg/mL) or MoAb IF1 (IO pg/mL) and developed
with affinity-purified goat antirabbit IgG or goat antimouse IgG
conjugated to alkaline phosphatase (Sigma, St Louis, MO). Purified
human erythrocyte CD59 served as molecular weight standard.
Complement assembly on human endothelial cells. Confluent
endothelial cells were washed three times with Hanks-HEPES-BSA.
The cells were then incubated with rabbit anti-endothelial IgG ( 5
mg/mL) for 15 minutes at 23"C, washed once, and incubated with
C8-deficient serum for 10 minutes at 37°C. The cells were then
washed three times with buffer chilled to 4"C, and incubated with
antLCD59 (IgG or Fab fragments) or Hanks-HEPES-BSA only
(see figure legends) at 4°C for 30 minutes. Finally, cells were washed
twice and incubated at 37°C with C8 and/or C9 at the concentrations described in the figure legends. In some experiments, radiolabeled C8 or C9 was used to measure cell surface binding (see legend
to Fig 6).
Quantitation of CSb-9-induced changes in membrane permeability using 3H-2-deoxy-Dglucose (-'H-DOGJ. For 24 hours before an
experiment, cells were grown in culture medium (300 p L ) containing
3H-DOG (ICN Radiochemicals, Irvine, CA, 33 Ci/mmol/L) 4
&/well for 24 hours. Cells were then washed and complement
complexes assembled as described above, including incubation with
antLCD59 IgG (0 to 1 mg/mL) before addition of C8 and C9. Ten
minutes after addition of C8 and/or C9, supernatants were removed
and pooled with two subsequent washes (100 pL). Cells were lysed
with 125 PL of 2% Triton X-100 (Calbiochem, San Diego, CA), each
well was washed twice, and washes pooled with lysates. Supernates
and lysates were counted after addition of 4 mL of Aquasol (New
England Nuclear Research Products, Boston, MA).
Assay ofprothrombinase activity. Complement-inducedchanges
in the ability of the endothelial membrane to support assembly of the
prothrombinase enzyme complex was assayed as reported previ~usly.~
Briefly, after complement assembly, bovine prothrombin, factor Va,
and factor Xa were added in the final concentrations of 1.4 pmol/L,
2 nmol/L, and 10 pmol/L, respectively. After a 6-minute incubation, aliquots were removed and the reaction terminated using
EDTA. Thrombin generation was assayed using the synthetic
substrate Spectrozyme TH (American Diagnostica, Greenwich,
CT) .
Assay for v WFsecretion. After complement assembly, supernatants were removed and assayed for released vWF by methods
previously described." Pooled normal human plasma was used as the
standard, and results were expressed in units per milliliter where
normal pooled plasma is defined as containing 1 U/mL.
RESULTS
Expression of the CD59 antigen in HUVE. Human
erythrocytes, platelets, and leukocytes have been shown to
express membrane proteins that serve to restrict activation of
the terminal complement proteins at the surface of these
cell^.'^-'^ In the case of platelets, neutralization of this
C5b-9-regulatory function with an antibody against CD59
has been shown to potentiate complement-induced cell activation. The resistance of human endothelial cells in culture to
the pore-forming and cytolytic effects of these proteins
suggested the possibility that these cells also express C5b-9regulatory proteins.'~~To explore this possibility, we first
demonstrated that '251-anti-CD59(IgG) bound specifically
to endothelial monolayers, with saturation observed at 500
pg/mL and half-maximal binding observed at approximately
100 pg/mL (data not shown). Moreover, two MoAbs reactive with erythrocyte CD59 (1F1 and 1F5) were found to
bind specifically to the endothelial plasma membrane (Table
1). The presence of CD59 antigen in these cultured human
endothelial cells was confirmed by Western blotting with
both polyclonal and MoAbs against puried human erythrocyte CD59 (Fig 1). Immunohistochemical evidence for the
expression of the 1F5 epitope on human vascular endothelium has been recently reported by Nose et al." The binding
of these MoAbs at saturation suggests that human endothelial cells in culture express approximately 2 x IO5 molecules
of CD59 antigen per cell, equivalent to 100 molecules/pm*of
lumenal surface. By comparison, human erythrocytes are
estimated to express approximately 25,000 CD59 molecules/
cell, equivalent to approximately 200 molecules/pm2 membrane surface." Whether this endothelial antigen is structurally identical to the 18- to 21-Kd phosphatidylinositol-linked
protein isolated from human erythrocytes remains to be
determined.
Table 1. Binding of CD69 MoAbs 1F1 and 1F5 to
Endothelial Monolayers
Specific Binding.
(molecules laG/cell)
Antibodv
1251-1F 1
1251-1F5
151,000
185,000
* 26,000
* 6,000
Winding of each antibody was performedat a saturating concentration
of 5 wg/mL. Nonspecific binding was measured in the presence of a
20-fold excess of each unlabeled antibody and was 15% of total ( 1F 1)
and 2 5 % of total ( 1F5). Data are mean SD.
*
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HAMILTON ET AL
2574
n
B
kD
2009769'
16-
3021-
Fig
lmmunoblotting t
m a n eni thelia1
cells with antibody t o CD59. lmmunoblotting of
purified erythrocyte CD59 and second-passage
HUVE with polyclonal anti-CD59 (A) and murine
MoAb 1F1 (6) was performed as described in
Experimental Procedures.
ldEffect of anti-CD59 on C5b-9 pore formation. The
expression of CD59 epitopes by human endothelial cells
suggested a complementary C5b-9-regulatory function of
the endothelial plasma membrane. We therefore examined
whether functionally inhibitory antibody to the CD59 antigen (anti-CD59) altered the assembly and activation of the
complement pore on these cells. In these experiments, C5b67
complexes were first deposited on the endothelial plasma
membrane, followed by incubation at 4OC with anti-CD59
(IgG or Fab fragments). C5b-9 assembly was then completed by addition of C8 and C9, with incubation at 37OC
(see Experimental Procedures). The efflux of 'H-DOG from
the endothelial cytoplasm was used to monitor formation of
the C5b-9 pore in the plasma membrane. As illustrated by
Fig 2, C5b-9 pore formation was potentiated by addition of
anti-CD59, with apparent saturation of this effect at 0.5
mg/mL IgG. At the submaximal concentration of C9 used in
this experiment, anti-CD59 caused a fourfold increase in the
C5b-9-induced efflux of 'H-DOG. By contrast, this antibody
had no effect on the efflux of 'H-DOG from C5b-8 cells
(omitting C9).
Potentiation of C5b-9 induced stimulatory responses by
anti-CD59. The capacity of anti-CD59 to increase the
C5b-9-mediated change in plasma membrane permeability
(Fig 2) suggested that this antibody might also potentiate the
cell-activation responses induced on C5b-9 binding to the
endothelial surface. In cells exposed to anti-CD59 (Fab
fragments), CSb-9-induced prothrombinase activity was
increased approximately threefold when compared with
identically treated cells not exposed to antibody (Fig 3). By
contrast, the prothrombinase activity measured for control
cells was unaffected by incubation with anti-CD59.
In addition to inducing expression of membrane prothrom-
binase sites, the C5b-9 proteins have been shown to stimulate
secretion of vWF multimers from endothelial storage
granules.' As shown in Fig 4, this secretory response to C5b-9
increased with increasing anti-CD59, and was augmented
approximately fourfold at saturating concentrations of Fab
(>250 pg/mL). As shown in Fig 5A, antLCD59 (Fab)
potentiated the C5b-9-induced secretory response at all
W
0)
0 =O;;/
W
-
;l o - -
C5b-8
<?.................................................................... 0
c3
0
Q
O
Fig 2. Potentiation of C5b-9 pore formation in the endothelial
plasma membrane by antibody t o CD59. 'H-DOG was equilibrated
into the endothelial cytoplasm by incubation for 24 hours. After
washing, C5b67 was assembled (see Experimental Procedures).
Monolayers were then incubated with anti-CD59 (IgG) at 4°C
(concentrations shown on abscissa). Following washing t o remove
free antibody, C8 (100 nglwell) and C9 (62.5 ng/well) were added
and 3H-DOG release quantitated after 10 minutes, 37°C (0).(0)
indicate data obtained in absence of C9. Results shown are mean f
SD (n = 3) and are representative of three similar experiments.
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ENDOTHELIAL CELL COMPLEMENT C ~ B - 9INHIBITOR
2575
c
-
C5b-8.I6 Therefore, we examined the possibility that the
endothelial surface protein recognized by anti-CD59 also
affected CSb-9 pore formation by limiting the amount of C9
that can bind to membrane C5b-8. As shown in Fig 6, we
were unable to detect any effect of anti-CD59 on the binding
of either 1251-C8to C5b67 or '251-C9to C5b-8 deposited on
the endothelial surface. In these experiments, antLCD59
(Fab) was used at concentrations that maximally augmented
CSb-9-induced prothrombinase activity and vWF secretion.
a,
I
2
.-
i?ZJ +Anti-CD59
E
Fob
1.0
>0
:
0.5
Q
W
.E
0.0
n
E
2
1
DISCUSSION
I
-0.5
1
C5b-9
C5b67
kFig 3. Effect of antLCD59 on C5b-9-induced exposure of
catalytic surface for prothrombinase assembly. C5b67 complexes
were deposited on endothelial monolayers and these cells then
incubated at 4°C with anti-CD59 (0 or 0.35 mg/mL Fab). After
washing, C8 (0.5 pglwell) and C9 (12.5 ng/well) were added, cells
incubated 10 minutes at 37°C. and prothrombinase activity measured as described in Experimental Procedures. C5b67 denotes
cells incubated in the absence of C8 or C9. Results are reported as
change from C6b67 controls (mean f SD, n = 3). and are representative of three similar experiments.
concentrations of C9 tested. By contrast, this antibody had
no effect on histamine-induced vWF secretion at any concentration of histamine (Fig 5B). The data of Fig 5 suggest that
anti-CD59 potentiates the CSb-9-induced response by directly affecting activation of the complement pore, and does
not potentiate receptor-mediated endothelial secretory responses.
Effect of anti-CD59 on C5b-9 assembly. The data of
Figs 2 through 5, suggest that anti-CD59 specificallyinhibits
a CSb-9-regulatory protein on the endothelial surface. In red
blood cells, CD59 has been shown to restrict hemolysis by
C5b-9 and to reduce the incorporation of C9 into membrane
C5b-9, thereby decreasing the number of C9 bound to
0-0
7
20
T
X
E
C5b-9
0- - OC5b67
l
I/*
3 0 0i- O- o -100- o - - ,200
. _ - -
r\
v
_, - - --0
- 8
300
400
500
Anti-CD59 Fab (,ug/ml)
Fig 4. Anti-CD59 potentiates CBb-9-induced secretion of
endothelial vWF. C5b67 endothelial monolayers were incubated
with anti-CD59 (Fab) at concentrations indicated on abscissa
before addition of C8 (0.5 pg/well) and C9 (20 ng/well: 0).
Secreted vWF was quantitated after 10 minutes, 37°C (see
ExperimentalProcedures). Data for C5b67 controls are also shown
(0).
Results are mean f SD, n = 3, and are representative of two
experiments so performed.
Our data suggest that the human endothelial plasma
membrane contains an 18- to 21-Kd inhibitor of the terminal
complement proteins that shares both functional and antigenic properties with the 18- to 21-Kd CSb-9-inhibitor,
CD59, previously detected in the human erythrocyte and
leukocyte plasma membrane. In erythrocytes, this protein
appears to serve a key role in restricting the cytolytic
consequence of C5b-9 assembly.12*16
In addition to contributing to the normal resistance of human endothelial cells to
lysis by complement, our data suggest that this membrane
component serves directly to attenuate the capacity of the
C5b-9 proteins to evoke thrombogenic responses from these
cells, which normally express predominantly anticoagulant
properties.
When CSb67-containing endothelial cells were exposed to
anti-CD59, C5b-9 pore formation (as indicated by 'H-DOG
release, Fig 2) was enhanced fourfold, suggesting that this
antigen limits C8 and C9 binding to C5b67 sites or limits
subsequent activation of the pore. Similarly, anti-CD59
enhances hemolysis of CSb67-erythrocytes exposed to C8
and C9, and enhances secretion and prothrombinase activity
of CSb67-platelets exposed to C8 plus C9. The protective
effect of CD59 in CSb-9-mediated hemolysis appears to be
related to this protein's capacity to limit the number of C9
that bind to C5b-8 deposited on the erythrocyte surface.I6
Human platelets exposed to anti-CD59 show exhanced
susceptibility to the stimulatory effects of the C5b-9 complex
and increased binding of C9 to C5b-8,I5 suggesting that
epitopes recognized by anti-CD59 include a functional domain@) which interfere(s) with the binding and/or activation of C9 by membrane bound C5b-8. In endothelial cells,
we did not observe increased C9 binding to C5b-8 on cells
exposed to anti-CD59 (Fig 6). One interpretation of these
data is that in endothelial cells the cell surface complement
regulatory protein(s) that are inhibited by anti-CD59 restrict
a conformational change in the C5b-9 complex necessary for
pore formation and cell activation, rather than reducing C9
binding to C5b-8 per se. Alternatively, we have demonstrated previously that C5b-9 induces the formation of
membrane microparticles that are shed from the endothelial
surface and contain the membrane-inserted CSb-9 protein^.^
It is likely, therefore, that our inability to detect increased
binding of lZ5I-C9to C5b-8 on anti-CD59-treated endothelial monolayers is related to a concomitant increase in this
vesiculation of membrane-embedded C5b-9 complexes from
the surface of these cells. Of note in platelets, increased
membrane incorporation of C9 after exposure to anti-CD59
has been shown to be largely accounted for by C5b-9
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HAMILTON ET AL
T
A
B
aT
/
---+
'4
0.
08
0
100
50
C9 Input (ng/weII)
10-8
,
I
1'0-7
1'0-6
1'0-5
1'0-,
[Histamine] (M)
Fig 5. Effect of anti-CD59 on histamine-stimulated vWF secretion. Endothelial monolayers were incubated with either 0 (0)or WO
pg/mL anti-CD59 Fab (0)and the dose-dependent stimulation of vWF secretion determined for C5b-9 (A) and histamine (e). (A) After
incubation of C5b67 monolayers with anti-CD59, C8 (0.5 pglwell) and C9 (0 t o 125 ng/well) were added, and secreted vWF quantitated
after 10 minutes, 37°C. Data shown are mean SD, n = 3. and are representative of two experiments performed. (B) After incubation of
endothelial monolayers with anti-CD59. histamine (0 t o 0.1 mmol/L) was added, and secreted vWF quantitated after 10 minutes, 37°C.
Data shown are mean f SD, n = 2, and are representative of three experiments so performed.
*
complexes present on microparticles that were released from
the platelet plasma membrane.15
The capacity of anti-CD59 to potentiate the C5b-9induced vWF secretion and prothrombinase activity of endothelial cells suggests that a deletion or inactivation of the
membrane protein recognized by this antibody might potentiate the procoagulant response of these cells exposed to low
I
I
levels of complement activation. Alternatively, increased
expression of CD59 on the plasma membrane of vascular
endothelialcells, as might potentially be achieved by transfection with the human CD59 gene, might render these cells
resistant to the cytolytic and cell-stimulatory effects of the
terminal complement proteins. Such resistance to activated
complement would be of particular benefit in protecting
c
-
:
\
0
c
v
a 5
C
3
0
m
cn
0
I
-
In
N
0
1 .o
'251 -C 8 Input ( p g / w el I)
.5
. _ . _ .:-.-.-
- 0
I
100
200
1251-C9 Input (ng/weII)
Fig 6. Effect of anti-CD59 on the incorporation of C8 and C9 into membrane C5b-9. C5b67 was deposited on endothelial monolayers
and the binding of 1zsCC8(A) and "%C9 (B) determined for endothelial monolayers treated with either 0 ( A ) or 500 pg/mL (A)anti-CD59
Fab. (A) After incubation with anti-CD59. 1261-C8was added at concentrations on abscissa and incubated 12 minutes, 37°C. After washing
five times with ice-cold Hanks-HEPES-BSA, the monolayer were solubilized in 4% SDS. and radioactivity determined. Nonspecific binding
was determined by omitting C8-deficient serum (0: see Experimental Procedures). (B) The binding of "%C9 was performed at
concentrations shown on abscissa, measured in the presence of 0.5 pg C8 per well (4 pg/mL). Nonspecific binding was determined in the
absence of C8 (0).
Data shown are mean ? SD, n = 2, and are representative of two experiments so performed.
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ENDOTHELIAL CELL COMPLEMENT C58-9 INHIBITOR
2577
xenogeneic organ transplants, or in ameliorating hyperacute
allograft rejection.
ACKNOWLEDGMENT
The authors gratefully acknowledge the advice and suggestions of
Dr Therese Wiedmer (Oklahoma Medical Research Foundation)
and the excellent technical assistance of Elizabeth A. Smith,
Darlene Schwartzott, and Janet Heuser. Factors Va, Xa, and
prothrombin were generous gifts from Dr Charles T. Esmon (Oklahoma Medical Research Foundation), and MoAbs IF1 and 1F5
were generously provided by Drs Motowo Tomita (Showa University, Tokyo, Japan) and Hidechika Okada (Nagoya City University
School of Medicine, Nagoya, Japan).
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blood platelets: Evidence for reversible depolarization of membrane
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2. Wiedmer T, Ando B, Sims PJ: Complement CSb-9-stimulated
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3. Hattori R, Hamilton KK, McEver RP, Sims PJ: Complement
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of endothelial von Willebrand factor and translocation of granule
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2649053,1989
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proteins C5b-9 induce vesiculation of the endothelial plasma membrane and expose catalytic surface for assembly of the prothrombinase enzyme complex. J Biol Chem 265:3809,1990
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6. Kirszbaum L, Murphy BF, Walker ID: Molecular studies on
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8. Scolding NJ, Morgan BP, Houston WAJ, Linington C, Campbell AK, Compston DAS: Vesicular removal by oligodendrocytes of
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10. Sims PJ, Wiedmer T, Esmon CT, Weiss HJ, Shattil SJ:
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11. Fugate RD, Hamilton KH, Sims PJ: Fluorescence imaging of
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C5b-8 complex. Biophys J 57:283A, 1990 (abstr)
12. Sugita Y, Nakano Y, Tomita M: Isolation from human
erythrocytes of a new membrane protein which inhibits formation of
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13. Okada H, Nagami Y, Takahashi K, Okada N, Hideshima T,
Takizawa H, Kondo J: 20 kDa homologous restriction factor of
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From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
1990 76: 2572-2577
Regulatory control of the terminal complement proteins at the surface
of human endothelial cells: neutralization of a C5b-9 inhibitor by
antibody to CD59
KK Hamilton, Z Ji, S Rollins, BH Stewart and PJ Sims
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