Consecutive Enzyme Cascades: Complement Activation at the Cell Surface Triggers
Increased Tissue Factor Activity
By Steven D. Carson and Donald R. Johnson
Complement activation at the cell surface initiates cell
damage through a series of reactions occurring at the cell
membrane and, after assembly of the terminal membrane
attack complex, produces leakage of cytoplasmic contents
from the cell. It has been documented that chemical or
physical damage to cell membranes can cause a rapid
increase in the expression of tissue factor procoagulant
activity. In this study, antibody-mediated complement activation at the cell surface resulted in increased tissue factor
activity, which correlated with cytolysis, as measured by
51-chromium release. Therefore, complement fixation on
the cell surface can have a direct and immediate stimulatory effect on the coagulation cascade at the point of its
initiation, with formation of a fibrin clot requiring only three
consecutive proteolytic reactions after immunologically
mediated cell damage.
0 1990 by The American Society of Hematology.
T
seeded into 96-well microtiter plates at least 24 hours before
beginning each experiment.
Experiments were begun by replacing the complete culture
medium with DMEM lacking phenol red (GIBCO) and containing
only bovine serum albumin at 40 mg/mL (DMEM-BSA). The
fibroblasts were incubated with sodium 5 1-chromate(Amersham) at
100 pCi/106 cells (1 Ci = 3.7 x 10” Bq) for 3 hours. The labeled
cells were washed five times with DMEM-BSA and then incubated
with 100 pL serial dilutions of antibody. After 45 minutes, 50 pL of
rabbit complement (newborn rabbit serum; Cedar Lane Laboratories, Hornsby, Ontario) diluted 1/10 in DMEM-BSA was added to
each culture well. Complement activation was allowed to proceed for
2.25 hours at 37OC. The cell supernatants were sampled and counted
fqr chromium released from the cells, residual reaction media was
drained from the cells, and the wells were rinsed and assayed for
tissue factor activity.
Chromium release from the cells was determined by removing 100
pL of the culture supernatant from each well and measuring
radioactive 51-chromium in a Beckman Gamma 5500 counter
(Beckman Instruments, Irvine, CA). The machine background was
recorded for each experiment. In addition, supernatants were collected from cells maintained in DMEM-BSA without antibody or
complement to determine the spontaneous release of chromate, and
other supernatants were sampled after detergent lysis (1% Tween20) of the cells for determination of total chromium label in the cell
cultures.
Before determination of tissue factor activity, the remaining 50 pL
of DMEM-BSA was drained from the wells. The cells were gently
rinsed with 100 pL of reaction buffer (0.05 mol/L Tris, 0.1 mol/L
NaC1, 0.02% sodium azide, pH 7.6 with 1 mg/mL BSA) and
drained, and 60 pL of reaction buffer was added to each culture well.
Tissue factor activity was determined using a microplate readerbased chromogenic assayz3and bovine factors VIIa and X (provided
by Drs Yale Nemerson and Arabinda Guha, Mt. Sinai Medical
H E COMPLEMENT A N D coagulation systems share
several protein-protein and enzymatic interactions. For
example, C4-binding protein interacts with both complement
component C4 and protein S, which functions in the anticoagulant protein C-thrombomodulin
Vitronectin interferes with assembly of the terminal C9 complex’ and can
modulate the function of heparin cofactor IL4 C1 esterase
inhibitor is a principal inhibitor of activated complement
component C1, and inhibits factor XIJa, kallikrein, and
factor XIa, as
Further, complement components
C5b-9 accelerate the rate of platelet-catalyzed blood clotting, apparently by increasing the ability of the platelet
membranes to support prothrombinase (factor Va-factor
Xa) assembly.”*” While these examples show that multiple
interactions intertwine the coagulation and complement
reaction schemes, we postulate that complement fixation at
the membranes of cells may directly initiate coagulation by
evoking tissue factor expression.
Tissue factor is a membrane protein that serves as the
essential cofactor for factor VI1 in the initiation of coagulation via factors I X and X.I2-l4 Some cells, such as
and cells of the g l o m e r ~ l u s , may
~ ~ ~ routinely
’~
contain tissue factor. Studies with cells in culture have
established that “healthy” cells may express tissue factor
functional activity, but the procoagulant activity increases
.’~
dramatically upon cell “damage” or d i s r u p t i ~ n . ’ ~While
monocytes and endothelial cells are believed to contain no
tissue factor in the absence of inflammatory stimuli, complement activation may induce synthesis of tissue factor by
these cells, possibly in response to fragments of C5.20-22
Insofar as complement fixation a t the cell surface can result
in “membrane damage,” there may be a direct and immediate link between the complement and coagulation cascades
when they meet at the surface of a cell containing tissue
factor. To examine this possibility, we conducted the following studies.
MATERIALS AND METHODS
Human fibroblast lines used in this study (GM5756 and GM5758)
were obtained from the National Institute of General Medical
Sciences (NIGMS) Human Genetic Mutant Cell Repository. They
were maintained in 25 cmz T-flasks in Dulbecco’s modified Eagle’s
medium (DMEM) with high glucose (GIBCO, Grand Island, NY)
supplemented with 10% fetal calf serum (Dutchland, Denver, PA),
antibiotics, and NCTC 109, as previously detailed.” The cells were
dispersed in trypsin-EDTA (Flow Laboratories, McLean, VA) and
Blood, Vol 76,No 2 (July 15). 1990:pp 36 1-367
From the Department of Pathology and Microbiology. and the
Eppley Institute for Research on Cancer and Related Diseases,
University of Nebraska Medical Center, Omaha, NE.
Submitted December 21, 1989; accepted March 21,1990.
Supported in part by Grants No. HL31408 and RCDA HL02072
from the National Institutes of Health,
Address reprint requests to Steven D. Carson, PhD. Department
of Pathology and Microbiology. University of Nebraska Medical
Center. 600 S 42nd, Omaha, NE 68198-6495.
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 1734 solely to
indicate this fact.
0 1990 by The American Society of Hematology.
0006-4971/90/7602-0007$3.00/0
36 1
362
CARSON AND JOHNSON
School, New York, NY). The factor VIIa and factor X were used at
1 nmol/L and 100 nmol/L, respectively, in each microplate well.
This assay has been used to measure tissue factor activity on intact
cells,24and the relative activity measured and expressed in terms of
A405/min2 (x106) has been shown to be extremely sensitive to
changes in known concentrations of tissue factor.25
The rabbit antithymocyteglobulin (RATG) was prepared according to Bieber et alZ6and Davis et ai?’ and provided by Dr Doug
Smith (University of Nebraska Medical Center). BaSO, (10 mg/
mL) was added for 1 hour and removed by centrifugation to adsorb
most of the factor X(a) and factor VII(a) from the RATG. This
antiserum was used in these experiments because of its availability
and proven strong immunologic reactivity with surface antigens of
human cells. Additionally, since RATG was developed using thymocytes as antigen, no antibodies against human tissue factor were
present.
Human brain “thromboplastin” was prepared as a source of crude
tissue factor using the protocol described for bovine brain.’* The
-
N
1000
I
1
.-c
0
a
W
o
;2000
4
600
J
$
a
a
8
0
a
3
1000
5
0
10
20
30
40
RELATIVE ANTIBODY CONCENTRATION
1000
0
1500
I
a
a
400
a
n
B
Cl
0
P0
3000
E 2500
800
F
Physical disruption of cells by freeze-thaw cycles or
sonication clearly elevates the tissue factor activity of cultured fibroblasts, but procedures such as these are, in
general, not very relevant to biological processes. Immunologic damage to cells, mediated by antibodies and complement, is a much more relevant physiologic process that has
not been directly evaluated for immediate “damage”-related
effects on tissue factor activity. We hypothesized that antibody-mediated complement fixation, which caused measurable injury to cultured fibroblasts, would also cause an
increase in tissue factor activity. To test this hypothesis, we
have used a rabbit antibody directed against human cells and
n
W
c5
RESULTS
1
A
E
washed suspension was diluted in reaction buffer and stored frozen at
- 20oc.
I
10
20
30
40
RELATIVE ANTIBODY CONCENTRATION
0
I
N
.-c
E
2
C
800
W
600
2
c
0
0
4
8
400
2
2
200
3
m
v,
t
Fig 1. Tissue factor activity (A) and cytolysis (B) are dependent
on antibody concentration and complement and are correlated
( r = .9104) (C). The symbols refer t o measured values in experiments in which complement (0, 0 ) or media (no complement; 0 )
was added after incubation with RATG. Confirmation of the procoagulant activity as tissue factor was accomplished by adding monoclonal antibody HTFI, which inhibits human tissue factor activity?’
before the assay of some experimental samples ( 0 ) .In this experiment, the cells accumulated 15,852
1,738 cpm (n = 6) of
51-chromium, and spontaneous release from control cells (no
antibody or complement) averaged 1,013 + 60 (n = 6 ) . The chromium release data presented ere (raw cpm) - 700. Cytotoxicity30
ranged from - 1 % t o 15.6% in this experiment.
*
-
0
0
1000
2000
3000
CHROMIUM RELEASE (cpm)
363
COMPLEMENT ACTIVATION INCREASES TISSUE FACTOR
rabbit complement, which has potent lytic activity when
directed against human cells. Incubation of human fibroblasts with rabbit antibody against human thymocytes
(RATG)26*27
followed by complement produced significant
release of chromium from the cells. Measurements of factor
X activation by the same cells in the presence of factor VIIa
revealed a strong correlation between the amount of chromium released (and inferred cytolysis) and the amount of
tissue factor activity expressed by the cells. As shown in Fig
1A and B, both tissue factor activity and chromium release
were dependent on the antibody concentration and the
presence of complement. As expected by their similar dependencies on antibody concentration, tissue factor activity and
cytolysis were highly correlated (Fig 1C) ( r values for three
experiments ranged from .8457 to .9770).
An enzyme-linked immunosorbent assay (ELISA) conducted on cell cultures with antibodies against beta-2microglobulin (DAKO, Santa Barbara, CA) demonstrated
that very little of this antibody bound to the cells. Incubation
with this antibody followed by complement produced little
chromium release from the cells and, similarly, little increase
in tissue factor activity. These experiments showed that, in
the absence of measurable antibody binding to the cells, no
increase in tissue factor activity or antibody-related chromium release was observed in the presence of complement.
Since the complement source and antibody were expected
to contain some factor VI1 that might contribute to measured
tissue factor activity, assays were conducted to ascertain the
presence of enzymatic activities and the extent to which they
could influence our results. As shown in Table 1, both the
complement source and the antibody contained some factor
VII(a) activity, but little or no factor X activity, and neither
produced direct hydrolysis of the chromogenic factor Xa
substrate. The heat-treated complement source retained its
factor VII(a) activity (“stable factor”) but no longer retained complement activity. The additional factor VI1 increased the measured tissue factor activity less than 15% and
should contribute even less in the experiments that included a
buffer rinse before tissue factor assay. Equally important,
neither reagent inhibited the activity of added tissue factor.
The heat-treated complement served as a reagent to verify
the dependence of tissue factor activity increase on the action
of complement. Aliquots of reconstituted newborn rabbit
serum were warmed to 56OC and maintained at that temperature for 10 to 60 minutes. Other aliquots were held on ice.
Fibroblast cultures incubated with RATG were treated with
the various complement aliquots and evaluated for chromium release and tissue factor activity. As shown in Fig 2A,
chromium release was virtually eliminated by the heat
inactivation of the complement, while the unheated complement again produced antibody-dependent cytolysis. Tissue
factor increased in an antibody-dependent manner in the
presence of active complement, but remained at much lower
levels in cells treated with the inactivated complement (Fig
2B). There was a small but significant antibody-dependent
increase in tissue factor activity in the cells treated with
complement heated for only 10 minutes (coefficient of
regression = 1.13, t = 5.84, df = 12), even though chromium release was not significantly related to antibody
Table 1. Enzyme Activities Present in Reagents
Reaction Components.
Reagent
Tested
None
Complement source$
Factor
Factor
Vlla
X
+
+
+
+
+
+
+
+
+
+
+
+
+
-
Complement source
(56OC 6 0 min)
+
+
-
-
Antibody5
+
+
-
-
Tissue
Factor
+
+
+
+
+
+
+
+
+
+
+
+
+
-
Tissue
Factor Activityt
4,226
4.844
35
1,397
23
2
4,771
31
1.47 1
20
1
4,389
6
347
5
23
“Bovine factors X and Vlla were added for the tissue factoc assay to
give 100 nmol/L and 1 nmol/L in the reaction, respectively. When
omitted, they were placed with reaction buffer (0.05 mol/L Tris, 0.1
mol/L NaCI, pH 7.6 with 1 mg/mL bovine albumin). The tissue factor
added was prepared as human brain ”thromboplastin,”2B diluted 1/400
in reaction buffer and used at 5 0 p l per assay.
+Tissue factor activity is expressed in relative units of A405 nm/min’
( x lo6),as de~cribed.’~.’~
$Newborn rabbit serum, expected to retain factor Vll(a) activity
(previously known as ”stable factor”), was used at 10 pL per reaction
after dilution of 1/9 with reaction buffer.
§Antibody was barium sulfate-adsorbed RATG.26.27used at 10 pL per
reaction after a dilution of 1 /9 in reaction buffer.
concentration with this complement (coefficient of regression = 0.4, t = 0.88, df = 12). This may reflect the relative
sensitivities of these two assays. Alternatively, some tissue
factor increase may occur in response to activation of complement a t sublytic levels. The remaining treatments (no
complement and complement heated for 60 minutes) did not
produce antibody-dependent increases in tissue factor activity or chromium release. Overall, tissue factor activity
correlated strongly with cytolysis (Fig 2C), and increased
tissue factor activity was clearly related to complement
fixation.
The hypothetical model under examination presumed the
presence of tissue factor in the cell membranes in a low
activity state that must become more active in response to
antibody-dependent activation of complement and consequent membrane damage. Further validation of this model
required demonstration of a temporal relationship between
complement action and tissue factor expression. To this end,
we conducted a final series of experiments in which antibody
and complement were added at the same time, and the
cultures were evaluated for cytolysis and tissue factor activity a t varied times thereafter. The details of this experiment
and results are shown in Fig 3. The graphs shown in Fig 3
demonstrate that both chromium release and tissue factor
activity were dependent on the presence of active complement, the concentration of antibody, and the time allowed for
complement fixation. Figure 3 further shows the agreement
364
CARSON AND JOHNSON
n
n
*Oo0
E
a 15001
0
0
A
0
W
0
0
W
3
1000
1
rY
1
,
I
I
I
C
0
I -
€
2
’
400
0
W
2
8
’0.
W
I
500
nl
300
1
1
500”
I
200 -
1
22
0
0
0
1
0
LT
r
0
0
0
0
A
20
10
30
40
L
-
0
500
1000 1500 2000
CHROMIUM RELEASE (cpm)
RELATIVE ANTIBODY CONCENTRATION
500
n
0
“
.-, 400
0
E
2 300
W
c2 200
t-
2
P
‘0
2
100
7 5 ~
A
50
w
3
rv,
n
A
A
A
A
:A
25
-I
0
8 0
0‘
0
0
10
20
30
40
RELATIVE ANTIBODY CONCENTRATION
obtained in duplicate culture wells, and the qualitative
agreement obtained on consecutive days. To facilitate discussion of conclusions drawn from these experiments, the
duplicates have been averaged and the results segregated and
summarized in Fig 4. Increased tissue factor activity was
demonstrable before released chromium (Fig 3, columns 5
and 6 , corresponding to 35 or 39 minutes in Fig 4A through
D), indicating that measurable tissue factor increases preceded the measurable release of chromium. Wigzel13’showed
that chromium release subsequent to complement damage
occurred in two stages after an initial lag, and is slow relative
to the occurrence of cell damage. The results shown in Figs 3
and 4,considered with Wigzell’s findings, are consistent with
our conclusion that tissue factor activity increases in re-
Fig 2. Antibody-dependent increases in cytolysis (A) and tissue
factor activity ( 8 )require functional complement and are correlated
( r = .9553) (C). The symbols refer to experimental samples that
contained active complement (01,complement treated at 56°C for
10 minutes ( A ) or for 1 hour (A),or no complement (0).In this
experiment, the cells accumulated 2,311 i. 345 cpm i n = 6 ) of
51-chromium, and spontaneous release averaged 285 f 24 cpm
(n = 6 ) . The chromium data presented are (raw cpm) - 250. The
inset in C shows points clustered near the origin on an expanded
scale. Cytotoxicitym ranged from -0.7% t o 76.3% in this experiment.
sponse to complement-mediated membrane damage, and
chromium is released relatively slowly after membrane
damage. This observation may be somewhat exaggerated by
differences in the sensitivities of tissue factor and chromium
determinations. Measurable increases in tissue factor activity were observed between 20 and 40 minutes after addition
of antibody and complement. At the highest concentrations
of antibody, tissue factor activity reached its maximum levels
within about 1 hour, whereas chromium release continued
throughout the experiment. This suggests that either maximum amounts of complement were fixed or tissue factor was
maximally increased within this period a t high concentrations of RATG. Alternatively, microscopic examination of
the cells showed severe damage in cultures with the greatest
365
COMPLEMENT ACTIVATION INCREASES TISSUE FACTOR
chromium release. Tissue factor may have been lost when
these wells were rinsed before assay. Such losses would
diminish the correlation of cytolysis and tissue factor activity, a correlation that remains convincing a t each time point
where both responses are measurable even with this qualification (Fig 4E and F).
DISCUSSION
We have shown that increased tissue factor activity is one
consequence of complement fixation on tissue factor-bearing
cells. While reactions of complement activation preceding
cytolysis may be responsible for the stimulation of tissue
factor expression, the observed stimulation correlates strongly
with chromium release, which is presumed to measure
cytoly~is.’~
These results support our hypothesis that complement fixation on cells may directly trigger blood coagulation
at a principal initiation reaction, with no requirement for
induction of tissue factor synthesis before expression.
By selecting RATG and rabbit serum as the sources of
antibody and complement, respectively, we intentionally
conducted experiments that were expected to cause immune
damage to human cells in order to test our hypothesis. We
have recently conducted additional experiments in which
human serum was used as the complement source. Even
though human complement should be less lytic than rabbit
complement when tested against human cells, these experiments have also shown increased tissue factor activity that is
dependent on both antibody concentration and the presence
of complement.
While the molecular mechanisms by which tissue factor
activity is increased after complement activation remain to
be elucidated, the observation itself may be particularly
relevant to pathologic processes in which fibrin deposition is
colocalized with immune complexes, or in conditions in
which the presence of specific antibodies is correlated with
thrombosis. Recent reports that complement fixation is
Fig 3. Time dependence of antibody-mediated complement fixation effects on chromium release and tissue factor activity. Cells were
plated into 96-well plates and used in these two experiments 48 hours (A and C ) and 72 hours (B and Dl later. Each graphed response
represents chromium released (A and Bl or tissue factor activity ( C and D) of an individual well of the 96-well plate. Conditions were run in
duplicate by row leg, wells a1 and a2). After the chromium labeling, all wells were emptied of liquid, and DMEM-BSA was added t o the cells.
At timed intervals. antibody and complement were added quickly thereafter t o selected wells. All incubations ended concurrently when the
wells were sampled for released chromium and then assayed for tissue factor activity. Row h was used for determination of spontaneous
release (over 190 minutes) and total incorporation of 51-chromium by the cells. Each row (a through g) contained constant dilutions of
antibody from l/M in row a (correspondst o relative concentration of 32 in Figs 1 and 2) t o in row 1. Row g received no antibody. Wells in
columns 11 and 12 were incubated with antibody but no complement for 190 minutes. Paired columns were incubated with antibody and
complement for varied times: columns 9 and 10.3 minutes (A. CI and 2 minutes (B, D); columns 7 and 8,22 and 20 minutes; columns 5 and
89 cpm (n = 4)
6,35 and 39 minutes; columns 3 and 4,67 minutes; and columns 1 and 2,190 minutes. Total chromium uptake was 2,292
(A. C) and 3,644
192 cpm In = 6) (B. D). The spontaneous release at 190 minutes was 322 k 53 cpm (n = 6) (A, C) and 453 2 26 cpm
(n = 6) (B, 0). The chromium release in the absence of antibody (row g) indicated a time-dependent release of chromium that was
independent of antibody-mediated complement fixation. The chromium data presented has been corrected for this time-dependent,
nonspecific release, and the data presented is (raw cpm) 0.629 x min - 141 (A. C) and (raw cpm) - 0.994 x min -141 (8, D).
*
*
-
366
CARSON AND JOHNSON
0
x
v
250
2
W
3
v)
E
o
15W
LL
W
h
0
600 I
,
400
,
/’
m
3
1250
1600
1200
800
/
@
c
v
Yd
U
400
loo
750
I
2 5 0 0
H6
251)
0
&
-/
/
50
!Y//
- 1IO0
150
200
TME ( m h )
600
w
lY
I
400
r
200
0
200
4M)
600
800
1000
1200
CHROMIUM RELEASE (cpm)
,.”
2
F
P
//’
W
V
- 0n
100
TME (min)
150
zoo
Fig 4. Time course of antibody and complement-dependent
chromium release and tissue factor activity. Data points are the
averages of duplicates shown in Fig 3. Panels A and 6 are derived
from the experiment conducted 48 hours after cells were plated (Fig
3,A and C ) and C and D are derived from the experiment conducted
72 hours after plating (Fig 3,B and D). In A, 6, C, and D, the varied
symbols correspond to RATG dilutions of
(0).l/,M (01, 1/2M ( A ) ,
(A),l/m ( 0 ) .l/,m (W), and no antibody (VI.Panels E and F show
the relationship between chromium release and tissue factor activity for A, B and C, D. respectively, at 190 min (0).67 minutes (O)#and
35 or 39 minutes ( A ) .
apparent in regions of arterial cholesterol a c c u m u l a t i ~ n ~ ~cated in tissue factor-expressive malignancies, since the
and that lipoproteins in human atherosclerotic plaques can
immune cytolysis can initiate coagulation and, hence, contribaccumulate immunoglobulins that activate ~ o m p l e m e n t , ~ ~ute to the production of local fibrin, which can isolate and
further protect the abnormal cells from the host r e ~ p o n s e . ~ ’ . ~ ~
considered with demonstration of tissue factor in such
vascular lesions,34show the potential relevance of our findACKNOWLEDGMENT
ings to important pathologic processes. Furthermore, it is
rational to propose that elimination of transformed cells by
We thank Barbara Switzer and Connie Anderson for their
technical assistance with these studies.
immune surveillance (and immunotherapy) may be compli-
367
COMPLEMENT ACTIVATION INCREASES TISSUE FACTOR
REFERENCES
1. Dahlback B, Smith CA, Muller-Eberhard HJ: Visualization of
human C4b-binding protein and its complexes with vitamin Kdependent protein S and complement protein C4b. Proc Natl Acad
Sci 80:3461, 1983
2. Dahlback B, Hildebrand B: Degradation of human complement component C4b in the presence of the C4b-binding proteinprotein S complexes. Biochem J 209:857,1983
3. Tschopp J, Masson D, Schafer S, Peitsch M, Preissner KT:
The heparin binding domain of S-protein/vitronectin binds to
complement components C7, C8, and C9 and perforin from cytolytic
T-cells and inhibits their lytic activities. Biochemistry 27:4203, 1988
4. Preissner KT, Sie P Modulation of heparin cofactor I1
function by S protein (vitronectin) and formation of a ternary S
protein-thrombin-heparin cofactor I1 complex. Thromb Haemost
60:399, 1988
5 . Colman RW, Schmaier AH: The contact activation system:
Biochemistry and interactions of these surface-mediated defense
reactions. CRC Crit Rev Oncol Hematol5:57, 1986
6. Schapira M, Scott CF, Colman RW: Protection of human
plasma kallikrein from inactivation by C1 inhibitor and other
protease inhibitors. The role of high molecular weight kininogen.
Biochemistry 20:2738, 1981
7. Lammle B, Griffin JH: Formation of the fibrin clot: The
balance of procoagulant and inhibitory factors. Clin Haematol
14:281, 1985
8. Weiss R, Silverberg M, Kaplan AP: The effect of C1 inhibitor
upon Hageman factor autoactivation. Blood 68:239, 1986
9. Meijers JC, Vlooswijk RA, Bouma BN: Inhibition of human
blood coagulation factor XIa by C-1 inhibitor. Biochemistry 27:959,
1988
10. Wiedmer T, Esmon CT, Sims PJ: Complement proteins
C5b-9 stimulate procoagulant activity through platelet prothrombinase. Blood 68:875, 1986
11. Sims PJ, Faioni EM, Wiedmer T, Shattil SJ: Complement
proteins C5b-9 cause release of membrane vesicles from the platelet
surface that are enriched in the membrane receptor for coagulation
factor Va and express prothrombinase activity. J Biol Chem 263:
18205,1988
12. Nemerson Y: The reaction between bovine brain tissue factor
and factors VI1 and X. Biochemistry 5:601, 1966
13. Jesty J, Nemerson Y:Purification of factor VI1 from bovine
plasma: Reaction with tissue factor and activation of factor X. J Biol
Chem 249:509,1974
14. Osterud B, Rapaport SI: Activation of factor IX by the
reaction product of tissue factor and factor VII: Additional pathway
for initiating blood coagulation. Proc Natl Acad Sci 74:5260, 1977
15. Zacharski LR, McIntyre OR: Procoagulant synthesis by
cultured fibroblasts triggered by cell adhesion. Nature 232:338,
1971
16. Maynard JR, Dreyer BE, Stemerman MB, Pitlick FA: Tissue
factor coagulant activity of cultured human endothelial and smooth
muscle cells and fibroblasts. Blood 50:387, 1977
17. Tipping PG, Dowling JP, Holdsworth SR: Glomerular procoagulant activity in human proliferative glomerulonephritis. J Clin
Invest 81:119, 1988
18. Neale TJ, Tipping PG, Carson SD, Holdsworth SR: Evidence
for the participation of cell-mediated immunity in the deposition of
fibrin in glomerulonephritis. Lancet 2:421, 1988
19. Gramzinski RA, Broze GJ Jr, Carson SD: Human fibroblast
tissue factor is inhibited by lipoprotein-associated coagulation inhibitor and placental anticoagulant protein but not by apolipoprotein
A-11. Blood 73~983,1989
20. Prydz H, Allison AC, Schorlemmer HU: Further link between complement activation and blood coagulation. Nature 270:
173,1977
21. Osterud B, Olsen JO, Benjaminsen AW: The role of complement in the induction of thromboplastin synthesis. Hemostasis
14:386, 1984
22. Muhlfelder TW, Niemitz J, Kreutzer D, Beebe D, Ward PA,
Rosenfeld SI: C5 chemotactic fragment induces leukocyte production of tissue factor activity: A link between complement and
coagulation. J Clin Invest 63:147, 1979
23. Carson SD: Computerized analysis of enzyme cascade reactions using continuous rate data obtained with an ELISA reader.
Comput Methods Programs Biomed 19:151,1985
24. Carson SD, Archer PG: Tissue factor activity in HeLa cells
measured with a continuous chromogenic assay and ELISA reader.
ThrombRes 41:185, 1986
25. Carson SD: Continuous chromogenic tissue factor assay:
Comparison to clot-based assays and sensitivity established using
pure tissue factor. Thromb Res 47:379,1987
26. Bieber CP, Griepp RB, Oyer PE, Wong J, Stinson EB: Use of
rabbit antithymocyte globulin in cardiac transplantation. Relationship of serum clearance rates to clinical outcome. Transplantation
22:478, 1987
27. Davis RC, Cooperband SR, Mannick J A Preparation and in
vitro assay of effective and ineffective antilymphocyte sera. Surgery
66:58, 1969
28. Pitlick FA, Nemerson Y: Purification and characterization of
tissue factor apoprotein. Methods Enzymol45:37, 1976
29. Carson SD, Ross SE, Bach R, Guha A: An inhibitory
monoclonal antibody against human tissue factor. Blood 70:490,
1987
30. Johnson TR, Massey RJ, Deinhardt F: Lymphocyte and
antibody cytotoxicity to tumor cells measured by a micro-5 1
chromium release assay. Immunol Commun 1:247, 1972
31. Wigzell H: Quantitative titrations of mouse H-2 antibodies
using Cr”-labelled target cells. Transplantation 3:423,1965
32. Seifert PS, Ferdinand H, Hansson GK, Bhakdi S: Prelesional
complement activation in experimental atherosclerosis: Terminal
C5b-9 complement deposition coincides with cholesterol accumulation in the aortic intima of hypercholesterolemic rabbits. Lab Invest
60:747, 1989
33. Hollander W, Colombo M, Small C: Lipoproteins in human
plaques behave like complement-fixing immune complexes. Circulation 80:84, 1989 (suppl)
34. Wilcox JN, Smith KM, Schwartz SM, Gordon D: Localization of tissue factor in the normal vessel wall and in the atherosclerotic plaque. Proc Natl Acad Sci USA 86:2839, 1989
35. Dvorak H F Thrombosis and cancer. Hum Pathol 18:275,
1987
36. Gunji Y,Gorelik E: Role of fibrin coagulation in protection of
murine tumor cells from destruction by cytotoxic cells. Cancer Res
48:5216, 1988