1. Art and Diseño

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Role of Free Protein S and C4b Binding Protein in Regulating the Coagulant
Response to Escherichia coli
By F.B. Taylor, Jr, B. Dahlback, A.C.K. Chang, MS. Lockhart, K. Hatanaka, G . Peer, and C.T. Esmon
Previous studies showed that infusion of C4b-binding protein with sublethal Escherichia coli (€. coli) in the primate
produced a consumptive coagulopathy followed by microvascular thrombosis and renal failure.The first objective of
this study was to characterize the pathophysiology and
mechanism of this phenomena following infusion of both
these agentswith emphasis ondefining the role of free protein S. The second objective was to examine the relevance
of this model t o the hemolytic uremic syndrome. Infusion
of C4b-bindingprotein alone reduced freeprotein S and decreased platelet concentration to 20% of baseline, whereas
infusion of the C4b-binding proteinlprotein S complex did
not. There was no activation of other inflammatory or coagulant factors. Infusion of sublethal E coli alone produced a
transient inflammatory response with no reduction of free
protein S. However, coinfusion of C4b-binding protein with
sublethal E coli reducedfree protein S andproduced a
thrombocytopenia, anemia, and a microvascularthrombotic
response, whereas infusion of the C4b-binding protein/protein S complex with sublethal Ecolidid not. Studies comparing the effects of neutralizing (S-163) and nonneutralizing
(S-145) antibodies with protein S coinfused with sublethal
E coli produced similar contrasting results. Therefore, we
concludedthat neutralization of freeprotein S, and not some
other property of C4b-binding protein influenced by protein
S, accounted for this microvascular thrombotic response.
This response is similar t o the hemolytic uremic syndrome
characterized by thrombocytopenia, anemia, shistocytosis,
and renal glomerularthrombosis with uremia. Comparison
of the respective renalhistopathologic appearance supports
this conclusion. This raisesthe possibility that inhibition of
protein S activity (possibly by one ofthe forms of C4b-binding proteins) might be one of the factors contributing to
microvascular thrombotic disorder, such as the hemolytic
uremic syndrome.
Q 1995 by The American Society of Hematology.
D
coli alone normally produces only a transient sympathomimetic,inflammatoryresponse
with no evidence ofa sustained release of cytokines,consumptive coagulopathy or
microvascular thrombosis. However, we found that coinfusion of C4bBP with sublethal E coli produced a lethal inflammatory, coagulopathic response.”
The purpose of this report is: (1) to study the mechanism
of this phenomena and determine
whether infusion of C4bBP
reduces free protein S in vivo, and whether or not the response to infusion of C4bBP plus sublethal E coli is related
to reduction of free protein S concentration; and (2) characterize more completely the pathophysiology of this response
with the goal of determining how closely this response mimics thehemolyticuremic
syndromet4and thethrombotic
thrombocytopenic purpuraI5 seen in clinical practice.
EFICIENCIES OF protein C’ and protein S2 have been
associated with deep vein thrombosis. These include
both
and acquired deficiencie~.~”
Acquired deficiencies of protein S have been associated with inflammatory
disorders suchaslupuserythematosus
in which thereare
elevations of acutephase proteins, one ofwhichis
C4b
binding protein.’ This protein binds to and neutralizes the
activated protein C cofactor activity of protein S.”” Protein
S circulates in plasma in both the free and bound forms.
70% is
Normally, about 30% to 40%
is free and 60% to
bound to C4bBP protein.’* It has beenpostulated that elevation of C4bBP to two to three timesnormal concentrations,
such as might occur in inflammation, could produce an acquired deficiency of protein S by binding and neutralizing
the remaining free protein
Infusion of C4b bindingprotein alone (20 mgkg) produces no changes from normal, except fora decline in platelet count at T +24 hours.” Infusion of sublethal Escherichia
From the Oklahoma Medical Research Foundation, Program of
Cardiovascular Biology and Howard Hughes Medical Institute,
Oklahoma City: University of Oklahoma Health Sciences Center,
Program of Cardiovascular Biology, Oklahoma City; University of
Lund, the Department of Clinical Chemistry, Malmo, Sweden; and
Developmental Research Laboratories, Shionogi & CO LTD. Osaka,
Japan.
Submitted August 19, 1994; accepted June 7, 1995.
Supported in part by National Institutes of Health Grant No. R37
HL30340 to C.T.E. and Grant No. 2ROI GM37704 to F.B.T.. C.T.E.
is an investigator at the Howard Hughes Medical Institute.
Address reprint requests to F.B. Taylor, Jr, MD, Curdiovascular
Biology Research, Oklahoma Medical Research Foundation, 825
NE 13th St, Oklahoma City, OK 73104.
The publicationcosts 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 1995 by The American Society of Hematologv.
0006-4971/95/8607-0034$3.00/0
2642
MATERIALS AND METHODS
Materials
C4b binding protein (C4bBP), protein S preparation: C4bBP and
protein S used in the infusion studies were prepared from human
plasma as described p r e v i o ~ s l y .Protein
’~
S is a cofactor for activated
protein C, which when added to the Xa one-stage assay, increases
theclottingtime by cleavageandinactivation of factors Va and
VIITa. The inhibition of protein S cofactor activity by C4bBP was
determined using the Xa one-stage assay to which activated protein
C was added as described previously.” Approximately 400
pg of
C4bBP per milliliter of plasma was sufficient to reduce the clotting
time from 100 seconds to the control clotting time (ie. no protein S
in assay) of 35 seconds.
S used in the
Thegoatpolyclonalantibodytohumanprotein
enzyme-linkedimmunosorbent
assay (ELISA)andimmunoblot
assays and a similar antibody to human C4bBP used in the ELISA
assays were affinity purified by adsorption to protein S, and C4bBP
coupled to affigel, respectively, and were then eluted with 0. I mol/
L glycine, ethylenediaminetetraacetic acid, pH 2.5.
Monoclonal antibody preparation. Balb/cmice(JacksonLabs,
Bar Harbor, ME) were immunized intraperitoneal (IP) three times
with 100 pg of human C4bBP or protein S per injection at 2-week
intervals. After a 2-month rest, they were then injected with 50 p g
Blood, Vol 86,
No 7 (October l ) , 1995: pp 2642-2652
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2643
ROLE OF FREE PROTEIN S AND C4bEP
of antigen and fusion performed 3 days later. A myeloma cell line
(P3-X-63-Ag8-653, 1.5 x lo7 viable cells) was mixed with 1.7 X 10'
viable spleen cells from one animal and fused by standard procedures
using polyethylene glycol (PEG) 1500. After selection in hypoxanthine, aminopterin, thymidine (HAT) medium, culture supernates
were tested for binding activity to the appropriate antigen(?.). Positive
clones were resubcloned twice by limit dilution. For production of
ascites fluid, approximately 5 X IO6 hybridoma cells were injected
per Balb/c mouse 2 weeks after Pristane treatment.
S-159, BP 91 isolation. Ascites fluid was diluted 1:1 with H20
and then precipitated by the addition of equal volumes of saturated
NH4S04. The precipitate was harvested by centrifugation and desalted into 0.27 mol/L Tris-PO.,, pH 6.3, before chromatography on
a QAE Sephadex Q50 column (Sigma Chemical CO, St Louis, MO)
(1 mL resin/mL ascites), equilibrated in the above buffer. The column was developed with a linear gradient from 0 to 0.4 mom NaCl
(total volume five X the column volume) over approximately 8
hours. The antibody was precipitated with 50% NH4S04,the pellet
harvested by centrifugation, resuspended in a minimum volume of
0.1 m o w NaCI, I mmol/L 3-[N-Morpholino] propane sulfonic acid
(MOPS), pH 7.5, before further purification on a Sephadex G-200
column, equilibrated in this buffer.
S-163, S-145. S-l63 is a neutralizing monoclonal antibody directed against the first epidermal growth factor (EGF) domain of
protein S. S-l45 is a nonneutralizing antibody directed against an
epitope that is not required for protein S cofactor activity. These
antibodies were prepared and isolated as described above for S-l59
and BP-9 1.
E coli organisms (ATCC 33 985 Type B7-086a:Kl) used in the
infusion study were isolated from a stool specimen at Children's
Memorial Hospital (Oklahoma City, OK). They were stored in the
lyophilized state at4°C after growth in tryptic soybean agar and
reconstituted and characterized as described previo~sly.'~
Preexperimentation and Experimentation Procedures
Papio c cynocephalus or Papio c anubis baboons were purchased
from a breeding colony maintained at the University of Oklahoma
Health Sciences Center (OUHSC) or from Charles Rivers, Inc
(Houston, TX). Animals weighed 4 to 9 kg, had leukocyte concentrations of 5,000 to 10,0W/mm3,and hematocrits exceeding 36%. They
were free of tuberculosis. These animals were held for 30 days at
the UOHSC animal facility where the infusion studies were done.
The animals were observed continuously during the first 6 hours
postinfusion and at 24 hours all animals were killed and tissues
examined.
Sampling
Mean systemic arterial pressure (MSAP) and heart rate were monitored with a Stathem pressure transducer and Hewlett Packard
(Avondale, PA) recorder. Rectal temperature was measured with a
Telethermometer (Yellow Springs Instrument CO, Yellow Springs,
OH). The above measurements were made, and blood samples were
collected at T -l/*, 0, + l , +2, +3, +4, +5, +6, and at 24 hours.
T -0 designated the point at which the infusion of E coli was started.
Not more than 10% of calculated blood volume (70 -g)
was
withdrawn over the 6-hour monitoring period. The blood collected
at the times indicated above included: 1 mL anticoagulated with
EDTA acid for complete blood count, including hematocrit, platelet
and differential counts; 2 mL anticoagulated with 3.8% sodium citrate for fibrinogenf6and for ELISA, immunoblot and gel filtration
analysis of C4b binding protein S, 1.0 mL in trasylollthrombin for
fibrin degeneration products," 3.0mLof clotted blood for blood
urea nitrogen," and creatinine." Two samples of 1.0 mL each were
taken at T -0 and T +2 hours for colony counts.*"All animals were
killed at T +24 hours after collection of the last blood sample.
Postmortem studies were performed and tissues were removed from
the lungs, kidney, liver, adrenal glands, heart, and spleen for routine
light microscopic examination.
The study protocol received prior approval by the Institutional
Animal Care and Use Committees of both the Oklahoma Medical
Research Foundation and the OUHSC.
Experimental Groups
Table 1 shows the five groups of animals studied. Group 1 was
infused with C4bBP alone (20 mgkg), group 2 C4bBP/protein S
complex (20/3 mgkg), group 3 sublethal E coli alone, group 4
sublethal E coli plus C4bBP, and group 5 E coli plus C4bBP/protein
S complex. T -0 was the time at which the 2-hour sublethal E coli
or saline control infusions werebegun. The C4bBP and C4bBP/
protein S complex were infused as a bolus at T -30 minutes. Both
C4bBP and the complex had half-lives of approximately 10 hours."
An additional two groups of animals were studied in which: (1)
a neutralizing monoclonal antibody to protein S (2 to 4 mgkg of S163) was substituted for C4bBP and coinfused with sublethal E coli
(n = 4), and (2) a nonneutralizing monoclonal antibody to protein
S (3 mgkg of S-145) was substituted for C4bBP and coinfused with
sublethal E coli (n = 2). These studies were performed to test the
conclusion that alteration of protein S function was responsible for
the response to coinfusion of C4bBP with sublethal E coli. The
infusion and sample collection procedures were identical to those
described for the C4bBP studies.
Infusion Procedures
Experiments were performed on 24 juvenile baboons. They were
fasted overnight before each experiment and administered water ad
libitum. Each animal was sedated with ketamine hydrochloride (14
mgkg, intramuscularly) on the morning of the study, and anesthetized with sodium pentobarbital (2 m a g ) via a percutaneous catheter positioned in the cephalic vein. Animals were intubated orally
and allowed to breathe spontaneously. The femoral artery and vein
were cannulated aseptically and used for measuring arterial pressure
and obtaining blood samples, respectively. The percutaneous catheter in the cephalic veinwasused
for infusions of anesthetic as
described previously13and a percutaneous catheter in the saphenous
vein was used for infusion of E coli organisms. Each baboon was
placed on its side in contact with controlled temperature heating
pads. A light level of surgical anesthesia was maintained for the 6hour observation period (2 mgkg sodium pentobarbital approximately every 20 minutes).
Assays
The crossed immunoelectrophoretic assay used to study the concentrations of protein S and C4bBP and the partitioning of free
versus bound protein S in humans was not applicable to the study
of baboon protein S. Therefore, it was necessary to (1) estimate total
protein S and C4bBP by ELISA, (2) develop a radioimmunoblot
assay to assess the concentrations of free and bound protein S, and
(3) to validate this assay by gel filtration estimates of free and bound
protein S run in parallel.
Protein S EWSA. Microtiter plates (Costar vinyl assay plates)
were coated overnight at 4°C. with a monoclonal antibody against
human protein S, (S159 or BP91). ( l pg/mL in 50 mmol/L carbonate
buffer, pH 9.6, 50 &/well). The wells were washed three times
with 20 m m o K Tris-HC1, 0.15 molL NaCI, 0.1% Tween-20, pH
7.5 (wash buffer) and then incubated at room temperature for 1 hour
with 100 p L of 10 mg/mL bovine serum albumin (BSA) in 20 mmoll
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2644
TAYLOR ET AL
Table 1. Experimental Groups
Experiment No.
Sex
Weight
(kg)
E coli (CFUAg)
E coli at T + 2 h
(CFU/mL blood)
C4bBPIPS
(mgikg)
-
-
-
-
20
20
20
20
-
-
-
-
20/3.0
20/3.0
20
lo6
106
105
-
lo6
-
105
lo5 ? 1.36
-
105
105
105
105
20
20
20
20
20
C4bBP
8
10
13
Average t SE
C4bBP/Protein S
16
17
Average t SE
Sublethal E coli
S-3
S-4
S-7
S-6
S-8
F
M
F
M
F
-
M
M
4.2
M
F
4.6
5.2
8.2
6.0 t 1 . 1
5.8
5.7
5.8 5 0.1
6.1
7.1
6.4
14.6
3.7 X 109
3.7 X 109
X 109
3.3 X 109
4.5 X lo9
M
5.7
-
8.0
3.2i- 1.7
X
1
M
2
3
7
M
3.9
2.7
2.3
5.2
3.5 t 0.6
4.9 X
38.0 X
3.0 x
2.1 X
12.0 X
7.7
1.4
3.2
7.3
4.9 t 1.5
19.3 X
23.7 x
4.3 X
6.1 X
13.3 X
Average t SE
C4bBP plus sublethal E coli
Average i SE
C4bBP/protein S plus sublethal E coli
F
M
-
4
5
14
15
M
F
Average t SE
-
M
F
L Tris-HCI, 0.15 m o w NaCI, pH 7.5. After the wells were washed
three times with wash buffer, diluted citrated plasma samples, which
had been incubated for 2 hours at 37"C, were added in duplicate to
the wells (50 pL/well). Each plasma sample was tested at two dilutions on the same plate. The diluting buffer was 20 mmol/L TrisHCI, 0.15 m o m NaCI, 1 mmol/L EDTA, pH 7.5, containing 1 mg
BSNmL. The plate was incubated overnight at 4°C. After washing
each well three times with wash buffer, a polyclonal antiprotein S
(affinity purified v human protein S) was added to each well, (2 pg/
mL in diluting buffer, 50 pL/well). The plate was incubated for 1
hour at room temperature. After washing each well three times with
wash buffer, rabbit-antigoat IgG antibody conjugated with peroxidase was added to each well, ( I : 1,000 dilution in diluting buffer, 50
pL/well). The rabbit-antigoat conjugate was incubated with an equal
volume of mouse serum for 5 minutes at room temperature before
dilution. The plate was incubated for 1 hour at room temperature
then washed three times with wash buffer. Fifty microliters of substrate (1: 1 dilution of 2,2-azino-di-(3-ethyl-benzthiazolinesulfonate)
in hydrogen peroxide (Kirkegaard and Perry Laboratories, Inc,
Gaithersburg, MD) was added to each well and the plate was incubated for 20 minutes at room temperature. The absorbance at 405
nm of the wells was measured using a Vmax microplate reader
(Molecular Devices, Inc, Palo Alto, CA), set to endpoint and analyzed with a 4-parameter curve fit of the normal baboon plasma
dilutions. A standard curve of normal baboon plasma (seven dilutions) was used to determine the percent of total protein S of the
experimental samples. The concentration of protein S in experimental samples were expressed as percent of the protein S plasma concentration at T -0.
C4bBP ELSA. Microtiter plates (Costar vinyl assay plates) were
1 0 9 2 0.2
109
109
lo9
109
lo9 t 8.6
109
109
109
109
IO9 i 4.8
3.2 x
1.0 x
2.8 X
7.60 x
6.1 X
2.54 X
4.8
X
8.7 X
0.3 X
1.1 X
3.73 X
1.43 X
6.45 X
17.0 X
20/3.0
0.87 X
6.44 X
105 t 1.93
105
105
105
lo5
lo5 i 20/3.0
3.74
-
-
20/3.0
20/3.0
20/3.0
coated overnight, at 4"C, with a monoclonal antibody to C4bBP,
(BWl), (2.5 pg/mL in 50 mmoVL carbonate buffer, pH 9.6, 50 pL/
well). The wells were washed three times with 20 mmol/L phosphate,
0.15 moliL NaC1, 0.05% Tween-20, 0.02% NaN3, pH 7.4 (wash
buffer) then incubated at room temperature for 1 hour with SO pL
of 1.0 mg/mL BSA in 20 mmol/L phosphate, 0.15 mol&. NaCI,
0.02% NaN3, pH 7.4. After three washes of each well with wash
buffer, 50 pL of diluted citrated plasma samples were added to the
wells in duplicate, two dilutions of each. The diluting buffer was
20 mmol/L Tris-HCI, 0.15 mom NaCI, 1 mmol/L EDTA, 1 mmoll
L benzamidine, 0.01% Tween-20, pH 7.4, containing 5 mg/mL BSA.
The plate was incubated overnight at 4°C. After three washes with
wash buffer, 50 pL of a polyclonal anti-C4bBP was added to each
well (10 pg/mL diluting buffer). The plate was incubated for 1 hour
at room temperature then washed three times with wash buffer. Next,
50 pL of rabbit-antigoat IgG conjugated with alkaline phosphatase
was added to each well (1:250 with diluting buffer). The plate was
incubated again for 1 hour at room temperature and washed three
times with wash buffer. Then SO pL of p-nitrophenylphosphate
(Sigma Chemical CO, St Louis, MO) (1 mg/mL 0.18 mol/L carbonate, 2 mmol/L MgClz, 0.02% NaN3, pH 8.6) was added to each well
in a darkened room. The plate was incubated, in the dark, for 20
minutes and the absorbance at 405 nm was measured. The standards
were analyzed using a 4-parameter curve fit of the standards. A
standard curve of purified baboon C4bBP was used to determine the
C4bBP concentration (micrograms per milliliter) of the samples. The
experimental samples were calculated as concentration of C4bBP,
= 14.1.*'
pg/mL, using @To
Thrombin-anrirhrombin ELISA. Thrombin-antithrombin (TAT)
complexes were detected by using a sandwich ELISA, which em-
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ROLE OF FREE2645
PROTEIN S AND C4bBP
Table 2. Comparison of Results of Clinical Laboratory Assays of Animals Infused With C4b Binding Protein Alone
and W Binding ProteirVProtein S Complex
Measurement
ExDerimental GrOUD
MSAP (mm Hg)
C4bBP
C4bBPlPS
Hct (mm)
C4bEP
Fibrinogen (% baseline)
FDP (pg/dL)
Platelets (x1o3/mm3)
WBC (x103/mm3)
BUN CR
C4bBPlPS
C4bBP
C4bBP/PS
C4bEP
C4bBP/PS
C4bBP
C4bBPIPS
C4bBP
C4bBP/PS
C4bBP
C4bEP/PS
T-0
111 t 6
104 t 21
41 t 2
40 t 2
93 t 4
100
<IO
<IO
250 t 40
344 t 13
6.2 2 2.3
7 t 2.0
22 t 5
0.3 e 0.05
16 t 2
0.5 t 0.1
T+Zhrs
T+6h
122 2 11
118 t 18
43 t 1
41 t 1
89 2 10
92 t 1.5
181 t 54
273 t 72
8.7 t 3.0
13.3 t 0.1
116 t 5
134 t 7
41 c 2
42 t 0
81 t 13
87 t 7
<IO
<IO
189 t 69
148 e 85
10.3 t 2.4
10.2 2 1.1
18 t 5
0.2 t 0.08
16 t 2
0.4 t 0.1
-
T+24h
39 t 1
40 e 2
158 t 28
130 t 23
<IO
<IO
95 t l*
327 t 40
12.6 t 1.6
12.0 t 0.4
19 t 4
0.6 e 0.1
14 2
0.6 t 0.2
_f
Abbreviations: MSAP, mean systemic arterial pressure; HCT, hematocrit; FDP, fibrin degradation products; WBC, white blood cells; BUN,
blood urea nitrogen; and CR, creatinine.
* Indicates that the volume is significantly different fromother values for that time point ( P = c.05).
ployed a murine monoclonal antibody to thrombin, HTH1281, to
trap the complex and rabbit polyclonal antibody against antithrombin
followed by peroxidase conjugated antirabbit antibody to detect
bound TAT complexes. The murine monoclonal antibody to thrombin was prepared against thrombin that had been inactivated with
diisopropyl fluorophosphate. The antibody was prepared and isolated
by standard methods described previously.22The TAT complexes to
be used as standards in the ELISA system were prepared immediately before use by incubating 4.1 pmoVL thrombin with 4.6 pmol/
L antithrombin I11 for 10 minutes at 37°C in 20 mmoYL Tris-HCl,
0.15 mol& NaC1, 0.02% NaN3.
Microtiter plates (Costar) were coated overnight at 4°Cwith a
murine monoclonal antibody against human thrombin, HTH1281, 1
pg/mL in 50 mmolL carbonate buffer, pH 9.6, 50 pL1well. The
wells were washed three times with 20 mmoVL Tris-HC1.0.15 mol/
L NaCI, 0.1 % Tween-20, pH 7.5, (wash buffer) and then incubated
at room temperature for 1 hour with 1% BSA, 20 mmoUL Tris-HC1,
0.15 m o m NaCI, pH 7.5, 100 pL/well. After washing, citrated
baboon plasma samples, diluted 1 :40 in 0.1% BSA, 20 mmoyL TrisHCI, 0.15 m o m NaCI, 5 mmovL EDTA, pH 7.5 (diluting buffer),
were added in duplicate to the wells, 50 pL/well. A standard curve,
5 to 500 pM TAT complex was made by diluting 4.1 pmoVL TAT
complex into citrated normal baboon plasma that had been diluted
1:40. The plate was incubated for 1 hour at 37°C. After washing,
50 pLof a 1:5,OOO dilution of rabbit antiserum to human antithrombin was added to each well. The plate was incubated for 1 h at 37°C.
After washing, 50 pLof a 1:5,000 dilution of peroxidase conjugated
goat-antirabbit antibody was added to each well. The plate was
incubated for 1 hour at 37°C. After washing, 3, 3', 5, 5"Tetramethylbenzidine (TMB) substrate, 50 pUwell, was added and the plate
incubated at room temperature for approximately 20 minutes. The
was measured with the microplate reader set to end point and
analyzed with a 4-parameter curve fitofthe standards. The data
were reported as pmol TAT complex.
Separation of complexed andfiee protein S by gel$itration. CiL, was added to a Sephacryl S-400
trated baboon plasma, 0.5 m
(Pharmacia LKB Biotechnology, Inc, Uppsala, Sweden) column, 0.9
X 44.5 cm, which had been equilibrated with 20 mmoVL TrisHCI, 0.15 m o m NaCI, pH 7.5. This column had previously been
calibrated with normal citrated baboon plasma to which a trace of
radiolabeled human protein S had been added. This plasma was
incubated for 2 hours at 37°C to allow complex formation before
addition to the column. The column was eluted with 20 mmol/L
Tris-HC1, 0.15 m o m NaC1, pH 7.5 at room temperature at 2.5 mU
h using a peristaltic pump (Microperplex, Pharmacia LKB Biotechnology, Inc). Fractions of 0.5 mL were collected and the absorbance
at 280 nm was measured (DU 7 spectrophotometer; Beckman Instruments, Palo Alto, CA). The fractions were analyzed for protein S
using the protein S ELISA.
Immunoblotting. Agarose gel electrophoresis was performed as
described on citrated baboon plasma samples using 0.8% agarose in
0.07 moVL Tris-HC1, 0.03 moVL tricine, 2 mmoVL EDTA, 0.02%
NaN,, pH 8.6.23Proteins were transferred to nitrocellulose (Transblot transfer medium; BioRad Laboratories, Richmond, CA), by
semidry blotting." The blot was blocked with 10%nonfat dried milk
(Carnation, Los Angeles, CA) in 20 mmoVL Tris-HC1, 0.1 moVL
NaCI, 0.02% NaN,, pH 7.5,
for 1 hour at room temperature. The
immobilized proteins were detected immunochemically using a 1251labeled monoclonal antibody to human protein S, S-159, (approxim o a ) in the blocking solution.
mately IO' cpm/mL, 2 to 3 X
After shaking overnight at room temperature, the blot was then
washed four times, for 3 minutes each time, with shaking in 20
mmoVLTris-HC1, 0.1 m o m NaCI, 0.1% Tween-20, pH7.5. The
blot was dried and analyzed using a radioanalytic imaging system
(Ambis Systems; Ambis Systems, Inc, San Diego, CA).
Radioiodination of human protein S and S-159. Protein S was
purified from human plasma as de~cribed?~
The IgG of a monoclonal
antibody against human protein S (S-159) was prepared by ammonium sulfate precipitation. Human protein S and S-159 were radioiodinated using Enzymobeads (Bio-Rad Laboratories, Richmond, CA)
according to manufacturer's instructions. Briefly, 35 pg human protein S or 100 p g S-l59 was added to a 1.5-mL microtube and the
volume brought to 100 pL with 20 mmoVL Tris-HC1, 0.15, moVL
NaCI, pH 7.5. Next, 2 p L of 3 m o m Tris-HC1, pH 7.5 buffer, 50
pL Enzymobead, 1 mCi or 100 pCi Na125I for protein S and S-159,
respectively, and finally 50 pL &glucose were added. The tube
contents were mixed and allowed to stand for 20 minutes at r w m
temperature. The reaction was stopped by desalting the solution
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2646
TAYLOR ET AL
Table 3. Comparison of Results of Clinical Laboratory Assays of Animals Infused W i h Sublethal E coli Alone and in Combination
With C4b Binding Protein and With C4b Binding Protein/Protein S Complex
Experimental
Group
Measurement
MSAP (mm Hg)
Het (mm)
43
Shistocytes (% of 400 RBC)
Fibrinogen (% baseline)
FDP (pg/dL)
Platelets ( x 103/mm3)
WBC ( X 1 0 3 1 ~ ~ 3 )
BUN (mg/dL)
CR (mg/dL)
8.1
SLEC
C4bBP/SLEC
C4bBP/PS/SLEC
SLEC
C4bBP/SLEC
C4bBP/PS/SLEC
SLEC
C4bBP/SLEC
C4bBP/PS/SLEC
SLEC
C4bBP/SLEC
C4bBP/PS/SLEC
SLEC
C4bBP/SLEC
C4bBP/PS/SLEC
SLEC
C4bBP/SLEC
C4bBP/PS/SLEC
SLEC
C4bBP/SLEC
C4bBP/PS/SLEC
SLEC
C4bBP/SLEC
C4bBP/PS/SLEC
T-0
T+2h
Tc6h
97 t 8
81 i- 8
102 -c 6
104 -t 3
124 -t 9
i- 1
70 t 6
89 _t 4
41 f 1
102 I 3
112 i- 15
43 i- 1
41 2 1
44 t 2
0.2
1.10 I
0.9 2 0.1
1.0 -t 0.2
100
100
100
10 i- 0
10 t 0
10 t 0
210
276 -t 30
173 % 51
155
-t 52
191
t 1.6
6.9 t 0.7
1.7
10.5 i- 2.8
15 t 2
0.5 i 0.1
29 -t 2
0.5 0.1
30 t 3
0.6 i- 0.1
39 i- 1
44 i 1
38 i l*
44 i- 1
-
-
-
-
-
-
95 t 5
88 2 3
67 _t 14
102 i- 8
2 t l*
60 -t 5
27 2 9
640 t O*
12 i 2.5
120
188 i 29
39 -t 13
150 -t 12
5.7 -c 2.9
3.1 t 1.0
6.0 t 1.1
17.3 -t 2
0.6 -t 0.1
39 2 6
3.51.4 2 0.4
27 i- 3
0.6 i- 0.1
-
-
i- 19
117 2 19
2 45
1.5 t 0.3
t 0.1
2.0 t 0.4
-
-
T t24h
37 t 1
27
44
1.2
7.6
1.0
146
2 3*
i- 1
t 0.4
2 1.8*
i- 0.3
2 39
41 i- 16
133 i- 7
15 i 5'
220 i- 50*
40 2 7
2 32
41 i- 9"
120 I 1 5
12.6 ? 4.2
11.9 i- 4.8
18 i7
25 1 9 . 5
0.6 2 0.1
93 l 19*
? 1.1*
28 lr 7.0
1.0 2 0.1
* Significantly different from other values for that time point ( P = <.05).
using aPD-10 column (PharmaciaLKB Biotechnology AB, Uppsala,
Sweden). The column had been washed with 20 mmol/L Tris-HC1,
0.1 m o m NaCI, 0.1%BSA, 0.02%NaN,,pH 7.5, then equilibrated
with 20 mmovL Tris-HC1, 0.1 m o m NaCI, 0.02% NaN3, pH 7.5.
The protein S or S-l59solutions were added to the column and the
labeledproteinselutedwithequilibrationbuffer.Fractions
of 0.5
mL were collected and aliquots counted in an Isodata
500 gamma
counter (Isodata 500; Iso-data, Rolling Meadows, IL). Fractions containing the highestcounts were pooled. The concentration was determined by measuring the absorbance at
280 nm (DU7 spectrophotometer, Beckman Instruments). The solutions weremade 5 mg/mL in
BSA and stored at 4"C, respectively.
Statistical Analysis
Students two-tailed t-test was used to determine significant differences ( P = s .05) between groups at a given time (in hours) for a
given variable. Standard errors of the mean are shown in Tables 2
and 3. Standard deviations of the mean are shown in the figures.
RESULTS
Response to Infusion of C4bBP Alone and to the C4bBP/
Protein S Complex
Figure 1 shows total protein S and C4bBP (Fig 1A) and
shows that infusion of 20 mgkg of C4bBP reduced the
endogenous free protein S to almost zero, while increasing
that portion of protein S that is bound to C4bBP (Fig 1B and
D). Gel filtration (Fig IC) indicates that the radioimmunoblot
accurately reflected the effect of C4bBP infusion onthe
free protein S concentration. Before infusion of C4bBP (- 1
hour), the added radiolabeled protein S exists in both free
and complexed form (open triangles). After infusion of
C4bBP ( + 2 hours), the added radiolabeled protein S exists
primarily in its complexed form (open triangles). Open
squares represent absorbance of protein filtrate at 280 mm.
The above results were taken from animal C4bBP No. 10.
Those of C4bBP No. 13 and No. 8 were similar. See Table
1 for list of animal groups.
Figure 2 shows total protein S and C4bBP (Fig 2A) and
shows that infusion of C4bBP/protein S complex (20 and 3
mgkg, respectively) did not reduce the endogenous free protein S (Fig 2B and C). The above results were taken from
animal C4bBP No. 16. That of C4bBP No. 17 was similar.
See Table 1 for list of animal groups.
Table 2 shows that the infusion of either C4bBP or the
C4bBP/protein S complex has little effect on vital signs or
clinical chemistries. Infusion of C4bBP alone over a period
of 24 hours, however, produced a profound fall in platelet
count. This was not accompanied, however, by other evidence of activation of the coagulant system, such as TAT
111 complexes. In contrast, neither infusion of the C4bBP/
protein S complex nor sham surgery produced anysignificant
decrease in platelet count.
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
2647
ROLE OF FREE PROTEIN S AND C4bBP
A
loo
Cn
60
o
40
L
0
20
Fig 1. Effect on infusion of
C4bBP on total protein S and
free versus bound protein S in
plasma. Comparison of plasma
concentrations of total protein S
and total C4b-binding protein
(A): infusion of C4bBP results in
a fivefold increase in C4bBP concentration (@ 142 pglmL at T
-60 to 840 pg/mL at T -0
plasmal and a transitory decrease in total platelet poor
plasma protein S as detected by
ELISA. Errorbars are standard
deviations. Antiprotein S antibody immunoblot of freeprotein
S (71 kD)versesC4b-binding
protein complexed t o protein S
(640 kD). (B1 Lanes areidentified
as follows: lanes 1 and 2,normal
humanplasma;lane 3, normal
baboon plasma; lane 4, human
protein S;lanes, 5 to 12 baboon
plasma at T -1.T -0, T +l, 3,
2,
4, 6, and 24 hours after infusion
with C4b binding protein 120
mglkg); lane 13, human protein
S; lane 14, normal baboon
plasma; lanes 15 and 16, normal
human
plasma.
Gel
filtration
profiles of free versus bound
protein S (Cl: gel filtration of radiolabeled protein S added t o
plasma showsthat atT -1 hour
before infusion of C4bBP. the tot a l protein S is distributed
equally between free andbound
forms and that at T +2 hours
after infusion of C4bBP. the total
protein S is almost all bound.
Free versus bound protein S as
determined from immunoblot
(Dl: the results of immunoblot
are similar to reJuks of gel filtration and show graphically
that at T -1 hour beforeinfusion
of C4bBP protein S is distributed
equally between the freeand
bound forms, whereas at T -0
hours after infusion of C4bBP.
protein S is almost all bound.
0
BI
-
I
2 3 4 5 6 7
1
-0B'
8 9
10 1112 13
14
1516
1.00
m
N
4
0.W
0.00
20
50
M
60
R a d i a n no
30
60
U)
W
Fraction no
l2
D
Response to Infusion of Sublethal E coli and to Sublethal
E coli PIUS C4hBP Versus Sublethal E coli PIUS C4bBP/
Protein S Complex
Sublethal E coli alone, sublethal E coli with either C4bBP
or with C4bBP/protein S complex were infused. Figure 3A-
l0
2
0
-
1
0
1
2
3
4
5
8
24
Time (h)
C shows totalprotein S and C4bBP concentration, andFig
3D-F show the free versus bound protein S following infusion of these three different combinations. Only when sublethal E coli is coinfused with C4bBP is the free protein S
concentration reduced (Fig 3E) and the lethal coagulant re-
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
TAYLOR ET AL
2648
II
0
1
2
3
4
5
Y
1
24
Time (h)
B
C4bBP:PS+
Free PS+
1
c
2
3
4
S 6 7 8 9 10
11 12 13 1415
22‘
l6
16
14
z”
BOUND
6 -
4 -
.
,
1
2
I
0
3
4
Time (h)
sponse observed (Table 3 ) . When sublethal E coli is coinfused with the CQbBP/protein S complex, no reduction in
free protein S or lethal coagulant response is observed. The
above results are takenfrom animals S-3, 4, 6, 7, and 8;
C4bBP, 1, 2, 3, and 7; and C4bBP, 4, 5 , 14,and 15 (see
Table 1 for list of animal groups).
The pathophysiologic changes in the vital signs and clinical chemistries seen following infusion of the three reagent
combinations are summarized in Table 3. The results in this
table show that the fall in hematocrit, fibrinogen, and platelet
count is coincident with the appearance of shistocytes and
increase in bloodureanitrogen
following coinfusion of
C4bBP with sublethal E coli. While there is a reduction
in platelet count in all cases, it ismostprofoundwith
the
5
6
24
Fig 2. Etfeet on infusion of
C4bBP protein S complex ontot a l protein S andfreeversus
bound protein S in plasma. Comparison
of
plasma
concentrations of total protein S and total
C4b-binding protein (AI: infusion
of C4bBPlprotein S complex results in an
increase
in both
C4bBP andprotein S as detected
by ELSA. This is reflected byincreases of both free and bound
protein S in the immunoblot (B
and C). Antiprotein S antibody
immunoblot offree
protein S
versus C4b-bindingprotein complexedto protein S (B): lanes are
as follows: lanes l and 2, normal
human and baboon plasma; lane
3, human protein S; lane 4,
empty;lanes 5 to 14, baboon
plasma at T -0, T +l/,, l/*, 1,2,3,
4,6, 24, and 48 hours after infusion of C4bbinding protein complexed with protein S (20/3 m g l
kg]; lane 15, human protein S.
Free versus bound protein S as
determined from immunoblot
(Cl.
combination of C4bBP and sublethal E coli. Furthermore, it
is only with this combination that there also is a decrease
in hematocrit, appearance of shistocytes, consumption of
fibrinogen, and marked increase in creatinine and blood urea
nitrogen. This was also accompanied by the appearance of
TAT 111 complexes after the infusion of sublethal E coli
alone and C4bBP with sublethal E coli. In both cases, the
TAT 111 complex concentrations peaked at T +6 hours (6.3
and 6.4 5 2.0 pm, respectively). In contrast, coinfusion of
sublethal E coli with the C4bBP/protein S complex was not
accompanied by thrombosis or any of the above changes in
hemostatic factors.
Figure 4 compares the histopathologic appearance of the
renal glomerulae after infusion of the three reagent combina-
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2649
ROLE OF FREE PROTEIN S AND C4bBP
26
20
-
l6
.
24
I
i 5
11
12
-
24
28
20
t
18
.
-
BOUND
8
-
(
I
FREE
0
.-0
d
BOUND
0
-
1
0
1
2
1
3
*l
0
4
5
8
24
Time (h)
-
1
0
1
2
3
4
5
6
24
Time (h)
Fig 3. Effect on infusion of sublethal E coli alone, with C4bBP. or with C4bBPlprotein S complex on total protein S and free versusbound
protein S in plasma. Left column: total protein S and total C4b binding protein following infusion of sublethal E coli alone (A), sublethal E coli
plus C4b binding protein (B),and sublethel E coliplur C4b binding protein complexed to protein S (C). Right column: free versusbound protein
S following infusion of sublethal E coli alone (D),
sublethal E coli plus C4b binding protein (E), and sublethal E coli plus C4b binding protein
complexed to protein S (F).
tions with that of a patient who died of hemolytic uremic
syndrome. Note that the microthrombi and tubular necrosis
is associated only with the infusion of C4bBP along with E
coli. These changes are similar in appearance to that obtained
from a patient with hemolytic uremic syndrome.
Response to Infusion of Sublethal E coli Plus Neutralizing
and Nonneutralizing Antibodies to Protein S
These studies were performed to establish by another
means whether protein S neutralization by C4bBP was responsible for promoting the pathophysiologic response to
sublethal E coli. Coinfusion of S-l63 neutralizing antibody
with sublethal E coli resulted in a significant drop in fibrinogen concentration, platelet count and increase in creatinine
comparedwiththatfollowing
infusion of S-l45 nonneutralizing antibody with sublethal E coli. The fibrinogen concentrations at T +6 hours were 26% ? 15.9% versus 74%
? 17%, while the platelet concentrations at that same time
pointwere 89 ? 36 versus 202 ? 122 X 103/mm3.The
creatinine concentrations at T +24 hourswere 2.0 ? 0.8
versus 0.5 t 0.1. The decrease in hematocrit andappearance
of fibrin degradation products were also more pronounced
in the animals receiving the neutralizing antibody to protein
S (S-163). Histopathologic studies showed microthrombi and
From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
TAYLOR ET AL
2650
1.
D
Fig 4. Comparisonof renal glomerular lesions:
sublethal € coli alone (A), sublethal E co/i plus C4b
binding protein (B), sublethal€ coliplus C4bBP complexed to protein S (C), and patient with hemolytic
uremic syndrome (D). Arrows point to microvascular
thrombi.
renal tubular necrosis only in those tissues from animals
receiving neutralizing antibody to protein S .
DISCUSSION
It has been shown in vitro that increasing concentrations
of C4bBP relative to a fixed concentration of free protein S ,
not only binds, but also neutralizes cofactor activity of free
protein S for activated protein C.*." It also has been shown
that C4bBP saturated with protein S neither binds nor neutralizes free protein S.R"'This study demonstrates that (1) the
elevation of C4bBP two to five times normal concentration
decreases free protein S in vivo, as well as in vitro. This is
associated with a decrease in platelet count to one-half baseline concentration of 24 hours withno changes in other
coagulant or clinical chemical parameters assayed, (2) saturation of the C4bBP infusate with protein S prevents this
phenomenon, (3) coinfusion of C4bBP with sublethal E coli
reduces free protein S and converts a nonlethal inflammatory
response to E coli into a lethal inflammatory consumptive
coagulopathy microvascular thrombosis, and (4) coinfusion
of C4bBP saturated withprotein S plus sublethal E coli.
attenuates both the reduction of free protein S and the lethal
response to the C4bBP/sublethal E coli combination.
The reduction in platelet count by T +24 hours is common
to both the C4bBP alone and C4bBP plus sublethal E coli
infusion studies. However, this platelet response to C4bBP
alone is of particular interest because it does not occur in
infusion of C4bBP/protein S complex. Under normalcircumstances the high affinityof thrombin that might be generated
spontaneously in the microcirculation for thrombomodulin
prevails over other thrombin substrate/receptor interaction.'
This thrombin/thrombomodulin complex, in turn, converts
protein C to activated protein C. We believe that this activated protein C with its cofactor protein S , in turn, arrests
premature platelet activation and sequestration by inactivating the platelet Va produced during transit through the gen-
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2651
ROLE OF FREE PROTEIN S AND C4bBP
eral circulation. Neutralization of protein S , however, by
elevated levels of C4bBP, we believe, could prevent this
laundering of platelets in the microvasculative and lead to a
reduced life span and decrease in platelet count. Our observations comparing the responses to C4bBP with C4bBP/protein
S , and neutralizing with nonneutralizing antibodies against
protein S support this interpretation. However, our failure to
detect TAT 111 complexes after infusion of C4bBP alone is
of interest because antithrombin I11 has been shown to bind
and remove thrombin from its thrombomodulin receptor. The
limit of detection of our assay of TAT TI1 complex is 0.25
pmol/L. The concentration of this complex generated may
be below this limit. Infusion of C4bBP with an inflammatory
agent (sublethal E coli) amplifies the above phenomena to
the point where sufficient thrombin is generated so that not
only are these TAT I11 complexes detectable, but also a fullblown microvascular thrombosis ensues. That platelets play
a dominant role in this response is supported by our observation that coinfusion of antibody to GPIIb-IIIa inhibits this
response.z6 Finally, the observation that neutralizing antibody to protein S (S-163) produces an inflammatory coagulapathy when coinfused with sublethal E coli, whereas nonneutralizing antibody to protein S (S-145) does not, supports
by another means the conclusion that neutralization of protein S anticoagulant activity and not alteration of some other
property of C4bBP on binding to protein S is responsible
for the phenomenon observed.
Although this study may provide a molecular mechanism
explaining the results in this baboon model reported by us
previously,’3 it also coincides with the observation of
D’Angelo et a17 of low free protein S in patients with manifest disseminated intravascular coagulation. However, the
C4bBP levels in these patients were not elevated and the
connection, therefore, between low free protein S and high
C4bBP levels was not established. Furthermore, patients
with high C4bBP levels after orthopedic surgery were not
observed to have low free protein S concentration^.^' Thus,
whereas low free protein S concentrations have been associated with severe inflammatory states such as sepsis and disseminated intravascular coagulation, the role of C4bBP in
reducing free protein S has not been established. It is more
likely in these acute inflammatory states that the levels of
protein S, as well as protein C, may be reduced as a part of
a consumptive process, rather than binding and neutralization by elevated concentrations of C4bBP. Further documentation in patients of such C4bBP/protein S interactions is
complicated by difficulty in assay of free protein S and the
wide range of normal
Thus, while the results of
this study show that reduction of the components of the
protein C anticoagulant system can exacerbate the inflammatory coagulopathic response, C4bBP induction of free
protein S deficiency in inflammatory states may applyonly to
certain inflammatory disorders systemic lupus erythematosus
(SLE) or to the later stages of these disorders in which levels
of C4bBP have increased. These disorders might include
subacute disorders such as postdiarrheal hemolytic uremic
syndrome as opposed to acute disorders such as sepsis or
postoperative states.
With respect to hemolytic uremic syndrome, this model
may be ofparticular interest. It closely duplicates the physiologic changes seen in hemolytic uremic syndrome. In this
case infusion of C4b binding protein with sublethal E coli
produces a disseminated intravascular coagulopathy of varable intensity followed by a severe progressive microvascular thrombosis involving renal glomerular and pulmonary
alveolar capillaries. This is associated by a profound decrease in platelet count and hematocrit, with the appearance
of shistocytes, an increase in creatinine and a histopathologic
picture of multiple microthrombi in the renal glomerular
capillaries. Therefore, this primate model mimics the hemolytic uremic syndrome with the exception that the consumption of fibrinogen in this model ismore severe thanthat
usually seen clinically. Even in this case, however, the microvascular thrombosis is developing as the fibrinogen increases and in cases where animals are followed for 48 hours
(not reported here), the microvascular thrombosis continues
with a continual increase in blood urea nitrogen in the face
of fibrinogen levels that are near or above normal. However,
the question remains whether evidence for low free protein
S secondary to C4bBP elevation can be established in patients with hemolytic uremic syndrome, thrombotic thrombocytopenic purpura, and in other syndromes in which microvascular thrombosis is a dominant feature.
ACKNOWLEDGMENT
We acknowledge the contribution of Clendon Brown and Jefferey
Box in preparation of C4bBP and protein S, and of Joy Albert-Gorr
and Richard Irish in preparation of the manuscript and figures.
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From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
1995 86: 2642-2652
Role of free protein S and C4b binding protein in regulating the
coagulant response to Escherichia coli
FB Jr Taylor, B Dahlback, AC Chang, MS Lockhart, K Hatanaka, G Peer and CT Esmon
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