Radioimmunoassays for Protein C and Factor X
Plasma Antigen Levels in Abnormal Hemostatic States
DAVID J. EPSTEIN, M.D.,* PETER W. BERGUM, M.S., S. PAUL BAJAJ, PH.D.,
AND SAMUEL I. RAPAPORT, M.D.
Departments of Pathology and Medicine, School of
Medicine, University of California at San Diego,
San Diego, California
Specific radioimmunoassays, sensitive to plasma levels of less
than 1% of normal, were developed for protein C and Factor
X. In 31 normal subjects, mean plasma antigen levels were as
follows: protein C, 3.23 ± 0.79 Mg/mL (2 SD); Factor X, 7.74
±1.81 pg/mL. In patients on chronic warfarin therapy, protein
C and factor X were depressed equivalently: protein C, 42%
± 20% (of a pooled plasma reference); Factor X, 44% ± 24%.
Protein C antigen fell much more rapidly than Factor X antigen
when warfarin therapy was begun, creating an initial period of
potential hypercoagulability. In patients with severe liver disease, mean protein C antigen (25% ± 17%) was lower than
Factor X antigen (51% ± 29%). Protein C antigen levels did
not appear to be a sensitive indicator of compensated intravascular coagulation or systemic fibrinolysis induced by infusion
of streptokinase. Clinical implications of these findings are
discussed. (Key words: Protein C; Vitamin K dependent proteins;
Factor X; Warfarin; Oral anticoagulants; Disseminated intravascular coagulation; Coumarin skin necrosis; Thrombosis;
Liver disease) Am J Clin Pathol 1984; 82: S73-S81
PROTEIN C is a vitamin K dependent zymogen of a
serine protease, which, when activated, functions as an
anticoagulant. The physiologic activator of protein C is
believed to be thrombin in complex with an endothelial
cofactor, thrombomodulin.9 Activated protein C proteolytically degrades coagulation factors Va and Villa15
and also elicits fibrinolytic activity in plasma.7
Assays for bioactivity of plasma protein C just now
are being developed. Consequently, clinical investigations
of protein C as yet depend upon immunologic assays of
protein C antigen. From studies employing electroimmunoassay of protein C antigen, we know that plasma
protein C is depressed in patients receiving warfarin,410
in liver disease,14 in some postoperative patients,14 and
in some patients with disseminated intravascular coagulation."14 Moreover, hereditary deficiency of protein
Received January 3, 1984; received revised manuscript and accepted
for publication March 26, 1984.
Supported in part by Grant PHS HL27234 from the National
Institutes of Health. During this work Dr. Epstein was supported by
an NIH training grant, PHS HL07107. Dr. Bajaj is supported by an
Established Investigatorship (83-176) of the American Heart Association.
Address reprint requests to Dr. Rapaport: University of California
Medical Center H-811K, 225 Dickinson Street, San Diego, California
92103.
* Dr. David Epstein passed away after submission of this article.
C has been reported to cause a familial thrombotic
diathesis.610
Since the normal plasma concentration of protein C
approaches the sensitivity limit of conventional electroimmunoassays, we developed a specific radioimmunoassay (RIA) for protein C antigen. Using this radioimmunoassay together with radioimmunoassays for Factor
X antigen and Factor VII antigen, we have examined
the kinetics of protein C depression after administration
of warfarin. We also have measured protein C antigen
in several other clinical circumstances in which alteration
of vitamin K dependent clotting factors is known or
suspected.
Materials and Methods
Reagents
Congenital factor deficient human plasmas were purchased from George King Biomedical Inc., Overland
Park, Kansas. A sample of highly purified human protein
S was a generous gift from Dr. W. Kisiel. Agarose (BioGel-A15m) and Enzymobeads were obtained from Bio
Rad Laboratories, Richmond, California. Bacto gelatin
was a product of Difco Laboratories, Detroit, Michigan.
Rabbit anti-goat IgG and normal rabbit serum were
purchased from Research Products International Corp.,
Elk Grove Village, Illinois. All other reagents and chemicals were of the best available commercial grade.
Plasma Specimens
Use of volunteer blood donors was approved by the
Human Subjects Committee, University of California,
San Diego. Normal volunteers were students and staff
at the University of California, San Diego Medical
Center. Patient specimens were obtained at the San
Diego Veterans Administration Medical Center and at
573
574
EPSTEIN ET AL.
the University of California, San Diego Medical Center.
Additional clinical specimens were from the Special
Coagulation Laboratory, University of Southern California-Los Angeles County Medical Center. Blood samples
were anticoagulated with one part of 29 g/L sodium
citrate in nine parts blood. Platelet poor plasma was
separated by high-speed centrifugation and stored frozen
until use.
Coagulation Tests
Catalytic activity of purified activated protein C was
measured semiquantitatively by prolongation of the
activated partial thromboplastin time or by a chromogenic substrate assay.2 Coagulant activities of prothrombin, Factor VII, Factor IX, and Factor X were measured
as previously described by Bajaj and associates.3 Prothrombin times were measured using either rabbit brain
or human brain tissue factor. Fibrinogen was assayed
by a thrombin time method, and fibrinogen degradation
products were measured by the Thrombo-Wellcotest.®
Plasminogen was determined by a chromogenic substrate
assay (Protopath® Plasminogen Assay, Kimberly-Clark
Corp., Neenah, WI).
Purified Proteins
Protein C, Factor X, Factor IX, prothrombin, protein
S, and Factor VII were purified from normal human
plasma as detailed elsewhere by Bajaj and colleagues.2,3
After preparative gel electrophoresis, most protein C
preparations contained up to 10% contaminating Factor
X. Factor X was removed from protein C preparations
by passing the protein C through a goat anti-human
Factor X antibody column.2 Concentrations of pure
proteins were determined by optical density at 280 nm
using published extinction coefficients.812
Protein C and Factor X Antisera
Specific antisera against protein C and Factor X were
raised in goats. On day 1 of immunization, 25 to 30 ng
of purified protein in TRIS-buffered saline was emulsified
with an equal volume of Freund's complete adjuvant
and injected intramuscularly. Immunization was repeated
on day 8 and day 16 with intradermal injection of 25
to 30 ng antigen in incomplete Freund's adjuvant. One
week later, blood was drawn and the resulting serum
was centrifuged twice and heat inactivated for 30 minutes
at 57 °C. Sodium oxalate was added to 0.01 M, and the
serum was absorbed in the cold with 100 mg/mL barium
sulfate. The suspension was centrifuged, and the supernatant serum was used for radioimmunoassay without
further purification. Specificity of antisera was evaluated
by double immunodiffusion against purified vitamin K.
A.J.C.P. • November 1984
dependent proteins2 and by inhibition of coagulant
activity in specific factor assays.
Radiolabeled Proteins
Protein C and Factor X were labeled with 125I using
a commercial preparation of coimmobilized glucose
oxidase/lactoperoxidase (Enzymobeads). Prior to iodination, the purified protein was incubated for 30 minutes
at 25 °C in TRIS-buffered saline containing 5 mM diisopropyl fluorophosphate. The protein then was dialyzed
overnight in the cold against 1,000 volumes of 0.2 M
phosphate buffer, pH = 7.2. In a typical labeling reaction,
10-20 ng protein in 75 pL 0.2 M phosphate buffer, pH
= 7.2, was mixed with 50 fiL hydrated Enzymobeads®
and 1 mCi fresh 125I-KI. The reaction mixture was
incubated at 25 °C for 30-40 minutes. Iodination was
terminated by addition of 150 fiL TRIS-buffered saline,
containing 0.2 g/L sodium azide and 1.0 g/L gelatin.
Labeled protein was separated from free 125I by passing
the reaction mixture over a 0.5 X 10 cm column of
Sephadex G-25 M equilibrated with TRIS-buffered saline
containing 0.2 g/L sodium azide and 1.0 g/L gelatin.
Purity of radiolabeled protein preparations was established from radioactivity profiles after sodium dodecyl
sulfate (SDS) polyacrylamide gel electrophoresis as described elsewhere.213 Iodine-labeled proteins were stored
at -70 C C for up to six weeks.
Radioimmunoassays
The technic employed for radioimmunoassay of protein C and Factor X was adapted from the method of
Price and associates.18 All reagents for RIA were prepared
in an assay diluent containing 0.14 M NaCl, 0.01 M
sodium phosphate, 0.025 M EDTA, 1.0 g/L gelatin, 1.0
g/L Tween-20, pH = 7.4. Each RIA tube received, in
order of addition, 200 fiL of assay diluent, 100 nL of
diluted sample, 20,000 to 30,000 cpm of radiolabeled
protein C (or radiolabeled Factor X) in 100 ftL of buffer,
and 100 nL of the appropriate specific antiserum. In
nonspecific binding tubes, specific antibody was replaced
by buffer alone, and in maximal binding tubes the
sample was replaced by buffer. Tubes were incubated
16 h at 4°C followed by addition of 100 fiL diluted
normal goat serum and 100 pL of diluted rabbit antigoat IgG. After 2 hours incubation at 4°C, 2 mL of
cold TRIS-buffered saline containing 1 g/L bovine albumin was added to each tube, the tubes were centrifuged, and the supernatant was decanted. Radioactivity
in the pellets was counted for 1 minute.
All samples were assayed at least in duplicate and
usually in triplicate. In initial experiments, standards of
purified protein C were employed to determine the
concentration of protein C antigen in a pooled reference
Vol. 82 • No. 5
575
PROTEIN C AND FACTOR X ANTIGENS
plasma prepared from 15 healthy volunteers. In subsequent clinical studies, each RIA included serial dilutions
of this pooled reference plasma as standards. Each
unknown specimen was diluted 1:50 prior to assay.
Results were calculated by linear regression of the function logit(B/Bo) as described by Rodbard.19 Slope, intercept, and correlation coefficient of the logit fit were
monitored as indicators of assay performance.19 Results
were expressed as Mg/mL or as percentage of the pooled
reference plasma.
Initial evaluation of results revealed a consistent small
overestimate of antigen (not exceeding 20%) when the
normal pooled reference plasma itself was assayed at a
1:50 dilution as a test sample. This problem eventually
was traced to a discrepancy between the calibration of
the pipette used for the serial dilutions of the standard
curve and the pipettes used for the one step dilution of
test samples. Since all assay runs included nine control
samples of normal pooled plasma diluted with the same
technic as used for the unknowns, it was possible to
correct for this dilution error. To accomplish this, we
divided the logit derived antigen level for each unknown
test sample by the mean antigen level for the nine
control samples of pooled reference plasma included in
that run.
Similar technics were used for radioimmunoassay of
Factor VII antigen. Details will be presented in a subsequent publication.
Results
Validation of Protein C Radioimmunoassay
The specific activity of freshly prepared l25I protein C
was approximately 10 /iCi/jig. On nonreduced SDS gels,
the radiolabel migrated as a single peak with apparent
molecular weight identical to that of purified protein C
on stained gels (Mr = 62,000). On reduced SDS gels,
radioactivity was partitioned into two peaks with mobilities characteristic of the protein C heavy and light
chains. On double immunodiffusion against all of the
vitamin K dependent proteins, protein C antiserum
gave a precipitin line only with protein C. In a typical
fresh preparation of iodinated protein C, 85% of radioactivity was immunoprecipitable by saturating concentrations of protein C antiserum. Sixty percent of radiolabeled protein C was precipitated by a 1:25000 dilution
of protein C antiserum, and this dilution of antiserum
was used for the RIA.
Figure 1 is a representative standard curve for the
protein C radioimmunoassay. Radioligand binding was
completely inhibitable by saturating concentrations of
purified protein C, and the titrations of purified protein
and plasma were parallel over the full range of the assay.
Protein C Concentration, /ug/ml
LO1^
.156
-i
1
1
.0024
r
o
m
m
64
Plasma Dilution
4096
FIG. 1. Typical standard curves in the protein C radioimmunoassay.
Each point represents the mean of triplicate determinations.
Purified vitamin K dependent proteins other than protein
C gave negligible cross-reactivity in the assay (Table 1).
Increasing concentrations of purified protein C were
added to normal pooled plasma and the protein C
concentration determined by RIA was plotted against
the concentration of purified protein added. This plot
gave a straight line with a correlation coefficient of r
= 0.976. The slope of the line was 0.94 (the analytic
recovery of the assay) and the intercept was 3.44 fig/mh
(the endogenous plasma level of protein C). The working
range of the protein C assay extended to less than 0.02
Mg/mL, allowing us to quantitate protein C at levels of
1% of the normal plasma concentration. In this assay,
and in the Factor X assay described below, the withinrun coefficient of variation was 5-10% and the betweenrun coefficient of variation was less than 10% when the
radiolabeled protein was less than four weeks old.
Validation of Factor X Radioimmunoassay
The specific activity of freshly prepared l25I Factor X
was 30-40 fid/ng. On SDS gels the radiolabeled protein
migrated identically to native purified Factor X. In
Table 1. Negligible Cross-reaction of Other Vitamin K
Dependent Proteins in the Protein C
Radioimmunoassay
Specimen
Total Protein
in Sample
Gig/mL)
Protein C
Measured in
Sample (Mg/mL)
Protein C
Prothrombin
Factor VII
Factor IX
Factor X
Protein S
10
140
50
95
36
160
<0.06
0.17
<0.06
0.18
0.34
10
Percent of
Protein as
Protein C
100%
<0.04%
0.34%
<0.09%
0.5%
0.21%
576
EPSTEIN ET AL.
Factor X Concentration, /ug/ml
, n 2.5
.078
1.0i
1
1
1
1
1
.0024
1
1
1
A.J.C.P. • November 1984
/tg/mL. Sensitivity of the Factor X RIA extended to
0.06 Mg/mL, or about 1% of the normal plasma level.
• —
Protein C and Factor X Antigen Levels in a
Normal Population
Purified r
factor X /
5
.5m
/
-
J
/
/
/ Plasma
/
fll
/
M
~
1
i
'
'
'
I
I
32
Plasma Dilution
I
I
1024
FIG. 2. Typical standard curves in the Factor X radioimmunoassay.
Each point represents the mean of triplicate determinations.
double immunodiffusion experiments with purified vitamin K dependent proteins, Factor X antiserum gave
a strong precipitin line with purified Factor X and a
weak line with purified prothrombin. Over 90% of
radiolabeled Factor X was immunoprecipitable with
Factor X antiserum. Sixty percent of radiolabeled Factor
X was precipitated by a 1:40,000 dilution of Factor X
antiserum, and this dilution of antiserum was used for
the radioimmunoassay.
Figure 2 is a representative standard curve for the
Factor X radioimmunoassay. Radioligand binding was
completely inhibitable by saturating concentrations of
purified Factor X, and the titrations of purified protein
and plasma were parallel over the full range of the assay.
Cross-reactivity with other vitamin K dependent proteins
was not significant (Table 2). When purified Factor X
was added to normal pooled plasma and assayed by
RIA, analytic recovery of Factor X antigen was 93%
and the endogenous plasma level was calculated at 7.9
Table 2. Negligible Cross-reaction of Other Vitamin K
Dependent Proteins in the Factor X
Radioimmunoassay
Total Protein
in Sample
Factor X
Measured
in Sample
Specimen
(/ig/mL)
(/ig/mL)
Percent of
Protein as
Factor X
Factor X
Prothrombin
Factor VII
Factor IX
Protein C
Protein S
Congenital X
deficient plasma
10
100
5
10
2
20
10
0.02
0.014
<0.08
0.20
0.035
100%
0.02%
0.28%
<0.8%
1.0%
0.17%
—
<0.12
—
Protein C and Factor X antigens were assayed in
plasma from 31 healthy volunteers. A plot of the data
on normal probability axes indicated that the population
was described adequately by a gaussian distribution. The
mean value for protein C antigen was 3.23 /ig/mL
± 0.79 jug/mL (this value and subsequent population
values are expressed as mean ± 2 SD). Expressed as
percent concentration relative to our normal reference
pool, this corresponded to 94% ± 23%. The normal
range for Factor X antigen was 7.74 jtg/mL ± 1.81 ngj
mL or 98% ± 23% relative to the reference pool. For
each specimen we calculated the ratio of percent Factor
X antigen to percent protein C antigen (Xag/Cag). This
ratio also was distributed normally with a mean value
of 1.04 ±0.33.
In seven normal subjects, protein C and Factor X
antigens were measured in both plasma and serum.
When plasma values were corrected for dilution with
anticoagulant, the values for plasma and serum did not
differ significantly in either assay.
Antigen Levels in Pregnancy
We assayed plasma samples from eight women in the
third trimester of pregnancy. Mean protein C antigen
was 88% ± 11%, mean Factor X antigen was 112%
± 15%, and the ratio Xag/Cag had a mean value of
1.27 ± 0.09. Factor VII antigen also was measured by
radioimmunoassay, and a mean value of 144% ± 32%
was obtained.
Effect of Warfarin on Protein C Antigen and
Factor X Antigen
Factor X antigen and protein C antigen were assayed
in 62 plasma specimens from patients on long-term
warfarin therapy. The patients selected were on a stable
warfarin maintenance dose. The mean concentration of
protein C antigen was 42% ± 20%, and the mean concentration of Factor X antigen was 44% ± 24%. The
mean value for the ratio Xag/Cag was 1.09 ± 0.75. The
distributions for protein C antigen, Factor X antigen,
and the ratio Xag/Cag in this population are shown in
Figure 3.
Whereas most of the values for Xag/Cag in warfarin
patients were clustered around 1.0, the distribution was
not gaussian due to a tail extending to much higher
values of Xag/Cag. Five patients had values of Xag/Cag
greater than 1.5. Review of their medical charts failed
Vol. 82 • No. 5
577
PROTEIN C AND FACTOR X ANTIGENS
to reveal any common clinical features. When we secured
new plasma specimens from two of these patients, the
ratios were found to be in the normal range for warfarin
patients. One patient with a high value of Xag/Cag
(2.12) was a 30-year-old man with an unexplained
history of recurring deep-vein thrombosis in both legs,
pulmonary embolism, and normal antithrombin III
activity. Unfortunately, this patient was discharged from
the anticoagulant clinic before we could obtain a second
blood specimen.
CO
Q
Z
o
o
20
LU
CO
-L.
10
J
Factor X
Protein C
Factor VII
100
o
LU
Q.
LU
>
LU
50
Warfarin, 40 mg
LU
CD
r-
Z
<
50
TIME, HOURS
100
FIG. 4. Protein C, Factor X, and Factor VII antigen levels in serial
plasma samples after a single oral dose of 40 mg warfarin. The
relationship between the antigen levels and the prothrombin time (PT)
also is shown.
NOR
WAR LIV
LUP
FIG. 3. Distribution of protein C antigen, Factor X antigen, and the
ratio Xag/Cag in plasma from 31 normal subjects (NOR), 62 patients
on chronic warfarin therapy (WAR), 15 patients with hepatocellular
disease (LIV), and 23 patients with the lupus anticoagulant (LUP).
Kinetics of Warfarin Effect on Protein C Antigen
After the administration of warfarin, the plasma concentration of each vitamin K dependent protein declines
at a rate that reflects the plasma half-life of that protein.21
After one of us (SIR) took a single oral dose of 40 mg
warfarin, serial plasma specimens were assayed for protein C, Factor X, and Factor VII antigens. The results
of this experiment are illustrated in Figure 4. Protein C
antigen and Factor VII antigen declined at comparable
rates; the disappearance halftime was estimated at less
than six hours. Factor X antigen declined much more
slowly, consistent with its known plasma half-life of
over 30 hours.21 Because protein C declined more
rapidly than Factor X, the ratio Xag/Cag increased after
warfarin and reached a maximum value of 1.71 at 48
hours after the drug was administered.
We studied six hospitalized patients to determine the
rate of fall of protein C antigen when warfarin therapy
is initiated with usual clinical doses in the range of 10
mg/day. Analysis of these data revealed a consistent
pattern in which protein C antigen was depressed more
rapidly than Factor X antigen. This resulted in a transient
elevation of the ratio Xag/Cag in five of the six cases
analyzed. Significant depression of protein C antigen
coincided with the onset of a prolonged prothrombin
time (Fig. 5).
578
A.J.C.P. • November 1984
EPSTEIN ET AL.
100 r
2.5
» Xag/Cag
I PT Ratio
TJ
>TJ
d3)
mO
CD
O
co
X
LU
§£
LU
Om
z*
<
Protein C
Factor X
wcr
50
OLU
i=o-
oo
rr
1.0
90
45
TIME, HOURS
FIG. 5. Plots of the Xag/Cag ratio and the prothrombin time ratio
(patient prothrombin time/control) in a hospitalized patient. The
warfarin dose was 10 mg per day, and the first dose was given about
24 hours before the first data point shown.
z"
LU
O
o
It was noted that three of the hospital patients beginning warfarin had protein C antigen of less than 60%
before warfarin therapy was begun. These three patients
had undergone major surgery: cardiac surgery in two
patients and a bowel resection in the third. The three
patients who had normal protein C levels prior to
warfarin therapy were not surgical patients—two had
deep vein thrombosis of the leg and one had transient
cerebral ischemic attacks.
We also measured protein C and Factor X antigens
in serial plasma specimens from a patient who was given
intravenous vitamin Kl for warfarin-induced hemorrhage. Markedly depressed levels of protein C antigen
and Factor X antigen rose at comparable initial rates
after the administration of vitamin K (Fig. 6).
120
LU
I Protein C
i Factor X
§£
ZQ
LUrr
60
OLD
iTQ.
25
-//-
150
z
DC
CO
LL
30
TIME, HOURS
60
FIG. 7. Effect upon protein C and Factor X antigens of systemic
fibrinolysis induced by infusion of streptokinase.
Effect of Streptokinase Infusion on Protein C Antigen
Since plasmin has been shown to activate and then
degrade protein C in vitro,2 it was of interest to determine
whether activation of fibrinolysis alters plasma protein
C levels in vivo. Therefore, protein C antigen was
assayed in serial plasma specimens from eight patients
infused with intraarterial streptokinase to attempt lysis
of arterial thromboses. Plasminogen concentration declined during streptokinase infusion in all patients (mean
preinfusion plasminogen, 2.68 ± 0.67 U/mL, mean
postinfusion plasminogen, 0.70 ± 1.08 U/mL). Fibrinogen levels fell in all patients, and all patients had
positive tests for fibrinogen degradation products. In six
of eight patients, neither protein C nor Factor X antigen
was decreased. In two patients, protein C antigen, but
not Factor X antigen, fell modestly late in the course of
streptokinase administration, at a time when both plasminogen and fibrinogen levels were reduced substantially.
The data from one of these cases are depicted in Figure 7.
TIME, HOURS
FIG. 6. Effect of vitamin K. upon protein C and Factor X antigen
levels in a patient with warfarin-induced hemorrhage. Vitamin Kl (5
mg intravenously) was given at the time of the first plasma specimen.
No blood products were administered.
Protein C Antigen in Hepatocellular Disease
We measured protein C and Factor X antigens in 15
patients with severe hepatocellular disease and jaundice
Vol. 82 • No. 5
PROTEIN C AND FACTOR X ANTIGENS
(Fig. 3). The mean protein C antigen level was 25%
± 17%, the mean Factor X antigen was 51% ± 29%,
and the mean Xag/Cag ratio was 2.17 ± 1.86. Six of
the patients had advanced alcoholic cirrhosis; each had
differential depression of protein C antigen, with Xag/
Cag ratios ranging from 1.60 to 4.30.
Protein C Antigen in Lupus Anticoagulant Plasmas
The lupus anticoagulant may be associated in rare
patients with a specific antibody to prothrombin, which
causes severe hypoprothrombinemia.1 The lupus anticoagulant also is associated with an increased incidence
of thrombosis.17 These considerations prompted us to
measure protein C antigen in plasma from 23 patients
with the lupus anticoagulant, to determine whether
antibody-mediated protein C deficiency could contribute
to the pathogenesis of thrombosis in this syndrome. In
most instances the lupus anticoagulant, defined by criteria
described elsewhere,17 had been discovered as an incidental laboratory finding in patients with diverse underlying diseases. However, two of the patients had a
clinical history of thrombotic disease.
A wide range of protein C and Factor X antigen
levels was found in these patients. Mean protein C
antigen was 80% ± 57%, and mean Factor X antigen
was 85% ± 44%. The mean value of Xag/Cag was 1.12
± 0.42. The two patients with a history of thrombotic
disease had normal protein C levels. Four of the 23
patients had protein C antigen less than 60%, but in
each patient Factor X antigen was depressed comparably.
Thus, no evidence was obtained for selective, antibodymediated depression of protein C as a pathogenic factor
for thrombosis in patients with the lupus anticoagulant.
Protein C Antigen in Disseminated
Intravascular Coagulation
Protein C levels may decline in disseminated intravascular coagulation, including some cases of compensated disseminated intravascular coagulation with elevated fibrin degradation products but fibrinogen levels
above 1 g/L and platelet counts above 100,000/ML." 1 4
In two patients with acute defibrination secondary to
disseminated intravascular coagulation, we found differential depression of protein C antigen: a Xag/Cag ratio
of 2.4 in a patient with amniotic fluid embolism and a
Xag/Cag ratio of 1.6 in a patient with defibrination
secondary to traumatic brain injury. We also measured
protein C and Factor X antigen in two patients with
chronic, compensated disseminated intravascular coagulation manifested primarily by elevated fibrin degra-
579
dation products and a positive plasma protamine paracoagulation test. In one patient, who had adenocarcinoma of the stomach, protein C antigen was 78% and
the ratio Xag/Cag was 1.3. In the second patient, who
had the Kasabach-Merritt syndrome of giant hemangioma with intravascular coagulation, protein C antigen
was 75% and the ratio Xag/Cag was 1.4. Thus, the
protein C levels in these two patients were within our
normal range.
Discussion
The sensitivity and precision of the protein C radioimmunoassay described herein are appropriate for
measurements of protein C in clinical specimens. Our
mean plasma concentration of protein C antigen in 31
normal subjects of 3.23 n%/mL ± 0.79 Mg/mL agrees
reasonably well with the value of 4 Mg/mL obtained by
electroimmunoassay of a plasma pool of 15 normal
subjects.10 The lowest value of protein C antigen in our
31 normal subjects was 71% of our normal reference
plasma pool. Similarly, Bertina and associates found a
lowest measured value of 65% in 33 normal subjects,4
Griffin and associates found a lowest value of 67%
among 40 normal subjects (estimated from their Figure
l),10 and Mannucci and co-workers found a lower limit
of 72% (mean —2 SD) in 60 subjects.14 From these data
we conclude that a protein C antigen level less than
65% should be considered abnormal. It is important to
define this lower limit of normal because heritable
depression of protein C antigen to values in the 50%
range has been associated with thrombotic disease in
several families.6,10
Our data confirm and extend previous reports that
the plasma concentration of protein C falls in patients
with hepatocellular disease.14 Plasma protein C antigen
was depressed to lower levels than Factor X antigen in
the majority of our patients, resulting in abnormal
elevation of the ratio Xag/Cag. In patients with advanced
alcoholic cirrhosis, protein C levels may be lowered
strikingly, e.g., as low as 15%. The absence of thrombotic
complications at such low levels of protein C probably
reflects a protective effect of the deficiency of multiple
procoagulant clotting factors in liver disease.
Our data suggest that depression of protein C antigen
is not a sensitive indicator of either intravascular generation of thrombin or intravascular generation of plasmin. In two patients with severe disseminated intravascular coagulation we confirmed the depression of protein
C antigen reported earlier by Griffin and colleagues"
and Mannucci and colleagues.14 However, in two other
patients with more chronic, compensated disseminated
intravascular coagulation, protein C antigen was within
580
A.J.C.P. • November 1984
EPSTEIN ET AL.
our normal range. The normal protein C level in a
patient with Kasabach-Merritt syndrome was of particular interest because the large endothelial surface area
in this syndrome might provide an ideal setting for
thrombin binding to endothelial thrombomodulin with
consequent activation and consumption of protein C.
When patients were given intraarterial streptokinase to
induce fibrinolysis, protein C levels fell only in two
patients with fibrinolysis extensive enough to deplete
plasminogen and substantially depress plasma fibrinogen
(see Figure 7). In patients with lesser degrees of systemic
fibrinolysis, protein C antigen was not affected.
The rapid disappearance of protein C antigen after
warfarin administration suggests that protein C, like
Factor VII, has a short plasma half-life of about six
hours. In a recent abstract, Vigano and colleagues also
reported early depression of protein C after warfarin was
administered.20 When warfarin is started, protein C and
Factor VII levels will both be reduced substantially when
the prothrombin time first reaches a therapeutic level.
Levels of Factor IX (half-life 20-24 hours),21 Factor X
(half-life 32-48 hours)21 and prothrombin (half-life two
to five days)21 fall more slowly. Thus, reduction of
protein C anticoagulant activity prior to reduction of
the procoagulant action of Factor IX, Factor X, and
prothrombin may lead to a paradoxic initial increase in
coagulability. This may last for several days until Factor
IX, Factor X, and prothrombin are reduced to therapeutic levels. These observations provide additional support for the clinical strategy of continuing intravenous
heparin in patients beginning oral anticoagulants until
the prothrombin time has been prolonged for several
days.
Protein C and Factor X antigen levels were restored
rapidly at comparable rates after intravenous vitamin
Kl was given to a patient to reverse the effect of
warfarin. These data are in agreement with previous
data from this laboratory (unpublished observations) in
which the intravenous administration of 20 mg of
vitamin Kl to an over-anticoagulated patient resulted
in return of prothrombin, Factor VII, Factor IX, and
Factor X activities to above 50% within 14 hours.
Apparently, the rate of plasma reappearance of the
vitamin K dependent proteins is independent of their
widely differing intravascular half-times.
Transient paradoxic hypercoagulability caused by the
rapid fall of protein C after beginning warfarin therapy
appears to explain the rare syndrome of coumarininduced skin necrosis. This unusual side effect of oral
anticoagulants is seen in the first week of warfarin
therapy, and particularly in patients given a large loading
dose. Three patients with coumarin-induced skin necrosis
and hereditary depression of protein C antigen now
have been described.1516 In these patients who have only
40-50% of normal protein C antigen prior to anticoagulant therapy, the rapid fall of protein C after warfarin
administration results in very low protein C activity
before Factor IX, Factor X, and prothrombin are reduced
substantially. This then could promote the thrombosis
in skin venules characteristic of this syndrome.
An association has been reported between isolated
hereditary depression of protein C antigen in the range
of 40-60% of normal and recurring venous thrombosis.610 Hereditary deficiency of protein C antigen should
be suspected when protein C antigen is below 60% and
the other vitamin K dependent clotting factors are
within the normal range. Because of their thrombotic
diathesis, patients with suspected protein C deficiency
often are receiving oral anticoagulants at the time of
examination. Then, one must employ the strategy of
comparing protein C antigen levels with antigen levels
of another vitamin K dependent protein, e.g., prothrombin10 or Factor X.4 However, as our data clearly illustrate
(Figs. 4 and 5), there is differential depression of protein
C when warfarin therapy is initiated. This presumably
also occurs when warfarin dosage is increased and
probably accounts for the nonreproducible elevation of
the Xag/Cag ratio found in two of our anticoagulant
clinic patients (Fig. 3). Thus, finding an elevated ratio
of Factor X or prothrombin antigen to protein C antigen
can be taken as evidence of hereditary protein C deficiency only after the elevated ratio has been demonstrated
repeatedly in a patient stabilized on a constant warfarin
regimen. Similarly, one should not interpret an elevated
ratio of Xag/Cag as evidence of hereditary protein C
deficiency until underlying liver disease has been ruled
out as an explanation for this finding (Fig. 3).
Acknowledgments. The authors are grateful to Dr. William G.
McGehee and Ms. Kathy Donnelly for providing valuable plasma
specimens utilized in these studies.
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