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O rig in a l P a p e r
David Green
Kochurani Maliekel
Elena Sushko
Rasheed Akhtar
Gerald A. Soff
Haemostasis 1997;27:112-118
Received: January 28,1997
Accepted in revised form: February 6. 1997
Activated-Protein-C Resistance in
Cancer Patients
Key W ords
Protein C
Factor V Leiden
Background: Resistance to activated protein C (aPC) is usually linked to
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factor V Leiden, but may occur in other disorders associated with hyper­
coagulability. In this study, we investigated the frequency of resistance to
aPC in patients with advanced cancer and examined the relationship of
aPC resistance to other markers of coagulation activation. Methods:
Patients (n = 39) had an established diagnosis of advanced cancer; controls
(n = 20) were healthy persons. aPC resistance was measured as the ratio of
activated partial thromboplastin times with and without aPC (aPC-sensitivity ratio, aPC-SR). The factor V Leiden mutation was detected by a
polymerase-chain-reaction based technique. Other assays were performed
by standard laboratory methods. Data were analyzed using t tests and the
Pearson correlation. Results: aPC-SR was below 2 SD for 5 of the cancer
patients (13%), but none of the controls; only 1 of the 5 had the factor V
Leiden mutation. aPC-SR was inversely correlated (p < 0.01) with fac­
tor VIII and fibrinogen in patients and with prothrombin activation frag­
ment 1.2 (FI.2) in controls. Patient factorVIII, von Willebrand factor,
(vWF), fibrinogen, F I.2 and D dimer were all significantly increased (p <
0.01); anlithrombin III, protein C and proteins were similar to controls.
Factor VIII correlated with vWF (p < 0.001) and FI.2 with d-dimer (p <
0.001). Other associations (p < 0.05) were observed between factor V and
protein C, fibrinogen and protein C, factor V and antithrombin III and
protein C and antithrombin III. Four cancer patients had a history of
thromboembolism; their aPC-SR was similar to that of patients without
thrombosis. Of the several coagulation measures examined, only vWF was
higher in the patients with thrombosis (p = 0.01). Interpretation: Cancer
patients have evidence of intravascular coagulation and increases in pro­
coagulants and may have aPC resistance. The aPC resistance is not due to
factor V Leiden, but is rather associated with elevated levels of factor VIII
and fibrinogen, and in itself does not predict thrombosis.
€> 1997 S. Karger AG, Basel
David Green, MD. PhD
345 E Superior Street, Room 1407
Chicago, IL 60611 (USA)
Tel. (312) 908 4701
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Division of
Department of Medicine,
Northwestern University Medical
School, Chicago, 111., USA
PC Resistance in Cancer Patients
Haemostasis 1997;27:112-118
Activated protein C (aPC) resistance was
originally described by Dahlback et al. [ 1] as an
impaired responsiveness to aPC. This was
measured by examining the ratio of activated
partial thromboplastin times (aPTT) with and
without aPC (aPC-sensitivity ratio, aPC-SR).
Subsequently, resistance to aPC was found in
most instances to be due to a mutation in fac­
tor V (Arg506Gln; factor V Leiden), rendering
it partially resistant to proteolysis by aPC [2],
However, not all patients with aPC resistance
have the factor V Leiden mutation [3-5]. To
investigate this phenomenon, we examined
the APC-SR, as well as other hemostatic fac­
tors, in persons anticipated to have enhanced
coagulability, i.e. patients with cancer [6].
M aterials and M ethods
Study Subjects
Patients (n = 39) with an established diagnosis of
cancer attending an oncology clinic were the subjects
of this study. Their ages ranged from 42 to 85 years
(mean 65); 33 were men. The cancers included lung
(10), prostate (10), colorectal (6), head and neck (6),
stomach/pancreas (4) and other cancers (3). All pa­
tients had advanced metastatic cancer, and most were
receiving chemotherapy, radiation or hormonal thera­
py. A careful history was recorded for each patient, and
information regarding previous episodes of thrombo­
embolism was noted. Control subjects (n = 20) were
healthy persons between the ages of 26 and 55 years
(mean 35), equally divided between men and women.
The study was approved by the Institutional Review
Board, and all subjects provided written informed con­
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Coagulation Studies
Fibrinogen was assessed by the method of Clauss
using reagents from Dade Division, Baxter Healthcare
Corporation, Deerfield, 111., USA [7], The assay was
calibrated with standard normal plasma (SNP reagent;
Dade), and the results were calculated using the data
management system of the MLA-Electra 800 clot tim­
er. The technical error of the assay, expressed as a per­
centage of the mean, was 5.6 ± 2.4%.
Factor V and factor VIII coagulant activities were
assayed by a one-stage system using reagents from
Pacific Hemostasis, Huntersville, N.C. and George
King Biomedical, Overland Park, Kans., USA. The
standard curve was prepared using Universal Refer­
ence Plasma from Curtin Matheson Scientific, Woodale, 111., USA, and the results were calculated as a per­
centage of the standard with the data management sys­
tem of the MLA-Electra 800. The technical error of the
assay was 6.0 ± 2.3%.
von Willebrand factor (vWF) antigen was mea­
sured by an enyzme-linked immunosorbent assay
(ELISA) [8] obtained from American Bioproducts,
Parsippany, N.J., USA. In brief, a plastic support
coated with specific rabbit anti-human vWF anti­
bodies binds the factor in the test plasma. Rabbit antivWF antibody coupled with peroxidase binds to the
remaining free antigenic determinants of the factor,
and the bound peroxidase is revealed by its activity on
o-phenylenediamine in the presence of hydrogen per­
oxide. A standard curve was prepared using Universal
Reference Plasma, and the results were reported as a
percentage of the standard. The technical error of the
assay was 7.6 ± 1.5%.
Prothrombin activation fragment 1.2 (F1.2) was
measured by an ELISA [9] using the Enzygnost F 1+2
kit from Behring, Marburg, Germany, with an interas­
say coefficient of variation of 5-7.5%. D dimer was
measured using the Asserachrom D-Di (Diagnostica
Stago, Asnières, France) ELISA method. Antithrom­
bin III was examined using a synthetic chromogenic
substrate method (Stachrom AT III, Diagnostica Sta­
go) and the PAP-4C instrument (Bio/Data, Hatboro,
Pa., USA,). Functional protein C was determined us­
ing the activator extracted from A. contortrix venom
and the ST4 instrument (Staclot Protein C, Diagnosti­
ca Stago). The functional activity of free protein S was
assayed by a method based on the inhibition of factor
Va, using Staclot Protein S and the ST4 instrument
(Diagnostica Stago).
The aPC resistance assay was performed by a mod­
ification of the aPTT, as described by Dahlback et al.
[1], using the ST4 instrument and reagents distributed
by Pharmacia, Franklin, Ohio, USA. Comparison of
the aPTT obtained for a particular sample in the pres­
ence of aPC with the results obtained without aPC
yields an aPC-dependent sensitivity ratio (aPC-SR). A
decreased aPC-SR is observed with aPC resistance.
The technical error of the assay was 7.7%.
The factor V Leiden mutation was detected by the
method of Bcrtina et al. [2], The polymerase chain
reaction was used to amplify a 267-bp segment of the
Table 1 . Coagulation assay
results in cancer patients and
healthy controls (means ± SD)
Factor V ,%
Factor VIII, %
vWF, %
Fibrinogen, mg/dl
F1.2,nM /l
D dimer, ng/ml
Protein C, %
Protein S ,%
p value
(n = 20)
3.2r0.7 (39)
89 ±20 (39)
161 ±63 (39)
211 ±89 (39)
438 ± 152 (39)
1.55 ±0.99 (24)
1,674± 1,237 (24)
105 ± 19 (24)
95 ±31 (24)
120 ±31 (24)
81 ± 11
119 ± 32
92 ± 27
270 ±5
240 ±96
101 ± 13
114 ± 17
134 ±27
AT III = Antithrombin III.
factor V gene, which includes the G to A mutation at
nucleotide 1691, from leukocyte DNA. The primers
PR-6967 and PR-990 were from Bertina et al. [2], and
the PCR product was digested with Mnl 1 restriction
enzyme (New England Biolabs, Beverly, Mass., USA).
The 1691 <G-A>point mutation results in the loss of an
Mnl 1 restriction site, and the wild-type restriction pat­
tern thus yields 37-, 67- and 163-bp fragments, while
the 1691(G_A) mutant yields 67- and 200-bp bands.
Statistical Analysis
Data were expressed as the mean ± SD. Compari­
sons between groups were made with a t test for inde­
pendent samples. Pearson correlation coefficients were
calculated between clotting factors and the aPC-SR.
The statistical software was True Epistat, version 3.1
(Epistat Services, Richardson, Tex., USA). A p value
(two tailed) of <0.05 was considered significant.
Table 2. Pearson correlation coefficients for the
aPC resistance ratio
Factor V
Factor VIII
von Willebrand factor
F 1.2
D dimer
Antithrombin III
Protein C
Protein S
*p = 0.02;**p< 0.001.
Table 1 shows the results of the coagula­
tion assays in the patients and controls. The
values for factor VIII, vWF, fibrinogen, F 1.2
and D dimer were significantly higher in the
patients. The increase in factor V was of bor­
derline significance (p = 0.06), and antithrom­
bin III, protein C and protein S were similar
to control values. In the patients, strong corre­
Haemostasis 1997;27:112-118
lations (p < 0.001) were observed between fac­
tor VIII and vWF and F 1.2 and D dimer.
Other associations (p < 0.05) were observed
between factor V and protein C, factor V and
antithrombin III, protein C and antithrombin
III and protein C and fibrinogen.
aPC-SR did not differ significantly be­
tween cancer patients and controls. The 2 SD
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6 .0 0 n
Fig. 1. Correlation between the
aPC-SR and factor VIII in patients
with advanced malignancy. Pear­
son correlation; r = -0.37; p =
1 - 00-1 ------------------------1----------------------- 1------------------------1------------------------1----------------------- 1----------------------1—
Factor VIII (%)
0 .02.
Fig. 2. Correlation between the
aPC-SR and fibrinogen in patients
with advanced malignancy. Pear­
son correlation: r = -0.36; p =
Fibrinogen (mg/dl x 100)
cut-off value for aPC-SR was determined to
be 2.6 for the controls; none of the 20 controls,
but 5 of the 39 cancer patients, had ratios
below this value. Thirty-eight patients were
tested for the factor V Leiden mutation; only
the patient with the lowest aPC-SR (1.7) was
found to have this mutation.
To determine the factors responsible for
the low aPC-SR in some of the cancer pa­
tients, correlation coefficients between the
aPC-SR and the hemostatic factors were ex­
amined. Significant inverse correlations were
observed for factor VIII and fibrinogen in the
cancer patients (table 2, fig. 1, 2). Correlation
PC Resistance in Cancer Patients
Haemostasis 1997:27:112-118
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0 .02 .
An association between cancer and throm­
bosis has been recognized since the work of
Trousseau in 1865. As many as 90% of cancer
patients will have elevation of plasma-clotting
factor levels (fibrinogen, factors V, VIII, IX
and XI) and thrombocytosis [10]. In addition,
many will have evidence of activation of coag­
ulation, with elevated levels of D dimer, fibrinopeptide A, F 1.2 and thrombin-antithrom­
bin complexes [11, 12], Decreases in clotting
inhibitors have also been reported. Flowever,
the frequency of resistance to aPC and its rela­
tionship to thrombosis in the cancer patient
has thus far received little attention.
Dahlback [13] has calculated that while a
normal aPC-SR almost always (99%) predicts
the absence of the factor V gene mutation, the
predictive value of a low aPC-SR is only
about 69%. Thus, there are other influences
Haemostasis 1997:27:112—118
on the test for aPC resistance. Technical con­
siderations are important since the test is
based on the aPTT and may be affected by the
aPTT reagent, the preparation of aPC select­
ed and the instrument [14] used to record the
clotting times. All of these were standardized
in our study: the same instrument (ST4) and
reagent batch were used for all aPTT tests.
The method of collection and storage of pa­
tient samples may also affect aPTT results; for
example, freezing of samples decreases the
aPC-SR [ 15], perhaps by lysing residual plate­
lets [13]. To assure accurate aPC-SR results
when frozen stored samples are examined, the
pooled normal control plasma should also be
frozen and thawed, as was done in our investi­
gation. The duration of storage (up to 12
months) does not appear to influence the
assay [15, 16],
In most series, the lowest values for aPCSR occur in individuals with the factor V
Leiden mutation [ 17]; this was also true in our
study. However, there are other reasons for an
abnormal aPC-SR, such as heparin, the lupus
anticoagulant [18, 19], oral anticoagulant
therapy [20] and vitamin K deficiency [13].
These are suspected if the patients’ baseline
aPTT is prolonged. The aPTT was slightly
prolonged in 3 patients, but their aPC-SR
were within the normal range, and none was
receiving warfarin.
Since the aPC-SR test depends, in part, on
the aPTT and the inhibitory activity of the
protein C/S system, factors affecting these
measurements could alter the aPC-SR. The
aPTT is very sensitive to factor VIII levels
[21], and it is therefore not surprising that we
observed an inverse correlation between fac­
tor VIII and the aPC-SR. It was also of inter­
est that a similar correlation was noted be­
tween the fibrinogen concentration and the
aPC-SR. Both factor VIII and fibrinogen in­
crease two-fold during pregnancy [22], and
there is a significant reduction in aPC-SR [4],
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coefficients with factor V and vWF were not
significant in the cancer patients; in the con­
trols, the correlation with factor V was of bor­
derline significance (p = 0.07).
Four cancer patients had a history of
thromboembolism; their aPC-SR levels were
similar to those of patients without thrombo­
sis (p = 0.5). None of the 3 who were tested
had the factor V Leiden mutation. The levels
of factor VIII, vWF, fibrinogen, F 1.2, and D
dimer were also compared with those of pa­
tients without thrombosis. Only vWF was sig­
nificantly increased (315 vs. 199%; p = 0.01)
in the subpopulation with thrombosis. Nine­
teen patients received treatment that included
chemotherapy and/or radiation; their levels of
aPc-SR (3.0 ± 0.7), factor VIII (176 ± 66)
and fibrinogen (447 ± 184) did not differ sig­
nificantly from those who did not receive
these therapies (aPC-SR: 3.3 ± 0.7; factor
VIII: 147 ± 57; fibrinogen; 429 ± 118).
suggesting that the levels of these factors af­
fect the aPC-SR. With regard to the protein
C/S system, neither the levels of factor V
(within the range of 12.5-100%) nor protein S
(above 20%) have been found to influence the
test [23, 24],
The aPC-SR is a marker for thrombophilia
[25], While we did not observe a direct asso­
ciation between aPC-SR and thrombosis, we
did note that our cancer patients had signifi­
cant increases in F 1.2 and D dimer, indica­
tors of coagulation activation. Thus, aPC-SR
should be added to the spectrum of hyper­
coagulability measures characteristic of ad­
vanced malignancy.
Acknow ledgm ents
We thank D. CundilT, N. Reynolds and H. Kohl for
expert technical assistance. E.S. was a Thrombosis and
VascularTraining Center Fellow under the auspices of
the Council on Thrombosis of the International Soci­
ety and Federation of Cardiology and the International
Society on Thrombosis and Haemostasis. Grant sup­
port was received from the Feinberg Cardiovascular
Research Institute.
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