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Original Paper
Haemostasis 1997;27:163-167
Extrinsic Coagulation Factors and
Tissue Factor Pathway Inhibitor in
End-Stage Chronic Renal Failure
Takefumi Matsuo
Masanobu Koide
Kazuomi Kario
Shunji Suzuki
Miyako Matsuo
Key Words
End-stage chronic renal
Factor Xlla
Extrinsic coagulation factors
Tissue factor
pathway inhibitor
Diabetes mellitus
The relationship between extrinsic coagulation factors, tissue
factor pathway inhibitor (TFPI) and activated factor XII
(FXIIa) was examined in 71 patients with end-stage chronic
renal failure. They had chronic stable uremia due to regular
hemodialysis. The patients were divided into two age- and sexmatched groups with and without diabetes mellitus. As extrin­
sic coagulation parameters, FVIIa and FVII antigen (FVIIag),
tissue factor antigen and TFPI (the activity and antigen) were
measured. FXIIa was measured as a marker of contact activa­
tion, and thrombin generation was evaluated using the two
markers thrombin-antithrombin III complex and prothrom­
bin fragment 1 + 2. In both hemodialysis groups with and
without diabetes, significant elevations of FXIIa, FVIIa and
tissue factor, with high levels of TFPI, were found. Thus,
hyperactivation of the coagulation system was in part compen­
sated by TFPI, and a significant increase in FXIIa could not
directly affect FVIIa hyperactivation. No differences of these
parameters, except for FVIIag and fragment 1 + 2, were found
between the groups with and without diabetes. It is suggested
that the long-term hemodialysis might have masked any dif­
ferences due to the underlying disease in these two subgroups.
In patients undergoing regular hemodialy­
sis for end-stage renal failure (ESRF), the risk
of cardiovascular complication is increased
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with the progression of atherosclerosis. Blood
lipid and coagulation abnormalities are major
contributing factors in the progression of ath­
erosclerotic vascular change. In our previous
study [1], we found that ESRF is associated
Dr. Takefumi Matsuo
Department of Internal Medicine
Hyogo Prefectural Awaji Hospital, Shimogamo
Sumoto, 656 (Japan)
Fax +81 799 24 5704
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Hyogo Prefectural Awaji Hospital,
Sumoto, Japan
Subjects and Methods
Seventy-one consecutive ESRF patients aged 61.4
± 14.1 (mean ± SD) years, 35 males and 36 females,
who had been undergoing regular hemodialysis treat­
ment at the Awaji Hospital Kidney Center, Sumoto,
Japan, for at least 1 year were enrolled in this study.
Before hemodialysis, all subjects had uremic syn­
drome, hyperkalemia, metabolic acidosis, fluid over­
load and < 10 ml/min creatinine clearance. The coagu­
lation parameters in the 25 patients with diabetic
nephropathy (age 63.5 ± 11.5 years, 12 males and 13
females) were compared with those in 25 sex- and agematched nondiabetic patients (age 63.8 ± 12.1 years;
23 patients with chronic glomerulonephritis and 2 with
nephrosclerosis). Thirteen diabetic patients had been
diagnosed as having non-insulin-dependent and 12
insulin-dependent diabetes. All diabetic patients were
under the care of a diabetologist and had maintained
hemoglobin A|C levels under 8.5% (6.4±1.0%).
Eighty-six healthy controls (69.4 ± 17.8 years, 39
males and 47 females) who had shown no medical
problems at a medical check-up were also studied.
Blood samples were obtained in 0.1 vol of 3.8% tri­
sodium citrate in the disposable siliconized glass tubes
Haemostasis 1997;27:163-167
in the morning before the start of hemodialysis. Plas­
ma was immediately separated and stored at -30° C in
plastic tubes until each measurement. Measurement of
FXIIa was carried out using a direct enzyme immu­
noassay (Shield Diagnostics), employing antibody
which recognizes a-XIIa and P-XIIa. The intraindivid­
ual variation of the FXIIa assay in 31 patients with
ESRF was assessed using blood samples obtained at
6-month intervals. No significant intraindividual dif­
ferences were observed, with a positive correlation for
the two measurements (r = 0.867; p < 0.001). Tissue
factor antigen was measured by an enzyme immunoas­
say consisting of two monoclonal antibodies against
human tissue factor, one side of which was labeled
with horseradish peroxidase, biotinyl-tyramide to am­
plify the enzyme signal and 2,2'-azino-di-(3-ethylbenzthiazoline sulfonate) as a substrate [4], Plasma FVIIa
was directly measured using a previously described
fluorogenic assay [5] employing a fluorogcnic peptide
substrate for thrombin (Peptide Institute), congenital
human FVII-deficient plasma (George King Bio-Medi­
cal) and recombinant human soluble tissue factor ex­
pressed in yeast and purified. The reaction was started
by addition of soluble tissue factor. Fluorescence in­
tensity was transformed into reaction time, which was
defined as the time to reach an estimated fluorescence
intensity. The reaction time was converted to FVIIa
(ng/ml) by comparison with a standard curve produced
by serial dilution of purified FVIIa (Chemo-Sero-Therapcutic Institute). FVII antigen (FVIIag) was assayed
using a commercially available enzyme-linked immu­
nosorbent assay (ELISA) kit (Diagnostics Stago). A
modified method of that described by Sandset et al. [6]
was employed for tissue factor pathway inhibitor
(TPFI) activity (TFPla) determination. TPFI antigen
(TFPIag) detected as full-length TFPI was measured
using an ELISA kit (American Diagnostics). Commer­
cially available ELISA kits were employed to measure
the thrombin-antithrombin III complex (TAT) and
prothrombin activation fragment 1 + 2.
Data are showm as means ± SD. Statistical signifi­
cance was determined by the unpaired Student’s t test.
Pearson’s correlation coefficients were calculated for
the different variables.
In the patients with ESRF, FXIIa was sig­
nificantly increased and did not correlate with
the levels of blood urea nitrogen or creatinine.
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with hyperactivation of coagulation due to
increased factor VII (FVII) activity and anti­
gen level and that these abnormalities con­
tribute to the pathogenesis of cardiovascular
complications. The present study was carried
out to clarify in more detail the role of factors
in the extrinsic coagulation pathway, includ­
ing activated FXII (FXIIa). The blood of
these patients with ESRF is regularly exposed
to a foreign surface during hemodialysis un­
der anticoagulation. FXII undergoes contact
activation due to the extracorporeal circuit,
being converted to FXIIa [2], FXIIa directly
induces activation of FVII to FVIIa, a useful
marker of activation in the extrinsic pathway
[3]. Since diabetes mellitus is more frequently
associated with severe cardiovascular compli­
cations, we also examined whether the diabet­
ic patients showed hyperactivation in the ex­
trinsic pathway compared to nondiabetic pa­
Table 1. Contact activation, extrinsic pathway activation and inhibition in patients with ESRF
A Extrinsic factors
FXIIa, ng/ml
Tissue factor, pg/ml
FVIIa, ng/ml
FVIIag, %
8.6± 3.7** (n = 59)
2.0± 1.2 (n = 86)
387 ± 110** (n = 50)
130 ±41 (n = 79)
6.3 ± 3.1 ** (n = 69)
3.9 ± 1.4 (n = 86)
135 ±43 (n = 70)
139 ± 37 (n = 81 )
TFPIa, %
TFPIag, ng/ml
185 ±37** (n = 71)
99 ± 28 (n = 71)
289 ±22** (n = 69)
120 ±21 (n = 81)
TAT, ng/ml
Fragment 1+2, nmol/1
5.5 ±6.3* (n = 70)
2.7 ± 3.3 (n = 86)
2.3 ±0.8** (n = 50)
1.0 ±0.2 (n = 50)
C Thrombin generation
*p<0.01, ** p < 0.001 : compared to controls.
Tissue factor and FVIIa were also significant­
ly increased compared to those in the control
group, but FVIIag showed no significant in­
crease (table 1). TFPIa, TPFIag, TAT and
fragment 1 + 2 showed significant elevations
compared to the control. Although FXIIa was
markedly increased in ESRF, it showed no
significant correlation with either tissue factor
(r = 0.281; p < 0.1) or FVIIa (r = 0.268; p <
0.1); prothrombin activation was observed
despite the increase in TFP1. Therefore, there
was no significant correlation between each
extrinsic factor and thrombin generation
markers, and there was no evidence that the
increased FXIIa was directly related to the
increases in plasma tissue factor and FVIIa
ESRF patients were divided into two
groups according to whether they had un­
derlying diabetes or not. No differences were
recognized between the patients with and
without diabetes, except for TAT (table 2).
Significant increases in extrinsic coagulation
and inhibitory factors were observed in both
subgroups of ESRF patients compared to the
control group, except for FVIIag. The results
indicated that the marked hyperactivation in
the diabetic ESRF group was not more
marked than that in the nondiabetic group.
Extrinsic Coagulation Factors, FXIIa
and ESRF
Haemostasis 1997;27:163-167
In this study, FXIIa was found to be signif­
icantly increased in patients with ESRF. As
no correlations were found among extrinsic
coagulation factors, FXIIa and inhibitory fac­
tors, hyperactivation in the extrinsic pathway
is thought to occur independently of the in­
crease in FXIIa. In this regard, Kario et al. [7]
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Table 2. Hemostatic factors in patients with ESRF with and without diabetes mellitus
A Extrinsic factors
Diabetic ESRF
Nondiabetic ESRF
Age-matched control
FXIIa, ng/ml
Tissue factor, pg/ml
FVIIa, ng/ml
FVIIag, %
9.1 ±3.6° (n = 22)
7.6±2.4C(n = 23)
1.7 ± 1.1 (n = 25)
368 ± 105c (n = 20)
387 ± 116c (n = 19)
128 ±41 (n = 25)
5.8 ± 2.8b (n = 25)
6.6±2.6c (n = 24)
3.5 ± 1.3 (n = 25)
129 ± 27 (n = 24)
140±43 (n = 25)
143 ± 38 (n = 25)
TFPIa, %
TFPIag, ng/ml
232 ± 32c (n = 20)
217±38c (n = 24)
99 ±29 (n = 20)
266±48c (n = 25)
272±44c (n = 24)
121 ±22 (n = 21)
TAT, ng/ml
Fragment 1+2, nmol/1
3.3 ± 1.4ad (n = 24)
4.9±3.3C(n = 25)
1.7 ± 1.9 (n = 25)
2.1 ±0.6C(n = 20)
2.4±0.9C(n = 25)
1.2 ± 0.4 (n = 25)
Diabetic ESRF
Nondiabetic ESRF
Age-matched control
C Thrombin generation
Diabetic ESRF
Nondiabctic ESRF
Age-matched control
reported that the increase in FVIIa during
hemodialysis is probably caused by endothe­
lial damage, resulting in tissue factor expres­
sion, and that this increase is related to the
high prevalence of cardiovascular complica­
tions. This Finding that the increase in FXIIa
in patients with ESRF is not directly related
to hyperactivation in the extrinsic pathway is
inconsistent with a previously in vitro study
[8], In the case of FXII deficiency during car­
diac bypass surgery, thrombin generation, di­
rectly initiated by the formation of tissue factor-FVIIa complexes is observed [9], Since
plasma TFPI was significantly increased in
these patients, there appears to be a compen­
satory mechanism to control hyperactivation
caused by FVII activation.
Haemostasis 1997;27:163-167
The progression of endothelial damage
among patients with ESRF is believed to be
more rapid in diabetic than in nondiabetic
patients [10]. However, there is no evidence
that the hypercoagulability in ESRF patients
with well-managed diabetes is more marked
than that in nondiabetic patients with ESRF.
There were also no differences between these
two groups regarding the levels of TFPI. Dia­
betic patients with ESRF undergoing hemodi­
alysis did not show characteristic findings of
the underlying disease. The TFPI activity in
ESRF is reported to be increased due to long­
term use of heparin [11]. The increase in plas­
ma TFPI levels is often caused by endothelial
dysfunction, which is essentially the same
mechanism as that described for plasma
thrombomodulin levels. Plasma tissue factor
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a p < 0 .0 5 ,b p < 0.01,'c p < 0.001: compared to controls;d p < 0.05 compared to nondiabetic ESRF.
induced by tissue factor expression on the
endothelium seems likely to be involved in a
marker of endothelial damage and an activa­
tion signal of the extrinsic pathway [12]. The
tissue factor levels in ESRF with and without
diabetes showed the same degree of elevation,
suggesting that the severity of endothelial
damage in ESRF is not affected by the status
of the underlying diabetes.
Hypercoagulability in ESRF leads to en­
hanced thrombin generation [13]. The results
of this study indicate that the same degree of
hypercoagulability was present in both groups
with ESRF and that there was no direct con­
tribution from the increase in FXIIa as there
was no correlation between FXIIa and throm­
bin generation markers. Consistent with these
results, no correlation was found between
fragment 1+2 production and the level of
FXIIa in cardiopulmonary bypass surgery
[14]. Thus, this study provided no evidence
that the increase in FXIIa is directly related to
hyperactivation in the extrinsic pathway in
This research was in part funded by a grant from
the Heparin Conference (Tokyo, Japan).
Extrinsic Coagulation Factors, FXIIa
and ESRF
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