© 1996 American Heart Association, Inc.
Articles |
Measurement of the Free Form of TFPI Antigen in Hyperlipidemia
Relationship Between Free and Endothelial Cell–Associated Forms of TFPI
From the National Cardiovascular Center, Research Institute (T.K., K-i.E., M.H.-S., A.Y., H. Kato) and Hospital (Division of Atherosclerosis and Metabolism; H. Koh, M.T.), Osaka, and Chemo-Sera-Therapeutics Research Institute, Kumamoto (K.K., Y-i.K.), Japan.
Correspondence to Dr H. Kato, National Cardiovascular Center Research Institute, Fujishirodai-5, Suita, Osaka 565, Japan.
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Abstract |
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Abstract Tissue factor pathway inhibitor (TFPI), a protease with three tandem Kunitz-type (K1, K2, and K3) domains, inhibits the initial reaction of the TF-mediated coagulation pathway. TFPI occurs in a free and a lipoprotein-associated form in plasma as well as an endothelial cell–associated form on vascular walls. In a previous study we had demonstrated that free-form TFPI activity was lower in hyperlipidemic patients. In the present study we established a new enzyme immunoassay method for measuring free-form TFPI antigen; this new method uses a monoclonal antibody that recognizes the K3 domain of free-form TFPI but not lipoprotein-associated TFPI. Free-form TFPI antigen was significantly lower in hyperlipidemic patients compared with those in normolipidemic individuals. We applied this new method to measure the amount of endothelial cell–associated TFPI, which can be released by heparin injection, as "free-form TFPI." We found that free-form TFPI antigen in plasma was positively correlated with the endothelial cell–associated form. These results indicate that both of these forms of TFPI are in equilibrium in vivo and that our new method can be used for assessing changes in the levels of endothelial cell–associated TFPI antigen and, hence, for assessing thrombotic tendencies in various disease states.
Key Words: enzyme immunoassay • hyperlipidemia • endothelial cell • plasmapheresis • tissue factor pathway inhibitor
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Introduction |
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TFPI, a protease inhibitor with K1, K2, and K3,1 inhibits the initial steps of the extrinsic blood coagulation cascade by forming complexes with FXa, FVIIa, and TF.2 3 Most TFPI is synthesized by vascular ECs4 5 and is associated with these cells.6 7 8 TFPI from plasma can be separated by gel filtration into free and lipoprotein-associated forms, ie, an LDL/VLDL-associated form and an HDL-associated form. Although it was once believed that most TFPI was associated with lipoproteins in plasma and that only
5% of TFPI in plasma was in the free
form,7 9 10 11 we recently demonstrated that 45% of
plasma TFPI is in the free form by measuring each form of TFPI activity
by gel filtration.12 We also found that free-form TFPI
activity was lower in hyperlipidemic patients and in
those who were undergoing repeated plasmapheresis. To further examine
TFPI levels in various diseases we developed an EIA method. Using a
monoclonal antibody that recognized the K3 domain of TFPI, we were able
to detect only the free but not the lipoprotein-associated forms of
TFPI in plasma.13 Although TFPI antigen levels in various
diseases have been reported,14 15 16 17 only total plasma TFPI
antigen was measured in these studies. Efforts have also been made to
elucidate the relation between EC-associated TFPI and plasma TFPI in
various human diseases18 19 and in diet-induced
hypercholesterolemia in monkeys20 ;
however, no correlation between the two forms of TFPI has yet been
established. On the contrary, Hansen et al21 recently
reported that downregulation of LDL did not affect the levels of
EC-associated TFPI in hyperlipidemia. In the
present study we established an EIA method specific for the free
form of TFPI in plasma and applied this method to measure the levels of
EC-associated TFPI, which was released by intravenous
heparin injection. We then analyzed TFPI antigen in
hyperlipidemic patients by this new method and
compared these findings with those obtained by an earlier method. We
discuss the relationship between free-form TFPI antigen in plasma
and EC-associated TFPI antigen. |
Methods |
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Materials
BSA and bovine milk proteins (Block Ace) were obtained from Boehringer Mannheim and Dai-Nippon Seiyaku, respectively. Rabbit anti-human rTFPI polyclonal antibody and mouse anti-human rTFPI monoclonal antibody were prepared as described.13 Horseradish peroxidase–labeled goat anti-rabbit IgG was purchased from Bio-Rad Laboratories, and an ELISA kit for total plasma TFPI antigen produced by Kaketsuken was purchased from Sanko Pure Chemicals. Porcine mucosal sodium heparin was purchased from Novo-Nordisk A/S. Cholesterol concentration was measured with a commercially available kit, the Cholesterol E test from Wako Junyaku. Multiwell plates (type H) were obtained from Sumitomo Bakelite. As described in a previous article,12 TFPI activity was measured by the ability of TFPI complex to inhibit activation of FX by TF-FVIIa complex. Free-form TFPI activity was measured after gel filtration of plasma through a column of Superdex 200 HR (26/60 from Pharmacia LKB Biotechnology).
Blood Sampling and Plasma Preparation
Blood specimens were obtained from normolipidemic individuals
(41 healthy volunteers aged 22 to 64 years) and 45
hyperlipidemic patients (aged 8 to 80 years) at the
National Cardiovascular Center Hospital, Osaka,
Japan (Table
). Of the 45 hyperlipidemic
patients 34 had not undergone plasmapheresis; their ages ranged from 23
to 80 years. Plasma from normolipidemic individuals and
hyperlipidemic patients was collected 10 minutes after
injection of heparin (30 U/kg IV). Before heparin injection, oral
informed consent to participate in a clinical examination for
measurements of lipoprotein lipase and coagulation factors (including
TFPI) was obtained from all study subjects. The other 11/45 patients
(aged 8 to 64 years) with heterozygous familial
hyperlipidemia received repeated, biweekly
plasmapheresis treatment, during which an apheresis column (Evaflux 5A
from Kurare or Liposorber LA-15 from Kanegafuchi Kagaku) was used.
Blood from these 11 patients was collected before heparin was
infused. The diagnosis of hyperlipidemia was
established according to the criteria of the Japan
Atherosclerosis Society (total serum
cholesterol concentration >5.7 mmol/L; Table). Blood was
collected into evacuated tubes containing trisodium citrate (final
concentration, 0.38% [wt/vol]) and plasma was separated by
centrifugation. Plasma specimens were stored at
-80°C until assay.
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EIA of TFPI Antigen
TFPI antigen was measured by a sandwich EIA method with
monoclonal and polyclonal antibodies against human rTFPI as
described.13 Each microplate well was coated with 50 µL
of monoclonal anti-human rTFPI antibody (10 µg/mL in TBS). After
a 2-hour incubation at room temperature each microplate well was soaked
with 200 µL TBS containing diluted (x1/4) Block Ace. The plates were
left for 2 hours at room temperature and then washed with TBS–Tween 20
(20 mmol/L Tris HCl, pH 8.0, containing 0.5 mol/L NaCl and 0.05% Tween
20). The rTFPI standard was serially diluted with Tris HCl buffer
(0.5% BSA and 0.6 mol/L NaCl) and plasma was diluted (1/10 or 1/100)
with Tris HCl buffer containing 0.6 mol/L NaCl. After addition of 50
µL of sample and rTFPI standard to each well, the plate was incubated
overnight at 4°C and antigen measured as described.13
The amounts of TFPI antigen were calculated from a standard curve
obtained from the serially diluted rTFPI. The level of TFPI antigen in
the control plasma in each microplate was defined as 100%. The
concentration of standard rTFPI was determined by titration with
human FXa, with the assumption that 1 mol TFPI
(Mr, 42 kDa) interacts with 1 mol FXa.
The concentration of human FXa was determined by titration with
p-nitrophenyl p'-guanidinobenzoate.
Fig 1 shows the standard curve for the TFPI antigen
assay in which TFPI antigen was measured at a range of 0.5 to 5.0
ng/mL. The intra-assay coefficient of variation was estimated to be
9.08% from 8 duplicate plasma samples. The inter-assay coefficient
of variation was estimated to be 6.30% from 8 determinations of 1
plasma specimen.
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Statistical Analyses
Values were expressed as mean±SD. Pearson's correlation
coefficient was calculated for the different variables, and a value
of P<.05 was considered statistically significant. The
nonparametric Wilcoxon test was employed for
statistical comparisons, with P<.05 indicating statistical
significance.
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Results |
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Measurement of Free-Form TFPI Antigen in Human Plasma
In a previous study13 we had shown that a monoclonal antibody against rTFPI recognized the K3 domain of TFPI and that this antibody reacted with free-form TFPI only and not with lipoprotein-associated forms of TFPI. In the present study we first attempted to establish the optimum conditions for measurement of free-form TFPI antigen in human plasma by using monoclonal and polyclonal antibodies against rTFPI. BSA concentrations >0.5% in the buffer that was used for dilution of rTFPI were found to be optimal. For dilution of plasma there was no difference between BSA-free buffer and buffer with 0.5% BSA. This result indicated that albumin or other proteins in human plasma prevented the loss of TFPI antigen during the assay. In accordance with these results we selected the 0.5% BSA buffer for dilution of rTFPI and BSA-free buffer for dilution of plasma. However, BSA-containing buffer can also be used for dilution of both rTFPI and plasma.
Next we examined the effect of heparin on TFPI antigen level. When
plasma was diluted in buffer containing 0.15 mol/L NaCl, the absorbance
decreased with increasing amounts of heparin (Fig 2). In
contrast, when plasma was diluted with buffer containing 0.6 mol/L
NaCl, the effect of heparin was not significant at concentrations <1
U/mL heparin (Fig 2). We also examined the effect of salt concentration
in the buffers for dilution of rTFPI and plasma. The difference between
the buffer with 0.15 mol/L NaCl and that with 0.6 mol/L NaCl was not
significant. However, reaction rates in buffers with 0.8 mol/L or 1
mol/L NaCl were clearly lower than those in buffer with 0.15 mol/L
NaCl. In accordance with these results we selected the buffer with 0.6
mol/L NaCl for dilution of the samples to abolish the effects of
heparin.
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indicates in buffer
containing 0.15 mol/L NaCl; 
, in buffer containing 0.6 mol/L
NaCl.
In another previous study12 we demonstrated that
free-form TFPI activity could be measured quantitatively after gel
filtration of plasma in a buffer with 1 mol/L NaCl. We investigated
whether the same result could be achieved in a buffer with 0.6 mol/L
NaCl, and we also examined whether heparin-releasable TFPI could be
measured in its free form under these conditions. The elution profile
of TFPI activity after gel filtration of plasma in the buffer with 0.6
mol/L NaCl was identical to that for the buffer with 1 mol/L NaCl, as
we had reported previously12 (Fig 3). When
postheparin plasma was collected from 5 normolipidemic
individuals and 5 hyperlipidemic patients and then
subjected to gel filtration in buffer with 0.6 mol/L NaCl, an increase
in TFPI activity was found for free-form TFPI only (Fig 3). These
results are consistent with those reported for buffers with 1
mol/L NaCl.12 Because our EIA method for TFPI antigen
detects the free form only, these results indicate that
heparin-releasable TFPI, ie, EC-associated TFPI, can be measured as
"free-form TFPI" in a buffer with 0.6 mol/L NaCl.
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We compared the concentration of TFPI antigen in plasma (as determined
by our new EIA method) with the values for total plasma TFPI activity
or free-form TFPI activity (as measured by the earlier method) in
plasma from normolipidemic individuals (n=13),
hyperlipidemic patients (n=12), and
hyperlipidemic patients who had received repeated
plasmapheresis treatment (n=8). TFPI antigen was positively correlated
with free-form TFPI activity (r=.819,
P<.0001), but the correlation of TFPI antigen with total
plasma TFPI activity was poor (r=.488, P<.005)
(Fig 4A and 4B). On the other hand, we found no
correlation between free-form TFPI activity and total plasma TFPI
antigen (Fig 4C). The correlation coefficient for the total plasma TFPI
activity–total plasma TFPI antigen relation was calculated to be
.418 (P<.05; Fig 4D, dotted line). When two markedly
different values were excluded (circles), the correlation coefficient
increased to .615 (P<.0005; Fig 4D, solid line). These
results indicate that the best correlation was observed between
free-form TFPI activity and free-form TFPI antigen. The reason
for the relatively poor correlation between total plasma TFPI activity
and free-form TFPI activity or antigen and that between total
plasma TFPI activity and total plasma TFPI antigen will be discussed in
a subsequent section.
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). Dotted regression line in D was
calculated from the entire sample set (n=33). The solid regression line
was calculated after two outlying values were excluded (encircled).
We also compared TFPI antigen concentration, as determined by our new
EIA method, with TFPI antigen concentration as measured with an EIA kit
(Kaketsuken) that detects both lipoprotein-associated forms and the
free form of TFPI (Fig 5). The mean values for
free-form TFPI antigen and total plasma TFPI antigen were 19.2 and
39.4 ng/mL, respectively. The ratio of free-form TFPI antigen to
total plasma TFPI antigen was approximately 0.5. This result is
consistent with our previous finding that showed 45% of
total plasma TFPI activity to be in the free form.12
All of these results indicate that although the new EIA method
specifically detects free-form TFPI, it can also be used to measure
the EC-associated TFPI that is released after heparin injection.
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Relation Between Free-Form TFPI Antigen and EC-Associated TFPI
Antigen in Normolipidemic and Hyperlipidemic
Subjects
Fig 6 shows free-form TFPI antigen in plasma
from normolipidemic individuals (n=41), hyperlipidemic
patients (n=34), and hyperlipidemic patients who were
receiving repeated plasmapheresis treatment (n=11). The amount of
free-form TFPI antigen in hyperlipidemic patients
was significantly lower than that in normolipidemic individuals
(P<.001), whereas these values in
hyperlipidemic patients who were receiving repeated
plasmapheresis treatment were even lower (P<.0001). These
results are consistent with our previous findings on the
changes in free-form TFPI activity in
hyperlipidemic patients: in that study12
we speculated that the decrease in free-form TFPI activity
reflected a decrease in EC-associated TFPI activity. To confirm this
hypothesis, we performed an experiment in which normolipidemic
individuals (n=28) and hyperlipidemic patients who were
not receiving repeated plasmapheresis treatment (n=13) were given
heparin (30 U/kg IV). Blood was drawn 10 minutes after heparin was
administered, and the TFPI antigen that had been released from ECs was
assayed by our new EIA method. Free-form TFPI antigen in plasma was
positively correlated with TFPI antigen released from ECs
(r=.581, P<.0001; Fig 7A, solid line). Because
the value for one sample deviated markedly from the others, we excluded
this outlier and recalculated the correlation coefficient, which
increased to .707 (dotted line). On the other hand, the correlation of
EC-associated TFPI antigen with total plasma TFPI antigen was less
(r=.472; Fig 7B). Free-form and
EC-associated TFPI levels in normolipidemic individuals were 119±26%
and 870±213%, respectively, and the corresponding values in
hyperlipidemic patients were 85±22% and
679±226%. The difference in free-form TFPI level between
normolipidemic and hyperlipidemic individuals and the
difference in EC-associated TFPI level between them were found to be
significant (P<.05 and P<.01, respectively).
These findings led us to conclude that the changes in free-form
TFPI generally reflected the changes in EC-associated TFPI and that
both forms of TFPI in hyperlipidemic patients were
usually lower than those in normolipidemic individuals.
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Discussion |
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In a previous study12 we had demonstrated that free-form TFPI activity in plasma was significantly lower in hyperlipidemic compared with normolipidemic individuals. From this finding we speculated that the decrease in free-form TFPI activity in hyperlipidemic patients might reflect a decrease in EC-associated TFPI. To confirm this idea we developed a new EIA method for both free-form and EC-associated TFPI in plasma, which employed a monoclonal antibody that recognized only free-form but not lipoprotein-associated TFPI antigen in plasma. Lipoprotein-associated TFPI did not react with the monoclonal antibody because its epitope (the K3 domain) was "masked" in lipoprotein-associated TFPI.13 In the present study we established the optimum conditions for measuring both free-form TFPI in plasma and that released after heparin injection, ie, EC-associated TFPI. As demonstrated previously22 23 TFPI has two heparin-binding sites, one in the K3 domain and the other in the carboxy-terminal basic domain. Therefore, we presumed that heparin could interfere with the interaction between TFPI and the monoclonal antibody by binding to the K3 domain. In fact, levels of TFPI antigen were markedly decreased by adding heparin to a buffer containing 0.15 mol/L NaCl (Fig 2
). To
overcome this problem we examined the effect of salt concentration in
the reaction mixture on the TFPI level, and 0.6 mol/L NaCl was found to
be the optimal concentration.
As demonstrated in the present study, free-form TFPI antigen in
plasma was correlated with free-form TFPI activity (Fig 4). This
result indicates that TFPI antigen has TFPI activity, although a
monoclonal antibody against the K3 domain of TFPI was employed in the
EIA method. However, it is clear that the EIA method does not
discriminate full-length TFPI from TFPI with a deleted
carboxy-terminal basic domain. That is, the measured levels of TFPI
antigen in the present study include TFPI with a deleted
carboxy-terminal basic domain (if any) and full-length TFPI.
Recently Broze et al24 suggested that TFPI with a deleted
carboxy-terminal basic domain and even a deleted K3 domain was
present in normal plasma. However, for the following reasons, our
results unequivocally indicate that the majority of free-form TFPI
in normolipidemic and hyperlipidemic plasma is K3
domain–containing TFPI: (1) the TFPI antigen level in the
free-form TFPI fraction obtained by gel filtration of plasma was
not significantly different from that measured with the ADI kit for
total plasma TFPI antigen, in which antibodies against K1 and K2
domains were used (data not shown); and (2) the finding that
free-form TFPI antigen was 50% of the total TFPI antigen is
consistent with our previous measurement of TFPI
activity.12 Despite these results we cannot exclude the
possibility that truncated forms of plasma TFPI may be lipoprotein
associated in patients with other diseases, such as disseminated
intravascular coagulation.
In contrast to the strong correlation between free-form TFPI
antigen and free-form TFPI activity, the correlation between
free-form TFPI antigen and total plasma TFPI activity was
relatively poor (r=.488), as shown in Fig 4B. The
correlation between free-form TFPI activity and total plasma TFPI
activity was also poor (r=.548, data not shown).
Furthermore, the correlation between total plasma TFPI antigen and
total plasma TFPI activity was not as high as expected
(r=.615), as shown in Fig 4D. It has been suggested that the
anticoagulant activity of lipoprotein-associated forms of TFPI is
markedly lower than that of free-form TFPI.10 When we
compared the TFPI activity of LDL- and HDL-associated forms of TFPI
with that of free-form TFPI antigen (using the EIA kit for total
plasma TFPI after gel filtration of control plasma), we found that
free-form TFPI activity was 6.7 times higher than that of the
LDL-associated form and 2.5 times higher than that of the
HDL-associated form in terms of nanograms of TFPI antigen (data not
shown). We suspect that the discrepancy between TFPI activity and TFPI
antigen may have been caused by the different specific activities of
these forms in plasma. In particular, these levels are different in
each hyperlipidemic patient and are probably different
in normolipidemic individuals as well. Therefore, it is not surprising
that the correlation between free-form TFPI and total plasma TFPI,
in terms of their activity and antigen levels, was relatively poor. On
the basis of these speculations, we recommend that free-form TFPI,
and preferably lipoprotein-associated TFPI, be measured rather than
total plasma TFPI to reveal the significant role of TFPI in various
diseases.
It is generally agreed that most TFPI is synthesized in ECs and binds
to proteoglycans on the cell surface and that some portion of the
EC-associated TFPI is continuously released into plasma as the free
form. Some of the free-form TFPI released from ECs then binds to
lipoprotein particles (thereby becoming lipoprotein-associated
TFPI) by some as-yet-unknown mechanism. On the basis of this
consideration, we speculate that the decrease in free-form TFPI in
the plasma of hyperlipidemic patients may have been
caused by an increase in LDL-associated TFPI and/or a decrease in
EC-associated TFPI. The relatively high LDL cholesterol
levels in the hyperlipidemic patients would accelerate
the conversion of free-form TFPI to lipoprotein-associated
TFPI, as has been demonstrated in diet-induced
hypercholesterolemia in monkeys20
and in familial hyperlipidemia in
humans.25 26 The decrease in EC-associated TFPI may be due
to EC dysfunction and/or "consumption" of the EC-associated TFPI
owing to the hypercoagulability in these patients. Although
free-form TFPI can be reconverted to EC-associated TFPI,
lipoprotein-associated TFPI cannot bind to ECs because the
heparin-binding sites of TFPI have been masked, as evidenced by
their inability to bind to a heparin-conjugated
column.10 Hansen et al21 have recently
suggested that EC-associated TFPI and LDL-associated TFPI behave
independently in the plasma of hyperlipidemic patients
who are receiving lipid-lowering drugs. However, in
hyperlipidemia it is very difficult to detect a
decrease in the levels of EC-associated TFPI because they are markedly
higher than those of plasma TFPI and the decrease is restricted to
localized areas in the vascular wall. In fact, we found no significant
difference in EC-associated TFPI levels between normolipidemic and
diet-induced hypercholesterolemic
monkeys.20 We speculate that a combination of the changes
in the equilibrium between free-form and lipoprotein-associated
TFPI in plasma and EC-associated TFPI caused decreases in both
free-form TFPI in plasma and EC-associated TFPI. As demonstrated in
Fig 7A free-form TFPI antigen in plasma was positively correlated
with EC-released TFPI antigen, and an even stronger correlation was
calculated by excluding one outlier. These results indicate that the
free-form TFPI level in plasma generally reflects the level of
EC-associated TFPI, although some individual cases show a poor relation
between free-form TFPI and EC-associated TFPI owing to unknown
mechanisms. We believe that further experiments on these TFPI antigens
and use of our new EIA method may clarify the relation between
free-form TFPI levels and EC-associated TFPI levels in various
disease states.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported in part by a grant from the Japan Cardiovascular Research Foundation, by a grant-in-aid for Research Promoting Comprehensive Longevity Science, and by a Research Grant for Cardiovascular Diseases (5A-2) from the Ministry of Health and Welfare of Japan.
Received September 12, 1995; accepted March 26, 1996.
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