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1
Department of Laboratory Medicine, University of Washington, Seattle, WA 98195.
a Address correspondence to this author at: Department of Laboratory Medicine, Box 357110, University of Washington, Seattle, WA 98195. Fax 206-598-6189; e-mail wlc{at}u.washington.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: Active tPA was measured by use of an indirect amidolytic assay and immunofunctional assays. tPA/PAI-1, tPA/C1-inhibitor, and total tPA antigen were measured by use of microtiter plates coated with anti-tPA antibodies and, respectively, anti-PAI-1, anti-C1-inhibitor, and anti-tPA antibodies conjugated to peroxidase.
Results: The immunofunctional tPA assay detected 1 U/L (0.001 U/mL) tPA and recovered 108% ± 12% of active tPA added to samples containing high (mean, 60 000 IU/L) PAI-1 activities vs a detection limit of 10 U/L (0.01 U/mL) and 13% ± 25% recovery for the indirect amidolytic tPA activity assay. For measurement of tPA/PAI-1 complex, polyclonal anti-PAI-1 conjugates recovered 112% ± 20% of the expected tPA/PAI-1 vs recovery of only 38% ± 16% when monoclonal anti-PAI-1 conjugates were used. Of three methods tested, two total tPA antigen assays correlated well (r2 = 0.85) and showed recoveries near 100%, whereas the third method showed lower correlations, higher intercepts, and falsely high recovery. A single anti-tPA capture antibody that performed the best in the individual assay evaluations was used to measure the different forms of tPA in 22 samples with a range of tPA and PAI-1 values. The sum of the molar concentrations of active tPA, tPA/PAI-1, and tPA/C1-inhibitor using the optimized methods was equal to 94% ± 7% of measured total tPA.
Conclusion: Optimized assays based on a single anti-tPA capture antibody can be used to accurately measure the major forms of tPA in plasma.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To better understand the complex regulation of the fibrinolytic system, we have been developing kinetic models of the circulatory regulation of tPA (8)(9)(11)(12). Kinetic modeling puts heavy demands on the assays used to provide data. To model tPA regulation, several different assays are needed to specifically measure the different forms of tPA in plasma (active tPA, tPA complexed with inhibitors, and total tPA). Both types of assays, antigenic or activity, must be expressed as molar concentrations. Finally, the different assays must be directly comparable. For tPA this means that the sum of the different subtypes of tPA should add up to match the total tPA activity in the plasma.
Current assays are designed to compare the relative levels of fibrinolytic factors between different groups of subjects. Although they are adequate for that purpose, there are clear accuracy and calibration problems between different commercial sources for assays designed to measure the same factor. For example, Declerck et al. (13) found that although most assays for PAI-1 antigen produced results that were highly correlated, there were substantial variations in the calibration of the assays, with some methods producing results that were more than twice as high as other assays run on the same samples.
The purpose of this study was to evaluate four different methods used to measure tPA in plasma: (a) tPA activity assays; (b) tPA/PAI-1 complex assays; (c) tPA/C1-inhibitor assays; and (d) total tPA antigen assays. The study was designed to evaluate the ability of each assay to accurately measure a specific type of tPA in plasma and to determine whether the sum of the different subtypes of tPA was equal to the total tPA activity in plasma.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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materials
Human fibrinogen, cyanogen bromide, and Triton-X-100 were
obtained from Sigma. Cyanogen bromide-cleaved human fibrinogen
fragments were prepared as described previously (14).
Chromogenic substrate
D-valyl-phenyl-alanyl-lysyl-p-nitroanaline
(cat. no. S-2390) was obtained from Pharmacia Hepar. The enzyme
immunoassay (EIA) kits used in the study are shown in Table 1
. Immunofunctional kits for the measurement of tPA activity
(Chromolize tPA; kit H) and PAI-1 activity (Chromolize PAI-1) were
obtained from Biopool. Monoclonal anti-urokinase plasminogen activator
(anti-uPA; product no. 394), goat anti-human tPA (product no.
387), single-chain uPA, human glu-plasminogen, 10-mL evacuated blood
sample tubes containing 0.105 mmol of citrate and 0.25 µmol of
D-Phe-Pro-Arg chloromethyl ketone (PPACK), and
three noninhibitory monoclonal anti-human tPA antibodies [product no.
3696 (Ab I), product no. 3692 (Ab J), and product no. 3705 (Ab K)]
were obtained from American Diagnostica. Purified human C1-inhibitor
was obtained from Calbiochem. Peroxidase-conjugated sheep anti-human
C1-inhibitor F(ab')2 fragments were obtained from
The Binding Site. All other materials not described above were reagent
or analytical grade. Single-chain human melanoma tPA activity
calibrator (0.567 U/ng) was obtained from American Diagnostica.
tPA/PAI-1 calibrator and tPA/C1-inhibitor calibrator were prepared as
described previously (15)(16). Briefly,
single-chain tPA was added to an excess of either purified active PAI-1
or purified C1-inhibitor. Free tPA, PAI-1, and C1-inhibitor were
removed from the bound form by column chromatography. The total tPA in
the purified complex was then determined. tPA activity or antigen
concentrations in the calibrators were calibrated by comparison with
international tPA standard 86/670.
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blood sampling and sample preparation
Blood was obtained from a forearm vein. Blood samples for tPA
antigen assays were anticoagulated by the addition of 4.5 mL of whole
blood to 0.5 mL of 105 mmol/L sodium citrate for a final citrate
concentration of 10.5 mmol/L of whole blood or by the addition of 10 mL
of whole blood to a tube containing lyophilized PPACK (0.25 µmol) and
sodium citrate (0.105 mmol), for final whole blood concentrations of
10.5 mmol/L citrate and 25 µmol/L PPACK. The PPACK/citrate tubes were
stored at 4 °C before use. Blood samples for tPA activity were
anticoagulated and stabilized by the addition of 4.5 mL of whole blood
to 0.5 mL of 500 mmol/L acidified citrate, pH 4.0, for a final citrate
concentration of 50 mmol/L and plasma pH of 5.5 (17). All
samples were centrifuged for 15 min at 2000g at room
temperature. Citrate, PPACK/citrate, and acidified citrate plasma
samples were then removed and frozen at -80 °C until analyzed.
For the final comparison of the four assays, samples were obtained before, during, and after cardiopulmonary bypass. These samples had near-normal tPA and PAI-1 values before surgery, increased tPA during cardiopulmonary bypass, and increased postoperative PAI-1 (18).
tPA ACTIVITY ASSAY EVALUATION
An in-house immunofunctional tPA activity assay was developed
based on the capture of active tPA by a noninhibitory anti-human tPA
antibody bound to microtiter plates (19). Four different
anti-tPA capture antibodies were studied: kit A, Ab I, Ab J, and Ab K.
Anti-tPA antibodies Ab I, Ab J, and Ab K were reported to be
noninhibitory to tPA. Microtiter plates were coated with 10
mg/L antibody in 0.1 mol/L sodium bicarbonate, pH 9.0,
overnight at 4 °C. The anti-tPA antibody from kit A was already
coated on microtiter plates.
To measure tPA activity in the in-house immunofunctional assay, acidified plasma was diluted 1:5 in 0.1 mol/L phosphate buffer, pH 5.5, containing 5 mmol/L EDTA and 10 g/L Triton X-100. The diluted acidified plasma (200 µL) was then incubated in the microtiter plate coated with anti-tPA antibody for 90 min at room temperature to allow the tPA to bind. Nonbinding proteins were washed off the plate with 0.01 mmol/L phosphate-buffered saline, pH 7.4, containing 1 g/L Tween 20. Bound active tPA was detected by the addition of a plasminogen-chromogenic substrate reagent (14). The microtiter plate was incubated 90–240 min at 37 °C. After incubation, the plasminogen-chromogenic substrate reaction was stopped by addition of 50 µL of 250 mL/L acetic acid. Absorbance was read at 405 nm.
This in-house immunofunctional tPA activity assay was compared with an indirect amidolytic tPA activity assay and a commercial immunofunctional assay (kit H). In the indirect amidolytic tPA activity assay, acidified plasma was added to a plasminogen-chromogenic substrate reagent as described previously (14). This assay detects any form of plasminogen activator in plasma. To improve the specificity in plasma samples, monoclonal anti-uPA (25 mg/L) was added to suppress uPA activity in the assay (9). The commercial immunofunctional assay was similar to the assay developed in-house and used an anti-tPA capture antibody followed by a plasminogen-chromogenic substrate reagent (20).
Purified one-chain human melanoma tPA calibrator was used to calibrate all of the assays. The tPA and PAI-1 activities were converted to the molar concentration of active tPA or PAI-1 by use of an in vivo specific molar activity of 4.48 x 1013 U/mol (15).
tPA/PAI-1 ASSAY EVALUATION
Four different microtiter plates coated with anti-human-tPA
antibodies from kits A–D and four different anti-human-PAI-1
peroxidase conjugates from kits D–G were used to evaluate 16 different
antibody combinations for measuring tPA/PAI-1 complex by EIA. All four
anti-tPA antibodies were reported to bind all forms of tPA, including
tPA/PAI-1 complex. All four anti-PAI-1 antibodies were reported to bind
all forms of PAI-1, including tPA/PAI-1 complex. All assays were
calibrated with a purified tPA/PAI-1 complex calibrator as described
previously (15). The relative assay response to the
calibrator (change in absorbance per microgram of tPA/PAI-1 complex per
liter) and the detection limit were determined for each antibody
combination. The detection limit was defined as the tPA/PAI-1 complex
value 3 SD above the blank absorbance for that antibody combination.
The purified tPA/PAI-1 complex calibrator was compared to a commercial
plasma-based tPA/PAI-1 calibrator from kit D. Each antibody combination
was used to measure the tPA/PAI-1 complex in seven citrated plasma
samples containing increased PAI-1 activity [range, 25 000–115 000
IU/L (25–115 IU/mL); mean ± SD, 68 000 ± 34 000 IU/L
(68 ± 34 IU/mL)], before and after addition of an excess of
active tPA.
tPA/C1-INHIBITOR ASSAY EVALUATION
Three different microtiter plates coated with anti-human-tPA
antibodies from kits A, B, and C were used with anti-human-C1-inhibitor
peroxidase conjugate to evaluate three different antibody combinations
for measuring tPA/C1-inhibitor complex by EIA. All assays were
calibrated with a purified tPA/C1-inhibitor complex calibrator as
described previously (16). An excess (25 mg/L) of unlabeled
anti-tPA was used to blank the assay (21). The detection
limit was determined as described above.
tPA ANTIGEN ASSAYS
Three commercially available EIA methods for total tPA antigen (A,
B, and C) were compared. The tPA antigen calibrator in each method was
run along with a single-chain melanoma tPA calibrator that was added to
plasma. Each method was run according to the manufacturer’s
recommendations.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). All four anti-tPA antibodies were able to bind pure tPA from
sample buffer at a pH of 5.5 while retaining the activity of the tPA.
In contrast, only the anti-tPA antibody from kit A was able to bind and
stabilize active tPA in acidified plasma samples. When the anti-tPA
antibody from kit A was used, the assay showed a linear response to
increasing tPA activity up to a plasma concentration of 5000 U/L [(5
U/mL); equal to 1000 U/L (1 U/mL) in the microtiter plate well after
1:5 dilution with sample buffer, pH 5.5]. The in-house
immunofunctional tPA activity assay was compared to a commercial
immunofunctional tPA activity assay (kit H) and an indirect amidolytic
tPA activity assay.
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The analytic sensitivity of the three assays to low tPA activities was evaluated by adding 10 or 1 U/L (0.01 or 0.001 U/mL) active tPA into the sample buffer for each assay. The incubation time for the plasminogen-substrate reaction was increased to 4 h (37 °C). Only the in-house immunofunctional assay was able to detect 1 U/L (0.001 U/mL) pure tPA in buffer. The changes in absorbances (final absorbance minus final mean blank) were as follows: 0 U/L = 0 ± 8 milliabsorbance units vs 1 U/L = 35 ± 3 milliabsorbance units; P < 0.01, unpaired t-test. All three assays could detect 10 U/L (0.01 U/mL) tPA in buffer, but the absorbance change in the in-house immunofunctional assay was two- to sixfold higher than in the other assays.
To evaluate specificity, tPA and single-chain uPA were added to
acidified plasma, and the plasminogen activator activity was measured
in the three assays. Table 2
shows the specific absorbance change for each assay. The
in-house immunofunctional assay had the highest specific absorbance
change for tPA. A low but measurable uPA activity was detected by the
in-house immunofunctional assay. On average, the specific absorbance
change for the in-house immunofunctional assay was 365-fold higher for
tPA vs uPA. No uPA activity was detected when kit H or the indirect
amidolytic assay containing anti-uPA antibodies were used.
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The recovery of tPA activity was evaluated by adding purified
single-chain tPA to plasma containing moderate to high PAI-1 activity
(Table 3
). Both the in-house and the commercial immunofunctional assays
recovered ~100% of the added active tPA regardless of the PAI-1
activity. In contrast, the indirect amidolytic assay recovered variable
amounts of active tPA, ranging from 63% when PAI-1 activity was
13 000 IU/L (13 IU/mL) to <10% in most samples with increased PAI-1.
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The in-house immunofunctional assay was slightly less specific but more
sensitive than the other assays. Both immunofunctional assays were able
to recover active tPA added to plasma containing high PAI-1 activities,
this was not the case for the indirect amidolytic tPA activity assay.
As a final test, we compared tPA activity measured in the two
immunofunctional assays in 23 acidified plasma samples containing
various amounts of active tPA, total tPA, and PAI-1 activity (Fig. 2
).
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tPA/PAI-1 COMPLEX ASSAY EVALUATION
A total of 16 different assays for tPA/PAI-1 complex were
evaluated based on four different microtiter plates coated with
anti-human-tPA antibodies vs four different anti-human-PAI-1 peroxidase
conjugates. Good agreement was found between the purified tPA/PAI-1
complex calibrator added to plasma and the commercial plasma-based
tPA/PAI-1 complex calibrator from kit D for 15 of the 16 antibody
combinations. The commercial calibrator was reported to contained 10
µg/L tPA/PAI-1 complex; the measured value was 10.3 ± 0.9
µg/L (n = 15). One antibody combination (anti-tPA kit B with
anti-PAI-1 conjugate kit F) detected only 2.9 µg/L tPA/PAI-1 complex
in the commercial vs the purified calibrator.
The recovery of tPA/PAI-1 complex from plasma was assessed by measuring
tPA/PAI-1 in seven plasma samples containing high PAI-1 activities,
before and after addition of an excess of purified single-chain tPA.
Because the samples all contained high amounts of PAI-1, it was
expected that the majority of the tPA in the plasma would be in the
form of tPA/PAI-1 complex. In general, the use of mouse monoclonal
anti-PAI-1 conjugates (kits D and E) produced lower estimates of the
percentage of tPA in the form of tPA/PAI-1 complex (Table 4
) than did polyclonal anti-PAI-1 conjugates (kits F and G). Four
antibody combinations found 80–90% of the tPA in the form of
tPA/PAI-1 complex in plasmas with high PAI-1 activity: mouse monoclonal
anti-tPA F(ab')2-coated plates from kits A or D
used with goat polyclonal anti-PAI-1 conjugates from kits F or G.
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When excess tPA is added to plasma, it converts all of the active PAI-1
into tPA/PAI-1 complex. tPA/PAI-1 complex was measured before and after
the addition of excess tPA. Antibody combinations that utilized
polyclonal anti-PAI-1 recovered 112% ± 20% of the expected increase
in tPA/PAI-1 complex based on the original PAI-1 activity (Table 5
). In contrast, the antibody combinations that used a monoclonal
anti-PAI-1 conjugate recovered only 38% ± 16% of the expected
tPA/PAI-1. Thus, the source of tPA in the complex, native or exogenous,
did not affect the ability of different antibody combinations to detect
tPA/PAI-1 complex in plasma, but the type of anti-PAI-1 conjugate did.
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Correlations between the different tPA/PAI-1 assays were in general good. The correlation for one assay vs all the others was, on average, r2 = 0.89 (range, 0.6–0.98; n = 15 for each correlation). Two assays showed clearly lower correlations with all the others (r2 = 0.64 and 0.67). These two assays also recovered low amounts of tPA/PAI-1 from high PAI-1 plasma (29% and 11%, respectively) and after excess tPA was added back to plasma. When data from these assays were removed, the mean correlation for one assay vs all others increased to r2 = 0.95 (range, 0.88–0.98; n = 13 for each correlation).
The specific absorbance change for the tPA/PAI-1 calibrator was evaluated as a measure of assay response. The anti-tPA capture antibody had little effect on assay response to tPA/PAI-1. The assays fell into three groups depending on the anti-PAI-1 conjugate used. For anti-PAI-1 conjugates from kits D and E, the final absorbance change per µg/L of complex was 0.069 ± 0.011; for anti-PAI-1 conjugate from kit F, it was 0.027 ± 0.08, and for the anti-PAI-1 conjugate from kit G, it was 0.018 ± 0.003. The detection limits for the different assays were related to the degree of variation in the blank value vs the change in absorbance for the blank vs the calibrators. The best detection limit was for 0.1 µg/L for the anti-tPA plate from kit A paired with anti-PAI-1 conjugate from kit D; the worst detection limit, 0.5 µg/L, was for the anti-tPA plate from kit B paired with anti-PAI-1 conjugate from kit G.
Overall, the optimum combination was the mouse monoclonal anti-human tPA F(ab')2 capture antibody from kit A with goat polyclonal anti-PAI-1 peroxidase conjugate from kit F. This combination of antibodies had an acceptable absorbance response with consistent detection of a high percentage of tPA/PAI-1 complex in plasma containing high PAI-1 activities and complete recovery of tPA/PAI-1 complex when excess tPA was added to plasma. This assay was specific for tPA/PAI-1 complex, showing no response to purified tPA or PAI-1 added alone. The detection limit of the assay was ~0.1 µg/L.
tPA/C1-INHIBITOR ASSAY EVALUATION
Three different microtiter plates coated with anti-human tPA
antibodies from kits A, B, and C were used along with a single
anti-human-C1-inhibitor peroxidase conjugate to measure
tPA/C1-inhibitor complex. Capture antibodies from kits B and C showed
high background values with little or no response to increasing amounts
of tPA/C1-inhibitor complex; they were not studied further. When the
capture antibody from kit A was used, the assay demonstrated good
specificity with no detectable response to pure tPA concentrations up
to 50 µg/L or pure C1-inhibitor concentrations up to 100 mg/L. When 1
g/L pure C1-inhibitor was added, some nonspecific binding was noted. To
correct for this nonspecific interference, an antibody blank was
instituted using an excess (25 mg/L) of unlabeled anti-tPA antibody.
When the antibody blank technique was used, no interference was
detected with pure C1-inhibitor up to 1 g/L or in plasma. The detection
limit for the blanked assay was ~0.3 µg/L. When 5 µg/L
tPA/C1-inhibitor complex calibrator was added to plasma samples,
recovery was 5.0 ± 0.5 µg/L (n = 6). The final assay
detected on average <1 µg/L of tPA/C1-inhibitor in the seven samples
containing high PAI-1 activities. Higher concentrations were detected
in some samples with increased active tPA or low PAI-1 activity.
total tPA ANTIGEN EVALUATION
A set of 23 samples that contained high PAI-1 activity, high tPA
activity, a tPA/PAI-1 complex calibrator, and a tPA/C1-inhibitor
complex calibrator were compared in three total tPA antigen assays
(Fig. 3
). The Deming regression lines and correlation coefficients were
as follows:
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Kit B = [(1.025 ± 0.064) xkit A] + (1.98 ± 1.18 µg/L); r2 = 0.84; Sy|x = 2.55
Kit C = [(0.924 ± 0.058) xkit A] + (7.32 ± 1.06 µg/L); r2 = 0.86; Sy|x = 2.30
Kit C = [(0.896 ± 0.074) x kit B] + (5.65 ± 1.53 µg/L); r2 = 0.76; Sy|x = 3.02
The intercepts were significantly different from zero for all three comparisons (P <0.05). The recovery of tPA antigen from plasma was evaluated by adding 100 µg/L purified single-chain tPA into seven plasma samples: kit A = 106% ± 7%, kit B = 99% ± 10%, kit C = 131% ± 5%. Kit C overestimated the recovery of tPA antigen. The detection limits for the three kits were 0.3 µg/L for kit A, 0.8 µg/L for kit B, and 0.8 µg/L for kit C.
measurement of different forms of tPA IN PLASMA
Of the anti-tPA capture antibodies tested, the anti-tPA-coated
plate from kit A had the best overall characteristics: it was able to
bind and stabilize active tPA at low pH in the in-house
immunofunctional assay, was optimal for measuring tPA/PAI-1 complex and
tPA/C1-inhibitor complex, and showed acceptable correlation with the
other assays for measuring total tPA. Therefore, we selected the
anti-tPA-coated plate from kit A to measure the four forms tPA in
plasma: (a) active tPA in the in-house immunofunctional
assay; (b) with the polyclonal anti-PAI-1 conjugate from kit
F to measure tPA/PAI-1 complex; (c) with the
anti-C1-inhibitor conjugate to measure tPA/C1-inhibitor complex; and
(d) kit A to measure total tPA. The imprecision for the
immunofunctional tPA activity assay was as follows: within-run,
4.2 ± 0.3 pmol/L (n = 8; CV = 7%); between-run,
22.9 ± 1.8 pmol/L (n = 11; CV = 8%). The imprecision
for the total tPA antigen assay was as follows: within-run 194 ±
12 pmol/L (n = 8; CV = 6%); between-run; 260 ± 20
pmol/L (n = 23; CV = 8%). Because the tPA/PAI-1 complex and
tPA/C1-inhibitor complex EIAs used the same capture antibody and
methodology, their imprecision was similar to that for the total tPA
antigen assay.
These four assays were used to measure the different forms of tPA in 21
samples obtained before, during, and after cardiopulmonary bypass. This
provided samples with a wide range of tPA and PAI-1 values for
evaluation. Active tPA ranged from 1 to 171 pmol/L (mean, 41 pmol/L).
tPA/PAI-1 ranged from 116 to 437 pmol/L (mean, 221 pmol/L).
tPA/C1-inhibitor ranged from undetectable to 8 pmol/L. Total tPA ranged
from 148 to 537 pmol/L (mean, 282 pmol/L). Fig. 4
shows a comparison of the total tPA antigen concentration vs
the sum of the active tPA plus tPA/PAI-1 complex plus tPA/C1-inhibitor
complex for all 21 samples. Table 6
shows the individual values for each assay and the sum in six
samples. On average, the sum of the three subtypes of tPA accounted for
94% ± 7% of the total tPA measured (range, 77–103%).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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These assays were used to evaluate the amounts of different forms of
tPA in plasma under conditions of normal tPA and PAI-1 activities, high
tPA activity, and high PAI-1 activity. The sum of the three specific
assays accounted for 94% ± 7% of the total tPA measured
(r2 = 0.92). This indicated that over
a wide range of individual assay results (e.g., 1–171 pmol/L for
active tPA), the sum of the tPA subtype assays agreed well with the
total tPA measured. This was critical if the results from the assays
were to be used for kinetic modeling, where the model is being using
the track the various forms of tPA in the vascular system and the
results from the assays are used to develop and validate the model. We
noted that, on average, the sum of the tPA subtypes was slightly less
than the total tPA measured (P = 0.005, paired
t-test). Recently, an EIA for measuring
tPA/
2-antiplasmin complex has been described
by Nordenhem and Wiman (10), who reported finding 0–3
µg/L tPA/2-antiplasmin complex in plasmas
from 30 healthy patients. It is likely that part of the difference
between the total and summed tPA was unmeasured
tPA/2-antiplasmin complex.
In evaluating the different assays, we found the each had particular
characteristics that required optimization. In typical indirect
amidolytic tPA activity assays, acidified plasma is added directly to
plasminogen-chromogenic substrate reagent that is optimized to work at
pH 7–8 (14)(22). Once the sample is added to
the reagent, it is no longer acidified, and high concentrations of
PAI-1 can again inhibit active tPA in the sample during the incubation
period of the assay. The assay can measure tPA activity when PAI-1
activity is low, but high PAI-1 activity can destroy active tPA in the
assay, as shown in Table 3
. The indirect amidolytic tPA activity assay
was not accurate in the presence of high PAI-1 activity, was less
sensitive to low tPA activity, and was nonspecific toward other
plasminogen activators unless inhibitors were added (e.g., anti-uPA).
In immunofunctional tPA activity assays, tPA is bound to a microtiter plate under acidic pH conditions that block the reaction between tPA and PAI-1 (19)(20). Free PAI-1 and other inhibitors are then washed off. Bound active tPA is measured with a plasminogen-chromogenic substrate reagent. The immunofunctional tPA activity assays were able to measure tPA activity accurately in the presence of high PAI-1 activity, were specific for tPA, and were more sensitive than indirect amidolytic assays to low tPA activity. Immunofunctional tPA activity assays should probably replace indirect amidolytic assays in applications where tPA activity is being measured in plasma or culture media containing high activities of PAI-1 or other plasminogen activators, such as uPA.
Prior studies using a variety of methods reported that the majority of
tPA in plasma circulates as tPA/PAI-1 complexes and that as PAI-1
activity rises more tPA is converted to complex
(10)(15)(23)(24)(25)(26)(27). Previously, we
found that in subjects with PAI-1 activity averaging 4000 IU/L (4
IU/mL), 40% of tPA circulated in an active form, whereas 45% was in
the form of tPA/PAI-1 complexes (15). When PAI-1 increased
to a mean of 17 000 IU/L (17 IU/mL), only 12% of tPA was active,
whereas 79% was in the form of tPA/PAI-1 complex. Similar results were
found in this study, as shown in Table 6
.
The most interesting result in the tPA/PAI-1 complex assay comparison was the variation in the amount of tPA/PAI-1 complex detected in plasma samples although the same calibrators were used in all versions of the assay. The anti-PAI-1 conjugate was the most important factor in determining the amount of tPA/PAI-1 complex measured in plasma. When polyclonal conjugates were used, the expected amount of tPA/PAI-1 was found; when monoclonal conjugates were used, the amount of tPA/PAI-1 complex measured often was falsely low. It should be noted that although many assays showed good correlations, the absolute amount of tPA/PAI-1 measured in plasma was very different. The cause of this discrepancy is unknown, but it may relate to the different binding characteristics of monoclonal anti-PAI-1 antibodies to free PAI-1 vs tPA/PAI-1 complex. In the end, we selected a polyclonal anti-PAI-1 conjugate for measurement of tPA/PAI-1 complex in plasma.
Measurement of total tPA in plasma was more straightforward, but significant biases were noted among different commercially available total tPA antigen assays. This may in part relate to different assay strategies. Again, some kits used monoclonal antibodies, whereas others used polyclonal antibodies with unlabeled antibody blanking wells and non-immune IgG to reduce interference. The best correlation and least bias were between kits A and B. Kits A and B also showed near 100% recovery for purified tPA added to plasma. In contrast, kit C showed the least correlation and the largest bias compared with the other two kits and overestimated the recovery of added tPA antigen by ~31%. The large bias in tPA antigen results between different commercial kits necessitates the use of assay-specific reference ranges for each method.
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2-macroglobulin, and C1-inhibitor: studies in patients with defibrination and a fibrinolytic state after electroshock or complicated labor. Blood 1990;75:671-676.
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 W. L. Chandler, S. Jelacic, D. R. Boster, M. A. Ciol, G. D. Williams, S. L. Watkins, T. Igarashi, and P. I. Tarr Prothrombotic Coagulation Abnormalities Preceding the Hemolytic-Uremic Syndrome N. Engl. J. Med., January 3, 2002; 346(1): 23 - 32. [Abstract] [Full Text] [PDF]
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