© 1999 American Heart Association, Inc.
Original Contribution |
Gene Polymorphism of t-PA is Associated With Forearm Vascular Release Rate of t-PA
From the Clinical Experimental Research Laboratory (C.J., P.L., U.W., S.J.), Heart and Lung Institute, and Department of Neurology (C.J.), Institute of Clinical Neuroscience, Sahlgrenska University Hospital, Göteborg University, Göteborg, Sweden.
Correspondence to Christina Jern, MD, PhD, Clinical Experimental Research Laboratory, Sahlgrenska University Hospital/Östra, CK, S-416 85 Göteborg, Sweden. E-mail christina.jern{at}pediat.gu.se
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Abstract |
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Abstract—We have observed marked interindividual differences in release rates of tissue-type plasminogen activator (t-PA) among healthy subjects. The objective of the current study was to test the hypothesis that there is an association between a genetic variation at the t-PA locus and the in vivo release rate of t-PA. Fifty-one healthy males were studied at rest in the morning and 27 of these were also subjected to a mental stress test. Net release rates of total t-PA across the forearm vascular bed were calculated as the product of the venoarterial concentration gradient and forearm plasma flow. Zygosity for an Alu-repeat polymorphism in intron 8 of the t-PA gene was determined by a polymerase chain reaction. Basal t-PA release rates differed markedly by genotype (ANOVA, P<0.05); subjects homozygous for the insertion had a significantly higher release rate (mean 10.9 ng · min-1 · L-1, n=19) than both heterozygotes (4.5 ng · min-1 · L-1, n=26) and subjects homozygous for the deletion (0.9 ng · min-1 · L-1, n=6). After 2 minutes of mental stress release rates had increased approximately 2-fold in all groups. Arterial and venous plasma levels of t-PA were unrelated to genotype. In conclusion, the current results provide the first evidence of an association between a common genetic variation at the t-PA locus and interindividual differences in net release rates of t-PA in vivo. The relationship is not reflected by circulating steady-state plasma levels and can thus not be disclosed by conventional venous plasma sampling.
Key Words: genetics • tissue-type plasminogen activator • blood flow • secretion • stress
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Introduction |
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Genetic factors have been shown to influence plasma protein levels for several hemostatic factors.1 Common genetic variations (polymorphisms) at the fibrinogen, factor VII, and plasminogen activator inhibitor type 1 (PAI-1) loci have been associated with interindividual differences in the basal steady-state plasma level of the respective proteins.1 2 3 4 In addition, responses to environmental factors have been shown to differ by genotype.1
A polymorphism has been identified at the tissue-type plasminogen activator (t-PA) locus on chromosome 8, which consists of the presence or absence of a 311 bp Alu sequence in intron 8.5 The Alu insertion probably arose early in human evolution, and a number of populations have been found to be dimorphic for its presence or absence.6 7 A similar polymorphism in the angiotensin converting enzyme (ACE) gene has been shown to explain approximately 50% of the variability of plasma ACE levels between individuals.8 In contrast, it was recently reported that t-PA genotypes did not correlate with basal plasma levels of t-PA.9 10 11 However, in view of the complex regulation of the steady-state plasma level of t-PA, this observation does not exclude the possibility of an association between genotype and local t-PA release rates. The systemic concentration of t-PA not only is dependent both on secretion and clearance but also on its rate and degree of complex-formation with PAI-1.12 This is due to the fact that the t-PA/PAI-1 complex is cleared at a slower rate than free t-PA.13 It follows that an increased plasma concentration of PAI-1 will be paralleled by an increased plasma concentration of total t-PA.13 Consequently, the systemic plasma level of t-PA is unlikely to directly reflect its secretion rate.
Circulating t-PA is derived from vascular endothelial cells.14 It is the key enzyme in the initiation of an endogenous fibrinolytic/thrombolytic response.14 Recent data suggest that the local endothelial release rate, rather than the steady-state plasma concentration, of t-PA determines the thrombolytic potential.14 15 In 1994, our group has developed a modification of the perfused-forearm model to be able to directly measure rates of t-PA release in vivo.16 17 18 This method allows instantaneous determinations of local release rates of t-PA secreted from the endothelium without any interference of liver clearance or being confounded by shifts between complexed or free molecular forms. In addition, studying an intact vascular bed has the advantages of preserved innervation, circulation, blood flow pulsatility, and cell-to-cell interaction. In essence, the model is based on simultaneous arterial and venous sampling from the cubital region and a simultaneous plethysmographic measurement of forearm blood flow (FBF). In a series of studies, we have observed marked interindividual variations in net release rates of t-PA in healthy subjects.17 18 19 20 The aim of the current study was to test the hypothesis that part of this difference is related to genetic variations between the subjects.
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Methods |
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In vivo experiments of forearm t-PA release were performed in 51 apparently healthy, nonobese, nonsmoking, male subjects. All subjects were without any medication or history of cardiovascular disease. The investigation was based on earlier studies in our laboratory performed to investigate release mechanisms of t-PA. The nature, purpose, and potential risks of the study were carefully explained to each subject before informed consent to participate was obtained. The protocol was approved by the Ethics Committee of the University of Göteborg, and the study was conducted according to the Declaration of Helsinki. All procedures were performed in accordance with the guidelines of our laboratory. Experiments were performed after an overnight fast (at least 10 hours) and commenced at 8:30 AM.
Catheterization
An arterial polyethylene catheter (Viggo
Products, British Viggo) was introduced
percutaneously by the Seldinger technique into the
brachial artery of the nondominant arm and advanced 10 cm in the
proximal direction. An indwelling cannula (Venflon, Viggo) was
introduced retrogradely into a deep antecubital vein of the same arm
for venous blood sampling from the muscle vascular bed.
Hemodynamic Monitoring
Intraarterial blood pressure was recorded
continuously by an electrical transducer (EMT 35, Siemens-Elema) and a
Mingograph 82 (Siemens-Elema). Mean arterial pressure was
obtained by electrical damping of the pressure signal. The
electrocardiogram (ECG) was also continuously
monitored on the Mingograph. Venous occlusion plethysmography with a
mercury-in-rubber strain-gauge was used to assess FBF.21
FBF, in ml · min-1 ·
L-1 tissue, was calculated from 3 to 5 separate
recordings after each blood sampling. Intra- and inter-observer
coefficients of variation were, on the average, 5.6% and 4.6%,
respectively.
Experimental Design
After the catheterization and application of
recording devices, subjects rested 60 minutes in the supine
position in a dimly-lit and sound-proof room. Blood samples were then
obtained twice 10 minutes apart at rest. Blood sampling procedures were
strictly controlled and standardized to avoid sympathoadrenal
activation.16 17 18 In the subjects exposed to mental
stress, additional blood samples were obtained 2 minutes after the
start of the stress test.
Mental Stress
A subgroup of 27 subjects performed mental test (mental
arithmetic, MA) according to the highly standardized procedure we have
previously described in detail.22 23 The stress test has
been shown to induce a reproducible hemodynamic
activation.23
Blood Sampling
Arterial and venous blood samples were drawn
simultaneously by 2 persons from the catheters. The first 3
to 4 mL of blood were always discarded. Blood samples were collected in
tubes containing 1:10 0.45 mol/L sodium citrate buffer, pH 4.3
(Stabilyte, Biopool AB). Catheters were flushed with heparinized (5
IU/mL) saline after each blood sampling. The tubes were kept on ice,
and plasma was isolated within 60 minutes by
centrifugation at 4°C and 2000g for 20
minutes. Plasma was immediately frozen and stored in aliquots at
-70°C.
Determination of Plasma t-PA Concentration
An enzyme-linked immunosorbent assay (ELISA) was employed for
the quantitative determination of total t-PA antigen (TintElize® t-PA,
Biopool AB). The assays are based on the double-antibody principle.
Free t-PA and t-PA in complex with inhibitors are detected
with equal efficiency.24 The free, active fraction of t-PA
(t-PA activity) was determined by a bioimmunoassay (Chromolize(TM) t-PA,
Biopool AB). Active t-PA is expressed in µg/L using the specific
activity of 600 IU/µg.20 All samples from 1 individual
were assayed in duplicate on the same microtest plate. Intra-assay
coefficients of variation were 2.9% and 2.7% for total and active
t-PA, respectively.
Hematocrit
Hematocrit was determined in duplicate on arterial
blood using a microhematocrit centrifuge (Hettich Hematokrit,
Hettich Zentrifugen) with a coefficient of variation of <1%.
Net Release Rate of Total t-PA
Venoarterial concentration gradients (AV-gradients)
of each individual were computed by subtraction of the plasma level of
total t-PA measured in simultaneously collected venous and
arterial blood. A positive difference indicates a net
release and a negative net uptake. Individual forearm plasma flow was
calculated from FBF and arterial hematocrits corrected for
1% trapped plasma. Individual net release or uptake rates were
calculated from the AV-gradient times plasma flow per unit of time and
L forearm tissue.16 17 18 The following formulas were
used:
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Net Increment of Active t-PA
Forearm net increment of active t-PA was calculated with the
same formula as net release of total t-PA. However, as regards active
t-PA, local net flux may not only reflect tissue release/uptake but
also possible shifts between the complex-bound and free fractions of
t-PA on passage through the forearm vascular bed.19 25 To
signify this fact, the term net increment is used instead of net
release for active t-PA.19
Extraction and Amplification of Genomic DNA
Nucleated cells were prepared from frozen
EDTA-anticoagulated whole blood according to Sambrook et
al.26 Genomic DNA was extracted by a salting out
procedure27 and amplified by the polymerase chain reaction
(PCR) with oligonucleotide primers encompassing the
polymorphic region in intron 8 of the PA gene.5 The
forward and reverse primer used were 5'-TCCGTAACAGGACAGCTCA and
5'-ACCGTGGCTTCAGTCATGGA, respectively. The PCR was performed in a final
reaction volume of 50 µl containing 0.5 µg of genomic DNA; 20 pmol
of each oligonucleotide primer; 100 µM
deoxynucleotide triphosphates; 50 mmol/L potassium
chloride; 10 mmol/L TRIS-hydrochloric acid (pH 8.3 at 25°C);
1.5 mmol/L magnesium chloride; 0.001% (wt/vol) gelatin; and 1.25
U Taq DNA polymerase (AmpliTaq Gold, Perkin-Elmer/Cetus). The
thermocycling procedure (2400 Thermal cycler, Perkin-Elmer/Cetus)
consisted of 10 minutes of denaturation at 94°C, followed by 32
cycles of 1 minute at 93°C, 1 minute at 64°C, and 100 seconds at
72°C. The final extension step was prolonged to a total of 7 minutes.
A 15 µL sample was analyzed by agarose gel electrophoresis
and visualized with ethidium bromide and UV transillumination.
Amplification produces a 966-bp fragment from chromosomes with the
insertion (I allele) and a 655-bp fragment from those
without (D allele).
To avoid misclassification of ID genotypes as DD, an independent PCR with a forward primer that recognizes an insertion-specific sequence (5'-GATCACGAGGTCAGGAGAT) was performed on each sample classified as DD by the initial PCR. The reverse primer and PCR-conditions were identical as above except for a lower magnesium chloride concentration (1.0 mmol/L) and a slightly higher primer content (30 pmol) and annealing temperature (66°C). The reaction yields a 850 bp amplicon only in the presence of an I allele.
Southern Blot and Enhanced Chemiluminescence-Hybridization
To verify specificity of PCR-products, hybridization to a
probe directed to a sequence present in fragments from both
I and D alleles was performed. Amplified DNA
from several subjects of each genotype was electrophoresed on
an agarose gel and then transferred onto a nylon membrane (Hybond
N+, Amersham) using standard Southern blotting
methods.26 Hybridization was performed with 20 pmol
biotin-labeled probe (5'-GTCTGGTAGGCACACAGTCT, Scandinavian Gene
Synthesis) and 1 unit Streptavidin-POD conjugate (Boerhinger Mannheim).
After 3 stringency washes the membrane was transferred to a vessel with
detection reagents for enhanced chemiluminescence (RPN 2105,
Amersham).
Statistical Analysis
Mean values of the 2 baseline measurements are
presented. Standard statistical methods were used. Unless
otherwise stated, data are given as mean and SEM. Student's
t-test was used to test the probability that the
AV-gradients or the calculated net release/uptake indices were
different from zero. Responses to mental stress were evaluated by 1-way
analyses of variance (ANOVA) for repeated measures with subject
as random factor. One- and two-way ANOVA was also used to assess
whether baseline variables and responses to mental stress differed
by genotype. Post-hoc analysis was performed by
Fisher's protected least significant difference procedure. The
association between 2 variables was evaluated by the correlation
coefficient. The statistical analysis was performed after
logarithmic transformation of plasma levels of total and active t-PA.
Significance tests were considered significant at P<0.05
(2-tailed test).
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Results |
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Genotyping
Southern blot hybridization confirmed specificity of the PCR-products. When samples classified as DD by the initial PCR were subjected to a second amplification with an insertion-specific primer, 1 sample appeared that would have been mistyped if this additional PCR had not been applied. Frequency of the I allele was 0.63 (95% confidence intervals 0.53 to 0.72), which is in accordance with recently published data for other Scandinavian populations.6 The number of the II, ID, and DD genotypes were 19, 26, and 6, respectively, which was similar to the expected numbers as predicted by the Hardy-Weinberg equilibrium (II:ID:DD=20:24:7).
Association Between Genotype and Net Release Rate of
t-PA
The distribution of age, body mass index, or
hemodynamic variables did not differ between
genotypes (Table 1
). Net release
rate of total t-PA at rest showed a significant and graded relationship
to genotype (Figure 1). Net
release rate of t-PA was significantly higher in the
II-group as compared with both the ID- and
DD-group (P<0.05), whereas the difference in net
release rate between the DD- and ID-group did not
attain statistical significance. Forearm net increment of active t-PA
showed a similar graded relationship with genotype (Table 2). However, the step-up of net increment
of active t-PA across genotypes was less marked and fell short
of statistical significance. Arterial and venous plasma
concentrations as well as the AV-gradient of both total and active t-PA
were unrelated to genotype (Table 2).
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There was no significant correlation between the net release rate of total t-PA and the arterial plasma level of either total or active t-PA (r=0.10 and -0.16, respectively, NS for both). The arterial plasma concentration of total t-PA showed an inverse correlation both to the arterial plasma level and forearm net increment of active t-PA (r=-0.48 and -0.46, respectively, P<0.01 for both). In contrast, there was a direct correlation between the plasma concentration and net increment of active t-PA (r=0.45, P<0.01).
In the subgroup exposed to mental stress, a similar relation between
genotype and resting as well as stimulated net release rates of
total t-PA was observed (Figure 2). After
2 minutes of mental stress both the arterial plasma
concentration and forearm net release of total t-PA had increased as
compared with baseline (P<0.05 throughout). It is of note
that the relative increase in t-PA release was of the same magnitude
through genotypes. Induction of sympathoadrenal activation was
verified by increments in heart rate by on average 15 beats/min and
mean arterial blood pressure by 20 mm Hg
(P<0.001 throughout). Mental stress also induced a slightly
enhanced FBF in both arms (P<0.05) and an increase in
arterial and venous plasma concentrations of active t-PA
(P<0.01 throughout). The were no significant differences in
the response pattern to stress between the different genotypes
for either hemodynamic or t-PA variables.
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Discussion |
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This is the first study to show an association between a genetic variation at the t-PA locus and interindividual differences in the in vivo release rate of t-PA. The current results show that there is a graded increase in forearm t-PA release rate with the number of I alleles. This association is, however, not reflected by steady-state plasma concentrations of t-PA. This is likely to be due to the fact that the steady-state plasma concentration of t-PA not only is dependent on its rate of secretion in combination with the clearance rate but also on the degree of complex formation. That the systemic plasma level of t-PA is a poor marker of t-PA secretion is supported by the current findings of an absence of a correlation between net release rates of total t-PA and plasma levels of either total or active t-PA.
Interpretation of the current finding of a relation between local t-PA release rate and genotype is complicated by the fact that there are 2 pathways of t-PA secretion from endothelial cells: constitutive and regulated secretion.14 15 In constitutive secretion, the newly synthesized protein continuously leaves the Golgi compartment in transport vesicles to fuse with the cell membrane. By contrast, in regulated secretion, the protein is directed toward an intracellular storage compartment and is only secreted after the cell has been appropriately simulated. An endothelial granule for t-PA has recently been demonstrated,28 from which regulated secretion is mediated by activation of G-protein coupled cell surface receptors.14 While cultured unstimulated endothelial cells secrete t-PA by the constitutive pathway,29 it still remains to be determined whether basal t-PA secretion (and thus steady-state plasma levels) in humans is maintained by constitutive or regulated secretion.14 15
The assumption that regulated secretion is the main pathway of t-PA release in vivo, is supported by the finding that, in the intact rat, inhibition of protein synthesis does not result in a reduction of t-PA plasma levels until after several hours15.If this also holds true in humans, the current results may indicate that zygosity for the t-PA gene variation determines regulated rather than constitutive release. If so, however, it follows that DD-genotypes would completely lack stimulated t-PA release during baseline conditions. This could either indicate that endothelial cells of DD-genotypes are insensitive to the stimulatory mechanism that maintain basal regulated release in the other genotypes or have a diminished storage pool of t-PA. However, because DD subjects showed a t-PA response to sympathoadrenal stimulation of almost identical magnitude, as did individuals with ID or II genotypes, it is unlikely that unresponsiveness to stimulation explains the lack of basal release in the DD-group.
Instead, the similarity of the stimulated t-PA responses in combination with a marked gradient of basal release rates among the 3 genotypes seems to be more easily reconciled with the interpretation that zygosity is linked primarily to constitutive t-PA secretion. The possibility that constitutive secretion is the main pathway of basal t-PA release in vivo is also supported by the stability of steady-state plasma t-PA levels. Seemingly at variance with this interpretation of the current results, t-PA secretion from cultured umbilical vein endothelial cells has been reported to be unrelated to t-PA genotype.30 However, endothelial cells in vivo are constantly exposed to stimuli, such as fluid mechanical forces and humoral factors, known to enhance endothelial t-PA gene expression.31 32 If the observed relation between genotype and basal net release of t-PA is dependent on such a stimulation, it would not be expected to appear in endothelial cell culture systems.
Whereas the net release of total t-PA was significantly related to genotype, the net forearm increment of active t-PA showed a weaker association with genotype. This is not unexpected in view of our previous findings on local t-PA kinetics. In vitro studies show that t-PA is released in its free, active form.29 However, newly secreted t-PA will, when it enters the blood stream, very rapidly complex bind to PAI-1 (ie, second order rate constants above 107 mol · L-1 · s-1). It follows that the net flux of active t-PA across a vascular bed in vivo reflects not only its local release rate but also the kinetics of this local complex-binding to inhibitors.19 25 This phenomenon is illustrated by our recent observations that the arterial plasma level of PAI-1 is inversely correlated to forearm increment of free, active (but not total) t-PA.19 In fact, when the inflowing arterial blood contained sufficiently high levels of PAI-1, the abundant amounts of the inhibitor were shown to complex-bind almost all t-PA molecules that were released locally, and the output of free t-PA from the forearm approached zero.19 In the current study there was an inverse correlation between the plasma level of total t-PA and both the plasma level and net forearm increment of active t-PA. Again, this is most likely explained by local complex-formation, as it is well known that the plasma level of total t-PA show a direct correlation with plasma PAI-1.33 34 35 It follows that when assessing local t-PA secretion in vivo, measurements of total t-PA, that is not confounded by local complex-formation, is superior to that of active t-PA.
There are some earlier data to support the notion that the release rate of t-PA in vivo might be genetically determined. Several authors have reported familial clustering of low t-PA secretion as measured by venous occlusion (VO).36 37 38 In these studies, relatives to patients with both a history of venous thrombosis and a poor VO-response were investigated. A very modest increment of total t-PA in the brachial vein was observed in a high proportion of both symptomatic and asymptomatic relatives.38 As the increase in t-PA in response to VO mainly reflects the basal endothelial secretion in the occluded limb,39 these results are in line with the current findings of an association between genotype and forearm release rates of t-PA at rest.
The mechanism behind the observed association between t-PA genotype and net release rates of t-PA remains to be determined. It seems rather unlikely that an intronic Alu insertion polymorphism should have a direct effect on protein production. The most plausible explanation is that the insertion polymorphism is in linkage disequilibrium with functional variations elsewhere at the t-PA gene locus. Thus, the current findings prompt the search for other common polymorphisms at the t-PA locus and to relate such putative variations to expression and release rates of t-PA.
In conclusion, the current results provide the first evidence of an association between a common genetic variation in the t-PA gene and interindividual differences in net release rates of t-PA in vivo. A graded relationship between the number of the I alleles and forearm release rate of t-PA was observed. However, the genetic variation is not reflected in circulating plasma t-PA levels, and the altered local t-PA release rate cannot be disclosed by conventional venous plasma sampling. This is in line with the assumption that the level of circulating t-PA is a poor marker of t-PA secretion.
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Acknowledgments |
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We are grateful to Anne Ricksten, PhD, for valuable advice. The authors wish to thank Hannele Korhonen and Annika Johansson for excellent technical assistance throughout the study. The study was supported by grants from the Swedish Medical Research Council (09046) the Bank of Sweden Tercentenary Foundation, the Swedish Heart-Lung Foundation, the Magnus Bergvall Foundation, the 1987 Stroke Foundation, the Rune and Ulla Almlöv Foundation, the John and Brit Wennerström Foundation, and the Åke Wiberg Foundation. By the time of the study, C.J. was the recipient of a post-doc fellowship from the Berth von Kantzow Foundation.
Received January 19, 1998; accepted July 9, 1998.
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