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Thrombosis |
From the Institut für Pharmakologie und Klinische Pharmakologie, Universitätsklinikum Düsseldorf, Germany.
Correspondence to Artur-Aron WeberInstitut für Pharmakologie und Klinische Pharmakologie, Universitätsklinikum Düsseldorf, Moorenstr. 5, D-40225 Düsseldorf, Germany. E-mail weberar{at}uni-duesseldorf.de
| Abstract |
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The present study demonstrates that cultured human mammary artery smooth muscle cells (SMCs) very rapidly, transiently, and repeatedly release active, microparticle-bound TF when exposed to flow conditions. Thus, SMCs may become a pathophysiologically relevant source of TF that can be released into the circulation when SMCs come to close contact with the flowing blood. The re-exposure of releasable TF may result in repeated bursts of TF, which is known to be involved in cyclic variations of coronary flow after angioplasty.
Key Words: tissue factor microparticles vascular smooth muscle cells flow
| Introduction |
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Elevated levels of circulating TF have been observed in the blood of patients with acute coronary syndromes.57 Importantly, circulating TF is a major determinant of thrombosis in dissected coronary plaques.8
TF-containing microparticles in the circulation may derive from different cell types such as monocytes or platelets.912 Very recently, it has been shown that a soluble, alternatively spliced TF variant is released from cytokine-stimulated endothelial cells.13 In addition, TF has also been detected in vascular smooth muscle cells (SMCs).14 However, the release of TF from SMCs is a slow procedure that requires hours and, thus, cannot confer the rapid occurrence of TF in the blood of patients with acute coronary syndromes.15,16 Interestingly, studies on isolated rat arteries are suggestive for an involvement of flow in the rapid release of TF.17 Because SMCs are subjected to blood flow in situations in which the endothelium is damaged (such as after coronary angioplasty), these cells may represent a potentially significant source of circulating TF. The present study was designed to systematically investigate the effect of flow on the release of TF from human arterial SMCs.
| Materials and Methods |
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Flow Experiments
Cells were seeded on glass coverslips (24x60 mm), grown to confluence, and mounted into a parallel plate flow chamber. Cells were perfused with HEPES-buffered Tyrode at a constant flow rate (2 mL/min). The gap widths of the chamber were adjusted to expose the cells to different levels of shear rate (range 10 s1 to 1500 s1).
Endogenous Thrombin Potential
Perfusate fractions were centrifuged for 5 minutes at 3000g to remove cell debris. Procoagulant activity was measured using a modified thrombinoscope method.19,20 In brief, 60 µL of perfusate was incubated with 10-µL platelet membranes and 20 µL recalcification buffer (2 mmol/L Ca2+, 2 mmol/L Mg2+; final concentrations), containing the fluorogenic substrate (Z-Gly-Gly-Arg-7-amino-4-methylcoumarin; Bachem). The reaction was started by addition of 20 µL citrated human plasma, and thrombin generation was monitored for 60 minutes. In samples with low thrombin generation, complete endogenous thrombin potential (ETP) curves could not be obtained. Thus, time to peak (ttp1) was used as a quantitative parameter of thrombin generation. Original ETP curves are shown for all essential experiments to illustrate that all ETP parameters were influenced in a similar way.
Flow Cytometry
Microparticle-containing perfusates were stained with fluorescein isothiocyanateconjugated monoclonal anti-TF antibodies (Acris; BM740F) for 30 minutes. Then samples were diluted with Isotone and analyzed on an Epics-XL cytometer (Beckman Coulter). Microparticles were defined according to the scatter properties compared with detached SMCs. Detectors were set to logarithmic amplification, and 10 000 events were analyzed using the System II software.
Western Blot
SMCs and microparticle ultracentrifugation pellets were lysed in Laemmli buffer, and Western blotting was performed as described using polyclonal anti-TF antibodies (American Diagnostica; 4501; 1:1000).21
Immunofluorescence Microscopy
Cells were fixed for 20 minutes with freshly made 3.7% paraformaldehyde. These nonpermeabilized SMCs were blocked for 1 hour with 3% goat serum for 1 hour and stained with monoclonal anti-TF antibodies (Enzyme Research Laboratories; MAB TFE280; 1:100 in PBS/1% goat serum) for 1 hour and Cy3-conjugated secondary antibodies (1:200 in PBS) for 1 hour. Cells were washed with PBS between incubations, and nuclei were counterstained with Hoechst 33324.
Statistics
Data are mean±SEM of n independent experiments. Statistical analysis was performed by 1-way ANOVA followed by Bonferroni multiple comparisons test. P levels of <0.05 were considered significant.
| Results and Discussion |
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TF (full-length form only) was detected in the microparticle fraction of SMC perfusates by Western blotting (Figure 2A). The involvement of TF in the procoagulant activity of perfusates was studied using neutralizing antibodies (Figure 2B and 2C). A marked (
70%) reduction of thrombin generation was seen in the presence of the neutralizing antibodies (10 µg/mL), whereas control IgG antibodies were without any effect. At higher antibody concentrations, a complete inhibition of thrombin generation was observed, but at these concentrations, nonspecific effects of control IgG antibodies were measured (data not shown). However, flow cytometry studies detected microparticle-bound TF in perfusate fractions that were highly procoagulant (Figure 2D), whereas no TF was seen in fractions without procoagulant properties (Figure 2E). Together, these experiments demonstrate that TF is involved in the procoagulant activity of SMC perfusates.
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When perfusate fractions were sampled at different perfusion times, the procoagulant activity decreased with time and reached control (=buffer) levels after 10 minutes of perfusion (Figure 3A and 3B). Because no measurable reduction in total TF content in the SMCs was detected after perfusion (data not shown), the expression of cell surface TF was also studied by means of immunofluorescence microscopy using nonpermeabilized cells. TF staining on SMC surface did not measurably decrease after perfusion (Figure 3C). Thus, a total depletion of cell surface TF does not explain the transient release of procoagulant microparticles on perfusion. Therefore, we have hypothesized that only a small fraction of TF can be released on shear stress, possibly from membrane regions that are particularly susceptible to mechanical forces. This hypothesis implies that pure mechanical stress rather than "classical pathways" such as Ca2+-dependent lipid scrambling and calpain activation22 might be involved in flow-dependent release of TF-containing microparticles. Consistent with this hypothesis, neither the intracellular Ca2+ chelator (1,2-bis(2-aminophenoxy)ethane-N,N,N`,N`-tetraacetate-AM; 30 µmol/L) nor the calpain inhibitor (Calpain Inhibitor I; Calbiochem; 100 µmol/L) inhibited flow-dependent TF release (data not shown). It is known that oscillatory shear stress applied for 24 hours induces TF mRNA and protein expression in endothelial cells.23 In contrast, unidirectional shear resulted in a decrease of TF expression in these cells. However, in our experiments with SMCs, TF release occurred within a few minutes, thus excluding a transcriptional mechanism. In addition, no attenuation of TF mRNA expression in SMCs was observed after 30 minutes of flow at 500 s1 (data not shown).
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Because surface TF expression did not measurably decrease after exposure to flow, we have further hypothesized that the releasable fraction of TF may be re-exposed (eg, by redistribution) after a no-flow period. Interestingly, a time-dependent re-exposure of releasable TF was observed (Figure 3D and 3E). Similar to the initial perfusion, the re-exposed TF was rapidly but transiently released into the perfusate.
These data demonstrate that SMCs may become a pathophysiologically relevant source of TF that can be rapidly released into the circulation when SMCs come to a close contact with the flowing blood. One might speculate that the re-exposure of releasable TF may result in repeated bursts of TF, which is known to be involved in cyclic variations of coronary flow after angioplasty.24,25
In consequence, therapeutic strategies aiming to inhibit TF (such as TF pathology inhibitor nematode anticoagulant protein c2, factor VIIai) during acute coronary events warrant further investigation.26
| Acknowledgments |
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| Footnotes |
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Received December 8, 2005; accepted February 23, 2006.
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