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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2006;26:e34.)
© 2006 American Heart Association, Inc.

Thrombosis

Rapid Release of Active Tissue Factor From Human Arterial Smooth Muscle Cells Under Flow Conditions

Jan-Julius Stampfuss; Petra Censarek; Jens W. Fischer; Karsten Schrör; Artur-Aron Weber

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

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Circulating tissue factor (TF) is an important determinant of coronary thrombosis. Among other cell types, such as monocytes, vascular smooth muscle cells (SMCs) are capable of releasing TF. When studied under static conditions, SMCs do release TF, but this process is slow and, thus, cannot explain the elevated levels of circulating TF, as observed in patients with acute coronary syndromes. The present study demonstrates that cultured human mammary artery SMCs very rapidly (minutes) release active, microparticle-bound TF when exposed to flow conditions. There was a clear log-linear correlation between the shear rate (range 10 s^–1 to 1500 s^–1) and the procoagulant activity of SMC perfusates. Flow-dependent release of TF was transient (10 minutes) and did not measurably reduce cell surface TF content. Interestingly, a time-dependent (t_1/2 30 minutes) re-exposure of releasable TF was detected after a no-flow period. These data demonstrate that SMCs may become a pathophysiologically relevant source of TF that can be rapidly released into the circulation in situations in which endothelial damage occurs and SMCs come into a close contact with the flowing blood.

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

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Tissue factor (TF) is the principal initiator of coagulation.^1,2 Apart from its presence in the vessel wall, TF has also been detected in the circulation ("blood-borne TF").³ Microparticle-bound circulating TF has been demonstrated to contribute to fibrin formation during thrombus propagation.⁴

Elevated levels of circulating TF have been observed in the blood of patients with acute coronary syndromes.^5–7 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.^9–12 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

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Cell Culture
Human mammary artery SMCs were isolated by the explant technique and cultured as described previously.¹⁸ Before the experiments, SMCs were serum deprived for 24 hours.

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 s^–1 to 1500 s^–1).

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 (ttp^–1) 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 isothiocyanate–conjugated 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

Top Abstract Introduction Materials and Methods Results and Discussion References

 
Human mammary artery SMCs were washed and incubated with fresh serum-free medium for different time periods. Consistent with a previous report,¹⁵ a slow release of procoagulant microparticles containing active TF was observed (Figure 1A; data not shown). At shorter incubation periods (eg, 5 minutes), no procoagulant activity was detected in the supernatants (Figure 1B). In contrast, when SMCs were perfused for the same time at a shear rate of 500 s^–1, a marked stimulation of thrombin formation by the perfusate was observed (Figure 1B). In additional experiments, it was demonstrated that the procoagulant activity of SMC perfusates was abolished by ultracentrifugation and was recovered in the ultracentrifugation pellet, indicating that microparticle-bound TF was responsible for the thrombin generation activity (Figure 1C and 1D). Perfusion experiments have been performed at different shear rates ranging from 10 s^–1 to 1500 s^–1. A clear log-linear correlation between shear stress rate and procoagulant activity was established (Figure 1E).

View larger version (22K): [in this window] [in a new window]   Figure 1. Release of microparticle-associated procoagulant activity from SMCs under flow conditions. A, Time-dependent release of procoagulant activity under static conditions (means±SEM; n=6; *P<0.05). B, Original tracings demonstrating the effects of supernatants from nonperfused (stasis) compared with perfused (flow; 500 s–1; 0 to 5 minutes) SMCs. C, Original tracings demonstrating the effects of ultracentrifugation (100 000g; 1 hour) on thrombin formation induced by SMC perfusates (500 s–1; 0 to 5 minutes; con indicates perfusates; sn, ultracentrifugation supernatant; pellet, ultracentrifugation pellet). (Note that ultracentrifugation supernatants did not induce any visible thrombin formation). D, Quantitative effects of ultracentrifugation on thrombin formation (ttp; means±SEM; n=6; *P<0.05; abbreviations as in C). E, Correlation between shear rate and procoagulant activity of SMC perfusates (ttp; means±SEM; n=5).

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.

View larger version (28K): [in this window] [in a new window]   Figure 2. Release of microparticle-bound TF from SMCs under flow conditions. A, Western blot, demonstrating the presence of TF in microparticles (MP) derived from SMC perfusates (500 s–1; 0 to 5 minutes) compared with whole cell lysates (SMCs). Samples from 3 representative experiments are shown. B, Original tracings demonstrating the effects of neutralizing anti-TF antibodies (American Diagnostica; 4501; 10 µg/mL) compared with control IgG (10 µg/mL) on thrombin formation induced by SMC perfusates (500 s–1; 0 to 5 minutes). C, Quantitative effects of neutralizing anti-TF antibodies (American Diagnostica; 4501; 10 µg/mL) compared with control IgG (10 µg/mL) on thrombin formation induced by SMC perfusates (500 s–1; 0 to 5 minutes; ttp; means±SEM; n=6; *P<0.05). D, Fluorescence-activated cell sorter analysis demonstrating the presence of TF on microparticles in procoagulant active (inset) perfusate fractions (500 s–1; 0 to 5 minutes). E, Fluorescence-activated cell sorter analysis demonstrating the absence of TF on microparticles in nonprocoagulant active (inset) perfusate fractions (500 s–1; 10 to 15 minutes).

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 ^s–1 (data not shown).

View larger version (34K): [in this window] [in a new window]   Figure 3. Transient release and re-exposure of releasable TF on SMCs. A, Original tracings demonstrating the effects of SMC perfusate fractions (500 s–1) obtained at different times. B, Quantitative analysis of the effects of SMC perfusate fractions (500 s–1) obtained at different perfusion times (ttp; means±SEM; n=6). C, Effects of perfusion/stasis on the surface expression of TF on the surface of nonpermeabilized SMCs (Bar=10 µm). D, Effects of stasis (60 minutes) on the re-exposure of releasable (500 s–1) TF (ttp). E, Effects of different periods of stasis on the re-exposure of releasable (500 s–1) TF (means±SEM; n=6; *P<0.05).

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

 
This study was supported by the Forschungskommission of the Heinrich-Heine-Universität Düsseldorf.

	Footnotes

 
The first 2 authors contributed equally to this work.

Received December 8, 2005; accepted February 23, 2006.

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This Article Abstract Full Text (PDF) All Versions of this Article: 26/5/e34    most recent 01.ATV.0000216407.89528.b0v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Stampfuss, J.-J. Articles by Weber, A.-A. Search for Related Content PubMed PubMed Citation Articles by Stampfuss, J.-J. Articles by Weber, A.-A. Pubmed/NCBI databases Substance via MeSH Related Collections Acute coronary syndromes Arterial thrombosis Coagulation Thrombin