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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Division of Clinical Biochemistry and Division
of Angiology and Haemostasis, Department of Medicine, and the
Department of Morphology, Centre Médical Universitaire, Geneva,
Switzerland, and from the Department of Haematology, U474
INSERM, Hôpital Cochin and Faculté Paris-Ouest, Paris,
France.
Vascular endothelial cells are thought to be the main source of
plasma tissue-type plasminogen activator (t-PA) and von Willebrand factor (VWF). Previous studies have suggested that both t-PA and VWF
are acutely released in response to the same stimuli, both in cultured
endothelial cells and in vivo. However, the subcellular storage
compartment in endothelial cells has not been definitively established.
We tested the hypothesis that t-PA is localized in Weibel-Palade (WP)
bodies, the specialized endothelial storage granules for VWF. In
cultured human umbilical vein endothelial cells (HUVECs), t-PA was
expressed in a minority of cells and found in WP bodies by
immunofluorescence. After up-regulation of t-PA synthesis either by
vascular endothelial growth factor (VEGF) and retinoic acid or by
sodium butyrate, there was a large increase in t-PA-positive cells.
t-PA was exclusively located to WP bodies, an observation confirmed by
immunoelectron microscopy. Incubation with histamine, forskolin, and
epinephrine induced the rapid, coordinate release of both t-PA and VWF,
consistent with a single storage compartment. In native human
skeletal muscle, t-PA was expressed in endothelial cells from
arterioles and venules, along with VWF. The 2 proteins were found to be
colocalized in WP bodies by immunoelectron microscopy. These data
indicate that t-PA and VWF are colocalized in WP bodies, both in HUVECs
and in vivo. Release of both t-PA and VWF from the same storage pool likely accounts for the coordinate increase in the plasma level of the
2 proteins in response to numerous stimuli, such as physical activity,
Tissue-type plasminogen activator (t-PA) is a
key enzyme in fibrinolysis. When bound to fibrin, it catalyses the
cleavage of plasminogen to plasmin and thus initiates fibrin
degradation. Recombinant t-PA is widely used for the treatment of acute
myocardial infarction and other arterial thrombotic
disorders.1 Endothelial cells are thought to be the main
source of plasma t-PA. In cultured endothelial cells, t-PA synthesis is
up-regulated, usually at the transcriptional level, in response to
fluid shear stress, thrombin, histamine, retinoic acid, vascular
endothelial growth factor (VEGF), and sodium butyrate.2-6
In addition, there is both in vivo and in vitro evidence that t-PA is
acutely released from preformed stores in a regulated manner. A rapid
increase in plasma t-PA levels is observed in response to
The identification of the endothelial storage pool and the mechanism
controlling the regulated secretion of t-PA have been the focus of
several previous in vitro studies. t-PA expressed by transfection in
neuroendocrine cells is targeted to storage granules along with
catecholamines, suggesting that it is a bona fide regulated secretory
protein.14 In cultured human umbilical vein endothelial
cells (HUVECs), acute t-PA release has been shown in response to
calcium-raising agents such as thrombin, and to cAMP-raising agents
such as epinephrine and Iloprost.15 However, the
subcellular storage compartment of t-PA in the endothelial cells has
not yet been definitively identifed, and remains a matter of debate. We
and others have reported that t-PA is expressed in HUVECs and localized
in Weibel-Palade (WP) bodies, where it colocalizes with von Willebrand
factor (VWF) by immunofluorescence.16,17 However, in our
experiments the reliability of this finding was doubtful in view of the
very low level of t-PA expression in HUVECs, precluding measurements of
t-PA secretion. After transduction with a t-PA-expressing recombinant
adenovirus, t-PA was more convincingly localized to WP bodies, but in
these experiments heterologous t-PA was expressed at very high levels.
In contradiction to these findings, Emeis et al have provided evidence
for an alternative storage granule for t-PA, distinct from WP bodies,
characterized by smaller size, round shape, and more rapid release
kinetics.18 In their experiments, t-PA expression was
up-regulated by cell pretreatment with sodium butyrate. In vivo, t-PA
has also been mainly located to endothelial cells,19 but
again the subcellular t-PA storage compartment has not clearly been
identified. To clarify these issues, we have reinvestigated the
localization of t-PA both in HUVECs after up-regulation of endogenous
t-PA expression, and in endothelial cells in vivo.
RPMI 1640 and M199 media were from Gibco BRL (Gaithesburg, MD),
fetal calf serum (FCS) and collagenase were from Seromed (Berlin, Germany). Endothelial cell growth supplement (ECGS) was from Upstate Biotechnology (Lake Placid, NY). Anti-VWF antibodies were from Dako
(Glostrup, Denmark). Anti-t-PA antibodies (monoclonals ESP1, ESP4,
ESP5, PAM3, and goat polyclonal) were from American Diagnostica (Greenwich, CT). Fluorescein isothiocyanate (FITC)-conjugated goat
antimouse, Texas Red-conjugated goat antirabbit and Texas Red-conjugated donkey antigoat antibodies were from Jackson
laboratories (West Grove, PA). FITC-conjugated sheep antirabbit
antibodies were from Roche (Rotkreuz, Switzerland). Recombinant t-PA
(Actilyse) was provided by Dr J. Krause (Dr K. Thomae GmbH,
Biberach an der Riss, Germany); dilutions of t-PA were made in 0.2 M
L-arginine, 0.11 M phosphate, 0.01% Tween 80, pH 7.2. Histamine, human
thrombin, IBMX (3-isobutyl-1-methyl-xanthine), epinephrine, and
forskolin were from Sigma (St Louis, MO). VEGF, all-trans retinoic acid (ATRA), sodium butyrate, and cycloheximide were also from Sigma.
Cell culture
Secretion studies
t-PA and VWF measurements t-PA was measured using a novel ultrasensitive enzyme-linked immunosorbent assay (ELISA) procedure using commercially available antibodies. The 96-well plates were coated with monoclonal anti-t-PA antibodies ESP1 and ESP5, 1 µg/mL each in carbonate buffer (NaCO3 50 mM, pH 9.6), 100 µL/well. After 4 washes with TBS-T (Tris 20 mM, NaCl 140 mM, Tween-20 0.1%, pH 7.4), the remaining protein binding sites were blocked with 100 µL/well blocking solution (TBS-T plus 3% BSA) added for 2 hours at 37°C. Samples and standards diluted in blocking solution (0.1 mL/well) were then added and incubated overnight at 4°C. After 4 washes with TBS-T, a goat anti-human t-PA antibody (American Diagnostica, no. 381) diluted 1:2000, was added for 2 hours at 37°C. A third incubation with a rabbit anti-goat antibody conjugated to horseradish peroxidase (HRP) (Dako P 0449, diluted 1:10 000) was performed for 2 hours at 37°C. Finally, bound HRP activity was detected using o-phenylene diamine as a substrate and measuring optical density at 490 nm. A standard curve was constructed using purified recombinant t-PA. t-PA was reliably detected at concentrations of 0.025 ng/mL to 3.2 ng/mL. The assay recognizes t-PA both in free form and bound to PAI-1. Indeed, the optical signal obtained with recombinant t-PA was not modified by adding a 50-fold excess of purified PAI-1. The excess PAI-1 caused a quantitative shift of t-PA to a PAI-1-bound, high-molecular-weight complex as determined by Western blot (not shown).VWF was measured by ELISA as described previously.20 A standard curve was constructed from serial dilutions of normal pooled plasma, assuming a plasma concentration of 10 µg/mL. Results are expressed in ng/well per time unit. Unless indicated otherwise, results are shown as the mean ± SEM. Statistical analysis was done using the 2-tailed, paired Student t test. Cell immunofluorescence HUVECs grown on glass coverslips were fixed for 30 minutes in 3.7% formaldehyde and permeabilized for 15 minutes with 0.5% Triton X-100 in phosphate buffered saline (PBS). The coverslips were then incubated with the anti-t-PA monoclonal antibody ESP-4 diluted at 1:100 and a rabbit anti-human VWF antibody diluted at 1:30 000, and subsequently with FITC-conjugated goat antimouse and Texas Red-conjugated goat antirabbit antibodies, both diluted at 1:200. The slides were mounted with ProLong Antifade (Molecular Probes, Eugene, OR) and examined using a confocal microscope (Zeiss LSM 410, Germany).Cell transduction with recombinant adenovirus t-PA recombinant adenovirus (AdCMVt-PA) was propagated on a monolayer of 293 cells and titrated by plaque assay, as previously described.16 HUVECs grown to 50% confluency were transduced for 1 hour at 37°C in RPMI 1640/10% FBS with AdCMVt-PA at a titer of 108 pfu/mL, resulting in t-PA-positive staining in 30% to 50% of the cells. After transduction, the cells were washed and incubated for 48 hours at 37°C before fixation.Tissue immunofluorescence Samples of abdominal skin, gastrocnemius, and serratus anterior muscles were obtained at plastic surgery from unaffected patients who had given informed consent, in accordance with the guidelines of our institutional committee for clinical investigation. All samples were rapidly frozen in liquid nitrogen and cryosectioned. Sections were fixed in 4% paraformaldehyde containing 0.1% Triton X-100 and processed as previously described.21 Briefly, sections were rinsed, incubated for 2 hours at room temperature with either the rabbit polyclonal serum against von Willebrand factor (diluted 1:20 000) or the goat polyclonal serum against tPA (diluted 1:50), rinsed again and incubated for one hour at room temperature with either FITC-conjugated sheep anti-rabbit antibodies (diluted 1:500) to detect VWF or Texas Red-conjugated donkey anti-goat antibodies (diluted 1:200) to detect t-PA. After rinsing, sections were viewed with an Axiophot microscope (Zeiss, Oberkochen, West Germany). In all experiments, negative controls included exposure of sections during the first incubation to either normal rabbit or goat serum, fluorescein-conjugated anti-rabbit antibodies or Texas Red-conjugated anti-goat antibodies. None of these incubations resulted in a specific staining of the tisses examined (not shown).Immunoelectron microscopy HUVECs or tissue fragments were fixed in 1.5% glutaraldeyde in 0.1 M phosphate buffer, pH 7.4. Cryosections were made using an ultracryomicrotome (Reichert Ultracut S) and ultrathin sections mounted on Formvar-coated gold grids were prepared. During incubations at room temperature the grids were floated on the surface of droplets as previously described.22 Briefly, the sections were incubated for 15 minutes with PBS 15% glycine; for 5 minutes with PBS 15% glycine, 0.1% BSA; and for 20 minutes with PBS 15% glycine, 0.1% BSA, 10% normal goat serum followed by 1 hour incubation with a mixture of the mouse monoclonal anti-t-PA antibodies ESP-4 and PAM 3 and/or with a rabbit polyclonal antibody to VWF. The primary antibodies were diluted 1:50 in PBS 15% glycine, 0.1% BSA, 4% normal goat serum.23 After extensive rinsing in PBS 15% glycine, 0.1% BSA, sections were incubated for 30 minutes with gold-labeled secondary goat antimouse or goat antirabbit antibody or both in case of double-labeling, with a gold particle size of 10 nm (GAM 10) and/or 5 nm (GAR5), respectively (British Biocell, Cardiff, Wales). Sections were then washed for 30 minutes with PBS 15% glycine, stained with 2% uranyl acetate for 10 minutes, and air dried. Examination was performed in a Philips CM 10 electron microscope.
Up-regulation of t-PA synthesis from HUVECs The localization of t-PA in HUVECs, as well as the regulation of t-PA secretion are difficult to study given the low levels of t-PA expression in these cells. To circumvent this problem, we set up an ultrasensitive ELISA for t-PA. This ELISA allowed the detection of free or complexed t-PA at a concentration of 0.025 ng/mL (see "Materials and methods"). This high sensitivity permits studies on the regulation of t-PA synthesis and release. Basal t-PA release from confluent HUVECs was 0.71 ± 0.04 ng/well over 20 hours (mean ± SD). Incubation for 20 hours with VEGF (40 ng/mL), ATRA (10 6 M), or sodium butyrate (3 mM), caused a 3.3-, 1.9-, and 13-fold increase in basal t-PA release, respectively. VEGF and ATRA
added together had an additive effect, with a 5.1-fold increase in t-PA release. Estradiol (100 nM) had no effect. Thus, ATRA, VEGF, and sodium
butyrate all cause an increase in synthesis and constitutive release of
t-PA, in agreement with previous reports.3-5
Acute agonist-induced t-PA release from HUVECs Acute VWF release from WP bodies occurs in response to a variety of agonists, that act either via an increase in intracellular free calcium ([Ca++]i), or via activation of adenylate cyclase and an increase in cellular cAMP. Histamine is a potent activator of the [Ca++]i-dependent pathway.24 Forskolin, a direct activator of adenylate cyclase, induces VWF secretion when added together with IBMX (an inhibitor of the phosphodiesterases that degrade cAMP). Epinephrine, which acts via a receptor-mediated, G-protein-coupled pathway, also raises cAMP and induces VWF secretion, although its effect is weaker than that of forskolin (Figure 1B, and Vischer and Wollheim25). We tested whether these agonists also induce acute t-PA release.
Confluent HUVEC monolayers were incubated with histamine
(10 The acute release experiments were also performed after up-regulation of t-PA synthesis. After a combined VEGF and ATRA pretreatment, acute t-PA release increased 3.1-, 3.2-, and 1.5-fold in response to histamine, forskolin, and epinephrine, respectively (from 0.05 ± 0.002 to 0.17 ± 0.03, 0.17 ± 0.03, and 0.08 ± 0.01 ng/well per 30 minutes; n = 4; P < .003 for all 3 comparisons). After sodium butyrate pretreatment, the corresponding increases were 2.6-, 1.6-, and 1.3-fold, respectively (from 0.44 ± 0.1 to 1.14 ± 0.22, 0.74 ± 0.1, and 0.60 ± 0.13 ng/well per 30 minutes; n = 4; P < .004 for all 3 comparisons). IBMX added alone had no effect on t-PA release (data not shown). Thus, the up-regulation of t-PA synthesis by pretreatment with either VEGF and ATRA or sodium butyrate is associated with the induction of a rapidly releasable t-PA pool. In these experiments we verified that histamine, forskolin, and epinephrine induce acute VWF secretion as previously reported (Figure 1B). The rapidly releasable t-PA pool is thus responsive to the agonists that induce acute VWF release. To confirm that t-PA is released from a preformed store, the secretion
experiments were repeated in the presence of cycloheximide (CHX), an
inhibitor of protein synthesis. HUVECs were incubated with CHX (5 µg/mL) or medium alone for 4 hours and then subjected to acute
secretion experiments (Figure 2A). Basal
t-PA release was strongly decreased by CHX pretreatment, indicating an
inhibition of t-PA synthesis and constitutive release. Increases in
t-PA release were still observed in response to histamine, forskolin, and epinephrine. After substraction of the corresponding control value,
there was no decrease in t-PA release in response to these 3 agonists.
Similar results were obtained when the cells were pretreated with
sodium butyrate, added to increase t-PA synthesis (Figure 2B). CHX did
not affect basal or agonist-induced VWF release (not shown). These data
confirm the presence of an agonist-sensitive, preformed t-PA storage
compartment in HUVECs.
t-PA localization in HUVECs We studied the subcellular localization of t-PA in HUVECs by immunofluorescence. HUVECs grown on glass coverslips were preincubated with either VEGF and ATRA or sodium butyrate, to increase t-PA synthesis and storage. The cells were then fixed, permeabilized, and stained by double-labeling immunofluorescence for t-PA and VWF (Figure 3). In untreated HUVECs, only occasional cells were positive for t-PA. In these cells, t-PA was localized to WP bodies, identified as rod-shaped granules that were stained with anti-VWF antibodies (Figure 3A,B). No other structures were stained by anti-t-PA antibodies. After pretreatment with VEGF and ATRA, the number of cells positive for t-PA was markedly increased, and t-PA was again colocalized with VWF in WP bodies (Figure 3C,D). After pretreatment with sodium butyrate, the majority of cells were positive for t-PA, and the t-PA stain was again entirely localized to WP bodies (Figure 3E,F). t-PA was occasionally seen in the perinuclear area, but no particulate structure other than WP bodies was revealed by anti-t-PA antibodies. Thus, increased t-PA synthesis is associated with increased t-PA storage in WP bodies.
t-PA localization to WP bodies in HUVECs was further documented by
immunoelectron microscopy (Figure 4). To
increase t-PA expression, HUVECs were infected with t-PA-expressing
adenovirus, as previously described.16 After 48 hours, the
cells were trypsinized, pelleted, and fixed in 1.5% glutaraldehyde
(Figure 4). After immunostaining, t-PA was seen as 10-nm gold particles
over WP bodies, identified as elongated electron-dense organelles with
parallel tubular structures. Specific labeling was observed in no other
intracellular structures (Figure 4A). t-PA was not found in untreated
HUVECs, in accordance with the low levels of t-PA expression observed
by ELISA and by immunofluorescence (Figure 4B). In double-label
experiments, the elongated electron-dense structures that bound t-PA
antibodies (seen as 10-nm beads) also bound anti-VWF antibodies (seen
as 5-nm beads), confirming their identity as WP bodies (Figure 4C). We
also studied cells pretreated with sodium butyrate (Figure 5). WP bodies were again identified as
electron-dense elongated structures stained by anti-VWF antibodies
(Figure 5A). Similar structures were also labeled by anti-t-PA
antibodies (Figure 5B,C). These results confirm that both heterologous
and endogenous t-PA are localized to WP bodies in HUVECs.
The subcellular localization of t-PA in human tissues To evaluate whether t-PA is localized to WP bodies also in native tissues, we studied human skeletal muscle, since acute t-PA release has been demonstrated in human forearm perfusion studies. Muscle specimens obtained at surgery were quickly frozen, sectioned, and fixed with paraformaldehyde. t-PA and VWF were then visualized by double-label immunofluorescence (Figure 6). VWF antibodies stained the intimal layer of endothelial cells of arterioles and venules, whereas capillaries were usually not stained. t-PA antibodies identified only some of the VWF-positive arterioles and venules. In these cases, t-PA was localized in the intimal layer of these vessels, but again not in capillaries (Figure 6). Double labeling of the same sections with different fluorochromes showed that t-PA and VWF were expressed by the same set of cells (Figure 6). A similar distribution of both t-PA and VWF was observed in human skin specimens (not shown).
The subcellular localization of t-PA in these vessel segments was
studied by immunoelectron microscopy (Figure
7). Tissue fragments of skeletal muscle
were fixed in 1.5% glutaraldehyde, and thin sections were sequentially
incubated with anti-t-PA antibodies and anti-mouse Ig antibodies
coupled to gold particles. In arterioles, the gold particles indicating
the presence of t-PA were found concentrated over WP bodies, identified
as elongated, striated, electron-dense structures. WP body labeling was
highly specific; in particular, no gold particle concentration was seen
over other dense structures such as mitochondria. These results
indicate the presence of t-PA in WP bodies in vascular endothelial
cells in vivo.
Our results provide direct evidence that t-PA is localized to WP bodies, both in cultured HUVECs and in vivo. In previous work, we have found that t-PA is expressed at low levels in HUVECs. However, after heterologous expression via an adenoviral system, higher amounts of t-PA were also found in WP bodies.16 The present study extends these results and shows that endogenous t-PA is localized to WP bodies. In untreated HUVECs or after up-regulation of t-PA synthesis with VEGF, ATRA, or sodium butyrate, t-PA was found in WP bodies by immunofluorescence. By immunoelectron microscopy, t-PA was found in WP bodies after heterologous t-PA overexpression and after up-regulation of endogenous t-PA by sodium butyrate. We found no evidence for any other storage compartment. We also demonstrate agonist-induced t-PA release from HUVECs in less than 30 minutes, including after pretreatment with CHX, an inhibitor of protein synthesis. The rapid time course and the lack of inhibition by CHX indicate that agonist-induced t-PA release is due to release from a preformed store rather than to increased synthesis. Acute t-PA release occurred in response to histamine, forskolin, and epinephrine, which are all well-characterized agonists for VWF release from WP bodies. These morphologic and functional data indicate that both t-PA and VWF are stored in and released from WP bodies. Importantly, we demonstrate by immunoelectron microscopy that t-PA is localized to WP bodies of endothelial cells of skeletal muscle vessels, also in vivo. The existence of a t-PA storage compartment other than that represented by WP bodies has been proposed by Emeis et al.18 In cell fractionation experiments of rat lung, these authors found t-PA at a different density than VWF on sucrose gradients, although t-PA and VWF migrated at the same density on Nycodenz gradients. In HUVECs, they identified t-PA in small, round vesicles by immunofluorescence and immunoelectron microscopy. Although our approach was quite similar, we found no evidence for such a distinct vesicle type. We have no obvious explanation for these discrepancies. Like these authors, we performed t-PA immunolocalization after pretreating HUVECs with sodium butyrate, and we used culture conditions that are nominally quite similar. Datta et al reported t-PA localization to WP bodies in most HUVECs, even without pretreatment, again without obvious differences in cells or culture conditions used.17 These divergent results are likely due to subtle, unaccounted differences in culture conditions. The relevance of our results is strongly supported by our finding that t-PA is localized to WP bodies also in vivo, at least in the arterioles of native skeletal muscle. The conclusion that t-PA and VWF colocalize in WP bodies implies that
the 2 proteins are coordinately released in response to secretion
agonists. Indeed, our secretion studies in HUVECs indicate a coordinate
t-PA and VWF release in response to calcium-mobilizing agents such as
histamine, and to cAMP-raising agents such as forskolin and
epinephrine. In vivo, physical activity, In conclusion, we have shown that t-PA is colocalized in Weibel-Palade
bodies, along with VWF, both in HUVECs and in endothelial cells in
native skeletal muscle vessels. Release of both t-PA and VWF from the
same storage pool likely accounts for the coordinate increase in the
plasma level of the 2 proteins in response to numerous stimuli such as
physical activity,
The skilled technical assistance of Dominique Duhamel, Nicole Aebischer, and Dorothée Caille is gratefully acknowledged. We are grateful to Corinne Rosnoblet for her help with the adenoviral transduction experiments.
Submitted November 7, 2001; accepted January 11, 2002.
Supported by grants 3100-063754.00/1 (U.M.V.), 3200-061510.00 (E.K.O.K.) and 31-53720.98 (P.M.) from the Swiss National Science Foundation; QLG1-CT-1999-00516 from the European Union (P.M.); and by a research grant from the Department of Medicine, University Hospital, Geneva, Switzerland.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Ulrich M. Vischer, Division de Biochimie Clinique, CMU, 1 rue Michel Servet, 1211 Geneva 4, Switzerland; e-mail: ulrich.vischer{at}medecine.unige.ch.
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© 2002 by The American Society of Hematology.
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  J. H. Cleator and D. E. Vaughan Clinical Implications of the Contrasting Effects of In Vivo Thrombin Receptor Activation (Protease-Activated Receptor Type 1) on the Human Vasculature J. Am. Coll. Cardiol., May 6, 2008; 51(18): 1757 - 1759. [Full Text] [PDF]
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S. F. De Meyer, K. Vanhoorelbeke, M. K. Chuah, I. Pareyn, V. Gillijns, R. P. Hebbel, D. Collen, H. Deckmyn, and T. VandenDriessche Phenotypic correction of von Willebrand disease type 3 blood-derived endothelial cells with lentiviral vectors expressing von Willebrand factor Blood, June 15, 2006; 107(12): 4728 - 4736. [Abstract] [Full Text] [PDF]
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J. H. Cleator, W. Q. Zhu, D. E. Vaughan, and H. E. Hamm Differential regulation of endothelial exocytosis of P-selectin and von Willebrand factor by protease-activated receptors and cAMP Blood, April 1, 2006; 107(7): 2736 - 2744. [Abstract] [Full Text] [PDF]
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A. T. Wolf, R. L. Medcalf, and C. Jern The t-PA -7351C>T enhancer polymorphism decreases Sp1 and Sp3 protein binding affinity and transcriptional responsiveness to retinoic acid Blood, February 1, 2005; 105(3): 1060 - 1067. [Abstract] [Full Text] [PDF]
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K. Matsushita, M. Yamakuchi, C. N. Morrell, M. Ozaki, B. O'Rourke, K. Irani, and C. J. Lowenstein Vascular endothelial growth factor regulation of Weibel-Palade-body exocytosis Blood, January 1, 2005; 105(1): 207 - 214. [Abstract] [Full Text] [PDF]
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I. Oynebraten, O. Bakke, P. Brandtzaeg, F.-E. Johansen, and G. Haraldsen Rapid chemokine secretion from endothelial cells originates from 2 distinct compartments Blood, July 15, 2004; 104(2): 314 - 320. [Abstract] [Full Text] [PDF]
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U. Fiedler, M. Scharpfenecker, S. Koidl, A. Hegen, V. Grunow, J. M. Schmidt, W. Kriz, G. Thurston, and H. G. Augustin The Tie-2 ligand Angiopoietin-2 is stored in and rapidly released upon stimulation from endothelial cell Weibel-Palade bodies Blood, June 1, 2004; 103(11): 4150 - 4156. [Abstract] [Full Text] [PDF]
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S. M. Bates and J. I. Weitz Emerging Anticoagulant Drugs Arterioscler Thromb Vasc Biol, September 1, 2003; 23(9): 1491 - 1500. [Abstract] [Full Text] [PDF]
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S. L. Haberichter, P. Jacobi, and R. R. Montgomery Critical independent regions in the VWF propeptide and mature VWF that enable normal VWF storage Blood, February 15, 2003; 101(4): 1384 - 1391. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
-adrenergic agents, and 1-deamino-8d-arginine vasopressin (DDAVP) among others.
(Blood. 2002;99:3637-3645)
20°C until the assays.
All pharmacologic agents were either directly dissolved in incubation
medium or dissolved in dimethyl sulfoxide (DMSO). The final
concentration of DMSO in the incubation medium did not exceed 0.2%, a
concentration that has no effect on VWF release.
effect of interleukin 6.
Thromb Haemost.
1997;77:905-910