HEMOSTASIS, THROMBOSIS,AND VASCULAR BIOLOGY
Hepatic veno-occlusive disease (VOD) is
motherapy associated with bone marrow
transplantation. While the pathogenesis
of VOD is uncertain, plasminogen activa-
tor inhibitor-1 (PAI-1) has emerged as a
diagnostic marker and predictor of VOD
in humans. In this study, we investigated
the role of PAI-1 in a murine model of VOD
produced by long-term nitric oxide syn-
thase inhibition using L-NAME. After 6
weeks, wild-type (WT) mice developed
thrombi and biochemical evidence of he-
patic injury and dysfunction. In contrast,
PAI-1–deficient mice were largely pro-
tected from the development of hepatic
vein thrombosis. Furthermore, WT mice
that received tiplaxtinin, an antagonist of
PAI-1, were effectively protected from
L-NAME–induced thrombosis. Taken to-
gether, these data indicate that NO and
PAI-1 play pivotal and antagonistic roles
in hepatic vein thrombosis and that PAI-1
is a potential target in the prevention and
© 2006 by TheAmerican Society of Hematology
Hepatic veno-occlusive disease (VOD) is a common complication
tation.1It is characterized clinically by hyperbilirubinemia, hepato-
megaly, and fluid retention.2Histologic features of VOD include
fibrous occlusion of terminal hepatic venous lumen, dilatation, and
ultimately fibrosis of hepatic sinusoids and necrosis of zone 3
hepatocytes.3VOD develops in 10% to 60%4of patients undergo-
ing allogenic transplantation, and severe VOD is associated with a
mortality rate that approaches 100%.3VOD has been treated using
thrombolytics, such as tissue-type plasminogen activator,5and with
antithrombotic agents, such as the polydeoxyribonucleotide defib-
rotide,6with some success. However, the optimal treatment of
VOD would theoretically employ agents that address the cause as
well as the consequences of the disorder.
Several studies have provided evidence that injury to hepatic
sinusoidal endothelial cells by chemotherapeutic agents is the
initiating event in the pathogenesis of VOD.7,8In cell culture,
isolated sinusoidal endothelial cells were more susceptible to injury
than hepatocytes when incubated with dacarbazine, an agent
associated with the development of VOD.7Recently, it was
reported that decreased nitric oxide (NO) production contributed to
the development of VOD.9NO is the enzymatic end product of
nitric oxide synthase (NOS) and plays a diverse role in regulating
many physiologic systems.10In the liver, NO maintains the hepatic
microcirculation and endothelial integrity.11
Aside from the well-defined roles that endothelial NO plays in
regulating vascular tone and structure, NO suppresses plasminogen
activator inhibitor-1 (PAI-1) production.12PAI-1 serves as the
primary physiologic inhibitor of plasminogen activation and plays
a critical role in regulating endogenous fibrinolytic activity13and
resistance to thrombolysis.14In tissue, PAI-1 influences the re-
sponse to injury by impairing cellular migration15and matrix
degradation.16There is substantial evidence that PAI-1 may
contribute to the development of thrombosis and fibrosis after
chemical17or ionizing injury.18We reported that PAI-1 deficiency
effectively prevents the development of arteriosclerosis and hyper-
tension in mice treated with the NOS inhibitor L-NAME.19
Conversely, we have shown that the transgenic mice that express a
stable form of human PAI-1 develop spontaneous coronary arterial
thrombosis.20While the pathogenesis of VOD is largely unknown,
PAI-1 has emerged as both an independent diagnostic marker of
VOD and a predictor of the severity of the disease.21Taken
together, these data suggest that increased PAI-1 may contribute to
the pathophysiology ofVOD.To test this hypothesis, we developed
a murine model of hepatic vein thrombosis that involved adminis-
tration of L-NAME to mice for 6 weeks.We investigated the role of
PAI-1 in this model by characterizing the effects of L-NAME in
wild-type (WT) and PAI-1?/?mice and by administering a novel,
orally active PAI-1 antagonist, tiplaxtinin (PAI-039),22plus L-
NAME to WT mice.
PAI-1?/?mice23andWTmice on the same genetic background (C57BL/6J)
were purchased from the Jackson Laboratory (Bar Harbor, ME). Six male
animals were studied in each of 3 experimental groups. L-NAME (Sigma,
St Louis, MO) is a nonselective reversible inhibitor of NOS24and was
administered as described.19All control and untreated animals were fed a
From the Departments of Medicine and Pathology, Division of Cardiovascular
Medicine, Vanderbilt University Medical Center, Nashville, TN; and Wyeth
Research, Collegeville, PA.
Submitted July 12, 2005; accepted August 25, 2005. Prepublished online as
Blood First Edition Paper, September 13, 2005; DOI 10.1182/blood-2005-07-
Supported by grants HL 65192 and HL 51387 from the National Heart, Lung,
and Blood Institute. Two of the authors (H.E. and D.L.C.) are employed by a
company whose product was studied in the present work.
Reprints: Douglas E. Vaughan, Division of Cardiovascular Medicine,
Vanderbilt University Medical Center, 2220 PierceAve, PRB 383, Nashville, TN
37232; e-mail: firstname.lastname@example.org.
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.
© 2006 by TheAmerican Society of Hematology
132BLOOD, 1 JANUARY 2006?VOLUME 107, NUMBER 1
For personal use only. on December 30, 2015. by guest
regular unmodified chow diet. Tiplaxtinin (PAI-039) was administered by
mixing it into regular chow (1.0 mg/g chow) and administered in addition to
L-NAME ad libitum. This dose has previously been shown to produce
steady-state plasma levels of tiplaxtinin nearly equivalent to the in vitro
50% inhibitory concentration (IC50) against PAI-1.25Systolic blood pres-
sure was serially determined as described.19
Six weeks after the initiation of L-NAME treatment, animals were killed for
gross and microscopic hepatic analyses. After extensive saline perfusion,
livers were harvested, formalin fixed, and embedded in paraffin blocks.
Hepatic sections were stained with Masson trichrome and hematoxylin and
eosin stains and photographed under ? 20 to ? 80 magnification using an
Olympus BX40 microscope (Melville, NY) with an Optronics Magnafire
digital camera (Goleta, CA). Digital image analysis of each photomicro-
graph was performed with ImagePro Plus (Media Cybernetics, Silver
Spring, MD). The extent of hepatic venous thrombosis was determined by
calculating the vascular luminal area obstructed by thrombi divided by the
total vascular area in any given ? 20 field. For each liver, the obstructed
and total vascular areas were calculated from 5 random ? 20 fields. In total,
240 individual veins were analyzed in each of the treatment groups.
Sections were examined and characterized by a single blinded investigator.
Blood samples were taken by retro-orbital bleeding at week 0 and when the
animals were humanely killed. Samples were anticoagulated using acidified
3.8% sodium citrate. AST and bilirubin tests were performed at the
Vanderbilt Clinical Diagnostics Laboratory (Nashville, TN) per clinical
protocols. Plasma PAI-1 activity was measured using a functional enzyme-
linked immunosorbent assay (ELISA) assay26that identifies only the active
protein (Molecular Innovations, Southfield, MI).
Data were analyzed by analysis of variance (ANOVA), which was
performed by using SPSS 11.0 (SPSS, Chicago, IL). When ANOVA
indicated a statistically significant difference between treatment groups, the
Scheffe multiple comparison procedure was then used to determine which
pairs of treatment groups were significantly different. Data are reported as
the mean plus or minus standard error of the mean (SEM).
Results and discussion
NOS inhibition by L-NAME induces hepatic venous thrombosis
in WT mice
At baseline there were no significant differences in systolic blood
pressure between groups. After 6 weeks, systolic blood pressure
was significantly higher in L-NAME–treated WT mice com-
pared with L-NAME–treated PAI-1?/?mice (140.7 ? 5.0 mm
Hg in WT vs 121.4 ? 7.3 mm Hg in PAI-1?/?; P ? .001). This
observation is consistent with our previous studies and is
attributable to the formation of perivascular fibrosis in WT mice
receiving L-NAME. At the time the animals were killed, livers
from WT?L-NAME mice exhibited a significant number of
hepatic and portal veins occluded by fibrin thrombi compared with
the PAI-1?/??L-NAME mice (66.43% ? 8.7% occluded area in
WT vs 18.36% ? 5.6% occluded area in PAI-1?/?; P ? .001). WT
mice receiving L-NAME also exhibited other histologic changes
associated with VOD including hepatocyte necrosis and fibrosis
(data not shown). In contrast, these changes were not apparent in
PAI-1?/?mice receiving L-NAME (Figure 1G-I).
There were no significant differences inAST or bilirubin levels
between the treatment groups at baseline. After 6 weeks of
L-NAME treatment both AST (159.4 ? 13.5 U/L in WT vs
93.8 ? 20.5 U/LinPAI-1?/?;
(26.676 ? 8.55 ?M [1.56 ? 0.5 mg/dL] in WT vs 3.42 ? 0.855
?M [0.20 ? 0.05 mg/dL] in PAI-1?/?; P ? .001) levels were
increased in WT mice compared with PAI-1?/?mice (Figure 2A).
Elevated levels of AST and bilirubin are associated with VOD in
humans and reflect injury to hepatocytes and obstruction of the
liver. In this model, WT mice exhibit similar increases in serum
total bilirubin and AST levels, whereas PAI-1?/?mice do not,
suggesting that PAI-1?/?deficiency is sufficient to protect
against hepatic injury despite decreased NO. Furthermore, this
observation suggests that PAI-1 plays an early role in the
pathogenesis of VOD and may provide insight into the sequence
by which endothelial damage leads to hepatic thrombosis. It is
likely that increases in serum total bilirubin level reflect the
formation of obstructive hepatic and portal venous thrombi that
results from damage to endothelial cells, whereas changes in
AST level occur subsequently and reflect the resulting hepato-
P ? .018) andbilirubin
Figure 1. NOS inhibition and hepatic venous thrombosis. Masson trichrome
stains of representative livers from WT?L-NAME (A-C), WT?L-NAME?tiplaxtinin
(PAI-039) (D-F), and PAI-1?/??L-NAME (G-I) mice after 6 weeks of L-NAME
treatment at the indicated magnifications. Arrows illustrate the extent of venous
thrombi in WT mice. Images were visualized using an Olympus BX40 microscope
equipped with 10?/0.30, 20?/0.50, and 40?/0.90 Plan Apo objective lenses and an
Optronics digital camera (Optronics, Goleta, CA). Images were acquired with
Magnafire 1.0 software (Optronics) and were processed for publication with Image
Pro Plus 4.5 software (Media Cybernetics, Silver Spring, MD). (J) Calculated percent
occluded luminal area in all 3 treatment groups (P ? .001 for WT vs PAI-1 knockout
[KO]; and P ? .001 for WT vs WT?tiplaxtinin byANOVA). Values shown are mean ?
PAI-1 IN MURINE MODELOF HEPATIC VEIN THROMBOSIS 133BLOOD, 1 JANUARY 2006?VOLUME 107, NUMBER 1
For personal use only. on December 30, 2015. by guest
Effects of PAI-1 inhibition by tiplaxtinin (PAI-039) in
We have previously demonstrated that inhibition of PAI-1 by
tiplaxtinin protects against angiotensin II–induced aortic remodel-
ing.25This compound inhibits PAI-1 by binding directly to the
protein and inhibiting its activity.22As shown in Figure 1, L-NAME
induced numerous and extensive hepatic thrombi in WT mice.
Consistent with the data observed in PAI-1?/?mice, tiplaxtinin, a
small-molecule inhibitor of PAI-1, significantly attenuated the
number and extent of L-NAME–induced venous thrombi in WT
mice (41.55% ? 3.6% vs 66.43% ? 8.7% occluded area; P ? .05;
Figure 1J). As expected, plasma PAI-1 activity was decreased in
WT mice receiving tiplaxtinin?L-NAME compared with those
mice that received L-NAME alone (17.68 ? 1.6 ng/mL vs
36.05 ? 6.34 ng/mL; P ? .011; Figure 2B) and had no effect
on L-NAME–induced increases in systolic blood pressure
(136.6 ? 11.7 mm Hg vs 140.8 ? 11.74 mm Hg; P ? .05). The
consistency of the findings that both pharmacologic inhibition
and genetic deletion of PAI-1 reduce the extent and severity of
hepatic venous thrombi confirms that PAI-1 is directly involved in
the molecular pathogenesis of the disease.
In summary, this study provides direct evidence that PAI-1 is
more than a biochemical marker of VOD. Indeed, these results
establish that PAI-1 is essential to the pathogenesis of hepatic
veno-occlusive disease. Since both genetic deficiency and pharma-
cologic inhibition of PAI-1 provided protection against hepatic
thrombosis, this study also provides proof of concept for the
strategy of developing pharmacologic antagonists of PAI-1 for the
treatment of VOD. Importantly, while other chemical classes of
PAI-1 inhibitors have been reported that include both direct-acting
small-molecule inhibitors and antibodies,16,22none has shown the
oral activity and efficacy of tiplaxtinin or has been profiled in a
model of this disease. The present findings also suggest that PAI-1
is a rational and druggable target for the prevention and treatment
of VOD in humans.
1. Richardson P, Guinan E. Hepatic veno-occlusive
disease following hematopoietic stem cell trans-
plantation.Acta Haematologica. 2001;106:57-68.
2. Blostein MD, Paltiel OB, ThibaultA, Rybka WB.A
comparison of clinical criteria for the diagnosis of
veno-occlusive disease of the liver after bone
marrow transplantation. Bone Marrow Transplant.
3. Bearman SI. The syndrome of hepatic veno-oc-
clusive disease after marrow transplantation.
4. Richardson P, Bearman SI. Prevention and treat-
ment of hepatic venocclusive disease after high-
dose cytoreductive therapy. Leuk Lymphoma.
5. Bearman SI, Lee JL, BaronAE, McDonald GB.
Treatment of hepatic venocclusive disease with
recombinant human tissue plasminogen activator
and heparin in 42 marrow transplant patients.
6. FalangaA, VignoliA, Marchetti M, Barbui T. Defi-
brotide reduces procoagulant activity and in-
creases fibrinolytic properties of endothelial cells.
7. DeLeve LD. Dacarbazine toxicity in murine liver
cells: a model of hepatic endothelial injury and
glutathione defense. J Pharmacol Exp Ther.
8. DeLeve LD. Cellular target of cyclophosphamide
toxicity in the murine liver: role of glutathione and
site of metabolic activation. Hepatology. 1996;24:
9. DeLeve LD, Wang X, Kanel GC, et al. Decreased
hepatic nitric oxide production contributes to the
development of rat sinusoidal obstruction syn-
drome. Hepatology. 2003;38:900-908.
10. Moncada S, Palmer RM, Higgs EA. Nitric oxide:
physiology, pathophysiology, and pharmacology.
Pharmacol Rev. 1991;43:109-142.
11. Kuroki I, Miyazaki T, Mizukami I, Matsumoto N,
Matsumoto I. Effect of sodium nitroprusside on
ischemia-reperfusion injuries of the rat liver.
12. Bouchie JL, Hansen H, Feener EP. Natriuretic
factors and nitric oxide suppress plasminogen
activator inhibitor-1 expression in vascular
smooth muscle cells: role of cGMP in the regula-
tion of the plasminogen system.Arterioscler
Thromb Vasc Biol. 1998;18:1771-1779.
13. Loskutoff DJ, Sawdey M, Mimuro J. Type 1 plas-
minogen activator inhibitor. Prog Hemost Thromb.
14. Zhu Y, Carmeliet P, Fay WP. Plasminogen activa-
tor inhibitor-1 is a major determinant of arterial
thrombolysis resistance. Circulation. 1999;99:
15. Stefansson S, Lawrence DA. The serpin PAI-1
inhibits cell migration by blocking integrin alpha V
beta 3 binding to vitronectin. Nature. 1996;383:
16. Heymans S, LuttunA, Nuyens D, et al. Inhibition
of plasminogen activators or matrix metallopro-
teinases prevents cardiac rupture but impairs
therapeutic angiogenesis and causes cardiac fail-
ure. Nat Med. 1999;5:1135-1142.
17. Olman MA, Mackman N, Gladson CL, Moser KM,
Loskutoff DJ. Changes in procoagulant and fi-
brinolytic gene expression during bleomycin-in-
duced lung injury in the mouse. J Clin Invest.
18. Oikawa T, Freeman M, Lo W, Vaughan DE, Fogo
A. Modulation of plasminogen activator inhibitor-1
in vivo: a new mechanism for the anti-fibrotic ef-
fect of renin-angiotensin inhibition. Kidney Int.
19. Kaikita K, FogoAB, Ma L, Schoenhard JA, Brown
NJ, Vaughan DE. Plasminogen activator inhibi-
tor-1 deficiency prevents hypertension and vas-
cular fibrosis in response to long-term nitric oxide
synthase inhibition. Circulation. 2001;104:839-
20. Eren M, Painter CA,Atkinson JB, Declerck PJ,
Vaughan DE.Age-dependent spontaneous coro-
nary arterial thrombosis in transgenic mice that
express a stable form of human plasminogen ac-
tivator inhibitor-1. Circulation. 2002;106:491-496.
21. Lee JH, Lee KH, Kim S, et al. Plasminogen acti-
vator inhibitor-1 is an independent diagnostic
marker as well as severity predictor of hepatic
veno-occlusive disease after allogeneic bone
marrow transplantation in adults conditioned with
busulphan and cyclophosphamide. Br J Haema-
22. Elokdah H,Abou-Gharbia M, Hennan JK, et al.
Tiplaxtinin, a novel, orally efficacious inhibitor of
plasminogen activator inhibitor-1: design, synthe-
sis, and preclinical characterization. J Med Chem.
23. Carmeliet P, Kieckens L, Schoonjans L, et al.
Plasminogen activator inhibitor-1 gene-deficient
mice, I: generation by homologous recombination
and characterization. J Clin Invest. 1993;92:
24. HobbsAJ, HiggsA, Moncada S. Inhibition of nitric
oxide synthase as a potential therapeutic target.
Annu Rev Pharmacol Toxicol. 1999;39:191-220.
25. WeisbergAD,Albornoz F, Griffin JP, et al. Phar-
macological inhibition and genetic deficiency of
plasminogen activator inhibitor-1 attenuates an-
giotensin II/salt-induced aortic remodeling.Arte-
rioscler Thromb Vasc Biol. 2005;25:365-371.
26. Ngo TH, Verheyen S, Knockaert I, Declerck PJ.
Monoclonal antibody-based immunoassays for
the specific quantitation of rat PAI-1 antigen and
activity in biological samples. Thromb Haemost.
Figure 2. AST and bilirubin levels. (A) Total serum bilirubin and AST levels are
elevated in WT mice receiving L-NAME but not in PAI-1?/?mice or in WT mice
receiving tiplaxtinin (PAI-039). (B) Tiplaxtinin (PAI-039) reduced plasma PAI-1 activity
in WT mice receiving L-NAME.
134SMITH et alBLOOD, 1 JANUARY 2006?VOLUME 107, NUMBER 1
For personal use only.on December 30, 2015. by guest
September 13, 2005 Download full-text
originally published online
2006 107: 132-134
Kay Washington and Douglas E. Vaughan
Layton H. Smith, John D. Dixon, John R. Stringham, Mesut Eren, Hassan Elokdah, Dave L. Crandall,
Pivotal role of PAI-1 in a murine model of hepatic vein thrombosis
Articles on similar topics can be found in the following Blood collections
Updated information and services can be found at:
(2494 articles)Hemostasis, Thrombosis, and Vascular Biology
(1859 articles) Brief Reports
Information about reproducing this article in parts or in its entirety may be found online at:
Information about ordering reprints may be found online at:
Information about subscriptions and ASH membership may be found online at:
Copyright 2011 by The American Society of Hematology; all rights reserved.
of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036.
Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society
For personal use only. on December 30, 2015. by guest