HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Hemostasis and coagulation at a hematocrit level of 0.85: functional
consequences of erythrocytosis
Junpei Shibata, Jo Hasegawa, Hans-Joachim Siemens, Eva Wolber, Leif Dibbelt, Dechun Li, Dörthe M. Katschinski, Joachim Fandrey,
Wolfgang Jelkmann, Max Gassmann, Roland H. Wenger, and Klaus F. Wagner
We have generated a transgenic mouse
line that reaches a hematocrit concentration of 0.85 due to constitutive overexpression of human erythropoietin in an oxygen-independent manner. Unexpectedly,
this excessive erythrocytosis did not lead
to thrombembolic complications in all
investigated organs at any age. Thus, we
investigated the mechanisms preventing
thrombembolism in this mouse model.
Blood analysis revealed an age-dependent elevation of reticulocyte numbers
and a marked thrombocytopenia that
matched the reduced megakaryocyte
numbers in the bone marrow. However,
platelet counts were not different from
wild-type controls, when calculations
were based on the distribution (eg,
plasma) volume, thereby explaining why
thrombopoietin levels did not increase in
transgenic mice. Nevertheless, bleeding
time was significantly increased in transgenic animals. A longitudinal investigation using computerized thromboelastography revealed that thrombus formation
was reduced with increasing age from 1
to 8 months in transgenic animals. We
observed that increasing erythrocyte concentrations inhibited profoundly and reversibly thrombus formation and prolonged the time of clot development, most
likely due to mechanical interference of
red blood cells with clot-forming platelets. Transgenic animals showed in-
creased nitric oxide levels in the blood
that could inhibit vasoconstriction and
platelet activation. Finally, we observed
that plasmatic coagulation activity in
transgenic animals was significantly decreased. Taken together, our findings suggest that prevention of thrombembolic
disease in these erythrocytotic transgenic mice was due to functional consequences inherent to increased erythrocyte concentrations and a reduction of
plasmatic coagulation activity, the cause
of which remains to be elucidated. (Blood.
2003;101:4416-4422)
© 2003 by The American Society of Hematology
Introduction
A high hematocrit is expected to be associated with an increased
risk of thrombosis or embolism. Numerous reports from patients
with polycythemia vera and pseudopolycythemia confirm a
correlation of elevated hematocrit levels and the incidence of
thrombosis.1-3
The Framingham study established a positive correlation between the hematocrit value and the risk of cerebral infarction,4 and
in a prospective study a hematocrit level higher than 0.51 was
found to be an independent risk factor for stroke.5 In cyanotic
congenital heart disease, exceedingly high hematocrit values of up
to 0.80 have been recorded,6 and cerebral and pulmonary infarcts7
as well as cerebral venous thrombosis correlate with hematocrit
levels.8,9 Some studies even suggest a procoagulatory role for
erythrocytes put forth on platelets.10,11
A functional dissection of the relationship between the hematocrit,
the activation of hemostasis and coagulation, and the presence of
thrombembolism had been difficult to investigate due to the lack of an
adequate animal model. Thus, we generated a suitable mouse model by
establishing a transgenic mouse line characterized by an isolated
erythrocytosis.12 These transgenic mice, constitutively overexpressing
the human erythropoietin gene in an oxygen-independent manner, reach
a hematocrit plateau of 0.80 to 0.85 within the first 2 months without
altering their blood pressure, heart rate, or cardiac output.13 We showed
that in these transgenic mice, endothelial nitric oxide synthase levels and
nitric oxide–mediated endothelium-dependent relaxation was significantly increased12 despite concomitant expression of the vasoconstrictor
endothelin-1.14 Apart from this, another transgenic mouse line lacking
the receptor tyrosine kinase c-kit that exhibits hematopoietic defects
causing perinatal death could be rescued by breeding with our erythropoietin-overexpressing transgenic mouse.15 Of note, despite the fact that
erythropoietin was found to activate components of oxidative metabolism pathways in the brain of our transgenic mouse that could be related
to neuroprotective effects of erythropoietin,16 we found markedly
increased cerebral infarction volumes in our transgenic mice on
permanent occlusion of the middle cerebral artery.17
With aging, we observed a significantly increased mortality in
erythrocytotic transgenic mice after 6 to 8 months.13 Obviously,
thrombosis or impaired coagulation (or both) might contribute to
their reduced life expectancy. Therefore, the aim of this study was
to analyze the erythropoietin-overexpressing transgenic mouse line
for any signs of thrombosis or embolism, characterize its hemostasis and coagulation system, and functionally investigate the
From the Department of Anesthesiology, Clinic of Internal Medicine II, Institute
for Clinical Chemistry, and Institute of Physiology, University Lübeck, Lübeck,
Germany; Department of Anesthesiology, Johns Hopkins University, Baltimore,
MD; Institute of Veterinary Physiology, University Zürich, Zürich, Switzerland;
Institute of Physiology, University Essen, Essen, Germany; and Carl-LudwigInstitute of Physiology, University of Leipzig, Leipzig, Germany.
Shinshu University (K.F.W.) and a faculty grant of the University of Lübeck (K.F.W.).
J.S. and J.H. contributed equally to this study.
Reprints: Klaus F. Wagner, University of Lübeck, Ratzeburger Allee 160, D23538 Lübeck, Germany; e-mail: [email protected].
Submitted October 3, 2002; accepted January 24, 2003. Prepublished online as
Blood First Edition Paper, February 6, 2003; DOI 10.1182/blood-2002-09-2814.
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.
Supported by a grant of the Department of Anesthesiology and Resuscitation,
© 2003 by The American Society of Hematology
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BLOOD, 1 JUNE 2003 䡠 VOLUME 101, NUMBER 11
contribution of raised erythrocyte concentrations to clot characteristics and thrombosis.
Materials and methods
The transgenic mouse line termed tg6 carries the human erythropoietin
cDNA driven by the platelet-derived growth factor ␤ (PDGF-␤) promotor
as described earlier.12 The transgene is inherited as an autosomal dominant
trait. Hemizygous transgenic males were mated to wild-type C57Bl/6
females, and the wild-type littermates served as controls in all experiments.
Transgenic mice reached plasma erythropoietin levels of 123.8 ⫾ 29.0 U/L
compared with 8.4 ⫾ 17.9 U/L in wild-type control siblings. Animals were
kept in conventional housing conditions and had unrestricted access to food
and water up to the experiment. All investigations were performed with the
approval of the state animal ethics committee.
In anesthetized mice (inspiratory isoflurane concentration 1%-1.5%
supplemented with oxygen), subaquatic bleeding time was measured from
standardized dissection of the tail tip. It was cut at a diameter of 0.16 mm
and placed in 37°C sterile physiologic saline solution; the time until the
stream of erythrocytes stopped was recorded. If after 15 minutes the
bleeding had not stopped spontaneously, the wound was cauterized and a
bleeding time of 15 minutes was used for statistical calculations.
Histologic slides (1-3 ␮m) were cut from paraffin-embedded material
and stained with hematoxylin and eosin or Masson Goldner trichrome stain.
Blood was sampled from anesthetized mice from the retro-orbital
plexus for microhematocrit determination and from the caudal vena cava
for all other studies. After a median laparotomy, the caudal vena cava was
exposed, cannulated with a 26-gauge catheter (Abbocath-T, Abbott Ireland,
Sligo, Republic of Ireland), and the mouse exsanguinated at a rate of 1 mL
blood per minute. Typical blood volumes collected were 800 to 1200 ␮L
and 1500 to 2700 ␮L for wild-type and transgenic mice, respectively,13
gaining 300 to 450 ␮L and 200 to 350 ␮L plasma, respectively. Because
hypobaric pressures can lead to rupture of the erythrocyte membrane,
activate coagulation, and thus disturb coagulation measurements, particular
care was taken to avoid negative pressures during blood withdrawal.
The distribution volume of the coagulation inhibitor sodium citrate is
the plasma volume. For determination of the activated partial thromboplastin time, the prothrombin time, and the thrombin time, the amount of
sodium citrate added was calculated based on the plasma fraction (1 ⫺
hematocrit value) of the blood. Adding sodium citrate to whole blood in the
normal ratio of 1:10 had been reported to be the cause of false pathologic
coagulation test results in patients with polycythemia vera.18 Hematologic
parameters were measured with a VetABC blood counter (ABX Diagnostics, Montpellier, France); coagulation and clinical chemistry parameters
were measured with a KC-10 (Fa Amelung, Lemgo, Germany) and a Han
Aeroset analyzer (Abbott, Wiesbaden, Germany), respectively. Computerized thromboelastograms were performed with a TEG 5000 analyzer
(Haemoscope, Niles, IL). All tests described above were carried out in 5
groups of male mice aged 1, 2, 4, 6, and 8 months.
For thromboelastogram analysis performed on native whole blood, the
standardized blood withdrawal time was 1 minute, and the total elapsed
time until the beginning of the measurement was 2 minutes after the start of
the blood sampling. To investigate the functional role of the erythrocyte and
platelet concentrations on the thrombelastogram, the respective cell concentration of whole blood was adjusted by addition of platelet-rich plasma or
platelet-poor plasma. Platelet-rich plasma and platelet-poor plasma were
obtained after centrifugation of citrated whole blood (4°C 10 minutes) at
850g and 2550g, respectively.
To determine the osmotic fragility, washed erythrocytes from wild-type
and transgenic mice were resuspended in 0.9% NaCl and the hematocrit
level adjusted to 0.45. Erythrocyte suspension (10 ␮L) was added to 100 ␮L
lysis solution containing decreasing concentrations of NaCl (120-45 mM
corresponding to an osmolality of 210-82 mOsm/kg), incubated for 5
minutes at room temperature, and centrifuged at 2000g for 2 minutes. The
absorbance of the supernatant was determined at 550 nm.
Nitrate concentrations in plasma were measured with the Griess reaction (R
& D Systems, Minneapolis, MN); plasma concentrations of thrombopoietin and
HEMOSTASIS AND COAGULATION IN ERYTHROCYTOSIS
4417
erythropoietin were measured by enzyme-linked immunosorbent assay (ELISA;
Mouse TPO Immunoassay, R & D Systems, and EPO-ELISA Medac, Medac,
Wedel, Germany). Total RNA was extracted from liver,19 reverse transcribed, and
thrombopoietin mRNA quantified by competitive polymerase chain reaction
(PCR) normalized against glyceraldehydes-3-phosphate dehydrogenase (GAPDH)
expression. The primer sequences for thrombopoietin competitive PCR were
ctctgtccagccccgtagc (forward) and ccccaagaggaggcgaac (reverse) yielding a
product length of 314 bp.20
All data were tested with parametric tests (Student t test or ANOVA with
Bonferroni post hoc test as applicable) for statistical significance of
differences (P ⬍ .05), except erythropoietin and thrombopoietin plasma
protein concentrations, which were analyzed with the nonparametric
Mann-Whitney test. Results are presented as mean values ⫾ SD unless
stated otherwise.
Results
Excessive erythrocytosis implies increased numbers of young
erythrocytes and reticulocytes. Indeed, at 1, 2, and 4 months the
reticulocyte count in peripheral blood was increased significantly
in transgenic mice (Figure 1A), ultimately resulting in the marked
rise of the hematocrit level up to the plateau of 0.85 (Figure 1B).
Histology
Histologic sections from 8-month-old male transgenic mice suffering from excessive erythrocytosis were carefully evaluated for
signs of tissue infarction (Figure 1), because an elevated hematocrit
level is known to predispose to thrombosis and embolization. The
Figure 1. Lack of histologic evidence of thrombosis or emboli in any organ
despite a hematocrit value of 0.85. Reticulocyte numbers (A) were increased in
erythropoietin transgenic mice, most prominently in the first 4 months (n ⫽ 5 per
group). Time course of the hematocrit value (B) shows a plateau of 0.85 from 2
months onward in transgenic mice (n ⫽ 30 per group). BV indicates bronchial vein;
wt, wild-type; tg, transgenic. *P ⬍ .01, #P ⬍ .001, compared with age-matched
wild-type controls. Tissue sections from the lung and the heart of 8-month-old mice
(C,E, wild-type mice; D,F, transgenic mice) demonstrate the plethora of erythrocytes
in the vasculature of transgenic mice without signs of thrombosis or emboli. Original
magnification, ⫻ 200 for panels C and D, trichrome, scale bar 50 ␮m. Original
magnification, ⫻ 400 for panels E and F, hematoxylin and eosin, scale bar 20 ␮m.
4418
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SHIBATA et al
hemostasis was significantly impaired in transgenic mice (Figure
2A). Platelets are known to contribute to hemostasis and thus
concentrations thereof were subsequently measured. Apparently, at
1 month, platelet concentrations in transgenic mice were within the
normal range, but in older mice a more than 50% decrease of
platelet numbers was found (Figure 2B). However, considering that
erythrocytes contribute 85% to the blood volume, we calculated the
platelet concentration in the plasma volume fraction. As shown in
Figure 2C, platelet concentrations in transgenic mice were no
longer decreased when the platelet distribution volume in plasma
was taken into account. Platelets are released from megakaryocytes
mainly in response to the glycoprotein thrombopoietin. However,
neither thrombopoietin mRNA (wild-type, 15.0 ⫾ 4.2 versus transgenic, 10.6 ⫾ 3.5 aM/fM; not significant) nor plasma levels
(wild-type, 377 ⫾ 128 versus transgenic, 474 ⫾ 411 pg/mL; not
significant) were significantly decreased in transgenic mice.
Because nitric oxide inhibits hemostasis, we next measured the
plasma concentration of nitrate, the stable conversion product, or
nitric oxide. Notably, we found a significant increase of nitrate in
transgenic mice compared with controls (wild-type versus transgenic, 34.8 ⫾ 10.6 versus 54.3 ⫾ 14.3 ␮M; P ⬍ .01).
Bone marrow
Figure 2. Subaquatic bleeding time, platelet concentrations, and bone marrow
histology showing reduced megakaryocyte numbers. Subaquatic bleeding time
was measured to assess hemostasis (A). Apparently reduced platelet numbers in
transgenic mice (B) were calculated to be in the same range or even increased in
comparison with wild-type mice when the highly reduced distribution volume of
platelets; that is, the plasma fraction, was taken into account (C). Sternal bone
marrow was quantified for the presence of megakaryocytes per high-power (⫻ 400)
visual field (D) showing a progressive reduction of megakaryocyte numbers with
increasing age (n ⫽ 5 per group). *P ⬍ .01, #P ⬍ .001, compared with age-matched
wild-type controls. Panels E (wild-type) and F (transgenic) illustrate the reduced
number of megakaryocytes in 8-month-old transgenic mice compared with agematched wild-type controls. M indicates megakaryocyte. All sections were stained
with hematoxylin and eosin (original magnification, ⫻ 400), scale 20 ␮m.
Subsequently, bone marrow sections were evaluated for the presence of megakaryocytes. Whereas megakaryocytes were only
moderately reduced in transgenic mice until the second month
(Figure 2D), megakaryocyte numbers were markedly reduced in
8-month-old transgenic mice compared with wild-type control
mice (Figure 2E-F). Taken together, it is conceivable that the
constant acceleration of erythrocyte production resulted in the
displacement of thrombopoiesis in the bone marrow by red lineage
cells in erythropoietin transgenic mice.
Thrombus formation
plethora of blood vessels, most evident for veins and capillaries,
was prominent in all tested tissues. We did not find any evidence of
thrombosis, embolization, or tissue infarction in lung, heart (Figure
1), brain, liver, kidney, spleen, testes, or skeletal muscle (data not
shown). Of note, no hemorrhage was observed in any tissue.
Hemostasis
The lack of thrombosis despite the extremely high hematocrit value
of 0.85 in adult transgenic mice led us to further investigate
hemostasis and coagulation. Standardized subaquatic tail bleeding
time measurements provided evidence that from 2 months onward
To learn more about the lack of thrombosis and embolization in
transgenic mice, blood coagulation was further investigated by
thromboelastography. Thromboelastography allows the measurement of the complete clotting process, specifically the kinetics of
clot development (␣-angle), the strength of the clot (maximal
amplitude), and fibrinolysis (clot lysis). Most importantly, ex vivo
native whole blood from 2-month-old transgenic mice showed a
significantly reduced clot strength that declined further with
increasing age (Figure 3; Table 1, maximal amplitude). In addition,
the kinetics of clot formation were retarded in transgenic mice
(Table 1, ␣-angle).
Figure 3. Computerized thromboelastography of wild-type and transgenic mice blood: the effect of hematocrit and platelet concentration. Computerized
thromboelastography was used to investigate clot formation. Native whole blood from wild-type (dashed lines) and transgenic (solid lines) mice was analyzed and typical traces
of the different age groups are shown (A; n ⫽ 4-6 ). Clot strength was reduced as early as 1 month in transgenic mice and further declined with age compared with wild-type
controls. The erythrocyte concentration was increased in blood from transgenic mice and, at the same time, the concentration of platelets was decreased. To characterize the
effect on the thromboelastogram of the erythrocyte concentration separate from the effect of the platelet concentration, experiments were performed where the concentration of
one cell type was kept constant while the concentration of the other was varied. Increasing the hematocrit level when the platelet concentration was kept at 1000 (103/␮L) (B;
n ⫽ 3) resulted in a marked and progressive reduction of clot strength. When the hematocrit value was kept constant at 0.40 and the platelet concentration was varied (C;
n ⫽ 3), a small reduction of clot strength with decreasing platelet concentrations was found; wt indicates wild-type; tg, transgenic; m, month; Hct, hematocrit; Plt, platelet
number (103/␮L).
BLOOD, 1 JUNE 2003 䡠 VOLUME 101, NUMBER 11
HEMOSTASIS AND COAGULATION IN ERYTHROCYTOSIS
Table 1. Clot formation kinetics, clot strength, and clot lysis
in wild-type and transgenic mice
Age, mo
␣-Angle
Maximal amplitude, mm
Clot lysis, %
Wild-type
1
72.8 ⫾ 6.7
82.5 ⫾ 0.7
0⫾0
2
66.1 ⫾ 13.1
79.4 ⫾ 4.9
0.1 ⫾ 0.3
4
76.3 ⫾ 0.8
79.8 ⫾ 0.9
0⫾0
6
62.3 ⫾ 6.5
76.8 ⫾ 2.5
0⫾0
8
68.1 ⫾ 7.1
77.6 ⫾ 3.4
0⫾0
Transgenic
1
70.0 ⫾ 4.8
73.2 ⫾ 4.9
2.7 ⫾ 3.4
2
54.5 ⫾ 15.2
55.5 ⫾ 4.8*
12.2 ⫾ 11.3
14.7 ⫾ 10.3
4
40.3 ⫾ 13.5†
48.9 ⫾ 13.7†
6
30.1 ⫾ 14.5*
49.1 ⫾ 7.4†
4.5 ⫾ 10.1
8
32.5 ⫾ 6.9†
47.0 ⫾ 15.7†
9.7 ⫾ 14.6
At 1, 2, 4, 6, and 8 months, coagulation was assessed by computerized
thromboelastography of native whole blood from transgenic and wild-type animals.
The maximal amplitude, a parameter of clot strength, as well as the ␣-angle,
indicating the kinetics of clot formation, were significantly decreased in transgenic
mice aged 4 months and older. Clot lysis was virtually absent in wild-type blood, and
only a small percentage of clot lysis was found in transgenic mice. Clot lysis (%):
100 ⫺ (amplitude [mm] at 30 minutes after maximal amplitude normalized to maximal
amplitude [mm] ⫻ 100). Each group had 4 to 6 mice.
*P ⬍ .01, †P ⬍ .001 compared with age-matched wild-type control group.
We considered the effect of the hematocrit on the thrombus
formation as a possible cause of the coagulation defect of
transgenic mouse blood and in addition we were interested in
elaborating the contribution of the platelet concentration to clot
formation. In reconstitution experiments with wild-type blood, an
increase of the hematocrit with the platelet count kept within the
normal range resulted in a hematocrit-dependent decrease in clot
strength and retarded clotting kinetics, indicating the interference
of erythrocytes with clot formation (Figure 3). Of note, the clot
strength was reduced to approximately the same extent as in old
transgenic mice. When the platelet concentration was decreased in
wild-type blood at a constant hematocrit level of 0.40 (Figure 3), a
decrease of clot strength was noted, but that effect was much less
pronounced compared with the influence of the hematocrit elevation.
These findings indicate that the elevated erythrocyte concentration is
a major factor contributing to the retarded clotting in transgenic mice.
Conversely, a reduction of hematocrit level would be expected to result
in a normalization of the thrombelastogram and an increase in clot
strength. Indeed, when transgenic blood was diluted with transgenic
plasma to a hematocrit concentration of 0.40, the clot strength and the
clot development kinetics were found to be close to normal values
(Table 2). Similar results were obtained when wild-type plasma was
added to transgenic blood for dilution (Table 2).
Because the plasma from transgenic mice was tinted light
yellow, the presence of hemolysis products was investigated.
4419
Although the bilirubin level was normal and normal serum
potassium values (Table 3) indicated adequate blood sampling
techniques, the concentration of free hemoglobin (Table 3) and
lactate dehydrogenase (data not shown) was significantly elevated
in transgenic mice. In addition, the osmotic fragility of erythrocytes
from transgenic mice was markedly increased in animals aged 4
months and older (Figure 4B), thus providing further evidence that
hemolysis was present in these mice in vivo. To explore the impact
of hemolysis products on clotting, wild-type blood was hemolyzed
to yield the same concentration of free hemoglobin as in transgenic
mice, and thromboelastograms were recorded. As shown in Table 2,
no effects of elevated free hemoglobin levels on the clot strength
were observed, suggesting that hemolysis is not a likely cause of
the clotting deficiency in transgenic mice.
Plasma coagulation
Finally, we analyzed the plasma coagulation. Unexpectedly, clot
formation via the intrinsic (Figure 4C) and the extrinsic (Table 3,
prothrombin time) coagulation pathways was significantly impaired in transgenic mice. In addition, the thrombin time was also
prolonged (Figure 4D). A reduced fibrinogen level in transgenic
mice as cause of the impaired plasmatic coagulation was excluded
(Table 3). Thus, the plasmatic coagulation was found to be less
active in transgenic mice too.
Discussion
Hematocrit values of 0.72 to 0.91 have been reported in highaltitude inhabitants,21,22 erythrocytosis from congenital heart disease,23 neonatal erythrocytosis,24 polycythemia vera,18 and Chuvash polycythemia.25 From the published literature a hematocrit
value of 0.85 was expected to be associated with thrombosis and
embolization.2,3,26 Compared with sea-level population, long-term
high-altitude inhabitants have an increased risk of stroke, and
erythrocytosis is the common risk factor.27,28 In infants and young
children with cyanotic congenital heart disease, the risk of cerebrovascular accidents is increased.8,29 However, the exact role of an
increased hematocrit level as a risk factor per se for thrombembolic
accidents remains a matter of debate. Indeed it has recently been
reported that in adults with cyanotic congenital heart disease (mean
hematocrit, 0.70 ⫾ 0.11), the risk of stroke was not increased; that
is, no stroke occurred within 748 patient-years of observation.7 In
line with this observation is our finding that the erythropoietinoverexpressing transgenic mice with excessive erythrocytosis had
no signs of thrombembolic complications in any organ at any age.
In fact, preliminary data (not shown) indicate that the transgenic
Table 2. A reduction of the hematocrit resulted in a normalization of the clot formation kinetics and clot strength in transgenic mice
␣-Angle
Maximal amplitude,
mm
Clot lysis, %
Platelets, 103/␮L
HCT value
Tg blood diluted
58.0 ⫾ 7.6
57.5 ⫾ 6.5
0.0 ⫾ 0.0
732 ⫾ 93
0.40 ⫾ 0.01
Tg RBC ⫹ wt plasma
51.9 ⫾ 12.1
56.3 ⫾ 5.2
1.9 ⫾ 4.3
812 ⫾ 316
0.42 ⫾ 0.03
Wt blood ⫹ hemolysis
62.3 ⫾ 3.9
69.3 ⫾ 1.0
0.0 ⫾ 0.0
845 ⫾ 16
0.42 ⫾ 0.01
Wt 4 mo
76.3 ⫾ 0.8
66.0 ⫾ 6.8
0.0 ⫾ 0.0
1010 ⫾ 136
0.43 ⫾ 0.02
Tg 4 mo
20.1 ⫾ 5.3*
35.1 ⫾ 9.1*
2.1 ⫾ 2.3
338 ⫾ 194*
0.80 ⫾ 0.02*
Clot strength (maximal amplitude) and clot kinetics (␣-angle) were significantly increased after reduction of hematocrit by addition of plasma to red blood cells. For
comparison, values for blood (sampled with sodium-citrate anticoagulation) from 4-month-old wild-type and transgenic mice are shown. Hemolysis (free hemoglobin
concentration 3000 mg/L) did not impair the clot formation kinetics and clot strength. Clot lysis (%): 100 ⫺ (amplitude [mm] at 30 minutes after maximal amplitude normalized to
maximal amplitude [mm] ⫻ 100). n ⫽ 3-5, except for hemolysis n ⫽ 2.
HCT indicates hematocrit; tg, transgenic; RBC, red blood cell; wt, wild-type.
*P ⬍ .001 compared with all groups.
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SHIBATA et al
Table 3. Selected coagulation and clinical chemistry data
Age, months
Fibrinogen level, g/L
Prothrombin time, s
Total bilirubin level, ␮M
Potassium level, mM
Free hemoglobin level, mg/L
879.5 ⫾ 303.3
Wild-type
1
ND
ND
ND
ND
2
1.7 ⫾ 0.9
11.8 ⫾ 0.9
⬍2
4.2 ⫾ 1.1
747.7 ⫾ 778.6
4
1.3 ⫾ 0.4
11.6 ⫾ 0.9
⬍2
4.3 ⫾ 1.5
633.8 ⫾ 339.8
6
1.5 ⫾ 0.7
12.0 ⫾ 1.1
⬍2
4.5 ⫾ 1.2
199.3 ⫾ 96.4
8
1.4 ⫾ 0.5
11.6 ⫾ 0.9
⬍2
4.7 ⫾ 1.8
395.8 ⫾ 182.5
1531.3 ⫾ 1025.4
Transgenic
1
ND
ND
⬍2
ND
2
1.5 ⫾ 0.7
16.6 ⫾ 0.7*
⬍2
5.3 ⫾ 0.8
1794.6 ⫾ 1506.6
4
1.7 ⫾ 1.1
17.5 ⫾ 1.7*
⬍2
5.1 ⫾ 1.3
9000.0 ⫾ 2642.0*
6
1.5 ⫾ 0.7
17.1 ⫾ 0.9*
⬍2
4.8 ⫾ 1.2
3317.5 ⫾ 62.9*
8
1.3 ⫾ 0.8
17.6 ⫾ 2.3*
4.5 ⫾ 2.2
4.3 ⫾ 1.2
5613.3 ⫾ 3940.8†
Time course of some coagulation and clinical chemistry data of transgenic animals and age-matched controls; n ⫽ 3 to 6 per group.
ND indicates not determined.
*P ⬍ 0.01.
†P ⬍ 0.001 for age-matched intergroup comparison.
mice die from congestive cardiac failure. These findings need to be
validated by longitudinal controlled studies; if they are confirmed,
this observation extends the usefulness of the animal model to
nonerythroid diseases.
A limitation of the mouse model presented is the difference
in the molecular basis of the erythrocytosis in comparison with
the human disorders. Whereas polycythemia vera is a clonal
hematopoietic stem cell disorder often accompanied by an
increase in platelets and neutrophils, Chuvash polycythemia30,31
has recently been found to be caused by a mutation of the von
Hippel-Lindau protein leading to a constitutive elevation of the
transcription factor hypoxia-inducible factor 1 (HIF-1). HIF-1
not only induces the expression of erythropoietin resulting in
polycythemia, but also vascular endothelial growth factor and
other genes controlled by HIF-1. Therefore, factors in addition
to the erythrocytosis are likely to contribute to the thromboembolism in the human disorders discussed.
What mechanism might have prevented the clotting of the
highly viscous blood at a hematocrit level of 0.85? In this study we
compiled convincing evidence that high concentrations of erythro-
Figure 4. Increased erythrocyte osmotic fragility and decreased activity of the
plasmatic coagulation in transgenic animals. Osmotic fragility of erythrocytes
from transgenic mice (B) was markedly increased from 4 months onward compared
with wild-type controls (A). Prolongation of the activated partial thromboplastin time
(C) and the thrombin clotting time (D) revealed a decreased activity of the plasmatic
coagulation; wt indicates wild-type; tg, transgenic; OD, optical density. *P ⬍ .01,
#P ⬍ .001 compared with age-matched wild type controls.
cytes interfere with clot formation. Specifically, with a hematocrit
value of 0.80, clot strength was reduced and clot formation kinetics
were slowed down. Possibly, the high erythrocyte concentration
mechanically deters the interaction of platelets and fibrin with the
extravascular tissue or the endothelium. Indeed, our reconstitution
experiments showed that reversal of excessive erythrocytosis
resulted in a normalization of clotting performance and vice versa.
In keeping with this, in patients with hematocrit values over 0.60,
preoperative phlebotomy improved the coagulation abnormalities,
and blood loss at surgery was reported to be lower than in untreated
patients.32
Several mouse lines transgenic for different fragments of the
gene encoding erythropoietin33,34 or transgenic for a mutant human
erythropoietin receptor identified in a patient with erythrocytosis35
have been generated in the past. None of these publications
commented on the presence or absence of thrombosis or investigated the coagulation system. However, unlike the erythropoietinoverexpressing transgenic mouse line investigated here, the hematocrit levels of those mouse lines (0.45-0.69) did not reach the high
levels reported here.
Villeval et al36 induced erythrocytosis in mice by transplanting
bone marrow cells transfected with a retroviral vector carrying a
monkey erythropoietin cDNA into lethally irradiated mice, resulting in extremely high hematocrit values (0.90 ⫾ 0.05). In agreement with our observations, they found no histologic evidence of
thrombosis or emboli. Tissue damage and capillary vascular
permeability increase with increasing doses of irradiation.37 In
contrast to all mouse models of erythrocytosis discussed so far,
thoracic, muscular, and intestinal hemorrhage were noted in some
of the irradiated mice. Together with the markedly shortened mean
survival of 71 days, the findings suggest that irradiation effects in
addition to the high hematocrit values contributed to the
observed phenotype.
Another prominent hematologic abnormality in the erythropoietin transgenic mice was the reduced platelet concentration in
peripheral blood. However, the distribution space of platelets in
blood, in particular in flowing blood in vivo, is the plasma.38 The
extremely high hematocrit level in transgenic mice reduced the
distribution volume of platelets to 15%, whereas 85% of volume is
taken up by erythrocytes. Recalculating the platelet concentration
based on the plasma volume fraction revealed that in transgenic
mice the plasma platelet concentration was not different from that
in wild-type mice. Therefore, reduced clot strength or prolonged
BLOOD, 1 JUNE 2003 䡠 VOLUME 101, NUMBER 11
HEMOSTASIS AND COAGULATION IN ERYTHROCYTOSIS
hemostasis in transgenic mice in vivo cannot be attributed to
reduced platelet numbers. Based on the revised calculations, the
unaltered plasma thrombopoietin concentration is in agreement
with the plasma platelet concentration observed in wild-type and
transgenic mice.
Clot formation was further explored by analysis of plasma
coagulation. Surprisingly, we found a reduction of the activity of
both the extrinsic and the intrinsic pathways. In patients with
erythrocytosis caused by cyanotic congenital heart disease,
decreased levels of coagulation factors as well as thrombocytopenia have been reported.39 These deficiencies have been found
to be due to hepatic dysfunction from congestive cardiac failure
and chronic disseminated coagulation.40 Although the erythropoietin transgenic mice develop cardiac dysfunction during their
later life,13 no signs of hepatic dysfunction are known in these
mice. Alternatively, because the common final pathway of the
coagulation cascade, the generation of fibrin from fibrinogen
through thrombin, does affect the results of the activated partial
thromboplastin time as well as the prothrombin time, the
presence of a thrombin inhibitor in the plasma of transgenic
mice as the cause of the pathologic results cannot be excluded.
Although the fibrinogen concentration was within the range
reported for the mouse strain C57Bl/6,41 a relative deficiency of
fibrinogen with respect to the increased number of erythrocytes
might contribute to the impaired clot formation.
Thromboelastography has been shown to accurately detect
fibrinolysis.42 Hemolysis, which was observed in transgenic mice,
could have contributed to the coagulation defect by inducing a
fibrinolytic state. But computerized thromboelastography revealed
only minor fibrinolysis, present only in older transgenic animals,
and reconstitution experiments showed that acute hemolysis did
not affect clot formation. This is in agreement with a recent report
4421
on coagulation at high altitude43 and a study by Lugassy and Filin,44
who did not find hyperfibrinolysis in a cohort of patients with
polycythemia vera. Still, fibrinolysis as a factor contributing to the
coagulation abnormalities of the erythropoietin-overexpressing
transgenic mice cannot be completely ruled out.
Vasoconstriction and the formation of a white thrombus, among
other factors, contribute to hemostasis. Endothelial cells produce
nitric oxide in response to shear stress, promoting vasodilation and
inhibiting platelet activation, and nitric oxide deficiency enhances
hemostasis in endothelial nitric oxide synthase knock-out mice.45
Conversely, increased levels of nitric oxide products in our mice
might contribute to the defective hemostasis by weakening vasoconstriction12 and reducing responsiveness of platelets.
In this study we report the unexpected finding that erythropoietin transgenic mice living with a hematocrit level of 0.85, do not
have any thrombosis or embolization. We provided evidence that
hemostasis and plasmatic coagulation were significantly impaired
in transgenic mice. Important protective mechanisms were the
significantly reduced activity of the plasmatic coagulation cascade
and, surprisingly, an inhibition of the thrombus formation by
mechanical hindrance from the extremely high erythrocyte concentrations. As such, these mice may provide a useful animal model to
study the adaptive changes to erythrocytosis as well as the
interactions of rheology and coagulation.
Acknowledgments
The authors wish to thank Ann-Katrin Hellberg, Dunja Schumacher, Jenifer Ehlert, and Alex Jurat for expert technical assistance
and Claire Levine for valuable editorial help in the preparation of
the manuscript.
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