Large, Chronic Doses of Erythropoietin Cause Thrombocytopenia in Mice
By T.P. McDonald, Rose E. Clift, and Marilyn B. Cottrell
Both large, acute doses of erythropoietin (EPO) and shortterm hypoxia increase platelet counts in mice, but long-term
hypoxia causes thrombocytopenia. Therefore, we tested the
hypothesis that EPO injected in large, chronic doses (a total
of 80 U of EPO over a 7-day period) might cause thrombocytopenia. EPO caused increased red blood cell (RBC) production,
ie, increased hematocrits, RBC counts, mean cell volume
(MCV), and reticulocyte counts (from P < .05 to P < .0005),
and decreased thrombocytopoiesis, ie, decreased platelet
counts, percent 35S incorporation into platelets, and total
circulating platelet counts (TCPC) (Pe .OMS). Femoral marrow megakaryocytesize was unchanged, but megakaryocyte
number was significantly (P< .005) reduced in mice treated
with EPO. EPO-injected mice had increased spleen volumes
(Pe .0005), but blood volumes (BV) were unchanged. In
EPO-treated, splenectomizedmice, RBC production was also
increased (Pe .05 t o P < .0005) and platelet counts, TCPC,
and percent 36Sincorporation into platelets were decreased
(P< .05), but BV was not altered. Therefore, the decrease in
platelet counts observed in EPO-treated mice was not due t o
increased BV or t o an enlarged spleen. In other experiments,
mice were rendered acutely thrombocytopenic t o increase
thrombocytopoiesis, and platelet and RBC production rates
were determined. In mice with elevatedthrombocytopoiesis,
RBC counts, hematocrits, percent 59Fe RBC incorporation
values, and MCV were decreased (P .05 t o P < 4005).
Because =Fa RBC incorporation and MCV were not elevated,
the decrease in RBC counts and hematocrits does not appear
to be due to bleeding. Therefore, we show that large, chronic
doses of EPO increase erythropoiesisand decrease thrombocytopoiesis. Conversely, acute thrombocytopenia causes increased thrombocytopoiesis and decreased erythropoiesis.
These findings support the hypothesis of competition between precursorcells of the erythrocytic and megakaryocytic
cell lines (stem-cell competition) as the cause of thrombocytopenia in EPO-treated mice and the cause of anemia in mice
whose platelet production rates were increased.
8 1992by The American Society of Hematology.
P
thrombocytopenia in mice is not clear; however, it is well
established that the hypoxia-induced thrombocytopenia is
not caused by increased sequestration of platelets by an
enlarged spleen or by expanding blood volumes? In addition, previous w0rk4,6,9J3showed that the effects of hypoxia
are not directly on thrombocytopoiesis, but require stimulation of the erythroid cell line for reduced platelet production to occur. Based on these findings, it was hypothesized
that competition of a precursor cell between the erythrocytic and megakaryocytic cell lines (stem cell competition)
results in the thr~mbocytopenia?,~
Therefore, platelet counts of rodents exposed to hypoxia
have been shown to be biphasi~,3,~J~
ie, increased platelet
counts were observed at 1 to 3 days after enclosure in
hypoxic atmospheres, but the counts were significantly
decreased after 7 to 10 days of low O2 environment. It
seems clear that the increased erythropoiesis observed
during hypoxia is due to increased EPO levels, because
mice enclosed in hypoxia chambers have a sharp increase15
in EPO activity of about 2 U of EPO/mL of mouse plasma
at day 1.Thereafter, EPO activity in plasma of hypoxic mice
was elevated to a lesser degree, but remained above
normal, and the hematocrit levels of mice steadily increased
during a 10-day enclosure period.I5
Because EPO administered in large doses for short
periods of time will mimic hypoxia and increase platelet
counts at the early time period, it seemed possible that
long-term EPO treatment administered in doses that have
been shown to be released into the circulation by hypoxia15
might lead to thrombocytopenia. Therefore, we injected
both normal and splenectomized mice with a total of 80 U
of EPO over a 7-day period and found elevated erythropoiesis with thrombocytopenia with significantlyreduced platelet production rates. Conversely, stimulated platelet production in mice, caused by an acute episode of thrombocytopenia,
led to increased thrombocytopoiesis and decreased red
blood cell (RBC) production. These data lend credence to
the hypothesis of stem cell competition between the erythrocytic and megakaryocytic cell lines.
REVIOUSLY, acute studies have shown that high
doses of recombinant human erythropoietin (EPO)
will stimulate platelet production in
In addition,
hypoxia, a stimulator of EPO, causes increased platelet
counts in mice at 1 to 3 days after enclosure in silicone
rubber membrane hypoxia chambers? The increase in
platelet counts of mice after short-term hypoxia was absolute, because the total circulating platelet counts (TCPC)
were i n ~ r e a s e dIt. ~has, therefore, been established that in
short-term experiments large doses of either exogenous or
endogenous EPO can cause elevated platelet production in
mice.’-3
In contrast to these findings, it has also been shown that
long-term, chronic hypoxia results in decreased platelet
counts of rodent^.^-^ The thrombocytopenia appears to
result primarily from decreased platelet production, because decreases have been observed in megakaryocyte
colony-forming progenitors,10 megakaryocyte precursor
cells: megakaryocyte n u m b e r ~ , 7 , ~ and
J l , ~isotope
~
incorporation into platelet^!.^.^^ A recent study14 confirmed that
the decreased platelet production observed in mice kept in
hypoxic environments was the result of decreased differentiation of hematopoietic precursor cells into the megakaryocyte lineage. The mechanism by which hypoxia causes
From the Department of Animal Science, College of Veterinary
Medicine, Universiv of Tennessee, Knoxville, TN.
Submitted January 23, 1992; accepted March 17,1992.
Supported in part by Grant No. HL14637from the National Heart,
Lung, and Blood Institute, National Institutes of Health, Public Health
Service, Department of Health and Human Services.
Address reprint request to T.P. McDonald, PhD, The University of
Tennessee, College of Veterinary Medicine, PO Box 1071, Knoxville,
TN 37901-I071.
The publication costs of this article were defrayed in part by page
charge payments. This article must therefore be hereby marked
‘‘advertisement”in accordance with 18 U.S.C.,Section I734 solely to
indicate this fact.
0 I992 by TheAmerican Society of Hematologv.
0006-4971/92/8002-0005$3.00/0
352
Blood, VOI 80, NO2 (July 15), 1992: PP 352-358
EPO CAUSES THROMBOCYTOPENIA IN MICE
MATERIALS AND METHODS
Animals. Male C3H/HENHSD (C3H) mice weighing approximately 24 g were obtained from Harlan Sprague-Dawley(Indianapolis, IN).
EPO. Recombinant human EPO (TCerythropoietin) was purchased from R & D Systems (Minneapolis, MN). EPO had a
specific activity of greater than 10,OOO U / A m after formulation in
0.025% bovine serum albumin (BSA) in phosphate-bufferedsaline
(PBS). The final material was prepared by sequential chromatography and the concentration was approximately4,000 U of EPO/mL.
For controls, other mice were injected with BSA in PBS.
Experimental design. The dose of EPO administered to mice
was designed to approximate the EPO levels found in plasma of
mice after enclosure in silicone-rubber membrane cages.I5 In this
study,15high doses of EPO ( 2 U EPO/mL of plasma) were found
at 1 day after enclosure in the membrane cages; the EPO titers
were lower thereafter ( < 1 U EPO/mL plasma), but remained
significantly elevated. The overall half-life of EPO in rats was
reported to be about 2 to 3.5 hours.*J6Therefore, considering the
half-life and titer of EPO in plasma of rodents after hypoxia, we
administered mice subcutaneously (SC) at time 0,lO U EPO made
up in BSA in PBS (0.5 mL), followed by another injection of 10 U
EPO approximately 8 hours later. On days 1through 6, mice were
administered an injection of 5 U EPO in the morning and another
injection of 5 U in the afternoon (14 total injections of 80 U
EPO/mouse). The mice were killed on day 7.
At the time of killing, hematocrits and platelet counts were
determined on blood obtained from the retroorbital sinus using
standard techniques. RBC counts were determined using a Coulter
Counter (Hialeah, FL) and from these values RBC mean cell
volume (MCV) in cubic micrometers was calculated. Reticulocyte
counts were determined using new methylene blue and standard
techniques. Blood was also obtained via heart puncture for
determination of percent 35S incorporation into platelets,17 and
one of the femurs was removed for determination of megakaryocyte size and number.18
Percent 35Sincorporation intoplatelets. The 24-hour 35Sincorporation into platelets was obtained after an intravenous (IV)
injection of 30 FCi Naz3%04in 0.5 mL saline. Blood obtained via
cardiac puncture after an intraperitoneal (IP) injection of a
heparin-sodium pentobarbital solution was used for determination
of percent 35Sincorporation into ~1atelets.l~
Approximately0.5 mL
of blood was collected into syringes containing 1.0 mL of 1%
EDTA in 0.538% NaCl and the diluted blood was expressed into
plastic tubes. The blood was centrifuged at 4OOg for 4.5 minutes at
4°C to obtain platelet-rich plasma (PRP). The PRP was centrifuged at 8OOg for 15 minutes at 4°C to obtain a platelet button. The
platelets from each mouse were resuspended in 0.6 mL of 1%
ammonium oxalate, mixed, centrifuged (15 minutes at 8OOg), and
washed one additional time with 0.5 mL of 1% ammonium oxalate,
followed by a wash in 1.5 mL of saline. Platelet counts of
suspensions were made and the radioactivity was determined. The
percent 35Sincorporation into platelets was calculated as previously described,17 using the appropriate blood volume for the
corresponding treatment.
Megakaryocyte number and size. The number and size of megakaryocytes in the bone marrow of EPO-injected mice were determined by using histologic sections, as previously described.18 The
femurs were removed, fixed in 10% formalin, processed for light
microscopy by embedding in glycol methacrylate, and stained with
hematoxylin-eosin. The megakaryocytes were counted in sections
at 4OOX magnification, and the megakaryocyte number was expressed as cells per microliter. Megakaryocyte number per microli-
-
353
ter was corrected for errors due to section thickness and optically
lost profiles as described previously.18
Megakaryocyte size was determined from approximately 200
megakaryocyte profiles from randomly selected sections of each
femur, using section profile parameter measurements.ls Before
statistical analysis, all mean diameters and mean megakaryocyte
numbers were corrected for the 5% tissue shrinkage that occurs
during histologic processing.18
Blood volume (BV). Because the possibility existed that the
thrombocytopenia seen in mice treated with EPO might be due to
expanded BV, other mice were injected with EPO or BSA in PBS
as before, and their BV determined by a technique previously
described using 59Fe-labeled RBC from donor mice.' For this
measurement, a group of mice (seven normal mice) served as
erythrocyte donors and was administered a single IP injection of
0.5 KCi of 59Fe (ferric chloride; New England Nuclear Corp,
Boston, MA) 48 hours before the mice were killed and blood
collected into sodium citrate. After centrifugation, the RBC were
mixed with saline to the original volume. Both control mice (mice
injected with BSA in PBS for 7 days) and EPO-injected mice were
injected IV with 0.1 mL of 'Te-labeled RBC. After 15 to 20
minutes, the mice were bled from the retroorbital sinus and the
blood (100 FL) diluted into 2 mL of water was counted for
radioactivity using a gamma scintillation spectrophotometer. BV
was expressed as milliliters of blood per 100 g body weight (%).
TCPC. For each mouse, TCPC was determined by multiplying
the body weight in grams by the percentage of BV to give the total
blood volume of a mouse? This value was then multiplied by the
platelet count to give TCPC.3
Spleen volume. At the time of killing, spleens from treated mice
were placed in a IO-mL graduate cylinder that contained a small
amount of water and the displacement volume of the water was
recorded.
Splenectomy. The possibility also existed that the thrombocytopenia associated with EPO treatment might be due to sequestration of platelets in an enlarged spleen. Therefore, other mice were
splenectomized 2 weeks before being placed in experiments and
administered EPO or BSA in PBS as before. Their RBC and
platelet production indices were measured as outlined above.
Acute thrombocytopenia. It seems possible that if stem cell
competition is an appropriate hypothesis for explaining the thrombocytopenia in mice treated with large doses of EPO, then acute
thrombocytopenia should result in anemia, along with elevated
platelet production. Therefore, additional experiments were performed in which mice were rendered acutely thrombocytopenicto
increase thrombocytopoiesis. Mice were injected with rabbit antimouse platelet serum (RAMPS) that was shown previously19to be
specific for platelets, ie, at 4 hours after RAMPS injection RBC
and WBC counts were unchanged, but platelets were reduced to
less than 4% of the preinjection level. After RAMPS treatment (8
hours and 1, 3, 4, 5, and 6 days), RBC and platelet production
indices were measured as described above. Megakaryocytesize and
number20 have been extensively studied previously in RAMPStreated mice, and blood volumes3were found not to be different in
RAMPS-treated mice from normal mice, ie, values of 5.65 2 0.05
(20 micehreatment) mL/100 g for untreated mice versus 5.88 2
0.11 (12 micehreatment) mL/lOOg for RAMPS-treated mice were
found (measured at 3 to 7 days after RAMPS treatment). In
addition, RBC MCV was calculated using the hematocrits and
RBC counts of mice. For additional evidence that the anemia of
treated mice was not due to bleeding, but was due to decreased
RBC production, one experiment measured the incorporation of
59Feinto the RBC of treated miceJ1
Statzktics. The Student's t-test was used to statistically analyze
the data.
354
MCDONALD, CLIFT, AND COTTRELL
Table 2. Effects of EPO on Megakaryocyte Number and Size
RESULTS
Table 1shows a summary of three experiments in which
mice were injected with BSA in PBS or EPO made up in
BSA and PBS for 7 consecutive days, and the effects on
RBC and platelet production were measured. As shown,
EPO caused significant (P < .0005) decreases in platelet
counts, percent 35Sincorporation into platelets of mice, and
TCPC. In addition, RBC production was significantly increased, ie, elevated hematocrits (P < .0005), higher RBC
counts (P < .05),increased MCV (P < .005), and higher
reticulocyte counts (P < .005) were found in EPO-treated
mice, along with an obvious splenomegaly (P < .0005)
compared with BSA in PBS-treated mice. BV were not
significantly different in EPO-treated mice compared with
controls, illustrating that the decrease in platelet counts
was not the result of expanding blood volumes.
Table 2 shows the results of an experiment in which
megakaryocyte number and size were measured after EPO
injections. Significantly (P < .005) fewer megakaryocytes
were found in femoral marrow of EPO-treated mice compared with controls. However, megakaryocyte size was not
significantly altered.
Table 3 reports the results of an additional experiment in
which splenectomized mice were injected with BSA in PBS
or EPO. In agreement with the data from intact mice
(Table l), EPO-treated splenectomized mice showed a
significant (P < .05) reduction in platelet counts, decreased percent 35Sincorporation into platelets, and lower
TCPC values compared with controls. Although RBC
counts of EPO-treated splenectomized mice were not
significantly increased, hematocrits (P < .0005) and MCV
(P < .05) were increased compared with values of control
mice. As shown, the differences between values for control
and EPO-treated splenectomized mice were not as great as
those found in intact mice (Table 1v Table 3). However, as
Table 1. Effects of EPO on Platelet and RBC Production in Mice
Treatment
BSA in PBS
EPO
*
Platelet count/kL SE
0.83 f 0.03 (17)'
(x10-6)
1.07 f 0.04 (17)
% 355 incorporation into
platelets f SE (x103)
5.16 f 0.34 (6)*
7.17 ? 0.17 (6)
TCPC f SE ( x ~ O - ~ )
14.79 2 0.47 (17) 11.93 0.43 (16)'
44.4 f 0.4 (17)
56.9 2 0.7 (16)*
Hematocrits f SE (%)
RBCcountl~Lf SE ( x ~ O - ~ ) 7.26 f 0.25 (17) 7.93 2 0.22 (17)t
MCV 2 SE (pm3)
62.20 f 1.89 (17) 72.78 f 2.70 (17)*
Reticulocytecount f SE (%)
2.73 f 0.58 (6)
5.68 f 0.51 (6)*
Spleen volume f SE (mL)
1.9 f 0.1 (5)'
1.1 f 0.1 (5)
BV ? SE (%)
5.72 f 0.22 (5)
5.97 f 0.26 (5)§
*
Values are presented as means ? SE (number of micehreatment).
Mice were injected twice per day for 7 days with 0.5 mL of 0.025% BSA
in PBS or EPO in carrier protein. A total of 14 subcutaneous injections of
EPO (80 U) was injected into each mouse. TCPC was calculated as
described in the text. Values for mice treated with EPO were significantly different from mice treated with BSA in PES.
*f < .0005.
tf < .05.
S f < ,005.
O f is not significantly different.
Treatment
BSA in PBS
MegakaryocyteslKL k SE
(~10-3)
Megakaryocyte diameter f SE
(rm)
EPO
2.10 f 0.07 (6)
1.61
2
0.12 (6)*
36.62 f 0.51 (6) 35.30 f 0.48 (6)t
Values are presented as means k SE (number of miceltreatment).
Mice treated with EPO had significantly fewer megakaryocytes in their
bone marrow than other mice injected with BSA in PES, the carrier
protein for EPO. Megakaryocyte number was corrected for errors due
to section thickness, 5% tissue shrinkage, and optically lost profiles.18
Megakaryocyte size, obtained from perimeter measurements of from
127 to 224 section profiles for each mouse, was corrected for the 5%
tissue shrinkage that occurs during histological processing.18
*f < ,005.
tf is not significantly different.
was found in intact mice, BV were unchanged in splenectomized mice treated with EPO or BSA in PBS. These data
illustrate that the thrombocytopenia observed in EPOtreated mice was not due to dilution of the blood in an
enlarged spleen.
Because the data show that EPO increased erythropoiesis and decreased thrombocytopoiesis, additional experiments were performed to test the hypothesis that elevated
thrombocytopoiesis might cause a decrease in erythropoiesis. In the first experiments, mice were injected with
RAMPS and 5 days later, the RBC and platelet production
rates were measured. As shown in Table 4, the platelet
counts and TCPC of mice were significantly elevated
(P < .0005) compared with untreated mice. In addition,
there were significant decreases in the hematocrits and
RBC counts of RAMPS-treated mice (P < .0005) compared with controls, but no significant change was found in
the MCV of RBC, indicating that the anemia was probably
due to decreased RBC production. In addition, the peritoneal and thoracic cavities of mice were examined at the
Table 3. Effects of EPO on Platelet and RBC Production in
Splenectomized Mice
Treatment
BSA in PBS
*
Platelet count/kL SE (xl0-8)
% 35s incorporation into
platelets 2 SE ( x 103)
TCPC ? SE ( x 10-8)
Hematocrits f SE (%)
RBC count/bL f SE ( x ~ O - ~ )
MCV f SE (*m3)
BV f SE (%)
1.00 f 0.02 (6)
EPO
0.86 f 0.03 (7)*
7.15 f 0.64 (3) 4.93 f 0.42 (4)'
12.90 ? 0.40 (6) 11.20 f 0.40 (7)'
43.6 2 0.5 (6)
48.4 f 0.6 (7)t
6.64 f 0.24 (6) 6.89 f 0.11 (7)S
66.05 f 1.85 (6) 70.37 f 1.14 (7)*
5.17 2 0.16 (3) 5.56 2 0.42 (3)$
Values are presented as means f SE (number of mice/treatment).
Mice were splenectomized 2 weeks before injection of 0.025% BSA in
PBS or EPO in carrier protein. A total of 14 subcutaneous injections of
EPO (80 U) was injected into each mouse. TCPC was calculated as
outlined in text. Values for mice treated with EPO were significantly
different from mice treated with BSA in PES.
*f < .05.
tf < .0005.
S f is not significantly different.
355
EPO CAUSES THROMBOCYTOPENIA IN MICE
Table 4. Effects of RAMPS on Platelet and RBC Production of Mice
Treatment
Untreated Mice
RAMPS-Treated Mice
Platelet count/wL f SE ( x ~ O - ~ ) 0.97 f 0.02 (10) 1.40 f 0.03 (14)'
15.04 f 0.48 (10) 21.29 f 0.71 (14)'
TCPC f SE ( x 10-8)
44.5 f 0.3 (10)
41.7 f 0.2 (14)'
Hematocrits f SE (%)
RBCcount/pL f SE ( x ~ O - ~ ) 7.82 f 0.13 (10) 7.10 f 0.12 (14)'
58.8 f 1.0 (14)t
57.0 f 0.9 (10)
MCV f SE (pm3)
Values are presented as means f SE (number of mice/treatment).
RAMPS was injected IP and platelet and RBC values were measured 5
days later. Values were significantly different from values of untreated
mice.
' P < .0005.
t P is not significantly different.
time of killing and no evidence of bleeding was seen. We
interpret the data to show that RAMPS will cause an
increase in platelet production via thrombopoietin release
and action and that the increased thrombocytopoiesis leads
to a significant decrease in RBC counts and hematocrits.
Additional experiments were performed in which mice
were treated with RAMPS and their RBC and platelet
production indices were measured at 8 hours and on days 1,
3, 4, 5, and 6. Compared with untreated mice, platelet
counts of mice were significantly reduced (P < .0005) at 8
hours and on days 1 and 3, but the counts were significantly
elevated on days 4, 5, and 6 (P < .005 to P < .0005),
indicating that the thrombocytopenia led to increased
thrombocytopoiesis (Table 5). Likewise, TCPC of RAMPStreated mice were reduced at 8 hours to 3 days (P < .0005),
but elevated thereafter (P < .005 to P < .0005). Hematocrits of mice were significantly reduced (P < .05 to
P < .005) in mice administered RAMPS on days 3 to 6
compared with control values, but no significant changes
were observed at 8 and 24 hours. Likewise, RBC counts
were significantly reduced on days 4 and 5 (P < .05) after
RAMPS. Moreover, MCV was either not altered (at 8
hours and on days 4 and 5) or was significantly reduced
(days 1, 3, and 6) (P < .05 to P < .0005), as was found in
previous experiments. These data illustrate that increased
thrombocytopoiesis in mice led to a significant reduction in
RBC production as evidenced by decreased hematocrits
and RBC counts and a lower or normal MCV.
Table 6 shows a summary of two additional experiments
in which increased thrombocytopoiesis was produced in
mice by making them acutely thrombocytopenic with an
injection of RAMPS, and measuring 48-hour 59Feincorporation into RBC 5 days later. As before, platelet counts and
TCPC were increased (P < .0005) and hematocrits
(P < .0005) and RBC counts (P < .005) were decreased.
MCV was not altered by the thrombocytopenia, but percent
59Fe-RBC incorporation was decreased in thrombocytotic
mice (P < .05), indicating that the anemia was probably
caused by reduced production of RBC and not due to
bleeding.
DISCUSSION
Several previous studies4-14have shown that long-term
hypoxia increases erythropoiesis with concomitant decreases in platelet counts, along with a reduction in the
number of recognizable megakaryocytes in the bone marrow and a reduced percent 35Sincorporation into platelets.
The hypoxia-induced thrombocytopenia was not believed to
be caused by increased sequestration of platelets by an
enlarged spleen or by expanding BV.3 Moreover, the effects
of h y p o ~ i a ~were
, ~ ~not
, ~directly
~
upon thrombocytopoiesis,
but required stimulated erythropoiesis for decreased thrombocytopoiesis to occur. An obvious inverse relationship4J4,22,23
between the number of platelets and hematocrits of mice was found in mice enclosed in hypoxic
atmospheres. We hypothesized that competition between a
precursor cell of the erythrocytic and megakaryocytic cell
lines causes the thr0mbocytopenia.3,~
The present study shows that large, chronic doses of EPO
administered to mice caused increased erythropoiesis and
decreased thrombocytopoiesis (Table 1).Conversely, when
mice were made acutely thrombocytopenic, by an injection
of specific antibodies to mouse platelets, increased thrombocytopoiesis was found with significant reduction in hematocrits and RBCs (Tables 4 through 6). Neither EPO nor
RAMPS caused significant BV changes in mice (Table 1).
Splenectomized mice administered EPO had significantly
Table 5. Effects of RAMPS on Platelet and RBC Production of Mice
Time After
RAMPS
Treatment
0
8h
Id
3d
4d
5d
6d
PIateI et
CountlpL
No. of
Mice
12
10
10
10
10
10
10
2
SE (
~lo-~)
0.90 2 0.02
0.00 f 0.00'
0.01 f 0.01'
0.50 f 0.01'
1.10 f 0.055
1.43 f 0.06'
1.22 f 0.02'
TCPC r SE
Hematocrit
1x10-8)
r SE ( O h )
12.78
0.02
0.10
6.70
17.02
20.02
18.04
f 0.36
f 0.01'
f 0.08'
0.09'
2 1.145
& 1.11'
f 1.10,
f
44.6
45.3
43.6
42.2
43.4
42.6
43.4
f 0.2
f 0.8t
f 0.7t
0.8$
f 0.5S
2 0.51
f 0.31
&
RBC
CountlpL
r SE ( x
7.46 f 0.05
7.70 f 0.16t
7.72 f 0.18t
7.32 f 0.21t
7.14 f 0.09S
7.03 2 0.17$
7.54 f 0.15t
RBC
MCV
r SE (pm3)
59.8
58.9
56.6
57.9
60.8
59.6
57.6
f 0.4
f 0.7t
f 0.7'
f 0.9S
f 0.3t
2
&
0.9t
1.0$
Values are presented as means f SE. RAMPS was injected IP at time 0 and platelet and RBC values were measured at times indicated above.
Values were significantly different from values of untreated mice.
' P < .0005.
t P is not significantly different.
SP < .05.
5P < .005.
MCDONALD, CLIFT, AND COTTRELL
356
Table 6. Effects of RAMPS on Platelet and RBC Production of Mice
Treatment
Untreated Mice
RAMPS-TreatedMice
Platelet countlpl f SEC
(x10-6)
0.92 f 0.03 (13) 1.42 f 0.06 (13)*
12.92 k 0.29 (13) 19.80 f 0.86 (13)*
TCPC 2 S E ( X ~ O - ~ )
45.9 f 0.3 (13)
Hematocrits & SE (YO)
43.3 f 0.4 (13)'
7.67 f 0.10 (13) 7.19 f 0.10 (13)t
RBCcount/pL f SE (x10-6)
59.9 f 0.6 (13)
MCV f SE (km3)
60.3 f 0.9 (13)*
% 59Fe RBC incorporation f SE
(%I
49.7 lr 1.6 (11)
41.2 2 2.9 (11)5
Values are presented as means f SE (number of mice/treatment).
RAMPS was injected IP and platelet and RBC production values were
measured 5 days later. Values were significantly different from values
of untreated mice.
*P < .0005.
t P < ,005.
SP is not significantlydifferent.
§P < .05.
increased erythropoiesis with decreased thrombocytopoiesis (Table 3), but not to the same extent as found in intact
mice. Therefore, the thrombocytopenia observed in EPOtreated mice could not have been entirely due to increased
BV or to an enlarged spleen. The data presented herein are
consistent with the stem cell competition hypothesis as the
cause of decreased platelet production in EPO-treated
mice.
Previous work showed that large, acute doses of EPO
would increase percent 35S incorporation into platelets of
mice' and increase platelet counts of rats2 In the former
work,' significant increases in both platelet sizes and
percent 35S incorporation into platelets were found after
injections of 15 U EPO/mouse. However, Birks et aI5
showed that the daily administration of 6 U of Step I sheep
plasma EPO to mice increased hematocrits, but no effect on
platelet counts was noted. In rats: acute doses of EPO (30
to 90 U over 4 to 5 days) caused a dose-dependent increase
in platelet numbers, a 2.4-fold increase in the frequency of
small acetylcholinesterase-positive cells within 1 day, and
increased mean megakaryocyte size within 2 days.
Short-term hypoxia, a stimulator of large amounts of
endogenous EP0,15 resulted in similar platelet and RBC
production in mice3 as was observed in mice treated with
EP0.I The results of mice enclosed in hypoxic chambers
showed increased platelet counts, as well as elevated TCPC
values, after 1 to 3 days of hypoxia, and splenic release did
not account for the increase in platelet counts. It seems
possible that the cause of increased platelet counts of mice
at 1 to 3 days of hypoxia was due to the presence of large
amounts of endogenous EPO in mice.15 However, continued hypoxia results in elevated erythropoiesis and decreased thrombocytopoiesis."6
Chronic doses of EPO (10 U administered two times
daily over a 2-day period and daily doses thereafter for 22
days) administered to rats2 resulted in elevated platelet
counts at 4 to 6 days, but the counts returned to slightly
below normal at 18to 20 days, despite continued administration of EPO. We show here that when EPO is administered
to mice (10 U administered two times the first day and two
daily injections of 5 U each for the next 6 days) significant
decreases were found in platelet counts and TCPC on day
7. Differences in responses to chronic EPO administration
in our work and that of Berridge et alz may be due to
differences in dosage of EPO per gram of body weight.
It is unclear how hypoxia causes a biphasic platelet
response in rodents, and how EPO will mimic this response.
However, Berridge et a12 reported EPO receptors on
megakaryocytes, supporting the conclusion that the early
platelet response could be due to direct stimulation of
megakaryocytes already in the recognizable compartment.
Then, as precursors are depleted or shunted into the
erythroid pathway, megakaryocytopoiesis decreases.
In the present study, splenectomized mice responded to
the same EPO dosage with lower RBC production (Table 3,
11% increase in hematocrits over controls) than did intact
mice (Table 1 showed a 28% increase over controls). It
should also be noted that a less severe degree of thrombocytopenia (14% v 22.4%) was observed in splenectomized
mice than in intact mice, supporting the conclusion of an
inverse relationship between erythropoiesis and thrombocytopoiesis.23 The reason why splenectomized mice differed
in these responses from intact mice is probably because in
mice the spleen is an active site of erythropoiesis and its
removal decreases the mouse's ability to make RBC.% This
reduced erythropoiesis leads to a less severe thrombocytopenia, a finding that is consistent with the stem cell
competition hypothesis .
The present work showed that injections of EPO did not
cause significant increases in BV in both intact and splenectomized mice, whereas significant increases in BV were
found in previous studies3 after hypoxia. It should be
mentioned that the degree of erythropoiesis and thrombocytopoiesis was less in EPO-treated mice compared with
those exposed to hypoxia. Both a change in BV and a
greater RBC and platelet response might have been seen if
mice had been administered a larger number of EPO
injections, or a constant infusion of EPO that would have
achieved a consistently higher EPO level as was found in
hyp0~ia.l~
However, 80 U EPO, administered in 2 SC
injections/d for 7 days (days 0 through 6) gave significantly
increased erythropoiesis with significantly decreased thrombocytopoiesis.
RAMPS-treated mice showed a marked increase in
platelet production that was accompanied with a reduction
in hematocrits and RBC compared with values of control
mice. In addition to increased platelet counts and TCPC in
the present study, previous work showed that RAMPS
would also increase platelet sizesz and percent 35Sincorporation into platelet^,'^ along with increases in the small
acetylcholinesterase-positive cells26and megakaryocyte size
and
The decrease in hematocrits and RBC
shown in the present study by RAMPS was most likely due
to decreased erythropoiesis, because decreased percent
357
EPO CAUSES THROMBOCYTOPENIA IN MICE
59Fe-RBC incorporation and lower MCV were seen.
RAMPS did not directly affect RBC (Table 5 ) and the mice
did not appear to bleed from the acute, severe thrombocytopenia, as evidenced by the lack of an early decrease in
hematocrits and RBC counts. It appears, therefore, that
increased thrombocytopoiesis in RAMPS-treated mice led
to decreased erythropoiesis resulting in anemia, a finding
that is consistent with the stem cell competition hypothesis.
In support of the stem cell competition hypothesis, Han
et aIz8 identified a population of hemopoietic cells that
coexpress glycophorin A and glycoprotein IIIa antigens
from both erythrocytic and megakaryocyticlineages. Other
workers have presented evidence that these lineages also
share common transcription factor~,~~,30
indicating additional similaritiesin cells of the erythrocyticand megakaryocytic cell lines.
In addition to competition between precursors of
megakaryocytic and erythrocytic lineages presented herein
and eIsewhere,4-l4a recent study in newborn rats31 showed
that very high concentrations of EPO resulted in reduced
production of neutrophils that accompanied accelerated
erythropoiesis. In disagreement with this finding, Petursson
and Chervenickl' found that hypoxia, a condition with
elevated EPO levels, resulted in a marked granulocytosis.
We are unaware of any other previous studies showing that
EPO will suppress any other cell lines, but the data
presented herein support the stem cell competition hypothesis as the cause of thrombocytopenia in both hypoxic and
EPO-treated mice. This hypothesis would also explain the
decrease in erythropoiesis that was found to accompany
increased thrombocytopoiesis.
Although the levels of EPO used in the present work
were higher than those currently used in treating patients, a
recent report shows that EPO treatment may exacerbate a
pre-existing thrombocytopenia in patients with multiple
myeloma.32It also seems possible that large, chronic doses
of EPO may decrease platelet counts in other patients.
ACKNOWLEDGMENT
The authors are grateful to Patricia Greeter for technical
support, to Wanda Aycock for excellent stenographic help, and to
Dr C.W. Jackson for helpful advice.
REFERENCES
strains of mice with different modal megakaryocyte DNA ploidies
1. McDonald TP, Cottrell MB, Clift RE, Cullen WC, Lin Fl(:
after exposure to hypoxia. Exp Hematol20:51,1992
High doses of recombinant erythropoietinstimulate platelet produc15. McDonald TP, Lange RD, Congdon CC, Toya RE: Effect of
tion in mice. Exp Hematol15:719,1987
hypoxia, irradiation, and bone marrow transplantation on erythro2. Berridge MV, Fraser JK, Carter JM, Lin F K Effects of
poietin levels in mice. Radiat Res 42151,1970
recombinant human erythropoietin on megakaryocytes and on
16. Emmanouel DS, Goldwasser E, Katz AI: Metabolism of
platelet production in the rat. Blood 72970,1988
pure human erythropoietin in the rat. Am J Physiol247168,1984
3. McDonald TP, Cottrell MB, Clift R E Effects of short-term
17. McDonald Tp:Bioassay for thrombopoietin utilizing mice in
hypoxia on platelet counts of mice. Blood 51:165,1978
rebound-thrombocytosis. Proc SOCExp Biol Med 144:1006,1973
4. Langdon JR, McDonald Tp: Effects of chronic hypoxia on
18. Cullen WC, McDonald TP: Comparison of stereologic
platelet production in mice. Exp Hematol5:191,1977
techniques for the quantification of megakaryocyte size and num5. Birks JW, Klassen LW, Gurney CW:Hypoxia-inducedthromber. Exp Hematoll4782,1986
bocytopenia in mice. J Lab Clin Med 86:230,1975
19. McDonald TP, Cottrell MB, Clift RE: Hematologic changes
6. McDonald TP, Cullen WC, Cottrell MB, Clift RE: Effects of
and thrombopoietin production in mice after X-irradiation and
hypoxia on the small acetylcholinesterase-positive megakaryocyte
platelet-specific antisera. Exp Hematol5:291,1977
precursor in bone marrow of mice. Proc SOCExp Biol Med 183:114,
20. McDonald TP, Jackson C W Thrombopoietin derived from
1986
human embryonic kidney cells stimulates an increase in DNA
7. Jackson CW, Edwards C C Biphasic thrombopoieticresponse
content of murine megakaryocytes in vivo. Exp Hematol 18:758,
to severe hypobaric hypoxia. Br J Haematol35:233,1977
1990
8. Cooper GW, Cooper B: Relationshipsbetween blood platelet
21. McDonald TP, Lange RD, Lail S, Cooper G: Erythropoietin
and erythrocyte formation. Life Sci 20:1571,1977
bioassays utilizing silicone rubber membrane enclosures. J Lab
9. McDonald TP: A comparison of platelet production in mice
Clin Med 70:48,1967
made thrombocytopenic by hypoxia and by platelet specific anti22. McDonald Tp: Platelet production in hypoxic and RBCsera. Br J Haematol40299,1978
transfused mice. Scand J Haematol20213,1978
10. Rolovic Z, Basara N, Biljanovic-Paunovic L, Stojanovic N,
23. Cottrell MB, Jackson CW, McDonald TP: Hypoxia increases
Suvajdzic N, Pavlovic-KenteraV:Megakaryocytopoiesisin experierythropoiesis and decreases thrombocytopoiesis in mice: A commentally induced chronic normobaric hypoxia. Exp Hematol 18:
parison of two mouse strains. Proc SOCExp Biol Med 197:261,1991
190,1990
24. Bogs DR, Geist A, Chervenick P A Contribution of the
11. Petursson SR, Chervenick P: Effects of hypoxia on megamouse spleen to post-hemorrhagic erythropoiesis. Life Sci 8587,
karyocytopoiesisand granulopoiesis. Eur J Haematol39:267,1987
1969
12. Cullen WC, McDonald TP: Effects of isobaric hypoxia on
25. McDonald TP: Effect of thrombopoietin on platelet size in
murine medullary and splenic megakaryocytopoiesis.Exp Hematol
mice. Exp Hematol8:527,1980
17246,1989
26. Kalmaz GD, McDonald TP: Effects of antiplatelet serum
13. McDonald TP, Cottrell M, Clift R: Effects of hypoxia on
and thrombopoietin on the percentage of small acetylcholinesterasethrombocytopoiesis and thrombopoietin production of mice. Proc
positive cells in bone marrow of mice. Exp Hematol9:1002,1981
SOCExp Biol Med 160:335,1979
27. McDonald TP, Kalmaz GD: Effects of thrombopoietin on
the number and diameter of marrow megakaryocytesof mice. Exp
14. McDonald TP, Cottrell MB, Steward SA, Clift RE, Swearingen CJ,Jackson CW: Comparison of platelet production in two
Hematolll:91,1983
358
28. Han Z, Bellucci S , Pidard D, Caen JP: Coexpression in the
same cell in marrow culture of antigens from erythroid and
megakaryocytic lineage, in Kaplan-Gouet C, Schlegal N, Salmon
Ch, McGregor J (eds): Platelet Immunology: Fundamental and
Clinical Aspects, vol 206. Paris, France, Colloque INSERM/John
Libbey Eurotext Ltd, 1991, p 17
29. Romeo PH, Prandini MH, Joulin V, Mignotte V, Prenant M,
Vainchenker W, Marguerie G, Uzan G: Megakaryocytic and
erythrocytic lineages share specific transcription factors. Nature
344:447,1990
30. Martin DIK, Zon LI, Mutter G, Orkin S H Expression of an
MCDONALD, CLIFT, AND COTTRELL
erythroid transcription factor in megakaryocytic and mast cell
lineages. Nature 344444,1990
31. Christensen RD, Liechty KW, Koenig JM, Schibler KR,
Ohls R K Administration of erythropoietin to newborn rats results
in diminished neutrophil production. Blood 78:1241,1991
32. Cazzola M, Ponchio L, Beguin Y, Rosti V, Bergamaschi G,
Liberato NL, Fregoni V, Nalli G, Barosi G, Ascari E: Subcutaneous erythropoietin for treatment of refractory anemia in hematologic disorders. Results of a phase 1/11 clinical trial. Blood 79:29,
1992