1. Art and Diseño

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Thrombopoietin in Thrombocytopenic Mice: Evidence Against Regulation at
the mRNA Level and for a Direct Regulatory Role of Platelets
By Ruedi Stoffel, Adrian Wiestner, and Radek C. Skoda
Thrombopoietin (TPO), originally described
as an activity in
the serum of thrombocytopenic animals that leads to increased production of platelets, has recently been isolated
and cloned. Its closest relative in the cytokine superfamily,
erythropoietin (EPO),istranscriptionally regulated during
anemia, and it was expected that TPO would similarly be
regulated during thrombocytopenia. We induced thrombocytopenia in miceandconfirmed
that TPO activity was
upregulated, as determined by a bioassay. Liver and kidney
were found to be the major sources of TPO mRNA. Surprisingly, TPO mRNA in these tissues was not upregulated in
thrombocytopenic mice. Using a sensitive RNase
protection
assay that can distinguishbetween TPO isoforms, we found
no changein theprofile of mRNA for these isoforms. A semiquantitative reversetranscription-polymerasechainreaction assayalso did not demonstrate upregulation of TPO
mRNA in the spleen. Thus, the increase of TPOactivity during thrombocytopenia is not caused by regulation at the
level of TPO mRNA. Furthermore, isolated mouse platelets
absorbed high amounts of bioactiveTPO out of TPO-conditioned medium in a dose-dependent fashion. Our results are
consistent with TPO protein being regulated at a posttranscriptional level andlor directly through absorption andmetabolism by platelets.
0 1996 by The American Societyof Hematology.
E
immediately mixed with EDTA, and blood counts were performed
with an automated blood counter (model Tecnicon H-3; Miles Inc,
Territown, NY). The remaining blood was allowed to coagulate, and
serum was collected for the TPO proliferation assay. Bone marrow
cells were prepared by flushing two femurs from each mouse with
phosphate-buffered saline (PBS). Viable, trypan blue-excluding bone
marrow cells were counted. For morphologic examination, bone
marrow cells were concentrated on microscopic slides using a Shandon Cytospin 3 centrifuge (Life Science International, Ostmore, UK)
and stained with Wright stain. For histology, freshly dissected tissues
were fixed in Optimal Fix (American Histology Reagent CO, Stockton, CA). Fixed specimens were embedded in paraffin, sectioned,
and stained by the Transgenic Pathology Laboratory at the University
of California at Davis, CA.
Construction of plasmid vectors. TPO cDNA was generated by
reverse transcription-polymerase chain reaction (RT-PCR) using
first-strand cDNA synthesized from mouse liver RNAusingthe
sense primer 5’-TCGAAGCTTGGCCAGAATGGAGCTGACTG3‘ and the antisense primer 5’-ATAAGATCTGCGCTATGTTTCCTGAGACA-3’. The fragments were subcloned into pBluescript
KS (Stratagene, La Jolla, CA) and completely sequenced. For expression in COS cells, the full-length TPO cDNA was cut with
Hind111 and Bgl I 1 and subcloned into the pcDNAl vector (Invitrogen, San Diego, CA). For stable transfections, mouse TPO and
c-mpl cDNAs’ were subcloned into the pGD expression vecto?’ as
an XhoI-Not1 or BclI fragment, respectively.
RNA isolation, ribonuclease protection assay, and RT-PCR analysis. Tissues were homogenized in 4 mom guanidium isothiocyanate with a Polytron homogenizer, and RNA samples were prepared
by the acid phenol method.24For ribonuclease (RNase) protection
ARLY EXPERIMENTS have determined that the physiologic changes occurring in response to acute thrombocytopenia are mediated by a humoral factor called thrombopoietin (TPO).’ Such changes include increases in
megakaryocyte number, size, and ploidy and will result in
increased production of platelets. These experiments have
determined that TPO activity is inversely related to platelet
mass and have suggested that a feedback mechanism exists
that can sense a decrease in platelet mass and cause a reciprocal increase in circulating TPO a~tivity.’.~Recently, the orphan cytokine receptor c - m ~ l was
~ . ~used as a reagent to
isolate and clone a ligand that had biologic activities resembling TP0.1°”2 Several lines of evidence indicate that this
ligand is, in fact, TPO: the purified mpl-ligand is a potent
stimulator of thrombopoiesis in vivo,13 and soluble c-mpl
receptor can abrogate TPO activity.I4 Using bioassays for
TPO, two other groups independently purified and partially
sequenced the TPO protein and found it to be identical to
the sequence of the mpl-ligand.’53’6
TPO activity produced
by human embryonic kidney (HEK) cells is also identical to
mpl-ligand.”
In analogy to the transcriptional activation of erythropoietin (EPO) mRNA in response to anemia,18-” the increased
levels of circulating TPO protein during thrombocytopenia
may be due to upregulation of TPO mRNA. Alternatively,
a model has been proposed in which TPO production is
constant, and TPO activity is regulated by the binding and
metabolism of TPO by platelets,” which express c-mp1,22
the TPO receptor. Here we describe experiments that support
the latter model.
MATERIALS AND METHODS
Inductionof thrombocytopenia in mice. C57BW6.l mice were
purchased from BRL, Fiillinsdorf, Switzerland. Twelve mice were
injected intraperitoneally with 0.1 mL of antiplatelet serum generated
in rabbits (gift from Dr Jack Levin, University of California, San
Francisco, CA). Groups of three mice were killed after 4 hours, 24
hours, or 48 hours, and tissues were removed for analysis. As an
alternative method, pancytopenia was induced in six mice by total
body irradiation (TBI) with 8 Gy. Groups of three mice were sacrificed after 6 days and 9 days. The experiments have been approved
by the local animal welfare committee.
Blood and tissue analysis. Blood was obtained by cardiac puncture without anticoagulants. Approximately 300 pL of blood was
Blood, Vol 87, No 2 (January 15), 1996: pp 567-573
From the Department of Pharmacology, Biozenrrum of the University of Basel, Basel; and the Division of Hematology, Department
of Research, University Hospital, Basel, Switzerland.
Submitted April 26, 1995; accepted August 30, 1995.
Supported by Grants No. 31-37760.93 and 32-35.503.92 (to
R.C.S.) and 3135-040025.94 (to A. W . )from the Swiss National Science Foundation.
Address reprint requests to Radek C. Skoda, MD, Department of
Pharmacology, Biozentrum, University of Basel, Klingelbergstrasse
70, CH-4056 Basel, Switzerland.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section I734 solely to
indicate this fact.
0 1996 by The American Society of Hematology.
0006-4971/96/8702-0$3.00/0
567
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568
analysis:' we constructed a riboprobe for the detection of all of the
known TPO mRNA isoforms by subcloning a 398-bp Sal I-Sca I
fragment of mouse TPO cDNA into pBluescript. The resulting vector
was digested with XhoI, transcribed with l7 RNA polymerase, and
hybridized to 30 pg of total RNA at 50°C as described?' This riboprobe protects a 398-nucleotide (nt) fragment for TPO-I mRNA. In
addition, a 310-nt fragment can be detected for TPO-3, a 240-nt
fragment for both TPO-2 and TPO-4 isoforms, and a 147-nt fragment
for TPO-2. If genomic DNA was present in the RNAs, fragments
of 168, 160, and 72 nt would be generated, because two introns are
present in the genomic region spanned by this riboprobe. RNA loading was normalized with a riboprobe for mouse hypoxanthine-guanine phosphoribosyl transferase (HPRT), a housekeeping gene. A
Sca I-HindIll fragment representing nucleotides 679 to 840 of mouse
HPRT cDNAZ6was subcloned into pBluescript. This riboprobe protects a 161-nt fragment. HPRT riboprobe was mixed with the TPO
probe and added as an internal standard to each sample. Protected
fragments were separated on 6% polyacrylamide/8 moVL urea sequencing gels. Dried gels were exposed on film or on phosphorimager screens, and quantitations of radioactive bands were performed
on a PhosphorImager 425 using the ImageQuant software (Molecular
Dynamics Inc, Sunnyvale, CA).
For RT-PCR analysis, oligo(dT)-primed first strand cDNA was
synthesized from 2 pg total RNA using RNaseH- MuLV reverse
transcriptase (Stratagene), in a reaction volume of 20 pL under
conditions recommended by the manufacturer. This reaction mixture
was diluted to 50 pL with HzO, heated to 95°C for 5 minutes to
inactivate reverse transcriptase, and then quickly chilled on ice for
10 minutes. PCR was performed with 2 pL of the first strand cDNA
as a template in a final reaction volume of 20 pL. The reaction was
performed in 50-mmoy1 KCI, 1.5 mmoVL MgClz, 20 mmoVL Tris
pH 8.3, 0.25 mmoVL deoxynucleotide triphosphates (dNTPs), 0.75
p m o K of each primer, and 0.05 UIpL Taq polymerase (Life Technologies, Gaithersburg, MD). The primer pair for mouse TPO was
5"GTCTATCCCTG'ITCTG-3'
(forwad)
and 5"CAACAATCCAGAAGTCCT-3' (reverse), amplifying a 608-bp product, and
(forward) and 5'for HPRT, 5'-GCTGGTGAAAAGGACCTCT-3'
CACAGGACTAGAACACCTGC-3' (reverse), amplifying a 249-bp
product.*' We performed 30 cycles for TPO and 28 cycles for HPRT,
each consisting of 60 seconds at 94"C, 60 seconds at W C , and 60
seconds at 72°C using a DNA thermal cycler (Perkin Elmer, Norwalk, CT). The PCR products were electrophoresed on 1.5% agarose
gels and then transferred to Hybond N+ membrane (Amersham,
Buckinghamshire, UK). Blots were probed with internal 3ZP-labeled
oligonucleotides forTPO 5'-AGGACTTCTGGAITGTTG-3' or
HPRT 5'-GATATGCCC'ITGACTATA-3'.The hybridizations were
performed overnight at 55°C in 7% sodium dodecyl sulfate (SDS),
1 mmoK EDTA, 0.5 m o m sodium phosphate buffer pH 7.2, and
1% bovine serum albumin (BSA), and the blots were washed three
times for 15 minutes with 6X saline sodium citrate (SSC), 0.1%
SDS at 55°C.
Cell transfections and proliferation assay. Transient transfections of COS cells with the pcDNA1-TPO expression vector were
performed by the diethyl aminoethyl (DEAE)-Dextran method." To
generate a cell line responsive to TPO, BaF3 cells were electroporated with 40 pg of pGD-mpl DNA at 250 V/960 pF in PBS and
plated in serial dilutions. Clones were selected in 0.6 mg/mL G418
beginning at 24 hours, and (3418-resistant clones were assayed for
expression of mpl protein by Western blot using polyclonal rabbit
anti-mpl antibodies? BaF3/mpl clone TM17 expressed the highest
level of mpl protein and grew well in conditioned medium from
transiently transfected COS cells secreting mouse TPO. This clone
was chosen for the TPO proliferation assays. To assess the levels
of TPO in serum from thrombocytopenic mice, TM17 cells were
washed out of interleukin (IL)-3-containing medium, incubated for
STOFFEL,WIESTNER, AND SKODA
16 hours in medium without L-3, and plated in 96-well plates at
IO4 cells per well in 100 pL of medium containing dilutions of
mouse serum. After 22 hours, 1 pCi of 'H-thymidine was added to
each well, and incorporation of 'H-thymidine was measured after 6
hours in a &counter. Alternatively, XTT,a colorimetric tetrazolium
dye," was used to determine TPO activity in conditioned media.
Cells (5 X IO' per well) were seeded, and after 3 days of stimulation,
50 pL of a 1 mg/mLstock solution of XTT with 5 mmoVL phenazine
methosulfate (PMS), an electron coupling agent, was added to each
well. The product of X?T reduction by viable cells, reflecting the
number of cells per well, was measured at 4 hours at 450 nm. A
stable cell line producing mouse TPO was generated by electroporating NIW3T3 cells with a pGD-TPO expression construct followed
by selection in 0.5 mg/mL G418 as above. TPO-secreting clones
were identified using the TM17 proliferation assay.
Ahsorption of TPO by platelets. Mouse platelets were isolated
from five mice as de~cribed.~'The platelets were washed with PBS
and counted in a Neubauer chamber. The automated platelet count
of the purified platelet preparation was 166 X lO'/pL, with no detectable white blood cells (WBCs) and no red blood cells (RBCs).
In the stained cytospin of this purified platelet preparation with
approximately 500,000 platelets, we counted a total of 108 RBCs
and no WBCs. Specified numbers of platelets were then pelleted for
5 minutes at 1,300g and incubated while rotating for 1 hour at 37°C
with 50 pL of conditioned medium containing TPO or IL-3. Platelets
were removed by 5 minutes of centrifugation at 1,300g. and TPO
activity of the supernatants was assessed by the BaF3lmpl cell proliferation assay.
RESULTS
To examine the effects of thrombocytopenia on the levels
of TPO mRNA, we injected mice intraperitoneally with 0.1
mL of rabbit anti-mouse platelet serum (RAMPS). Groups
of three mice were killed at various times and analyzed (Fig
1A). After 4 hours, the platelet counts decreased more than
40-fold to a mean of 22 X 103/pLand remained low at 24
and 48 hours. Two of the three mice killed after 24 hours
had normal platelet numbers (not shown). These two nonresponders were not further examined. To assure that the animals were killed at the relevant time, we also determined
TPO activity in serum. We used a proliferation assay with
BaF3/mpl cells that express the mouse c-mpl and proliferate
in response to TPO. Only the transfected BaF3/mpl cell line
responded to the sera from thrombocytopenic mice, but not
the parental untransfected BaF3 cells. Proliferation of BaF3/
mpl cells was measured by incorporation of 3H-thymidine
(Fig 1A). The values for the untreated controls were indistinguishable from background incorporation into BaF3/mpl
cells in TPO-free medium. Thus, serum TPO concentrations
in normal miceare below the limit of detection of this assay.
TPO increased to measurable levels after 4 hours and was
elevated at 24 and 48 hours. RAMPS does not lead to destruction of megakaryocyte^.^' This was confirmed by cytospin analysis of bone marrow cells and histopathology of
the spleens from these mice (not shown). The average numbers of megakaryocytes per spleen section increased from
30 in the controls, and 33 at 4 hours, to 52 at 24 hours, and
to 109 at 48 hours (not shown).
To examine if megakaryocyte mass is important for the
regulation of TFQ mRNA, we also analyzed mice pancytopenic after T B 1 with 8 Gy. Platelet levels in mice treated with
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569
THROMBOPOIETINREGULATION
*'"l
1
T
0
0
4
24
40
6
9
hours
days
Fig 1. Platelet counts and serum TPOactivity in thrombocytopenic mice. Groupsof three micewere treated with RAMPS (A) or B Gy
TBI (B). Only one mouse was analyzed at 24 hours after RAMPS.
Solid line, platelet counts; broken line, 'H-thymidine incorporation
into BaF3/mpl cells. Mean values f SEM are given.
TB1 decreased slowly and reached a mean of 43 X 103/pL
at day 9 (Fig 1B). Conversely, TPO levels were measurable
at day 6 and increased further at day 9. No megakaryocytes
were found on histopathologic examination of spleens at
days 6 and 9 (not shown).
We devised a sensitive RNase protection assay that can
distinguish between four TPO isoforms (Fig 2). The TPO
riboprobe spans the region between nucleotides 330 and 728
of the mouse TPO cDNA sequence." RT-PCR from mouse
liver RNA yielded the full-length TPO cDNA (TPO-1) and
two shorter isoforms, TPO-2 and TPO-3, that have been
described p r e v i o ~ s l y . ~In
* *addition,
~~
we found a fourth isoform, TPO-4, which has a deletion of 197 bp from position
569 to 766 of the mouse TPO cDNA." We analyzed expression of the TPO isoforms and found the highest levels in
liver and kidney and smaller amounts in brain and testes
(Fig 2A). As an internal standard for RNA loading, a riboprobe for HPRT, a housekeeping gene, was added together
with the TPO riboprobe to each sample. The ratios between
the TPO isoforms in different tissues were constant, and
TPO-2 was the most abundant isoform (Fig 2B and Table
1). With this RNase protection assay, we determined TPO
mRNA levels in liver and kidney from thrombocytopenic
mice (Fig 3). Although TPO activity was elevated in serum
(Fig lA), TPO mRNA abundance did not increase in mice
treated with RAMPS (Fig 3A). Furthermore, mRNA for the
TPO isoforms also remained constant. The same result was
found in mice treated with 8 Gy TB1 (Fig 3B).
As megakaryopoiesis in mice occurs mainly in bone marrow and spleen, small amounts of P O produced in these
organs may regulate megakaryopoiesis in a paracrine fashion. By RNase protection assay, TPO was not detectable in
30 pg total RNA from spleen or bone marrow (Fig 2A) or
in 8 pg of polyA+ RNA from spleen (Fig 2B). Therefore,
we analyzed expression of TPO by RT-PCR. TPO mRNA
was detectable in bonemarrow.However,
the transcript
seems to be present at very low abundance, becauseamplification wasnot reliable despite consistant amplification of
the HPRT transcript in the same samples (not shown). In
contrast, TPO was amplifiedconsistently from spleen. Therefore, we assessed the expression of TPO in spleens from
thrombocytopenic mice (Fig 4). We used 30 cycles of PCR
with the TPO primers and normalized the results with the
PCR products obtained with HPRT primers after 28 cycles.
We could not detect any significant increase in TPO mRNA
in spleens during thrombocytopenia inducedwith either
RAMPS or TBI.
We examined if platelets can remove TPO activity when
preincubated with TPO using the BaF3/mplcell line (Fig 5).
To control for any inhibitory activityreleased during the
incubation with isolated platelets, we used IL-3-containing
WEHI-3 conditioned media that were treatedthe same way.
We observed a dose-dependent decrease in TPO activity in
supernatants incubated with mouse platelets. IL-3 activity
was unaffected under the same conditions, and no inhibition
wasobserved.Untransfectedparental
BaF3 cells did not
respond to the TPO-conditioned media (not shown). To exclude the possibility that platelets incubated in mediumcontaining calcium and other platelet-activating agents may release proteases to which TPO might be more sensitive than
to IL-3,weperformed
the following control experiment.
Purified platelets were incubated with medium for 1 hour.
This medium was then separated from platelets by centrifugation, mixed 1:l with TPO or IL-3-containing conditioned
media to give a final concentration of 2.5%, and assayed for
activity. No decrease in activity of either TPO or IL-3 was
observed, indicating that the decrease in TPO activity after
incubation with platelets is not due to release of proteases
or other agents that interfere with theTPO assay (notshown).
Therefore, platelets bind TPO, presumablythrough mpl, and
may be involved in regulating the free circulating TPO concentration.
DISCUSSION
We have analyzed TPO mRNA levels during thrombocytopenia in mice and have found no upregulation despite a
measurable increase of TPO activity in serum (Fig 1). EPO,
the closest homologue of TPO, can be upregulated more
than 100-fold at the mRNA leve1,'8-20 and it was suspected
that a similar mechanism would exist for TPO. Liver and
kidney, which express the highest levels of TPO (Fig 2),
were examined by an RNase protection assay (Fig 3). With
the same RNase protection assay, we tested the possibility
that TPO isoforms, which are believed to be generated by
alternative or aberrant splicing, mightplaya role inthe
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STOFFEL,WIESTNER, ANDSKODA
570
A
B
B
TPOl
-
--
T P 0 3 -+
TPO 2+4
TPO 1
TPO 3
+
TPO 2+4
+
TP02
-D
.
Fig 2.
HPRT
-+
C
5‘
intrcn
SD
3’
Analysis of TPO mRNA expression in
mouse tissues by RNase protection assay. (A) RNA
loading was normalized by adding
an HPRT riboprobe togetherwith theTPO riboprobe t o each sample. The band present in all lanes below TPO 2 + 4
represents the undigested HPRT probe. (B) Tissues
expressing TPO were analyzed with the TPO riboprobe alone t o visualize the 147-nt band specific for
TPO-2, which in (A) wasobscured by theHPRT band.
(C) Position of theTPO riboprobe in respect t o TPO
cDNA. Open box, region encoding the €PO-like domain; hatched box, region encoding the C-terminal
glycosylated domain; SD, cryptic splice donor site
used t o generate TPO-3. The length and position of
protected fragments for the TPO isoforms are indicated.
;
riboprobe
I
TPO 1
TPO 2
-+
1
-
0.1 kb
400 nt
240 + 147 nt
TPO 3 I
310nt
TPO 4
240 nt
regulation of TPO activity (Fig 3A and B). Two TPO isoforms have been described. TPO-2 has a deletion of the four
amino acids LPPQ at position I 12 to I 15, and TPO-3 is
produced by an internal splice in the last exon..’*”’ The proteins for TPO-2’’ and TPO-3” were expressed but not secreted by transfected cells lines, suggesting that these proteins are retained in the secretory pathway. As these isoforms
are conserved between humans, pig, and mouse,32.33
it was
suspected thattheymightplay
a regulatory role. Interestingly, no splice variants have been described for EPO, which
shares a highly homologous gene structure with TP0.34However, we found no changes in mRNA levels for TPO-2 or
TPO-4 and TPO-3 during thrombocytopenia (Fig 3Aand
B). Therefore, regulation of TPO does not occur at the
mRNAlevel
in liver andkidney and does not involve
changes in the ratios of TPO isoform mRNAs.
Because we found no expression of TPO in spleen and
bone marrow by RNase protection (Fig 2) and these organs
Table 1. Relative Amounts of TPO lsoforms in Mouse Tissues
Liver
100
TPO-1
TPO-2
TPO-3
TPO-4
100
44
7
9
Kidney
100
28 52
3
7
Testis
Brain
100
28
9
5
6
5
Radioactive bands from Fig 2A and B were quantified witha phosphorimager. The relative abundance of TPO isoforms isexpressed as
percent of TPO-l for each organ. The values were normalized with
the number of uridines in the protected fragments. Values for TPO-4
were calculated by subtracting TPO-2 from TPO-2 + 4.
are the sites of megakaryopoiesis in mice, upregulation of
TPO produced in situ in a paracrine fashion could have a
major effect on megakaryopoiesis. By RT-PCR, we were
able to detect TPO mRNA in spleen (Fig 4) and, less reliably,
also in bone marrow (not shown). However, there was no
significant increase of TPO mRNA detectable by RT-PCR
(Fig 4). Although this assay is not as quantitative as RNase
protection, we should have been able to detect a 5- to 10fold mRNA increase. Plasma from thrombocytopenic animalswas sufficient to induce accelerated plateletproduction,3s and purified thrombopoietin caused an up to fivefold
increase in platelet count when injected into normal recipients.’.’ This demonstrates that TPO is a potent humoralstimulator. As the levels of expression were verylowandwe
could not detect upregulation, the physiologic importance of
locally produced TPO in the spleen remains unclear.
It has been proposed that platelets might be directly involved in the regulation of circulating TPO activity.*’Platelets express mpl protein.” Purified fractions of megapoietin,
which was shown to be identical with TPO, lostactivity
whenfirst incubated with sheep platelets andtestedafter
removal of platelets by centrifugation.*’Megapoietin activity
was assessed measuring increase in megakaryocyte ploidy
of isolatedrat megakaryocytes. Weusedtheproliferation
assay with BaF3/mpl cells to more directly measure TPO
exposed to various concentrations of isolated mouse platelets
(Fig 5). When TPO-conditioned media were exposed to 1 X
I Oh platelets per microliter, the optical density 450-nm reading, reflecting the number of proliferating BaFYmpl cells,
decreased by 40% (Fig 5). As in this range thedose response
curve of the assay is linear with the logarithm of the TPO
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THROMBOPOIETIN REGULATION
57 1
kidney
liver
A
TPO 3
+
"
"
"
"
TPOl
100*20 70*10
100*30 80220 120130*40
TP03
TPO2+4
5
10*5
20 20f10
20k10
10*5
60*20 50*20 70 60k20
60*10
50k10 40 30t10
10*5
1025
liver
B
60 60*20
kidney
d9
CO d6
"
C
d9
O d6
"
TPO 1 -e
Fig 3. Determination of TPO mRNA levels in liver
and kidney from thrombocytopenic mice. TPO isoforms were detected by RNase protection analysis
total
of
RNA. The bands were
quantified
on
a phosphorimager andare expressed in arbitrary units. The
values for TPO-1 in the controls wereset t o 100 for
each organ. The mean values from
three
mice
? SEM
are given except for RAMPS at 24 hours postinjection. CO, untreated controls. (A) Numbers
above the
lanes indicate timein hours after injection
of RAMPS.
(B) Mice treated with 8 Gy TBI: d6 and d9, days 6
and 9 after irradiation, respectively.
TPO 3
TPO 2+4
-D
-
m
"
HPRT -e
"
TPOl
TPO2+4
TPC
140 150
f 10
50
f 20
W
100*30
40k10
10k2
20i10
-
6d
9d
100
*30
40t10
90e30
90*20
10f4
1Of1
40f10
40f10
of platelets to downregulate the free serum TPO concentration. The capacity of mouse platelets to adsorb TPO in our
assay appears to be higher than the calculated binding capacity of sheep platelets." However, these results are not directly comparable, because the assays used to measure TPO
were not the same. Isolated platelets may be activated and
display a higher binding than platelets under physiologic
conditions.
We show that the increase in TPO activity during thrombocytopenia isnotmediated
by changes in TPO mRNA
abundance. Our data is consistent with TPO regulation at a
translational or posttranslational level and/or with regulation
0"
-- --
"---
"
"
-
________I
TPO 100
SEM *20
.
-
"
-
"
"
" 0 .
HPRl
,.
.P
A
100k30 20f10
60f30
800 rad
RAMPS
-
.
10f3
10k5
5k1
TP03
10k3
concentration, this represents removal of more than 40% of
TPO activity. Using the more sensitive "-thymidine assay,
we measured an incorporation in the range of 6,000 cpm
with thrombocytopenic sera (Fig 1) and 60,000 cpm with
saturating concentrations of 5% TPO-conditioned medium
(not shown). Because the values measured with our thrombocytopenic sera are in the region where the curve becomes
nonlinear, we cannot accurately assess the TPO concentration. However, we can make an approximate estimate and
find that our TPO-conditioned medium at 2.5% contained
at least a 100-fold excess of TPO activity compared with
thrombocytopenic sera. These results demonstrate the ability
CO
4824 4
""
I
120
130
f 10
k50
Fig 4. Determinationof TPO mRNA levels in
spleens from thrombocytopenic mice. TPO mRNA
was detected by RT-PCR and compared with HPRT
mRNA. Autoradiograms Southern
of
blots
hybridized with 32P-labeledTPO, and HPRT-specific internal
oligonucleotides are shown. CO, untreated controls;
numbers above the lanes indicate timein hours after
injection of RAMPS; d6 and d9, days 6 and 9 after 8
Gy irradiation; -, notemplate DNA. The bands were
quantified on a phosphorimager and are expressed
in arbitrary units. The values for TPO-1 in thecontrols
were set t o 100. The mean values from
three
mice
? SEM are given, except for RAMPS at 24 hours postinjection.
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572
STOFFEL, WIESTNER, AND SKODA
120,
control
0.02
0.1
0.5
1
x106/vl
Fig 5. TF'O activity insupernatantsafterpreincubation
with
mouse platelets. CondRioned medie containing mouse TF'O or IL-3
(WEHI-3) were incubated for l hour with increasing concentrations
of isolated mouse platelets. After centrifugation, the supernatants
a proliferationassay with BaF3/mpl cells.Proliferation
were tested in
of BaF3/mpl cells was assessed measuringthe product of X l T reduction at 450 nm. The optical density at 450 nm reading for TPOor
WEHI-3 conditioned medianot exposed to platelets wasset as 100%.
Solid bar, lMEHl-3-conciitioned media; open bar, TPO-conditioned
media. The data represent the mean of duplicates f SEM.
directly through metabolism by platelets. A number of other
steps of TPO biosynthesis and metabolism may be important
as well, as will be determined through detailed studies of
the TPO protein in the future.
ACKNOWLEDGMENT
We thank Michael V. Wiles for help with the TB1 andmany
helpful discussions, Robert D. Cardiff for reviewing the histopathology, Jack Levin for the antiplatelet antiserum, Andr6 Tichelli for
the automated blood counts, and David C. Seldin for helpful comments on the manuscript.
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1996 87: 567-573
Thrombopoietin in thrombocytopenic mice: evidence against
regulation at the mRNA level and for a direct regulatory role of
platelets
R Stoffel, A Wiestner and RC Skoda
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