1. health and fitness
2. disease

Effects of Recombinant Human Megakaryocyte Growth and Development
Factor on Platelet Activation
By Giuseppe Montrucchio, Maria F. Brizzi, Giovanni Calosso, Stefania Marengo, Luigi Pegoraro,
and Giovanni Camussi
The c-Mpl receptor for thrombopoietin and its recombinant
related protein, the megakaryocyte growth and development factor (MGDF), is also expressed on circulating platelets. In the present study we evaluated the effect of MGDF
on platelet aggregation in platelet-rich plasma (PRP) and in
whole blood. The results obtained indicate that MGDF by
itself did not affect platelet aggregation. However, when
added before other agonists suchasadenosinediphosphates (ADP), epinephrine (EPI), and thrombin (THR), it rendered platelets more sensitive. This "priming" effect of
MGDF wasdependent on the dose and on the timeof platelet preincubation, and it occurred both inPRP and in wholeblood platelet aggregation. MGDF also "primed the release
of adenosine triphosphates and the production of thromboxane B, by platelets stimulated with ADP, EPI, and THR. When
added 15 seconds after the preincubation of platelets with
subthreshold concentrations ofADP,EPI, and THR, MGDF
exhibited a synergism with these agonists. Moreover, we
observed a "priming" effect of MGDF on the phosphorylation of p-42 mitogen-activated protein kinase promoted by
ADP, EPI, and THR. These observations suggest that thrombopoietin may play a physiologic role in modulating the response of platelets to several stimuli and thereby their hemostatic potential.
0 1996 by The American Society of Hematology.
P
tors were expressed onhuman platelets.('.'('
Therefore, in light
of the potential clinical use of the MGDF, the question of
whether it may affect platelet functions arises."
In the present study we evaluated the effect of MGDF on
plateletaggregationinplatelet-rich
plasma(PRP)and
in
whole blood. The results obtained indicate that platelet activation in response tocertain agonists was enhancedon exposure to MGDF. This observation suggeststhat thrombopoietin serves a spectrum of functions beyond the maturation of
megakaryocytic lineage, including modulation of hemostatic
properties of platelets.
HYSIOLOGIC PRODUCTION of plateletsis thought
to require atleast two humoral growth factors
that regulate proliferation and maturation of megakaryocytes.' One
is a megakaryocytecolony-stimulating factor (meg-CSF),
which induces the proliferation and differentiation of megakaryocyteprogenitors.',2Thesecond
isthrombopoietin,a
novel megakaryocyte growth and development factor,
which
promotes the maturation of megakaryocyte^.^.^ It has been
suggested that meg-CSF and thrombopoietin regulate megakaryocyte maturation by binding to the c-Mpl, a member of
thehematopoietic receptorfamily encoded by theprotooncogene c-mpl."'
After cloning of human and murine complementary DNA
for thrombopoietin,""'4 several groups have investigated in
vitro and in vivo the biologic properties of the recombinant
megakaryocyte growth and development factor (MGDF), a
to thrombopoietin.TheMGDF
hasbeen
proteinrelated
shown to bind the c-Mpl re~eptor'""~
and to stimulateplatelet
production when injected in mice'""3 and in nonhuman primates.I5
Recently, it has been reported that thrombopoietin recep-
From the Dipartimento di Fisiopatologiu Clinicu, the Dipartimentvdi Scienze Biomediche e Oncologia Umana, Universita di
Torino; and Istituto di Medicina e Sanita Pubblica, 11 Fucolta di
Medicina, Universita di Paviu, Varese, Italy.
Submitted June 12, 1995; accepted November 10, 1995.
Supported by the National Research Council (CNR), Targeted
Project "Prevention and control of disease factors," Subproject
" C a u ~ e of
s infective diseases" (CT 95.00778.PF4) and the Associazione Italiana per la Ricerca sui Cancro (to G.C.) and by the Targeted Project "Clinical applications of oncologic research, Subproject "Selective Therapies" and the Associazione Italiana per la
Ricerca sul Cancro (to L.P.).
Address reprint requests to Giovanni Camussi, MD, Cattedra di
Nefrologia, Istituto di Nefro-Urologia, Ospedale Maggiore S. Giovanni Battista, Corso Dogliotti, 14, 10126 Torino, Italy.
The publicution costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordunce with 18 U.S.C. section 1734 solely to
indicate this fuct.
0 1996 by The American Sociev of Hematologv.
"
0006-4971/yh/8707-0003$3.00/0
2762
MATERIALS ANDMETHODS
Reagents. RecombinanthumanMGDFwasagiftof
Dr J.L.
Nichol (Amgen Inc, Thousand Oaks, CA). Adenosine diphosphates
(ADP)andbovinethrombin(THR)wereobtainedfromSigma
Chemical CO (St Louis. MO). Epinephrine (EPI) was obtained from
Helena Laboratories (Beaumont, TX). Luciferin and luciferase were
purchased from Mascia Brunelli (Milano, Italy). The ELISA kit for
thromboxane Bz (TXB2) determination was purchased from Biotrac
(Amersham International, Amersham, UK). Polyclonal and monoclonal anti-p42""'k antibodies and antiphosphotyrosine monoclonal
antibodywerefrom Affinity ResearchProductsLtd(Nottingham,
UK).
Plateletpreparation und aggregation studies. Venous blood was
obtained by venipuncture from healthy adult donors (age range, 24
to 30) that had not taken any medications for at least 2 weeks. Nine
volumes of blood were withdrawn in 1 v01 of 3.8% trisodium citrate.
PRP was prepared by centrifugation for 15 minutes at 18Og. Plateletpoor plasma (PPP) was obtained by centrifugation at 2,000g for 10
minutes.I8 Platelet aggregation in PRP was performed as previously
described,Ix according to Born's method, at 37°C with constant rate
of stimng at1,000 rpm in the lumi-aggregometer (Chronolog, Haveras reference. The maximal aggregation was
town, PA) using PPP
quantified according to the Weiss formula."
Platelet aggregation in whole blood was assessed in whole-blood
lumi-aggregometer (Chronolog) by recording the increase in electrical impedance in I - m L sample of blood diluted onefold with Tyrode's buffer containing 2 U/mL of heparin and stirred at1,000 rpm
at 37"C.z" Thesystem was calibrated so that a 5-ohmchange in
impedance corresponded toa 20-mm deflection of the pen. Aggregation was quantified as ( I ) the surface area under the curve relating
electric impedance to time (ohms per seconds),(2) maximum amplitude (ohms). and (3) maximum rate of increase (ohms per seconds)
Blood, Vol 87, No 7 (April l), 1996:pp 2762-2768
THROMBOPOIETIN AND PLATELET ACTIVATION
6 minutes after addition of aggregating agents.” Similar results were
obtained using either methods.
PRP or whole-blood samples were preincubated with MGDF (0.25
to 120 ng/mL)directly in the lumi-aggregometerfor variable periods
of time at 37°C. before a second agonist was added. The following
agonists were used: ADP (0.1 to IO pmol/L), EPI (0.8to 24 pmoll
L), andTHR (0.02 to 1.2UlmL). The “priming” index (PI) was
calculated according to Gay’* as the responseto MGDF and agonists
together divided by the sum of the individual stimulus response. By
this calculation a PI > I .0 indicated synergism, a PI = 1 indicated
additive response, and PT < I indicated inhibition.22Moreover, the
synergic effect of MGDF on ADP, EPI, and THR was evaluated
by usingsubthreshold concentrations oftheseaggregating agents
followed, 15 seconds later, by the addition of MGDF.
When platelet aggregationin PRP and whole blood were measured
repeatedly the variation versus the previous measurement was, respectively, within 5% and 8%.
In selected experiments luciferin-luciferase reagent was added to
PRP 60 seconds before the addition of aggregating agents.” Luminescence caused bythe release of adenosine triphosphates (ATP)
during aggregationwascomparedwiththeluminescenceinduced
by 2 nmol of ATP standard as described by Feinman et al.’3
Measurement ofthromhoxane B? (TXB?)production. The generation of TXBz by stimulated platelets in PRP was evaluated as previously described.2JBriefly, at the indicated time after the addition
of agonists the reaction was stopped by addition of 5 pmol/L indomethacin, platelets were centrifuged at 12,000g for 2 minutes, and
plasma was removed and stored at-60°C. After appropriate dilution
TXBz levels were determined by ELISA.
Western blot analvsis and immunoprecipitationstudies. Platelets
(4 X IOx/ mL) were incubated at 37°Cfor 5 minutes with or without
ADP (0.5 pmol/L), EPI (2 pmol/L), or THR (0.6 UlmL) in the
presence or absence of MGDF (40 ng/mL). At the end of incubation,
platelets were extracted with cold DIM buffer (50 mmol/L PIPES
pH 6.8, 1 0 0 mmol/L NaCI, 5 mmol/L MgCL, 300 mmol/L sucrose,
5 mmol/L EGTA, 2 mmol/L orthovanadate) containing 1% Triton
X-100(Sigma Chemical CO)and a mixture of proteinase inhibitors
(I mmolL phenylmethylsulfonylfluoride, IO pg/mL leupeptin, 0.15
U/mL aprotinin, I pglmL pepstatin A) for 20 minutes at 4°C. and
centrifuged at I5,OOOg for 20 minutes. The protein concentration of
platelet lysates was determined according to Bradford’s technique”
and the protein contentof the samples was normalizedby appropriate
dilution with cold DIM buffer. The supernatant was precleaned for
I hour with 50 pL of Sepharose protein A (Sigma Chemical CO) (3
mg/sample). The samples (50 pg) were then absorbed by antisera
coupledto Sepharose protein A.Boundproteinswererepeatedly
washed in DIM buffer and eluted in boiling Laemmli buffer. Thirtymicroliter samples of eluted proteins were separated by 12% sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
and then transferred to nitrocellulose. The filters were incubated for
1 hour with a blocking solution containing 10% low-fat milk in 20
mmol/L Tris-HCI pH 7.6 and 17 mmol/L NaCI. Antisera were then
added at the same solution, and the incubation was carried out for
2 hours at room temperature. For detection the filters were washed
three times with phosphate-buffered saline (PBS) containing 0.05%
Tween 20 and reacted for I hour at room temperature with horseradish peroxidase-conjugated protein A. After washing the filters were
reacted for 1 minute with chemiluminescence reagent (ECL; Sigma)
and exposed to an autoradiography film for 1 to 15 minutes.
RESULTS
Effect of MGDF on platelet aggregation in PRP. MGDF
by itself did not affect platelet aggregation at all the concentrations tested (0.25 to 80 ng/mL). However, when added 5
2763
B
0.25
0.5
1
2
10
40
80
nghnl MGDF
Fig 1. Effects of MGDF on platelet function in PRP. (A) Platelets
preincubated 5 minutes with several doses of MGDF (0.25. 0.5, 1.0,
or 40 nglmL) or with thevehicle alone (RPM1containing 0.5% bovine
serum albumin [BSAI) were stimulated with 0.4 pmollL ADP, 1 pmoll
L EPI, or 0.2UlmL THR and the aggregation was measured in a lumiaggregometer as described in Materials and Methods. Plateletswere
preincubated with the vehicle in the curves labeled with ADP, EPI,
orTHRalone.These
results are a representative example of four
individual experiments. (B) “Priming“ index calculated on aggregation of PRP stimulated with 0.8 pmollL ADP, 1.6 pmol/L EPI, or 0.6
UlmL THR after pretreatment (5 minutes) with variousdosesof
MGDF. The results are expressed as mean f SE of four individual
experiments.
minutes before ADP, EPI, or THR, MGDF enhanced in a
dose-dependent manner the platelet aggregation (Fig 1A).
As shown in Fig IB the minimal “priming” dose of MGDF,
added 5 minutes before the agonists, was 0.25 ng/mL for
the stimulation with EPI and 0.5 ng/mL for stimulation with
ADP and THR. The “priming” effect reached a plateau at
40 ng/mL. However, the preincubation time also affected
the response of platelets to MGDF (Figs 2 and 3). When
low concentrations of MGDF were used the maximum
“priming” effect was observed after a 5- to 15-minute incubation depending on the agonist used to induce the aggregationof platelets. The “priming” effect persisted up to 3
hours. MGDF-induced “priming” was detectable at different concentrations of aggregating agents (ADP 0.2 to 0.8
p m o l k , EPI: 1 to 24 p m o l k , THR: 0.2 to 1.2 U/mL; data
not shown). However, optimal “priming” was observed for
the lower concentrations of these agonists (Fig IA). As
shown in Table 1, MGDF also enhanced the release of ATP
induced byADP, EPI, and THR. MGDF, which by itself
did not stimulate the synthesis of TXB2 when added before
ADP, EPI, and THR, induced a significant enhancement of
2764
g
MONTRUCCHIO ET AL
tn,
0.5
IS
5
30
60
1 8 0 (minutes)
B
.
-//
0.5
5
I
.
I
.
I 5
I
//"I/-
180
60
30
Time
(minutes)
TXB2 production (Fig 4). A "synergic" effect of MGDF
on ADP, EPI,and THR was evident when subthreshold concentrations of these agents were added l5 seconds before
MGDF (Fig 5 ) . The minimal effective dose of MGDF required for the "synergic" effect was 20 to 40 ng/mL. All
these effects of MGDF were abrogated by 5 minutes of
boiling. No effects were observed when platelets were incubated with the vehicle alone instead of MGDF.
Effect of MGDF on platelet aggregation in whole blood.
MGDF (1 to 80 ng/mL) byitself did not affect platelet
aggregation in whole blood but, as for PRP, "primed" the
action of ADP and EPI. As shown in Fig 6, the "priming"
effect of MGDF on aggregation induced by ADP and EPI
was dependent on the dose and on the time of preincubation.
The minimal effective dose of MGDF added to blood 5
minutes before stimulation with the agonists was 0.5 ng/mL.
Effect of MGDF on p-42 mitogen-activated protein kinase
(MAPK)tyrosinephosphorylation.
Platelets contain high
levels of tyrosine kinase activity and show many changes in
the pattern of protein tyrosine phosphorylation upon agonistinduced activation.26.28
Recently, tyrosine phosphorylation of
p42mpkisoform of MAPK has been involved in the signal
transduction pathway of aggregation induced by THR.29To
evaluate the phosphorylation of MAPK, platelets were stimulated in the lumi-aggregometer with ADP, EPI, or THR in
the presence or the absence of MGDF. Platelets recovered
from the aggregation assay were lysed and immunoprecipitated with antiphosphotyrosine antibody. The filterswere
then probed either with a monoclonal or a polyclonal antibody anti-p42"Opkisoform of MAPK. In parallel experiments
platelet lysates were immunoprecipitated with monoclonal
antibody anti-~42~P'
and the filters were primed with the
same antibody to detect the band corresponding to the
p42mpkisoform of MAPK that comigrates with the tyrosine
0.5
Fig 3. Effect of time of preincubationwith MGDF
on platelet aggregation induced by EPI in PRP. (A)
Platelets preincubated for various times with MGDF
(0.25 and 1 ng/mLl or with the vehicle alone (RPM1
containing 0.5% BSA)were stimulated with 1 pmoll
L EPI end aggregation was recorded. These results
are a representativeexampleof four individual experiments. (B) "Priming" index calculated on aggregation of PRP stimulated with 1 p m o l f l EPI after
pretreatmentfor various period of time with 0.25 (0)
or l ng/mL (0)
The
MGDF.
results
expressed
are
as
mean SE of four individual experiments.
*
Fig 2. Effect of time of preincubationwith MGDF
on the aggregation of platelets induced by ADP in
PRP. (A) Plateletspreincubated for various times
with MGDF (0.25 and 1 ng/mL) or with the vehicle
alone (RPM1 containing 0.5% BSA) were stimulated
with 0.4 pmol/L ADP and aggregation was recorded.
These resutts are a representative example of four
individual experiments. (B) "Priming" index calculated onaggregationof
PRP stimulated with 0.8
pmol/L ADP after pretreatment for various period of
time wRh 0.25 (m) or 1 nglmL (0)MGDF.
The
results
are expressed as mean
f SE offour individual expariments.
15
5
30
60
1 B O (minutes)
B
g
g
*
g
1
I /
0.5
.
I/-//-
30
15
Time
(minutes)
60
180
THROMBOPOIETIN AND PLATELETACTIVATION
2765
Table 1. Effects of MGDF on the Release of ATP From Platelets
Stimulated With ADP, EPI, and THR
Pretreatment (5 m i d
Stimuli
Vehicle
Vehicle
Vehicle
MGDF
MGDF
MGDF
ADP
EPI
THR
ADP
EPI
THR
2.22
4.65
Release
ATP
(nmol)
0.10 2
0.04 2
0.05 5
1.98 2
?
0.03
0.01
0.02
0.3'
0.2'
-c 0.3'
Platelets preincubated 5 minutes with 40 nglmL MGDF or with the
vehicle alone (RPM1 containing 0.5% BSA) were stimulated with 0.4
pmol/L ADP, 1 pmol/L EPI, or 0.2 UlmL THR and the release of ATP
was measured in lumi-aggregometer as described in Materials and
Methods. These results are a representative example of four individual experiments.Student'st-testwas
performed between vehicle
alone plus stimulus and MGDF plus stimulus: * P i .05.
phosphorylated band observed after immunoprecipitation
with anti-phosphotyrosine antibody. As shown in Fig 7, the
level of phosphorylated ~ 4 2 ' was
' ~ low in platelets stimulated with MGDF, ADP, EPI, or THR alone as well as in
unstimulated platelets. In contrast, when platelets were costimulated with MGDF and ADP, EPI, or THR a higher level
of tyrosine phosphorylated p42""'pkwas detected.
DISCUSSION
Thrombopoietin is considered a lineage-dominant cytokinef>-8.3fl.>l
that regulates the proliferation and maturation of
committed cells leading to the production of megakaryocytes
andplatelets."-x Recently, complementary DNAs for both
human and murine thrombopoietin have been cloned and
shown to encode a glycoprotein that has selective actions
on megakaryocyte proliferation."'." This glycoprotein has a
novel two-domain structure with an amino-terminal domain
homologous with erythropoietin and a carboxy-terminal domain rich in serine, threonine, and proline residues and con-
12
I
i i
Alone
MGOF
ADP
MGDF
ADP
EPI
MGDF
EPI
mR
YGDF
THR
Fig 4. "Priming" effect of MGDF (40 ng/mL) pretreatment (30 seconds) on TXBl generation by PRP stimulated with 0.8 pmol/L ADP,
1.6 pmol/L EPI, or 0.6 UlmL THR. The results are expressed asmean
2 SE offour individual experiments. One-way ANOVA with NewmanKeul's multicomparison test was performed Aloneversus MGDF,
ADP, EPI, THR: +, P < .05; ADP versus MGDF plus ADP; EPI versus
MGDF plus EPI; THR versus MGDF plus THR: *, P < .05.
Fig 5. Synergic effect of MGDF on subthreshold concentrations
of ADP (0.1 pmol/LI, EPI (0.8 pmol/L), and THR (0.1 U/mL) added 15
seconds before MGDF.
taining seven potential N-linked glycosylation sites." Several lines of evidence strongly suggest that the c-Mpl protein
encoded by the c-mlp proto-oncogene acts as a receptor for
thrombopoietin.GRThe c-Mpl receptor is expressed in the
megakaryocytic lineage from late progenitors to mature
platelets?h.'h In c-mpl-deficient mice a decreased number
of platelets butnotof other hematopoietic cell types was
observed.'*
The impressive feature of MGDF, the recombinant thrombopoietin, is its ability to increase the number of platelets'".
''.lsso that it may find several potential clinical applications
in thrombocytopenic states."
We report herein that MGDF was able to affect the function of mature platelets. We observed that MGDF by itself
did not induce platelet aggregation. However, when added
before other agonists such as ADP, EPI, and THR, it rendered platelets more sensitive. This "priming" effect of
MGDF was dose dependent and it occurred not only in PRP
but also in whole-blood platelet aggregation, which better
reflects normal physiology. The minimal effective dose was
similar to that reported for proliferation of c-mpl-expressing, responsive cell lines3' A "synergic" effect of MGDF
on ADP, EPI, and THR was also evident when subthreshold
concentrations of these agents were added 15 seconds before
MGDF. However, the minimal effective dose of MGDF required for the "synergic" effect was 80 to 160 times higher
then the minimal "priming" dose. Similar effects on platelets were previously observed with human stem cell factor
which is a hematopoietic growth factor produced by mesenchymal cells acting on c-Kit receptor."
The concept that proliferative cytokines may affect the
function of the mature end cells has been demonstrated by
studies on the role of granulocyte-macrophage colony-stimulating factor and of granulocyte colony-stimulating factor in
"priming" the leukocyte response to activating agents.'s4"
The results of present study suggest a modulatory role of
thrombopoietin on the function of circulating platelets. This
may imply that production of thrombopoietin could be stimulated not only by the reduction of circulating platelets but
also by platelet dysfunction.
Recently, it has been suggested that thrombopoietin is
released in response to changes in the platelet mass4' and to
poorly functioning platelets." In these conditions the humoral factor stimulates megakaryocytes to increase platelet
production.J2
MONTRUCCHIO ET AL
2766
Fig 6. Effect of time of preincubation with MGDF
on platelet aggregation induced by ADP or EPI in
whole blood. Whole blood preincubated for various
periods of time withMGDF (0.5 and 1 ng/mL) or with
the vehicle alone (RPM1 containing 0.5% BSAl was
stimulated with 1 pmollL ADP or 1.6 pmollL EPI and
MCDF".Ond*u
".
.LB.,
11m.1.1
5
60
30
aggregation
was
recorded as variation of impedance.
These results are a representative example of three
individual experiments.
180
Tlme
(minuter)
In the present study we have evaluated tyrosine phosphorylation of ~ 4 2 as
" a~marker of increased tyrosine kinase
activity. The results obtained indicate that MGDF enhanced
phosphorylation of ~ 4 2 " ' "promoted
~~
by ADP,EPI,and
THR.
It has been suggested that in platelet p42"""' could modulate cytoscheletal protein functions in secretion and adhesion.'" Another potential substrate for p42"'1'pkin platelets is
cytosolic phospholipase A2.s3,s4It hasbeenshownthat
MAPK belongs to the platelet signal transduction pathway
involved in the activation of phospholipase A2and in the
mobilization of arachidonic acid." Miyakawa et al" reported
that MGDF induces rapid protein tyrosine phosphorylation
of Janus kinase 2 and Shc in humanblood platelets. Our
results indicating that MGDF "primes" the phosphorylation
of
further support the contention of Miyakawa et a15"
that the ligand binding to c-mpl activates signal transduction
in human platelets. It is possible that Jak2, Shc. and p42"""
maybe involved in the increase in platelet sensitivity to
physiologic agonists induced by MGDF.
In conclusion, these results suggest that thrombopoietin
has pleiotropic effects, because, besides acting as an hematopoietic growth factor. it potentiates platelet activation. There-
In our study the priming effect of MGDF was dependent
on the time of platelet preincubation as it was already detectable after 30 seconds, reached its maximal after 5 to 30
minutes, and lasted up to 3 hours. This mayhave clinical
implication as treatment with MGDF will involve exposure
to this hormone for prolonged period of time. It remains to
be determined whether such an increase in platelet responsiveness may be beneficial or detrimental in various clinical
thrombocytopenic states.
The intracellular events that occur after thrombopoietin
binding to its receptor are poorly understood. Tyrosine phosphorylation of specific intracellular proteins are essential for
theaction ofmany hematopoietic growth factors that are
known to share common signal transduction ca~cade.~'"~
Recent studies have shown an increase in tyrosine kinase activity onTHR-,ADP-,
tromboxane A2- or collagen-induced
platelet
with phosphorylation of several members of Src and Syk families''"s2 and of MAPK." It has been
recently shown that platelets express two forms of MAPK,
p 4 p p h and p w p k .ZcJ During THR-induced platelet activa-
p42'Ih
tion,
P44"'"1'A
not
but
becomes phosphorylated on
serine, threonine, and tyrosine, and functionally activated."
1
Fig 7. Effect of MGDF on p42""pk tyrosine phosphorylation in human platelets. (Top) Platelets were
incubated for 5 minutes in the absence (lane 1) or in
the presence of MGDF (40 nglmL) (lane 21,THR (0.6
UlmL) (lane3). or THR (0.6 UlmL) andMGDF (40 n g l
mL) in combination (lane 4). lysed and immunoprecipitated with the antiphosphotyrosine monoclonal
antibody. The filter was probed with
anti-p42""Ph
polyclonal antibody. (Bottom) Platelets were incubated for 5 minutes in the absence (lane 1) or in the
presence of THR (1 UlmL) (lane 2). MGDF (40 nglmLl
(lane 3). ADP (0.5 pmollL) (lane 41, ADP (0.5 pmoll
L) andMGDF (40 ng/mL) in combination (lane 5). EPI
(2 pmol/L) (lane 61, EPI (2 pmol/LI and MGDF (40
nglmL) in combination (lane 7 ) . lysed, and immunoprecipitated with the antiphosphotyrosinemonoclonal antibody. The filter was probed with anti~42"'~
monoclonal
~'
antibody.The platelet werealso
immunoprecipitated with anti-p42"'P* monoclonal
IP
antibody t o evaluate the migration of the ~ 4 2 " ' ~ ~ '
(lane 8). The positions of immunoglobulins (lgl and
IB
of ~42"'" are indicated.
2
3
4
gl"
, t p 42 MAPK
anti P-lyr
IP
IB
1
2
anti p 42
3
4
MAPK
5
6
anti p 42MAPK
7
8
THROMBOPOIETIN AND PLATELETACTIVATION
fore, thrombopoietin may playa physiologic role in modulating the response of platelets to several stimuli and thereby
their hemostatic potential.
ACKNOWLEDGMENT
We thank Dr J.L. Nichol (Amgen Inc, Thousand Oaks, CA) for
providing us the recombinant human MGDF.
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