1. Art and Diseño

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Relative Reactivity of Platelets From Thrombopoietin- and
Interleukin-6 - Treated Dogs
By Jinpeng Peng, Paul Friese, Roman F. Wolf, Paul Harrison, Tamyra Downs, Si Lok, George L. Dale,
and Samuel A. Burstein
Previous reports have shown that interleukin-6 (IL-6) enhances the responsivenessof platelets to thrombin stimulation and has modest thrombocytopoietic effects in vivo.
Thrombopoietin 1TPO; mpl ligand) has been shown t o have
dramatic thrombocytopoietic effects in vivo, but little is
known of its capacity t o alter platelet function. In this study,
a direct comparison of the effects of IL-6 and TPO on platelet
function in dogs has been performed, with modest doses of
TPO (1pg/kg/d) chosen t o match or moderately exceed the
platelet counts achieved with IL-6 (40pg/kg/d) for 10 days.
Platelet responsiveness to thrombin stimulation was assessed in TPO-treated, IL-6-treated, and control dogs by
flow cytometric measurement of P-selectin expression. On
day 5, the dose of thrombin promoting half maximal stimulation (E&) of platelets was not significantly changed in TPOtreated dogs, whereas in IL-6-treated dogs the ECm decreased t o 73.1% 2 6.1% (mean l SD; n = 5) of control
values ( P c 0.01). These experiments were performed on
both gel-filtered platelets and washed whole blood, indicat-
ing that the observed changes in E& were caused by cytokine-mediated alteration of platelets rather than plasma
components. Because it has been shown that thiazole orange specifically labels a subpopulation of dog platelets that
is less than 24 hours old, the thrombin responsiveness of
these young, newly synthesized platelets was determined.
The ECso of thiazole orange-positive platelets from IL-6treated dogs decreased dramatically by day 5 to 46.5% &
13.1% (n = 4) of control values 1P < 0.001), whereas TPOtreated dogs did not significantly change. When TPO was
directly incubatedwith platelets ex vivo, no effects on either
thrombin-mediated P-selectin expression or adenosine diphosphate-induced fibrinogen binding were observed.
These data show that IL-6 alters platelet function, as measured by reactivity t o thrombin, whereas TPO does not. This
divergence in function is observed even though TPO is
equally, or more, effective at promoting platelet production
under these experimental conditions.
0 1996 by The American Society of Hematology.
I
approach in experimental animals to transiently increase the
number of young platelets in the circulation. Furthermore,
we have recently described a method to identify platelets
less than 24 hours old in the dog.” With these developments,
it is now feasible to directly compare the effects of IL-6
and TPO on platelet reactivity and to ascertain if the IL-6mediated alterations in platelet function previously described’ reflect only an increase in young platelets or perhaps
an alteration in platelet reactivity.
*
NTERLEUKIN-6 (IL-6), a cytokine with numerous biological consequences,’ shows a variety of effects on the
hemostatic mechanism, including inducing a modest elevation in the platelet count in normal and thrombocytopenic
animals as well as in human^,"^ an augmentation of plasma
fibrin~gen,~
and an enhancement of platelet size and platelet
f~nction.6.~
These alterations of platelet function may originate at the level of the megakaryocyte, because direct incubation of canine platelets with IL-6 has no effect on the activation capacity of platelets in response to t h r ~ m b i nSince
. ~ IL6 increases the platelet production rate,8 it is conceivable
that the observed changes in platelet function reflect an increase in young, more hemostatically effective platelets,
rather than a specific alteration caused by the cytokine.’
The recently described growth factor thrombopoietin
(TPO; mpl ligand), has been shown to have very potent
thrombopoietic effects in mice, but little is known about its
influence on platelet function.1°-16 Because the elevation in
platelet count caused by TPO is almost certainly a result of
increased production, the hormone provides an alternative
From the Department of Medicine and the W. K. Warren Medical
Research Institute, University of Oklahoma Health Sciences Center,
Oklahoma City, OK: and the Zymogenetics Corp, Seattle, WA.
Submitted May 1, 1995; accepted October 27, 1995.
Supported by the W.K. Warren Medical Research Institute, an
award from the American Heart Association (Oklahoma Affiliate),
and Grants No. HL50059 and AGO8545 from the National Institutes
of Health.
Address reprint requests to Samuel A. Burstein, MD, University
of Oklahoma Health Sciences Center, Room 271, College of Health
Bldg, PO Box 26901, Oklahoma City, OK 73190.
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 1734 solely to
indicate this fact.
0 1996 by The American Society of Hematology.
0006-4971/96/87I0-0060$3.00/0
4158
MATERIALS AND METHODS
Materials. Bovine thrombin, prostaglandin E, (PGE,), and bovine serum albumin (BSA) were obtained from Sigma Chemical Co
(St Louis, MO). Fluorescein isothiocyanate (FITC) and phycoerythrin-labeled streptavidin (streptavidin-PE) were obtained from Calbiochem (La Jolla, CA). NHS-biotin was obtained from Pierce
Chemical Co (Rockford, IL). Caltag Laboratories (South San Francisco, CA) provided the streptavidin-TRI-COLOR, and rabbit antihuman fibrinogen was from Dako Laboratories (Carpinteria, CA).
Thiazole orange (TO) was supplied as Retic-COUNT Kit from Becton Dickinson (San Jose, CA). Formalin was obtained from Fisher
Scientific (Fairlawn, NJ).
Bujfers. The buffers used were as follows: BSGC, pH 7.3 (buffered saline-glucose-citrate: 129 mmol/L NaCl, 13.6 mmol/L Na?
citrate, 11.1 mmol/L glucose, 1.6 mmol/L KHZP04,8.6 m m o w
NaH,PO, [pH, 7.31; ACD (acid citrate dextrose: 38.1 mmom citric
acid, 74.8 mmol/L Na7citrate, 136 mmol/L glucose); and PBS (phosphate buffered saline: 150 mmol/L NaCI, 10 mmol/L NaH,P04 [pH,
7.41).
Cytokines. Recombinant human IL-6 was expressed in Eschericheria coli and purified as previously described.6Concentration and
purity were assessed by the IL-6-responsive B9 cell bioassay’’ and
sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Endotoxin levels were measured using the limulus amebocyte lysate test
at Endosafe Inc (Charleston, SC). The specific activity of the purified
product was 2 to 5 x IOs U/mg protein and had an endotoxin level
of less than 2 U/mg. Recombinant human TPO (0.1 19 mglmL in 20
mmol/L Tris, 0.15 mol/L NaCl [pH, 8.51) was provided by Dr D.
Foster (Zymogenetics, Seattle, WA).
Blood, Vol 87, No 10 (May 15), 1996 pp 4158-4163
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41 59
REACTIVITY OF CYTOKINE-DRIVEN PLATELETS
Monoclonal antibodies (MoAbs). To detect canine platelets in
whole blood, antibody 2F9, recognizing a component of the glycoprotein IIbDIIa complex, was used as described.” To detect activated
canine platelets, antibody G5 (subtype IgGI; cell line provided by
R. McEver, Oklahoma Medical Research Foundation, Oklahoma
City, OK) raised against human P-selectin and cross-reacting with
the canine counterpart, was used. 2F9 was directly fluoresceinated
with FITC and G5 was biotinylated with NHS-biotin using standard
techniques. Biotinylated Tab (provided by R. McEver; an IgG,
MoAb recognizing human but not dog platelets) was used as an
isotype-specific control to set gates for resting versus activated platelets. MoAbs to detect canine fibrinogen were produced by immunizing BalblC mice with purified dog fibrinogen.” Antigen was injected
bimonthly 3 times into multiple subcutaneous and intraperitoneal
sites of Balb/C mice. Serum antifibrinogen titers were determined
by enzyme-linked immunosorbent assay (ELISA). Four days after a
final injection was administered intravenously, the mice were killed,
and the spleen cells were fused in 50% polyethylene glycol-1500
(Sigma) at pH 7.0 to Sp2/0 murine myeloma cells according to
standard techniques.” Hybrids were identified after hypoxanthine,
aminopterin, and thymidine (HAT) selection, expanded, and assayed
for antifibrinogen antibody by ELISA. Positive clones were subcloned twice from single cells by limiting dilution. One hybridoma,
designated 2B11, was used in these studies.
Animals. Beagles (8 to 13 kg; Hazleton Research Products,
Cumberland, VA) were obtained and housed according to the regulations of the Institutional Animal Care and Use Committee (IACUC)
of the University of Oklahoma Health Sciences Center (Oklahoma
City, OK; accredited by the American Association for Accreditation
of Laboratory Animal Care).
In vivo protocol. Blood samples for baseline determinations
were drawn followed by administration of TPO (diluted in 0.15 mol/
L NaCl supplemented with 5% autologous dog plasma) at 1 pg/kg/
d or IL-6 at 40 pg/kg/d in the same diluent buffer, both in a single
subcutaneous dose for 10 days (days 0 through 9). Control animals
received the diluent buffer on the same schedule.6 Blood samples
for analysis of cell counts, fibrinogen assay, quantitation of TO+
platelets, and platelet activation were drawn 2 to 3 times weekly.
Geljiltration ofplatelets. Gel filtration was used to deplete platelets of plasma components. For thrombin dose-response studies, 5
mL of blood was drawn into 1/10 vol ACD-A with prostaglandin
El (final concentration in blood, 0.5 pmom); for fibrinogen binding
analysis, blood was drawn into 1/10 vol 3.8% sodium citrate. Blood
was mixed with 5 mL of BSGC (pH, 7.3) and centrifuged at 200g
for 10 minutes to prepare platelet-rich plasma. Platelet-rich plasma
(1 mL) was passed through a 20 mL Sepharose 4B column equilibrated with BSGC (pH, 7.3), and the fraction containing platelets
was collected. Platelets were counted on a Baker System 9000 cell
counter and adjusted to 50,000/pL with BSGC.
Assessment of platelet activation in response to thrombin. Platelet activation was assessed in washed whole blood as previously
d e ~ c r i b e dor
~ ,by
~ the following adaptation of those methods. A total
of 20 pL of gel-filtered platelets was added to each of seven 15mL polypropylene tubes containing 928 pL of BSGC (pH, 7.3)
supplemented with 2 pL of G5-biotin (1 mg/mL) and 50 pL of
various concentrations of diluted bovine thrombin in BSGC (pH,
7.3). The tubes were incubated in a 37°C water bath for 10 minutes,
followed by the addition of 1 mL of 0.6% formaldehyde in PBS.
The tubes were then incubated at room temperature (RT) for 20
minutes, followed by the addition of 10 mL BSGC (pH, 7.3) supplemented with 0.1% BSA and 2 mmoVL EDTA (BSGC-BSA), and
centrifuged at 1,250g for 18 minutes at RT to remove the fixative.
Pelleted cells were resuspended in 0.4 mL of BSGC (pH 7.3). A
total of 200 pL of the resuspended cells was transferred to another
tube, followed by the addition of 2 pg streptavidin-PE and 2 pg of
FITC-2F9. After incubation at RT for 30 minutes, samples were
washed with BSGC-BSA as described above. After resuspension in
0.5 mL BSGC-BSA, samples were analyzed by flow cytometry.
Assessment of activation of TO+platelets in response to thrombin.
To determine the thrombin-responsiveness of a subpopulation of
platelets less than 24 hours old,17 thrombin dose-response curves
were performed on gel-filtered platelets as detailed above, and the
platelets were then labeled with the RNA-binding dye, TO. Specifically, after the thrombin activation, formalin-fixation, and wash steps
described above, 200 pL of sample were incubated with 4 pL streptavidin-TRI-COLOR for 30 minutes at RT to label the biotinylatedG5 antibody. After incubation with 1 mL TO solution for 30 minutes
at RT, samples were analyzed by flow cytometry as described below.
Streptavidin-TRI-COLOR was used in these experiments, because
its emission spectrum does not overlap significantly with that of
~0.17
Adenosine diphosphate (ADP)-induced jibrinogen binding.
Fibrinogen binding to activated platelets was performed according
to a modification of the method of Warkentin et aLZ2A total of 400
pL of gel filtered dog platelets was mixed with 0.2 mL of citrated
dog plasma and 1.2 mL of HEPES buffer (10 mmol/L HEPES, 145
mmoVL NaCI, 5 mmom KCI, 1 mmom MgSO, [pH, 7.41). A total
of 180 pL of the mixture was added to each of seven wells of a 96well plate, followed by the addition of 20 pL of various concentrations of ADP (final concentration,
mol/L to lo-, mom), and
was incubated for 40 seconds at RT. Next, 100 pL of the reaction
mix were transferred to tubes containing 2 mL of 0.3% formaldehyde
in PBS and fixed for 20 minutes. A total of 10 mL of BSGC-BSA
was then added, and the tubes were centrifuged at 1,250g for 18
minutes. The pellets were resuspended in 0.2 mL of BSGC. A total
of 5 pg of FITC-conjugated chicken antidog fibrinogen9was added
to each tube and allowed to incubate at RT for 30 minutes with
occasional agitation, followed by washing in BSGC-BSA. The pellets were resuspended in 0.5 mL of BSGC, and the samples were
analyzed by flow cytometry (settings: forward scatter [FSC], log
EOO; side scatter [SSC], log 332; fluorescence 1 [FLI], log 520;
FSC threshold, 260). In some experiments, gel-filtered platelets were
first incubated with 100 ng/mL of active TPO or 100 ng/mL of heatinactivated TPO (100°C for 5 minutes) for 45 minutes at RT before
the ADP-induced fibrinogen-binding study.
Flow cytometry. The percentage of platelets activated at the various concentrations of thrombin was determined with a FACScan
flow cytometer (Becton Dickinson) according to previously described method^.^.^ A total of 10,OOO events were analyzed for Pselectin expression. For analysis of the thrombin dose-response characteristics of TO’ platelets, the flow cytometer was set as follows:
FSC, log Em; SSC, log 350; FLl, log 700; and FL3, log 500. A
total of 50,000 total platelets were analyzed, and the percentage of
TO’ platelets within the activated ((359 and nonactivated populations was determined.
ELISA for dogjbrinogen. Dog plasma fibrinogen was measured
by ELISA. Briefly, 96-well microtiter plates were coated with rabbit
antihuman fibrinogen IgG at a dilution of 1:1,000 in carbonate buffer
(pH, 9.6). After blocking, diluted samples and standards were applied
to washed plates and bound fibrinogen was detected with biotinylated
MoAb 2Bll (diluted 1:200) raised against dog fibrinogen. Bound
antibody was detected by incubation with streptavidin-peroxidase.
The plates were developed by addition of o-phenylenediamine dihydrochloride/H20, and the absorbance at 492 mp (OD492)measured
after the reaction was stopped with 1 m o m H,S04.
Statistical analysis. The statistical significance of differences in
means was tested with a two-tailed Student’s t-test.
RESULTS
Platelet counts. To compare platelet function under circumstances in which platelet counts were not drastically
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4160
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PENG ET AL
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Time (days)
Fig 1. Peripheral blood platelet counts ( x 10-’/mL) after administration of IL-6 (A),
TPO (MI, snd buffer (0).Platelet counts are shown
on the ordinate (mean f 1 SEI; time (days)after initiation of cytokines
is shown on the abscissa. IL-6, TPO, and buffer were administared
from day 0 through 9 as indicated by the hatched bar (m). For all
time points, n = 5 except for days 4 to 5 where n = 4.
different between treatment groups, a dose of TPO was used
to promote elevations in platelet counts that would approximate or modestly exceed that expected with IL-6 (Fig 1).
After the administration of TPO (1 pgikg/d) or IL-6 (40 pg/
kg/d), platelet counts were monitored several times per week.
For TPO-treated animals, a maximal increase of 115% above
the pretreatment count was observed on days 11 to 13,
whereas an increase of 72% above the pretreatment count
occurred in IL-6-treated dogs at this same time. Although
the increment in platelet count for the IL-6-treated dogs
was less than that observed with TPO, the difference was
not statistically significant ( P = .09).
In vitro responsiveness of platelets to thrombin stimulation. At various times during the course of cytokine administration, platelet responsiveness to graded doses of thrombin
was determined by flow cytometry; results are reported as a
thrombin ECsO,the effector concentration (EC) resulting in
50% a~tivation.~.’
A summary of these EC50 values from five
separate sets of animals is presented in Fig 2. As early as
day 2, a decrease in the EC5,, for thrombin in IL-6-treated
dogs was observed, although the decrease was not statistically significant until day 5 when the EC50 had dropped to
73.1% t- 6.1% (mean t- 1 SD; n = 5 ) of control ( P < .01).
By day 15, the thrombin ECsohad returned to normal in the
IL-6-treated animals; IL-6 administration was stopped on
day 9. In contrast, there was no significant difference in the
EC5o for thrombin between TPO-treated and control dogs at
any time point. These data indicate that less thrombin is
required to activate the platelets from IL-6-treated animals
when compared with that for either TPO-treated or control
animals. It is noteworthy that the change in ECso in IL-6treated animals first occurred at day 5 when platelet counts
for IL-6 and TPO animals were essentially identical (Fig 1)
and before these platelet counts were significantly elevated
above control levels ( P > .12).
IL-6 markedly augments plasma fibrinogen that might
compete for thrombin and alter the thrombin dose-response
curve in an unpredictable fashion. In contrast, TPO does not
affect fibrinogen concentrations (mean, 1.28 ? 0.06 mg/mL;
range, 1.03 to 1.57 mg/nL throughout the treatment course;
n = 4). All of the experiments shown in Fig 2 were performed with plasma-depleted platelets, either by washing
whole blood (one experiment) or by gel filtration (four experiments). No differences were noted with the two techniques.
For example, in Fig 2, the IL-6-treated animals at day 5
had a mean change in thrombin EC50to 73.1% t- 6.1% of
control values when all samples were analyzed; if only the
gel filtered samples are considered, the change was 74.9%
2 5.4%.
Cytokine-induced production of TO+ platelets. Previous
studies have shown that TO’ platelets in the dog are less
than 24 hours old.17 Before treatment, the mean percentage
of TO+ platelets in all animals was 6.5% ? 0.8% (n = 15),
and Fig 3A shows the percentage of TO+ platelets during
cytokine administration. By day 3 to 4,a slight increase in
TO+ platelets was observed in both the TPO- and IL-6treated dogs followed by a modest decrease after cessation of
the cytokine, although none of the changes were statistically
significant. Figure 3B shows the absolute levels of TO’
platelets during cytokine administration. TPO resulted in statistically significant elevations in TO+ platelet count on days
5:
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6
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Time (days)
Fig 2. Alterations of the activation capacity of platelets derived
from five sets of dogs treated with 11-6 (A),TPO (MI, or buffer (0).
For each time point, the thrombin El& WM determined and normalized to the pretreatment E L ; the ordinate show the percantaw of
the pretreatment E C , (mean k 1 SE; n = 5). Time (days)after initiation of 11-6 and TPO is shown on the abscissa; cytokinw were administered through day 9, es indicated by the hatched bar ( 0 ) .For the
IL-6-treated dogs, a significant decrease in thrombin El& was observed on deys 5 and 8 ( P < .01; noted by asterisk [*I). Startlng ECm
values for these three experimental groups were 44.6 k 7.4, 54.4 k
10.1, and 46.2 f 13.0 mU thrombinlmL (mean f 1 SD; n = 5 ) for
control, TPO, and 11-6 groups, respectively.
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4161
REACTIVITY OF CYTOKINE-DRIVEN PLATELETS
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Fig 3. Changes in TO+ platelets after IL-6 (AI, TPO (HI, and buffer (0)administration. (A) The percentage of TO* platelets in the peripheral
circulation (percentage 2 1 SE; n = 5) is plotted versus the time (days) after initiation of the cytokines 1B). None of the differences h e e n
groups are statistically significant. (BI The absoluta levels of TO+ platelets during the experiment is shown. These velues were calculated from
the percentage of TO+ platelets and the total platelet count. For TPO, there was a significant elevation in TO+ platelets as compared with
For IL-6, the elevation in absolute TO+ counts we. significantly above control only for day
control values on days 5 to 6,9, and 12 ( P < .M).
9 ( P < .MI. The difference between TPO and IL-6 was significant only for day 12 ( P < .05).
5 to 6, 9, and 12 after initiation of the cytokine (P < .05);
IL-6 also produced increases in TO' platelet counts, although only the day-9 value is significantly different from
control values.
It is noteworthy that the increases in both the percentage
of TO+ platelets (Fig 3A), and the absolute numbers of TO'
platelets (Fig 3B) do not correlate well with the increase in
total platelets (Fig 1). Considering the dramatic increase in
total platelet count elicited by these cytokines, large increases in the percentage of TO' platelets, in particular,
would be e x p e ~ t e d . *The
~ * ~reason for this apparent discrepancy is not clear but may reflect either an inability of TO to
detect all newly synthesized platelets or the difficulty of
quantitating all TO+ platelets, because there is some overlap
with TO- platelets during flow cytometric anal~sis.'~
Thrombin responsiveness of TO+ platelets. Flow cytometric analysis permits an assessment during IL-6 and TPO
administration of the thrombin responsiveness of the very
young, TO+ subpopulation of platelets (Fig 4). By day 5,
the ECS0for TO+ platelets in IL-6-treated dogs decreased
to 46.5% ? 13.1% (mean ? 1 SD; n = 4) of control values
(P < .001); after cessation of IL-6, EC50 values returned
to normal. In TPO-treated dogs, no significant changes in
thrombin EC5o values were observed. These divergent
changes in the EC5o occurred despite the fact that there are
no significant differences in either the percentage of TO+
platelets or the total platelet counts between the TPO- and
IL-6-treated dogs for days 5 or 8 (Fig 1 and 3). After discontinuation of TPO, TO+ platelets in these animals showed a
trend of being less sensitive to thrombin with slightly in-
creased EC5o values, although no statistically significant differences were observed.
Effects of TPO on platelet responsiveness to thrombin and
ADP in vitro. The above data show that IL-6 has a greater
effect than TPO on platelet reactivity for the total platelet
population and, in particular, for TO' platelets. Previous
studies from our laboratory have suggested that the IL-6induced alteration in platelet function in dogs is mediated
via an effect of the cytokine on megakaryocytopoiesisrather
than via a direct effect on platelets per se.' To determine if
TPO has any direct effects on platelet responsiveness to
thrombin or ADP, the hormone (100 ng/mL) was incubated
with normal gel-filtered platelets for 45 minutes at RT, followed by performance of a thrombin dose-response study or
an ADP-induced fibrinogen-binding study as described in
Materials and Methods. No significant differences were
noted between TPO-incubated platelets and control platelets
(incubated with heat-inactivated TPO) for either the EC5o of
ADP-induced fibrinogen binding (TPO, 0.94 t 0.30 pmoY
L; control, 0.77 t 0.09 pmol/L; P > .3; n = 3) or for the
EC5o of thrombin-induced activation (TPO, 0.046 ? 0.006
U/mL; control, 0.048 ? 0.005 U/mL; P > .5; n = 3).
DISCUSSION
Previous studies from our laboratory have indicated that
administration of IL-6 leads to the production of platelets
with increased reactivity to thrombin: suggesting that cytokines that augment thrombocytopoiesis quantitatively may
also alter platelet function. Analogous effects on the granulocyte-macrophage lineage have been described, with granulo-
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4162
PENG ET AL
bin. The data show that the responsiveness of the total population to thrombin was greater in the IL-6-treated animals,
140
despite the fact that platelet counts and the percentage of
TO+ cells were similar or modestly higher in the TPO-treated
dogs. Furthermore, when a direct analysis of thrombin reactivity of the TO+ platelets was performed in both groups, it
was observed that the TO+ platelets in the TPO-treated dogs
c
behaved similarly to TO+ platelets derived from normal aniz 100 E
mals, whereas the TO+ platelets from the IL-6-treated ani2!
mals were substantially more reactive to thrombin.
80These data suggest that IL-6 has an effect on platelet
EP
function that is clearly different from that of TPO. Although
the molecular mechanism of this alteration is unknown, it is
60 clear that platelet activation is a sensitive balance of inhibitory and activating pathway^,^' and the results presented
herein
suggest that these two arms of platelet homeostasis
40 7
/
/ / / / / / / / / / / / / / / / / / A
may
be
regulated differentially. In IL-6-driven platelets, it
I
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appears that the activation pathway predominates as indi0
3
6
9
12
15
cated by the hypersensitivity of these TO’ platelets relative
to TO+ platelets from control or TPO-treated animals. EluciTime (days)
dation of the mechanisms regulating the expression of these
Fig 4. The percentage of pretreatment, thrombin EC, for TO+
putative pathways awaits further investigation.
platelets. The ordinate shows the percentage of pretreatment EC,
The analysis of platelet activation in this study has focused
for TO+ platelets (meen f 1 SE; n = 4) after administration of 11-6
on
one marker of activation, P-selectin expression on the
(A),TPO (HI,and buffer (0).whereas the abscicse shows the time
(drys) after initiation of treatment. Cytokines were administered
cell surface. Although P-selectin expression is a widely used
from day 0 through 9 as indicated by the hatched bar (El). For the ILindicator of 0-granule secretion, other possible activation
6 treatment, a significant decrease in thrombin ECm was observed
markers of physiological significance include, but are not
on days 5 and 8 ( P < .001; noted by asterisk [*I). None of the other
limited to, eicosanoid production and binding of various adchanges were statistically significant. Starting EC, values for the
hesive proteins.” Although disagreement may exist as to the
TO+ platelets in these three experimental groups were 28.0 f 3.2,
27.3 2 7.6, and 32.5 f 8.0 mU thrombinlmL (mean f 1 SD; n = 4)
most relevant marker of platelet activation, the significance
for control, TPO, and IL-6 groups, respectively.
of a-granule secretion in platelet function is supported by
the occurrence of bleeding disorders associated with abnormal a-granule f~nction.~’
The potential clinical significance of these findings are
cyte-macrophage colony-stimulating factor (GM-CSF) and
speculative at this time. TPO augments platelet production
granulocyte colony-stimulating factor (G-CSF) having been
but does not alter function to a significant extent; these findshown to alter function of mature g r a n ~ l o c y t e s .The
~ ~ .phys~~
ings argue for the potential safety of this hormone when
iological mechanism for the IL-6-stimulated increase in
used for clinical purposes. In contrast, IL-6 appears to alter
platelet sensitivity to thrombin is unknown but may be
platelet function (at least in response to thrombin) in a mancaused by (1) stimulation of platelet production leading to
ner disproportionate to the overall age of the population,
an influx of young platelets into the circulation, (2) an alterraising the potential that pathological thrombosis might ocation of the platelets that is unrelated to platelet age, and/or
cur with this cytokine. Nevertheless, under conditions in
(3) both mechanisms. Earlier literature has suggested that
young, newly produced platelets may be hyperfunctional27228; which the platelet count is very low, bleeding is occurring,
and the risk of thrombosis is not an issue, IL-6 might be
however, methodological considerations related to the capacapable of ameliorating bleeding precisely because of the
bility of clearly distinguishing young from old platelets have
increased reactivity of the platelets. Direct testing of these
not permitted definitive conclusions. Using biotinylation
hypotheses in bleeding and thrombosis models will be retechnology, we have shown recently that young canine platequired.
lets have an increased sensitivity to thrombin stimulation.’
Nonetheless, the possibility existed that the functional
ACKNOWLEDGMENT
changes induced by IL-6 were not entirely related to the
The authors are indebted to Dr Don Foster of Zymogenetics Corp
influx of young platelets into the circulation. The availability
for supplying recombinant human thrombopoietin.
of TPO, likely the physiological regulator of platelet product i ~ n , ’ ~ . ~has
’ . ’ ~provided a novel means to evaluate platelet
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1996 87: 4158-4163
Relative reactivity of platelets from thrombopoietin- and interleukin-6treated dogs
J Peng, P Friese, RF Wolf, P Harrison, T Downs, S Lok, GL Dale and SA Burstein
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