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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 12 (June 15), 1998:
pp. 4427-4433
Treatment of Thrombocytopenia in Chimpanzees Infected With Human
Immunodeficiency Virus by Pegylated Recombinant Human Megakaryocyte
Growth and Development Factor
By
Laurence A. Harker,
Ulla M. Marzec,
Francis Novembre,
I. Birgitta Sundell,
Edmund K. Waller,
Simon Karpatkin,
Harold M. McClure,
Andrew B. Kelly, and
Richard B. Stead
From the Division of Hematology and Oncology, Yerkes Primate Research
Center, Emory University School of Medicine, Atlanta, GA; the New York
University Medical Center, New York, NY; and Amgen Inc, Thousand Oaks,
CA.
 |
ABSTRACT |
Three chimpanzees experimentally infected with human
immunodeficiency virus (HIV) developed significant chronic
thrombocytopenia after 5, 4, and 2 years, with peripheral platelet
counts averaging 64 ± 19 × 103/µL (P = .004 compared with 228 ± 92 × 103/µL in 44 normal control
animals), mean platelet volumes of 11.2 ± 1.8 fL (P > .5 compared with 10.9 ± 0.7 fL in normal controls), endogenous
thrombopoietin (TPO) levels of 926 ± 364 pg/mL (P < .001 compared with 324 ± 256 pg/mL in normal controls), uniformly elevated
platelet anti-glycoprotein (GP) IIIa49-66 antibodies, and
corresponding viral loads of 534, 260, and 15 × 103 RNA
viral copies/mL. Pegylated recombinant human megakaryocyte growth and
development factor (PEG-rHuMGDF) was administered subcutaneously (25 µg/kg twice weekly for 3 doses) to determine the effects of stimulating platelet production on peripheral platelet concentrations in this cohort of thrombocytopenic HIV-infected chimpanzees.
PEG-rHuMGDF therapy increased (1) peripheral platelet counts 10-fold
(from 64 ± 19 to 599 ± 260 × 103 platelets/µL;
P = .02); (2) marrow megakaryocyte numbers 30-fold (from 11.7 ± 6.5 × 106/kg to 353 ± 255 × 106/kg;
P = .04); (3) marrow megakaryocyte progenitor cells fourfold (from a mean of 3.6 ± 0.6 to 14.1 × 103 CFU-Meg/1,000
CD34+ marrow cells); and (4) serum levels of Mpl ligand
from 926 ± 364 pg/mL (endogenous TPO) to predosing trough levels of
1,840 ± 353 pg/mL PEG-rHuMGDF (P = .02). The peripheral
neutrophil counts were also transiently increased from 5.2 ± 2.6 × 103/µL to 9.9 ± 5.0 × 103/µL (P
= .01), but neither the erythrocyte counts nor the reticulocyte counts were altered significantly (P > .1). The serum levels
of antiplatelet GPIIIa49-66 antibodies exhibited reciprocal
reductions during periods of thrombocytosis (P < .07).
PEG-rHuMGDF therapy did not increase viral loads significantly (395, 189, and 53 × 103 RNA viral copies/mL; P > .5 compared with baseline values). The striking increase in peripheral
platelet counts produced by PEG-rHuMGDF therapy implies that
thrombocytopenia in HIV-infected chimpanzees is attributable to
insufficient compensatory expansion in platelet production resulting
from HIV-impaired delivery of platelets despite stimulated
megakaryocytopoiesis. These data suggest that PEG-rHuMGDF therapy may
similarly correct peripheral platelet counts in thrombocytopenic HIV-infected patients.
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INTRODUCTION |
CHIMPANZEES ARE reproducibly infected
with human immunodeficiency virus type 1 (HIV) by injecting cell-free
virus, infected peripheral blood mononuclear leukocytes, or homogenates
of infected tissues from HIV-infected donors.1-3 Infected
chimpanzees develop humoral responses similar to HIV-infected patients,
and viral loads gradually decrease during the first year of infection
due to immunologic and cellular clearance mechanisms, analogous to asymptomatic human HIV carriers.4,5 Chronic
thrombocytopenia in 1 HIV-infected chimpanzee has been
reported6 and was associated with chronic lymphocytopenia
affecting both CD4+ and CD8+ T-cell counts, an
eightfold increase in HIV-specific antibody titer, and the presence of
cell-free virus in plasma.
Three additional chimpanzees have now developed chronic
thrombocytopenia and lymphocytopenia at Yerkes Regional Primate
Research Center (Atlanta, GA). The present study evaluates the effects of stimulating megakaryocytopoiesis in this cohort of HIV-infected thrombocytopenic chimpanzees by administering pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) and
measuring (1) peripheral platelet counts, mean platelet volumes (MPVs), and platelet thrombopoietin (TPO) receptor
numbers; (2) marrow megakaryocyte numbers, volumes, ploidy
distributions, and CD34+ megakaryocyte progenitor cells;
(3) trough levels of PEG-rHuMGDF; (4) antiplatelet glycoprotein (GP)
IIIa49-66 antibodies7; and (5) viral loads.
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MATERIALS AND METHODS |
Animals studied.
Three chimpanzees (Pan troglodytes) previously infected with HIV and
maintained at Yerkes Regional Primate Research Center were included in
this study.6 All procedures were approved by the
Institutional Animal Care and Use Committee and conducted in accordance
with federal guidelines (Guide for the Care and Use of Laboratory
Animals, National Institutes of Health, Bethesda, MD, NIH Publication
No. 86-23). Ketamine hydrochloride (5 to 20 mg/kg intramuscularly) was
administered to achieve short-term immobilization for obtaining blood
samples, bone marrow aspirates, and biopsies.
Study design.
The effects of administering PEG-rHuMGDF (25 µg/kg subcutaneously
twice weekly on Monday and Thursday) were assessed in 3 thrombocytopenic HIV-infected chimpanzees. A twice weekly dose regimen
was selected because animal accession by darting was practically limited to once every few days, and every other day administration of
PEG-rHuMGDF had previously been shown to stimulate megakaryocytopoiesis adequately.8,9 Before the initial dose of PEG-rHuMGDF,
baseline blood and marrow samples were obtained. Before each subsequent dose of PEG-rHuMGDF, blood was obtained for complete blood counts (including leukocyte counts with differential counts and platelet counts), serum antiplatelet GPIIIa49-66 antibodies, and
serum PEG-rHuMGDF levels. When the platelet counts reached normal
values, dosing was discontinued and blood samples were obtained for
lymphocyte counts, antiplatelet GPIIIa49-66 antibodies,
serum PEG-rHuMGDF levels, and viral loads. In addition, bone marrow
cells were obtained for repeat morphologic evaluation, flow cytometric
quantitation of megakaryocytopoiesis, and in vitro cell culture
assessment of megakaryocytic progenitor responsiveness to PEG-rHuMGDF.
Therapy was discontinued when the peripheral platelet counts reached
normal levels.
PEG-rHuMGDF reagent.
PEG-rHuMGDF, a gift from Amgen Inc (Thousand Oaks, CA), is a
nonglycosylated polypeptide produced in Escherichia
coli-transfected with a plasmid containing cDNA that encodes for
the 1- to 163-residue aminoterminus of human Mpl ligand. The resulting
polypeptide is covalently coupled to poly ethylene
glycol.10,11 After extraction, refolding, and purification,
this truncated protein was supplied as a sterile, clear, aqueous
solution.
Laboratory procedures.
Peripheral platelet counts, mean platelet volumes, red blood cell
counts, and total white blood cell counts were determined in whole
blood collected in Na2EDTA (2 mg/mL) using Serono/Baker model 9000 whole blood analyzer (Allentown, PA).12-14
HIV serologic status was determined using a commercially available
enzyme-linked immunosorbent assay (ELISA) and confirmed with Western
blot assay.15
Plasma viral load was assayed by means of a reverse transcription
polymerase chain reaction (PCR).3,6,16 The quantitative HIV
RNA PCR assay was performed according to the manufacturer's instructions (Amplicor HIV-1 Monitor Test; Roche Diagnostic Systems, Branchburg, NJ). RNA was extracted from heparinized samples using silica.17 Fifty microliters of each prepared RNA sample was used for the PCR assay. After amplification and detection of the PCR
product, the initial HIV RNA load in each sample was calculated by
comparing it with the internal quantitation standard, and the results
were expressed as HIV RNA copies per milliliter of plasma.
Antiplatelet GPIIIa49-66 antibody profile was performed as
recently described.7 In prior studies, the strong
correlation between antiplatelet GP IIIa49-66 antibody
levels and thrombocytopenia has been interpreted to reflect direct
binding of these antiplatelet GPIIIa49-66 antibodies to
platelet GP IIIa49-66 epitope7 as well as
contributing to the binding of immune complexes to
platelets.18
Serum levels of endogenous TPO and PEG-rHuMGDF were determined using
ELISAs involving initial polyclonal antibody capture procedure followed
by enzyme product formation and determination.19,20
The baseline number of platelet receptors was obtained before and 10 days after initiating PEG-rHuMGDF therapy using purified rHu-TPO (a
gift from Amgen Inc) radiolabeled by Iodo-beads iodination reagent
(Pierce, Rockford, IL). rHu-TPO was incubated with 50 mmol/L sodium
phosphate buffer, pH 7.2, and 125I for 15 minutes.
Platelets were obtained from blood drawn in acid-citrate-dextrose (ACD)
anticoagulant, pelleting platelets from platelet-rich plasma by
centrifuging at 500g for 15 minutes, and resuspension in
Tyrode's buffer containing 1:7 vol/vol ACD, pH 6.2, 1% bovine serum
albumin (BSA), and 0.01% Tween. Binding isotherms were determined by
incubating platelets in plasma-free Tyrode's buffer, 1:7 vol/vol ACD,
1% BSA, 0.01% Tween, pH 6.2, and various amounts of
125I-TPO (40 to 640 ng/mL final
concentrations) for 1 hour at room temperature.
Nonspecific binding was assessed comparing the effects of adding
100-fold excess unlabeled TPO 30 minutes before the addition of
125I-TPO. Nonspecific binding ranged from 10% to 20%.
Binding isotherms were analyzed using the Biosoft Ligand Program
(Cambridge, UK) to compute the number of binding classes, the number of
molecules bound per platelet, and the dissociation constant
(Kd).
The appearance of activated platelets in the peripheral blood was
evaluated by flow cytometry using fluoresceinated monoclonal antibodies
(MoAbs) against neoantigen(s) comprising conformationally altered
activated GPIIb/IIIa, ligand-induced binding sites (LIBS; a gift from
Dr E. Plow, La Jolla, CA).21,22 In addition, enhanced binding to platelets by fluoresceinated annexin V (a gift from Dr T. Yokoyama, Tokyo, Japan) was also examined using flow
cytometry.23-25 Flow cytometric analysis was performed
using FACScan (Becton Dickinson, San Jose CA).
Measurements of platelet production.
Megakaryocyte number, size, and ploidy were measured by flow cytometry
using a previously reported method for multiparameter correlative
marrow analysis with a single-argon ion laser FACScan analyzer (Becton
Dickinson).26-31 Cell DNA in aspirated marrow was stained
with propidium iodide, and surface membrane receptors were analyzed
using specific MoAbs labeled with fluorescein. Megakaryocytes expressing GPIIb/IIIa were enumerated in relation to the nucleated erythroid precursors expressing glycophorin A.29,32
Measurements of megakaryocyte diameters were based on the time of
flight principle, ie, time required for a cell in suspension to pass
through a focused light beam.26,27,29,31 Megakaryocytes
were selected on the basis of their distinct immunofluorescence at
levels above that of control cells labeled with an unrelated MoAb. In
each sample, 2,000 to 3,000 megakaryocytes were analyzed. Bone marrow
aspirates were obtained baseline and after peaking of the platelet
counts after the administration of PEG-rHuMGDF.
Estimates of marrow megakaryocyte mass were used to represent the
marrow substrate giving rise to circulating platelets and were
calculated as the product of megakaryocyte numbers and mean megakaryocyte volumes.33,34 Normal chimpanzee marrow values (n = 10) averaged megakaryocyte diameter of 39 µg (range, 21 µg for
2N to 56 µg for 64N cells), volume of 28 ± 4.5 × 103 fL, and megakaryocyte number of 11 ± 2.1 × 106/kg, giving a total megakaryocyte mass of 31 ± 5.3 × 1010 fL/kg. The normal modal ploidy was
16N.
Marrow megakaryocyte progenitors.
The assays for megakaryocyte colony-forming units (CFU-Meg) was based
on a plasma clot matrix formed from human citrated AB plasma.35 Aliquots of 5 to 10 mL of bone marrow were
collected in heparin. Cells were diluted in modified Hank's buffered
saline solution (HBSS) layered over Ficoll-Hypaque and centrifuged at 2,000 rpm in a Sorvall RT6000 at room temperature for 30 minutes. The
mononuclear layer was collected, diluted with HBSS, washed twice by
centrifugation at 1,500 rpm for 5 min/wash, and then counted.
CD34+ cells used in the megakaryocyte assay were selected
using the Miltenyi Biotech MiniMACS magnetic cell separation kit
(Miltenyi Biotech, Sunnyvale, CA). Postcolumn purity of the
CD34+ cell fraction was determined by staining an aliquot
of cells with phycoerythrin-conjugated HPCA-2 MoAb (Becton Dickinson
Immunocytometry Systems, San Jose, CA) and subsequent FACS
analysis. PEG-rHuMGDF was used at a final concentration of 10 ng/mL and
cells were plated in a modified Iscove's modified Dulbecco's medium
(IMDM) at 2 × 104 cells/mL in 15% human AB plasma.
Cells were cultured in a 24-well microtiter plate with 300 µL/well
volumes in triplicate for 8 days in a 37°C incubator with 5%
CO2 humidity. Cultures were fixed with methanol:acetone
(1:2) and stained with antiplatelet CD41/42 (GPIIb/IIIa) MoAbs,
followed by goat antimouse fluorescein isothiocyanate (FITC). Nuclei
were stained with propidium iodide. A CFU-Meg colony was defined as  3
brightly fluorescent cells by inverted fluorescence microscopy.
Morphology.
Marrow biopsies were obtained from the posterior superior iliac spine
before and immediately after the last dose of PEG-rHuMGDF therapy. The
biopsies were fixed in 10% buffered formalin solution, embedded in
paraffin, sectioned, and stained with polychromatophilic dyes for
examination at the light level.
Data analysis.
Data were analyzed using SIGMA STAT (Jandel Scientific Software, San
Rafael, CA). Comparisons between two groups were performed using the
two-tailed Student's t-test, unless the data were not distributed randomly, in which case nonparametric analysis was performed. Analysis of variance was used to compare values for a
particular group at various time points.36 Unless otherwise stated, variance about the mean is given as ±1 SD.
| |
RESULTS |
Three chimpanzees, 2 males and 1 female, experimentally infected with
HIV developed significant chronic thrombocytopenia 5, 4, and 2 years
after infection. Peripheral platelet counts averaged 64 ± 19 × 103/µL (P = .004 compared with 228 ± 92 × 103/µL in 44 normal control animals;
Table 1), and the mean platelet volume was
11.2 ± 1.8 fL (P > .5 compared with 10.9 ± 0.7 fL in controls). Endogenous TPO levels were substantially increased, averaging 926 ± 364 pg/mL (P < .001 compared with 324 ± 256 pg/mL in normal controls; Table 1). The circulating load of
HIV was 534, 260, and 15 × 103 RNA viral copies/mL,
respectively (Table 1). Antiplatelet GPIIIa49-66 antibodies
were readily detected in the sera of all 3 animals (Table 1).
PEG-rHuMGDF (3 doses of twice weekly of 25 µg/kg) increased the
levels of Mpl ligand from the basal levels of 926 ± 364 pg/mL (endogenous TPO) to predosing trough levels of 1,840 ± 353 pg/mL PEG-rHuMGDF (Table 1; P = .02). Three doses of
PEG-rHuMGDF amplified peripheral platelet counts 10-fold
(Fig 1), peaking at 599 ± 260 × 103 platelets/µL on day 12 (P = .02 compared with
64 ± 19 × 103 platelets/µL pretreatment; Table
1), and gradually returned to baseline values over the subsequent 2 weeks (Fig 1). During thrombocytosis, the mean platelet volume was
reciprocally reduced to 9.5 ± 0.9 fL (P = .1 compared with 11.2 ± 1.8 fL pretreatment), analogous to the
decrease observed in baboons after dosing with PEG-rHuMGDF.37,38 Resting unstimulated platelets did not
express membrane activation markers during PEG-rHuMGDF therapy (ie,
LIBS were 290 ± 17 v 429 ± 146 LIBS/platelet baseline,
and annexin V binding sites were 3,900 ± 786 v
2,570 ± 1,040 annexin V binding sites/platelet baseline; P > .3 in both cases), concordant with the absence of platelet
activation in baboons receiving PEG-rHuMGDF.37,38 Similarly, PEG-rHuMGDF therapy did not change platelet TPO receptor numbers significantly (99 ± 43 v 152 ± 74 receptors/platelet baseline; P = .11).
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| Fig 1.
Elevation of peripheral platelet counts in
thrombocytopenic HIV-infected chimpanzees by PEG-rHuMGDF. Three doses
of PEG-rHuMGDF (25 µg/kg on days 1, 4, and 7) produced peripheral
platelet counts that peaked at 599 ± 260 × 103
platelets/µL (P = .02 compared with pretreatment value of
64 ± 19) 3 days after discontinuing therapy. The timing of the 3 doses of PEG-rHuMGDF is indicated by the downward arrows. Platelet counts returned to baseline thrombocytopenic levels during the subsequent 2 weeks.
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The serum levels of antiplatelet GPIIIa49-66 antibodies
decreased reciprocally during the period of thrombocytosis (baseline of
333 ± 278 v nadir of 169 ± 164 arbitrary OD units;
P < .07; Fig 2 and Table 1).
Presumably, the decline in circulating levels of antiplatelet
GPIIIa49-66 antibodies represented transient depletion from
plasma by high-affinity binding to the 10-fold increase in the
circulating concentration of platelets during thrombocytosis.
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| Fig 2.
Effects of PEG-rHuMGDF therapy on circulating levels of
antiplatelet GP IIIa49-66 antibodies. Stimulation of
megakaryocytopoiesis by PEG-rHuMGDF (3 subcutaneous twice weekly doses
of 25 µg/kg) increased the peripheral concentration of platelets
10-fold ( ), peaking at day 12 and followed by a gradual return to
baseline values. The mean concentration of circulating antiplatelet GP IIIa49-66 antibodies exhibited a reciprocal reduction ( )
during the period when the peripheral platelet counts were elevated. This transient decrease in antibody levels is attributable to depletion
resulting from high-affinity binding to the greatly expanded pool of
platelets in the circulation.
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The administration of PEG-rHuMGDF did not significantly alter viral
loads (395, 189, and 53 × 103 RNA viral copies/mL;
P > .5 compared with baseline values; Table 1) or
CD4+ T-cell counts (Table 1).
Marrow megakaryocyte numbers expanded 30-fold to 353 ± 255 × 106/kg (P = .04 compared with 11.7 ± 6.5 × 106/kg pretreatment; Table 1), and megakaryocyte
progenitor cell numbers increased more than fourfold to 14.1 × 103 CFU-Meg/1,000 CD34+ marrow cells (compared
with 3.6 ± 0.6 × 103 CFU-Meg/1,000
CD34+ marrow cells pretreatment; Table 1). However, when
peripheral platelet counts peaked, mean megakaryocyte volumes were
significantly decreased to 18.7 ± 2.1 × 103 fL (P = .014 compared with 38 ± 1.9 × 103 fL pretreatment; Table 1) and megakaryocyte ploidy was
reduced in concert (Fig 3; P = .013). The calculated overall megakaryocyte mass was expanded 15-fold
to 624 ± 373 × 1010 fL/kg (P = .04 compared with 45.2 ± 27.2 × 1010 fL/kg
pretreatment; Table 1).
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| Fig 3.
Shift in megakaryocyte ploidy distribution induced by
PEG-rHuMGDF treatment of HIV-infected thrombocytopenic chimpanzees. Baseline megakaryocyte ploidy distribution ( ) was shifted to a modal
ploidy of 32N (P = .5 compared with a modal ploidy of 16N in
normal controls). By contrast, 2 weeks after initiating high-dose
PEG-rHuMGDF therapy (subcutaneous injections of 25 µg/kg for 3 doses
administered twice weekly), there was a 30-fold expansion of
megakaryocyte numbers with a modal ploidy of 8N (P = .01).
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Peripheral neutrophil counts were also transiently increased from 5.2 ± 2.6 × 103/µL to 9.9 ± 5.0 × 103/µL, also peaking on day 12 (P = .01; Table 1). Neither the erythrocyte nor the reticulocyte
counts were altered significantly (P > .1; Table
1).
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DISCUSSION |
HIV-infected chimpanzees exhibit many of the features observed in
HIV-infected patients, including (1) reproducible infection with HIV
(type 1) by injecting cell-free virus, infected peripheral blood
mononuclear leukocytes, or homogenates of infected tissues from
HIV-infected donors; (2) comparable viral loads; (3) analogous patterns
of cellular and humoral immunologic responses; and (4) similar
hematologic complications.1-6 The present study confirms chronic thrombocytopenia among the hematologic alterations to be
expected.
Chronic thrombocytopenia develops in approximately one third of humans
infected with HIV at some time during the course of acquired
immunodeficiency syndrome (AIDS).39-41 The pathophysiologic basis for the development of thrombocytopenia in HIV-patients has been
ascribed to variable and changing proportions of accelerated platelet
destruction, enhanced splenic platelet sequestration, and impaired
platelet production.18,39,40,42-45 Kinetic studies have
demonstrated shortened platelet lifespan in thrombocytopenic HIV
patients, indicating that platelet production was not sufficiently expanded to compensate for antibody-mediated platelet destruction in
those patients.43,46 In a recent study of HIV-infected
patients, thrombocytopenia was due to a combination of three factors:
accelerated platelet removal from the systemic circulation, enhanced
platelet sequestration in the splenic circulation, and inadequate
compensatory increases in platelet formation despite a threefold
expansion in marrow megakaryocyte mass.47 This threefold
disparity between marrow platelet substrate and circulating platelet
product is attributed to ineffective platelet formation by HIV-infected
megakaryocytes or HIV-induced inhibitory cytokines. The possibility of
HIV having detrimental effects on platelet formation by megakaryocytes
is supported by observations that thrombocytopenia in HIV-infected patients responds to antiviral therapy.48,49 In addition,
in situ hybridization studies have demonstrated HIV infection in marrow
megakaryocytes from thrombocytopenic HIV patients50 and morphologic abnormalities in megakaryocytes from thrombocytopenic HIV
patients, including naked nuclei and broad peripheral cytoplasmic rims.
Moreover, accelerated apoptosis has been documented in megakaryocytes obtained from thrombocytopenic HIV patients, and the degree of programmed cell death was inversely proportional to the severity of
thrombocytopenia.51 Thus, impaired platelet production in thrombocytopenic HIV patients may be due to premature apoptosis developing in HIV-infected megakaryocytes before the formation of
platelets by the dying megakaryocytes.
Platelet production in HIV chimpanzees with chronic thrombocytopenia
(Table 1) resembles platelet production in thrombocytopenic HIV-infected patients in several important respects.47
First, antiplatelet GPIIIa49-66 antibodies form and bind to
circulating platelets, producing accelerated platelet removal and
enhanced splenic sequestration.7 Second, circulating levels
of endogenous TPO are increased threefold over normal controls. Third,
marrow megakaryocytes are significantly increased in number, volume, and ploidy distribution compared with normal controls; enhanced megakaryocyte volume and ploidy are particularly characteristic of
TPO-driven augmentation of megakaryocytopoiesis.38
Three doses of PEG-rHuMGDF in thrombocytopenic HIV-infected chimpanzees
increased peripheral platelet counts 10-fold by amplifying marrow
megakaryocyte progenitors fourfold and marrow megakaryocyte numbers
30-fold, thereby expanding overall megakaryocyte mass 15-fold without
mobilizing HIV reservoirs. The reciprocal decrease in circulating
levels of antiplatelet GPIIIa49-66 antibodies during the
period of thrombocytosis is attributable to binding-depletion resulting
from the 10-fold increase in the concentration of peripheral platelets
(Table 1). Surprisingly, PEG-rHuMGDF therapy was associated with a
reduction in megakaryocyte volume and ploidy in these animals. Previous
studies in nonhuman primates and patients receiving PEG-rHuMGDF therapy
showed enlargement of megakaryocyte volume and ploidy in a log-linear
dose-dependent manner.9,37,38,52,53 One possible
explanation for the 30-fold increase in megakaryocyte number (Table 1)
with a reduction in megakaryocyte ploidy (Fig 2) may be that high-dose
PEG-rHuMGDF stimulation of chronic pre-existing TPO-stimulated
megakaryocytopoiesis may favor the proliferation of megakaryocytes from
progenitors, rather than fostering endoproliferation of already formed
megakaryocytes. Alternatively, the matured megakaryocytes may have been
selectively lost by cytoplasmic fragmentation into circulating
platelets and nuclear processing without a sustained stimulus for high
ploidy replacement during the 3 days between final dosing and marrow
sampling.
The striking elevation of the peripheral platelet count after
administering PEG-rHuMGDF therapy (3 doses of 25 µg/kg over 7 days)
to thrombocytopenic HIV chimpanzees constitutes convincing evidence of
therapeutic benefit. Based on PEG-rHuMGDF's pharmacokinetics and
log-linear dose-response38 and presumed platelet kinetic profile,47 it seems likely that a single 25 µg/kg dose of
PEG-rHuMGDF would have transiently normalized peripheral platelet
counts and that twice weekly injections of 5 µg/kg would have
maintained the peripheral platelet concentrations within the normal
range.
Thus, the extraordinary increase in peripheral platelet counts produced
by PEG-rHuMGDF therapy in thrombocytopenic chimpanzees infected with
HIV implies that the thrombocytopenia is largely due to insufficient
compensatory expansion in platelet production due to HIV-dependent
impairment in the delivery of platelets, despite stimulated
megakaryocytopoiesis. These findings suggest that PEG-rHuMGDF therapy
may be similarly corrective of peripheral platelet counts in
thrombocytopenic HIV-infected patients.
| |
FOOTNOTES |
Submitted October 6, 1997;
accepted March 19, 1998.
Supported in part by a grant from the National Institutes of Health to
Yerkes Regional Primate Research Center (RR-00165) and a Research Grant
from Amgen Inc.
Address reprint requests to Laurence A. Harker, MD, Division of
Hematology and Oncology, Emory University, 1003 Woodruff Memorial Bldg,
1639 Pierce Dr, Atlanta, GA 30322.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
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Abstract
Introduction
Abstract