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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 9 (May 1), 1998:
pp. 3239-3246
Ineffective Platelet Production in Thrombocytopenic Human
Immunodeficiency Virus-Infected Patients
By
James L. Cole,
Ulla M. Marzec,
Clifford J. Gunthel,
Simon Karpatkin,
Lydia Worford,
I. Birgitta Sundell,
Jeffrey L. Lennox,
Janet
L. Nichol, and
Laurence A. Harker
From the Department of Medicine, Emory University School of Medicine,
Ponce de Leon Center, Atlanta GA; New York University Medical Center,
New York, NY; and Amgen, Inc, Thousand Oaks CA.
 |
ABSTRACT |
Thrombocytopenia has been characterized in six patients infected
with human immunodeficiency virus (HIV) with respect to the delivery of
viable platelets into the peripheral circulation (peripheral platelet
mass turnover), marrow megakaryocyte mass (product of megakaryocyte
number and volume), megakaryocyte progenitor cells, circulating levels
of endogenous thrombopoietin (TPO) and platelet TPO receptor number,
and serum antiplatelet glycoprotein (GP) IIIa49-66 antibody
(GPIIIa49-66Ab), an antibody associated with thrombocytopenia in HIV-infected patients. Peripheral platelet counts
in these patients averaged 46 ± 43 × 103/µL
(P = .0001 compared to normal controls of 250 ± 40×
103/µL), and the mean platelet volume (MPV) was 10.5 ± 2.0 fL (P > 0.3 compared with normal control of
9.5 ± 1.7 fL). The mean life span of autologous
111In-platelets was 87 ± 39 hours (P = .0001
compared with 232 ± 38 hours in 20 normal controls), and immediate
mean recovery of 111In-platelets injected into the systemic
circulation was 33% ± 16% (P = .0001 compared with
65% ± 5% in 20 normal controls). The resultant mean peripheral
platelet mass turnover was 3.8 ± 1.5 × 105 fL/µL/d
versus 3.8 ± 0.4 × 105 fL/µL/d in 20 normal controls
(P > .5). The mean endogenous TPO level was 596 ± 471 pg/mL (P = .0001 compared with 95 ± 6 pg/mL in 98 normal control subjects), and mean platelet TPO receptor number was 461 ± 259 receptors/platelet (P = .05 compared with 207 ± 99 receptors/platelet in nine normal controls). Antiplatelet GPIIIa49-66Ab levels in sera were uniformly increased in
HIV thrombocytopenic patients (P < .001). In this cohort of
thrombocytopenic HIV patients, marrow megakaryocyte number was
increased to 30 ± 15 × 106/kg (P = .02
compared with 11 ± 2.1 × 106/kg in 20 normal
controls), and marrow megakaryocyte volume was 32 ± 0.9 × 103 fL (P = .05 compared with
28 ± 4.5 × 103 fL in normal controls). Marrow
megakaryocyte mass was expanded to 93 ± 47 × 1010 fL/kg
(P = .007 compared with normal control of
31 ± 5.3 × 1010 fL/kg). Marrow megakaryocyte
progenitor cells averaged 3.3 (range, 0.4 to 7.3) CFU-Meg/1,000
CD34+ cells compared with 27 (range, 0.1 to 84)
CFU-Meg/1,000 CD34+ cells in seven normal subjects
(P = .02). Thus, thrombocytopenia in these HIV patients was
caused by a combination of shortening of platelet life span by two
thirds and doubling of splenic platelet sequestration, coupled with
ineffective delivery of viable platelets to the peripheral blood,
despite a threefold TPO-driven expansion in marrow megakaryocyte mass.
We postulate that this disparity between circulating platelet product
and marrow platelet substrate results from direct impairment in
platelet formation by HIV-infected marrow megakaryocytes.
| |
INTRODUCTION |
CHRONIC THROMBOCYTOPENIA develops in
approximately one third of individuals infected with human
immunodeficiency virus (HIV) during the course of acquired
immunodeficiency syndrome (AIDS).1-3 The pathophysiologic
bases for the development of thrombocytopenia in HIV-infected patients
has been ascribed to changing proportions of three variables:
immune-mediated platelet destruction, enhanced platelet splenic
sequestration, and impaired platelet production.1,2,4-8 Kinetic studies demonstrate shortened platelet life span in
thrombocytopenic HIV-infected patients, suggesting that platelet
production was not sufficiently expanded to compensate for accelerated
platelet destruction in these patients.5,9 This study was
designed to characterize platelet production in a cohort of
HIV-infected patients with chronic thrombocytopenia by measuring the
relative contributions of impaired platelet production, increased
platelet removal, augmented splenic sequestration, marrow
megakaryocytopoietic responsiveness to stimulation by endogenous
thrombopoietin, and antiplatelet GPIIIa49-66Ab, an antibody
directed against the newly described immunodominant epitope in
HIV-infected patients with thrombocytopenia.10
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MATERIALS AND METHODS |
Patients studied.
Thrombocytopenic HIV-infected patients were recruited from the Ponce de
Leon Center, a free-standing infectious disease clinic affiliated with
Grady Memorial Hospital in Atlanta, GA. Six men with HIV-associated
thrombocytopenia of longer than 6 months duration were studied. Their
hematologic, viral, and overall clinical evaluation are presented in
Table 1. None of the patients was on any
medication known to affect platelet counts or function, nor had they
changed antiviral therapy within 3 months before entering the study.
Informed consent was obtained from each participant upon admission to
the Generalized Clinical Research Center (GCRC) at Emory University (Atlanta, GA). A detailed clinical history, physical examination, complete blood counts, serum chemistries, urinalysis, chest radiograph, and electrocardiogram were obtained at that time.
Study design.
The study was designed to characterize platelet production in these
patients by determining (1) autologous 111In-platelet mass
turnover, a measure of platelet delivery into the peripheral
circulation and defined in the steady state as effective platelet
production; (2) marrow megakaryocyte mass, the product of megakaryocyte
numbers and megakaryocyte volumes, represents the substrate from which
platelets are formed, and defined as total platelet production; (3)
marrow megakaryocyte progenitor cells obtained by stimulating
CD34+ marrow mononuclear cells with pegylated recombinant
human megakaryocyte growth and development factor (PEG-rHuMGDF); (4)
serum levels of endogenous TPO, representing the thrombocytopoetic
stimulus, and platelet TPO receptor numbers, a measure of TPO receptor
density on precursor marrow megakaryocytes; and (5) serum antiplatelet GPIIIa49-66Ab, a marker of immune platelet injury. These
results were related to measurements of plasma viral load, peripheral blood CD4+ T-cell counts, platelet, and marrow morphology.
Laboratory procedures.
Peripheral platelet counts, mean platelet volumes (MPV), red blood cell
(RBC) counts, and total white blood cell (WBC) counts were determined
in whole blood collected in Na2EDTA (2 mg/mL), using
Serono/Baker model 9000 whole blood analyzer (Allentown, PA).11-13
The HIV serologic status of these patients was determined using a
commercially available enzyme-linked immunosorbent assay (ELISA) and
confirmed with Western blot assay.14 Plasma HIV loads were
determined by means of a reverse transcription-polymerase chain
reaction (RT-PCR).15,16 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 A total
of 50 µL 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; the results were expressed as HIV RNA
copies per milliliter plasma.
Serum and platelet HIV-associated antibodies.
Antiplatelet GPIIIa49-66Ab profile was performed
as recently described.10 In prior studies, the strong
correlation between antiplatelet GPIIIa49-66Ab levels and
thrombocytopenia, have been interpreted to reflect direct binding of
these antiplatelet IgG antibodies directed against the immunodominant
epitope GPIIIa49-66,10 as well as contributing
to the binding of immune complexes to platelets.6
TPO levels in serum and TPO receptors on platelets.
Serum levels of endogenous TPO were determined using an ELISA involving
an initial polyclonal antibody capture procedure followed by
horseradish peroxidase (HPO)-linked signal antibody to generate color
using TMB substrate.18-20 The assay was sensitive to 30 pg/mL and reproducible with 15% coefficient of variation.
Mean platelet TPO receptor numbers were estimated from platelet binding
isotherms of 125I-labeled recombinant human (rHu) TPO
according to Scatchard analysis.21,22 In brief, TPO
receptors on platelets were determined using purified rHu-TPO, a gift
from Amgen (Thousand Oaks, CA), 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 with Iodo-beads
for 15 minutes. Platelets were obtained from blood drawn in
acid-citrate-dextrose (ACD) anticoagulant (1:7 vol/vol), pelleting
platelets from platelet-rich plasma by centrifuging at 500g for
15 minutes, resuspension in Tyrode's buffer containing ACD (1:7
vol/vol), pH 6.2, 1% bovine serum albumin (BSA), and 0.01% Tween.
Binding isotherms were obtained by incubating platelets in plasma-free
Tyrode's buffer, ACD (1:7 vol/vol), 1% BSA, 0.01% Tween, pH 6.2, and
various amounts of 125I-rHuTPO (40 to 640 ng/mL final
concentrations) for 1 hour at room temperature. Nonspecific binding was
assessed by comparing the effects of adding 100-fold excess unlabeled
rHu-TPO 30 minutes before 125I-rHuTPO was added.
Nonspecific binding ranged from 10% to 20%. Binding isotherms were
analyzed using the Biosoft Ligand Program (Cambridge, UK) to determine
the number of binding classes, the number of molecules bound/platelet,
and the dissociation constant.
Platelet kinetic measurements.
To measure platelet life span, autologous platelets were labeled with
111In-oxine, using the method described
previously.23 Labeling efficiencies averaged 90%, and the
labeled platelets functioned normally.24,25 After
reinjection, twice-daily blood samples were collected and analyzed for
111In-platelet activity to determine the rate at which
111In-platelets were cleared from the circulation. Platelet
life span (ie, the average time platelets remained in circulation) was
then calculated using computer least-squares fitting of the raw data to
a  -function modeling program.23 The immediate recovery of injected 111In-platelets in the circulation was
estimated by extrapolating the platelet radioactivity disappearance
curve to time zero and estimating the blood volume (70 mL/kg), using
the formula:
The
results were compared with two groups of thrombocytopenic patients: (1)
17 patients with megakaryocyte hypoplasia; and (2) 9 patients with
idiopathic thrombocytopenic purpura. Data regarding these two groups
have been reported previously.23
Steady-state platelet mass turnover was calculated by multiplying the
peripheral platelet concentration by the mean platelet volume and
dividing by platelet life span and the percentage recovery of injected
radiolabeled autologous platelets. In the steady state, platelet mass
turnover was a measure of the rate at which viable platelet mass was
delivered to the peripheral blood.26
Marrow megakaryocytopoiesis.
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 fluoroscein-activated cell sorter (FACS) analyzer (FACScan Becton Dickinson, San Jose, CA).27-31 Cell DNA in aspirated marrow was
stained with propidium iodide, and surface membrane receptors were
analyzed with antibodies labeled with fluorescein. Megakaryocytes
expressing platelet GPIIb/IIIa were enumerated in relation to the
nucleated erythroid precursors expressing glycophorin
A.30,32 Measurements of megakaryocyte diameters were based
on the time-of-flight principle, the time required for a cell
in suspension to pass through a focused light beam.27,28,30,31 Aspirated bone marrow (3 mL) obtained from the pelvic bones was collected into 10-mL plastic syringes containing equal volumes of ACD (formula A), 2.5 mmol/L EDTA and 2.2 µmol/L prostaglandin E1 (PGE1) (Sigma Chemical Co., St
Louis, MO), final concentrations. The marrow was gently pipetted,
passed through a 120-µm monofilament nylon filter, and diluted with
cold Ca2+-free and Mg2+-free phosphate-buffered
saline (PBS) containing 13.6 mmol/L sodium citrate, 2.2 µmol/L PGE1, 1 mmol/L theophylline (Sigma), 3% BSA (Fraction V; Calbiochem, La Jolla, CA), 11 mmol/L glucose, and adjusted
to a pH of 7.3 and an osmolarity of 290 mOsm/L. Megakaryocytes were
analyzed in marrow aspirates fractionated with 1.06 density Percoll
(Pharmacia Biotech, Piscataway, NJ). The nucleated erythroid marrow
cells were analyzed from marrow separated over 1.08 density Percoll
(Pharmacia Biotech). Megakaryocytes were selected on the basis of their
distinct immunofluorescence at levels above that of control cells
labeled with an unrelated monoclonal antibody (MoAb). In each sample,
at least 2,000 to 3,000 megakaryocytes were analyzed. Flow cytometric
analysis was performed using FACScan Lysis Program (Becton Dickinson).
Marrow megakaryocyte mass was the product of megakaryocyte number and
mean megakaryocyte volume; it was used to represent the marrow
substrate from which platelets are formed.26,33
Marrow CD34+ megakaryocyte progenitors.
The assays for colony-forming unit-megakaryocyte (CFU-Meg) was based on
a plasma clot matrix formed from human citrated AB plasma.34 Aliquots of 5 to 10 mL bone marrow were collected in heparin. Cells were diluted in modified Hanks' 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 (BDIS, 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 IMDM
medium 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 CD41/42 (GPIIb/IIIa) antiplatelet antibodies, followed by goat
anti-mouse FITC. Nuclei were stained with propidium iodide. A CFU-Meg
colony was defined as 3 or more brightly fluorescent cells by inverted
fluorescence microscopy.
Morphology.
Marrow aspirates and cores were obtained from the posterior superior
iliac crest. Core biopsy specimens were fixed in 10% buffered formalin
solution, embedded in paraffin, and sectioned. Aspirate and core
samples were 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 (ANOVA) was used to compare values for
a particular group at various time points.35 Unless
otherwise stated, variance about the mean is given as ±1 SD.
| |
RESULTS |
The hematologic, viral, and overall clinical characteristics of this
cohort of thrombocytopenic HIV-infected patients are presented in Table
1. HIV viral loads ranged from 0.16 to 366 × 103 RNA
viral copies/mL of plasma. In five of the six patients, the CD4+ T-cell counts were less than 250 cells/µL. The
peripheral erythrocyte counts were within the range for normals
(P > .1), but only two of the six patients had normal
leukocyte counts (Table 1).
The peripheral platelet concentrations in these six thrombocytopenic
HIV-infected patients averaged 46 ± 43 (range, 5 to 102) × 103/µL (P = .0001 compared with
250 ± 40 × 103/µL in 98 normal controls; Table
2). The MPV was 10.5 ± 2.0 fL, not
significantly different from the normal controls of 9.5 ± 1.7 fL
(Table 2; P > .3).
Antiplatelet GPIIIa49-66Ab levels were uniformly elevated
in the sera of these thrombocytopenic HIV-infected patients, averaging 129 ± 91 arbitrary OD units (P < .001 compared with less
than 21 units in 20 control subjects; Table 2), consistent with
previously published reports.1,2,4,6,7,10
TPO serum levels and TPO platelet receptors.
Endogenous TPO levels in serum were increased by more than sixfold; ie,
mean serum concentration of TPO was 596 ± 471 pg/mL compared with 95 ± 6 pg/mL in 98 normal subjects (Table 2;
P = .0001).20 Binding studies using
125I-labeled rHu-TPO demonstrated increased platelet TPO
receptors in thrombocytopenic HIV-infected patients (461 ± 259
receptors/platelet v 207 ± 99 receptors/platelet in seven
normal controls; Table 2; P = .04).
Platelet kinetic measurements.
The immediate recovery of autologous 111In-labeled
platelets in thrombocytopenic HIV-infected patients was decreased to 33 ± 16%, compared with 65 ± 5% in 20 normal controls
(Table 2; P = .0001). The single patient exhibiting normal
recovery of injected 111In-labeled autologous platelets
(Table 2) is presumably explained by the absence of clinical
splenomegaly (Table 1).
The life span of autologous 111In-platelets was shortened
to 87 ± 39 hours in thrombocytopenic HIV-infected patients
(P = .0001 compared with 232 ± 38 hours in 20 normal
controls; Table 2). Compared to another group of patients with
thrombocytopenia caused by megakaryocyte hypoplasia (Fig
1), platelet life span measurements in
three of the six thrombocytopenic HIV-infected patients were shortened
according to the severity of the thrombocytopenia (Table 2, Fig 1).
This concentration-dependent reduction in platelet life span has been
attributed to increasing proportions of peripheral platelets
undergoing utilization in maintaining platelet hemostatic function23,36 (Fig 1). At least two, and probably three,
HIV-infected thrombocytopenic patients had reduced platelet life spans
shorter than would have been predicted by the peripheral platelet
counts, implicating immune-mediated platelet destruction as an
extrinsic hazard, similar to patients with immune-mediated
thrombocytopenia typical of immune thrombocytopenic purpura
(ITP)23 (Fig 1). No identified clinical features were found
to discriminate those patients with shorter platelet life spans than
expected for the degree of thrombocytopenia from those with life spans
appropriate for the degree of thrombocytopenia.
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| Fig 1.
Relationship between autologous
111In-platelet life span and peripheral platelet
concentration. Platelet life span results are compared to peripheral
platelet counts in three groups of thrombocytopenic patients: (1) six
HIV-infected patients (depicted by large solid circles); (2) 17 patients with thrombocytopenia due to megakaryocytic hypoplasia
(identified by open circles); and (3) nine patients with
thrombocytopenia due to autoimmune destruction (clinical idiopathic
thrombocytopenic purpura, ITP, shown by open squares). Three
thrombocytopenic HIV-infected patients demonstrate
concentration-dependent shortening of platelet life spans attributable
to increased platelet utilization in maintaining platelet hemostatic
function. By contrast, two thrombocytopenic HIV patients show
shortening of platelet life span that is greater than expected on the
basis of peripheral platelet counts per se, thereby implicating
antiplatelet GPIIIa49-66Abs in the immune-mediated random
destruction of platelets. The markedly shortened platelet life span in
the sixth thrombocytopenic HIV-infected patient is also probably
attributable to immune destruction, although the above comparison of
platelet count to platelet life span does not clearly discriminate
between hemostatic utilization versus immune destruction for platelet
counts of less than 10,000 µL. The platelet life span data in
patients with thrombocytopenia secondary to marrow hypoplasia and
idiopathic thrombocytopenia represent results obtained from a prior
report.23
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Calculated platelet mass turnover was 3.8 ± 1.5 × 105
fL/µL/d, equivalent to 3.8 ± 0.41 × 105 fL/µL/d in
normal controls (Table 2; P > .5).
Marrow megakaryocyte measurements.
In these thrombocytopenic HIV-infected patients the number of marrow
megakaryocytes was increased nearly threefold, ie, 30 ± 15 × 106/kg compared with 11 ± 2.1 × 106/kg
(P = .02). Bone marrow biopsy specimens showed normal
cellularity with increased ratios of morphologic megakaryocytes to
nucleated erythroid cells (Fig 2).
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| Fig 2.
Increased megakaryocytes in bone marrow biopsy samples
obtained from HIV-infected patients with thrombocytopenia. In this cohort of thrombocytopenic HIV-infected patients bone marrow biopsy samples typically showed normal to abundant megakaryocytes;
megakaryocyte/erythroid ratios were typically increased.
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Marrow megakaryocyte volumes averaged 32 ± .9 × 103 fL
(P = .05 compared with
28 ± 4.5 × 103 fL in 20 normal controls).
Changes in megakaryocyte volume were caused by the relative increase in
high-ploidy megakaryocytes (Fig 3;
P = .0001).
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| Fig 3.
Comparison of marrow megakaryocyte ploidy distribution
for HIV-infected thrombocytopenic patients and normal subjects. The megakaryocyte ploidy distribution for 20 normal subjects is depicted by
the open bars. The ploidy distribution of marrow megakaryocytes for six
thrombocytopenic HIV patients is shown by the hatched bars. While there
is a significant increase in 64N megakaryocytes in patients with HIV
thrombocytopenia (P < .0001), the overall megakaryocyte
ploidy is nearly normal, because of the increase in 4N and 8N cells at
the expense of 16N cells. The ploidy distribution in HIV
thrombocytopenic patients is interpreted to represent TPO-driven stimulation of megakaryocyte growth and development.
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The median marrow megakaryocyte progenitor cell number in marrow
aspirates from HIV-infected thrombocytopenic patients was significantly
decreased by an order of magnitude, averaging 3.3 (range, 0.4 to 7.3)
CFU-Meg/1,000 CD34+ cells compared with 27 (range, 0.1 to
6.1) in thrombocytopenic HIV-infected patients (P = .02).
The mean marrow megakaryocyte mass, the product of megakaryocyte
numbers and megakaryocyte volumes, was increased threefold, i.e., 93 ± 47 × 1010 fL/kg compared with normal control value of
31 ± 5.3 × 1010 fL/kg (P = .007). Despite
this threefold expansion in megakaryocyte substrate available for
platelet formation, there was no increase in the delivery of viable
platelets to the peripheral blood (platelet mass turnover measurements
compared with results in normal controls; Table 2).
| |
DISCUSSION |
This study in six HIV-infected patients with chronic thrombocytopenia
shows that the low peripheral platelet counts were the result of a
combination of reduced life span of platelets in the systemic
circulation and enhanced sequestration of platelets in the splenic
circulation, coupled with ineffective compensatory responses in
platelet formation despite a threefold expansion in marrow
megakaryocyte mass. This disparity between circulating platelet product
and marrow platelet substrate may reflect impairment in platelet
formation resulting from HIV-infected marrow megakaryocytes, or
HIV-induced inhibitory cytokines.
Concordant with previous published reports,1,2,4,6,7,10
antiplatelet GPIIIa49-66Ab were uniformly elevated in the
sera of these thrombocytopenic HIV-infected patients (Table 2).
However, no reciprocal relationship was observed between antiplatelet
GPIIIa49-66Ab and peripheral platelet counts (Table 2).
Shortened platelet survival times observed in thrombocytopenic HIV-infected patients have generally been attributed to immune platelet
destruction induced by these antiplatelet GPIIIa49-66Abs directed against autologous platelet GPIIIa49-66
immunodominant epitope, analogous to immune-mediated thrombocytopenia
typical of ITP involving autoantibodies exhibiting gpIb/IX or
gpIIb/IIIa specificity, or both.5,9 The present
observations provide additional evidence that immune platelet
destruction is dominant in some, but not all, patients (Fig 1).
Platelet life span was substantially decreased in all thrombocytopenic
HIV-infected patients studied; on average, platelet survival time was
reduced by approximately two thirds, compared with results obtained in
normal control subjects (Table 2). At least two, and probably three, of
the six HIV-thrombocytopenic patients demonstrated ITP-like immune
platelet destruction mediated by antiplatelet
antibodies5,23 (Fig 1, and Table 2). By contrast, three of
the six patients convincingly demonstrated reductions in platelet life
span characteristic of the degree of thrombocytopenia, per se, typical
of the relationship between peripheral platelet counts and shortened
platelet lifespan observed in patients with thrombocytopenia secondary
to megakaryocyte hypoplasia23,36 (Fig 1). Accelerated
platelet removal in these three patients was mediated by
thrombocytopenia-dependent enhanced platelet utilization required to
maintain platelet hemostatic function, as opposed to the antiplatelet
antibody-mediated platelet removal typically occurring in patients with
chronic ITP. We postulate that the shortening of platelet survival
times reported by others may also reflect, at least in part,
utilization secondary to thrombocytopenia.5,9,23 This
interpretation assumes that circulating platelets may bear attached
molecules of IgG without inducing significantly accelerated platelet
removal, a well-documented observation.37
In the present study megakaryocyte number, volume and ploidy were
quantified in aspirated marrow using flow cytometric analyses. This
technique is well suited to measuring low-frequency cellular events in
complex cell suspensions.27,29,30 Highly efficient DNA
staining was combined with specific probes for megakaryocytes and
erythroid forms, to obtain measurements of megakaryocyte size, ploidy,
and number.29,30 Estimating the marrow megakaryocyte number
depended on determining the ratio of marrow megakaryocytes to nucleated
erythroid cells and assumed that erythropoiesis remained normal and
constant. Relatively normal steady-state peripheral erythrocyte counts
in these patients justifies that assumption (Table 1). This approach to
megakaryocyte quantitation also presumes that GPIIb/IIIa-negative
megakaryocyte progenitors do not constitute quantitatively important
proportions of total marrow megakaryocytes. In this regard less than
5% of marrow megakaryocytes are sufficiently immature that they fail
to express GPIIb/IIIa.32
Flow cytometric megakaryocyte quantitation documented a threefold
expansion in marrow megakaryocyte substrate available for platelet
production in these thrombocytopenic HIV-infected patients (Table 2).
Estimates of increased megakaryocytopoiesis were also evident from the
marrow biopsies (Fig 2). We attribute this amplification in marrow
megakaryocyte mass to stimulation by endogenous TPO (Table 2), because
endogenous TPO was significantly elevated in these patients (Table 2).
However, the reduction in the number of marrow megakaryocyte
progenitors in these patients implies a direct effect of HIV on
megakaryocyte development (Table 2). The decreased frequency of
megakaryocyte progenitors within the CD34+ population in
the marrow of HIV-infected patients is comparable to the reduction in
CFU-Megs found in the marrow of chemotherapy patients undergoing
autologous bone marrow transplantation. This reduction in marrow
progenitors may be due to inhibitory effects of HIV on the
proliferation of marrow hematopoietic progenitor cells.
The increase in endogenous TPO levels was modest, similar to levels
observed in ITP patients, a setting also characterized by enhanced
thrombocytopoiesis.20,23 By contrast, patients with severe
thrombocytopenia caused by marrow aplasia attain levels exceeding
normal values by at least an order of magnitude.18-20 It is
reasonable to speculate that the levels of free TPO in plasma may be
less than that observed in marrow aplasia caused by removal by
competitive binding of threefold increased TPO-receptor density on
platelets undergoing normal turnover and threefold expanded marrow
megakaryocyte cytoplasm. Presumably, the megakaryocytes also express
threefold increased TPO-receptor density, since the increased TPO
receptor density on platelets must have originated from higher TPO
receptor-density marrow megakaryocytes (Table 2). Thus, the
thrombocytopoietic stimulatory capacity of modestly elevated endogenous
TPO levels in HIV-infected thrombocytopenic patients may be
functionally equivalent to substantially greater circulating levels of
TPO, per se, as a result of enhanced binding and augmented
responsiveness of marrow megakaryocytes to free circulating TPO.
Preclinical evidence in nonhuman primates also supports the notion that
the increase in megakaryocytopoiesis is TPO driven by demonstrating
that increases in megakaryocyte ploidy constituted early, sensitive,
and quantitative morphologic indicators of TPO stimulation of
megakaryocytopoiesis, and that full expansion in megakaryocyte number
developed over weeks.1,5,9 Published results of marrow
changes in patients receiving injections of exogenous TPO or
PEGrHuMGDF report similar changes in megakaryocytes produced
by comparably increased levels of cytokine.
We conclude that the elevated TPO levels in sera from
HIV-thrombocytopenic patients are capable of stimulating
thrombocytopoiesis sufficiently to compensate for the modest shortening
in platelet life span and doubling of splenic pooling. The contrast
between the threefold expansion in megakaryocyte substrate available
for platelet formation and the unchanged normal platelet mass turnover (Table 2), represents a threefold disparity between marrow substrate and circulating product, a disorder known as ineffective production.
Thrombocytopenia in HIV-infected patients responds to antiviral
therapy,38-40 implying that the low platelet counts in
HIV-infected patients are directly related to HIV infection. In situ
hybridization studies demonstrate HIV infection in marrow
megakaryocytes from thrombocytopenic HIV patients. In addition,
megakaryocytes in thrombocytopenic HIV patients frequently show
morphologic abnormalities, including naked nuclei and broad peripheral
cytoplasmic rims.41 Accelerated apoptosis also develops in
megakaryocytes obtained from thrombocytopenic HIV patients, and the
degree of programmed cell death was inversely proportional to the
severity of thrombocytopenia.42 The present demonstration
of a threefold disparity between marrow substrate and circulating
product suggests that maturing marrow megakaryocytes may undergo
HIV-induced apoptosis before platelet formation has been completed by
prematurely dying megakaryocytes.
Thrombocytopenia in some HIV-infected patients also responds to
immunosuppressive therapies similar to those used in managing patients
with chronic ITP, including high-dose steroids, intravenous immunoglobulin (IVIG), splenectomy, anti-D antibody, vincristine, danazol, and interferon.43 These observations are
consistent with the present evidence implicating antiplatelet
GPIIIa49-66Ab in the mechanism underlying platelet
destruction in some patients, or at some stages in the course of their
illness. Antiplatelet GPIIIa49-66Ab produced
immune-mediated platelet destruction in at least two, and probably
three of the six patients in this study (Fig 1, Table 2), and IVIG
therapy transiently normalized their peripheral platelet counts.
However, some immunosuppressive therapies may complicate the clinical
course of AIDS. Because major bleeding seldom occurs in
thrombocytopenic HIV patients despite prolonged low platelet counts,
similar to the clinical course in chronic ITP patients, immune
thrombocytopenia in HIV patients may need therapy only when confronted
with some procedural, surgical, or traumatic event.44
Alternatively, peripheral platelet counts in thrombocytopenia in HIV
patients may be successfully treated by pharmacologic hyperstimulation
of megakaryocytopoiesis using PEG-rHuMGDF or rHuTPO. This possibility
is supported by the recent observation that the administration of three
twice-weekly doses of PEG-rHuMGDF to thrombocytopenic HIV-infected
chimpanzees increased the peripheral platelet counts 10-fold without
increasing HIV load.45 By contrast, other cytokines
effecting monocyte proliferation, such as GM-CSF, M-CSF,
and IL-3 have been shown to increase HIV replication in vitro.46
In summary, thrombocytopenia in HIV-infected patients results from
decreased formation of viable platelets despite TPO-expanded megakaryocytopoiesis, together with accelerated platelet removal from
the systemic circulation and increased platelet sequestration in the
splenic circulation. We attribute the threefold disparity between
marrow platelet substrate and circulating platelet product to impaired
formation of platelets by HIV-infected megakaryocytes, or HIV-induced
inhibitory cytokines. This formulation suggests that therapy with
PEG-rHuMGDF may be useful in HIV-infected patients with chronic
thrombocytopenia.
| |
FOOTNOTES |
Submitted September 30, 1997;
accepted December 18, 1997.
Supported in part by U.S. Public Health Service Grant No. MO1-RR-00039
from the GCRC program of the NIH NCRR and during a Clinical Research
Fellowship (to J.L.C.) provided by Amgen, Inc, Thousand Oaks, CA.
Address reprint requests to Laurence A. Harker, MD, Division of
Hematology and Oncology, Emory University, 1639 Pierce Dr, 1003 Woodruff Memorial Bldg, Atlanta, GA 30322.
| |
REFERENCES |
1.
Walsh CM,
Nardi MA,
Karpatkin S:
On the mechanism of thrombocytopenic purpura in sexually active homosexual men.
N Engl J Med
311:635,
1984[Abstract]
2.
Karpatkin S:
Immunologic thrombocytopenic purpura in patients at risk for AIDS.
Blood Rev
1:119,
1987[Medline]
[Order article via Infotrieve]
3.
Harbol AW,
Liesveld JL,
Simpson-Haidaris PJ,
Abboud CN:
Mechanisms of cytopenia in human immunodeficiency virus infection.
Blood Rev
8:241,
1994[Medline]
[Order article via Infotrieve]
4.
Karpatkin S,
Nardi M:
Autoimmune anti-HIV-Igp120 antibody with antidiotype-like activity in sera and immune complexes of HIV-1-related immunologic thrombocytopenia.
J Clin Invest
89:356,
1992
5.
Bel-Ali Z,
Dufour V,
Najean Y:
Platelet kinetics in human immunodeficiency virus induced thrombocytopenia.
Am J Hematol
26:299,
1987[Medline]
[Order article via Infotrieve]
6.
Karpatkin S,
Nardi MA,
Hymes KB:
Sequestration of anti-platelet GPIIIa antibody in rheumatoid factor immune complexes of human immunodeficiency virus 1 thrombocytopenic patients.
Proc Natl Acad Sci USA
92:2263,
1995[Abstract/Free Full Text]
7.
Gonzalez-Conejero R,
Rivera J,
Rosillo MC,
Cano A,
Rodriguez T,
Vincente V:
Association of autoantibodies against platelet glycoproteins Ib/IX and IIb/IIIa, and platelet-reactive anti-HIV antibodies in thrombocytopenic narcotic addicts.
Br J Haematol
93:464,
1996[Medline]
[Order article via Infotrieve]
8.
Stella CC,
Ganser A,
Hoelzer D:
Defective in vitro growth of the hemopoietic progenitor cells in the acquired immunodeficiency syndrome.
J Clin Invest
80:286,
1987
9.
Ballem PJ,
Belzberg A,
Devine DV,
Lyster D,
Spruston B,
Chambers H,
Doubroff P,
Mikulash K:
Kinetic studies of the mechanism of thrombocytopenia in patients with human immunodeficiency virus infection.
N Engl J Med
327:1779,
1992[Abstract]
10.
Nardi MA,
Liu L-X,
Karpatkin S:
GPIIIa is a major pathophysiologically relevant antigenic determinant for anti-platelet GPIIIa49-66 of HIV-1 related immunologic thrombocytopenia (HIV-1 ITP).
Proc Natl Acad Sci USA
94:7589,
1997[Abstract/Free Full Text]
11.
Hanson SR,
Pareti FI,
Ruggeri ZM,
Kunicki TJ,
Montgomery RR,
Zimmerman TS,
Harker LA:
Effects of monoclonal antibodies against the platelet glycoprotein IIb/IIIa complex on thrombosis and hemostasis in the baboon.
J Clin Invest
81:149,
1988
12.
Kelly AB,
Marzec UM,
Krupski W,
Bass A,
Cadroy Y,
Hanson SR,
Harker LA:
Hirudin interruption of heparin-resistant arterial thrombus formation in baboons.
Blood
77:1006,
1991[Abstract/Free Full Text]
13.
Cadroy Y,
Hanson SR,
Kelly AB,
Marzec UM,
Evatt BL,
Kunicki TJ,
Montgomery RR,
Harker LA:
Relative antithrombotic effects of monoclonal antibodies targeting different platelet glycoprotein-adhesive molecule interactions in non-human primates.
Blood
83:3218,
1994[Abstract/Free Full Text]
14.
Hollinger FB,
Bremer JW,
Myers LE,
Gold JWM,
McQuay L:
NIH/NIAID/DAIDS/ACTG Virology Laboratories: Standardization of sensitive human immunodeficiency virus coculture procedures and establishment of a multicenter quality assurance program for the AIDS Clinical Trials Group.
J Clin Microbiol
30:1787,
1992[Abstract/Free Full Text]
15.
Shearer WT,
Quinn TC,
LaRussa P,
Lew JF,
Mofenson L,
Almy S,
Rich K,
Handelsman E,
Diaz C,
Pagano M,
Smeriglio V,
Kalish LA:
Viral load and disease progression in infants infected with human immunodeficiency virus type 1.
N Engl J Med
336:1337,
1997[Abstract/Free Full Text]
16.
Fultz PN,
McClure HM,
Swenson RB:
Persistent infection of chimpanzees with human T-lymphotropic virus type III/lymphadenopathy-associated virus: A potential model for acquired immunodeficiency syndrome.
J Virol
58:116,
1986[Abstract/Free Full Text]
17.
Boom R,
Sol CJ,
Salimans MM,
Jansen CL,
Wertheim-van Dillen PM,
van der Noordaa J:
Rapid and simple method for purification of nucleic acids.
J Clin Microbiol
28:495,
1990[Abstract/Free Full Text]
18.
Marsh JC,
Gibson FM,
Prue RL,
Bowen A,
Dunn VT,
Hornkohl AC,
Nichol JL,
Gordon-Smith EC:
Serum thrombopoietin levels in patients with aplastic anaemia.
Br J Haematol
95:605,
1996[Medline]
[Order article via Infotrieve]
19.
Nichol JL,
Hokom MM,
Hornkohl A,
Sheridan WP,
Ohashi H,
Kato T,
Li YS,
Bartley TD,
Choi E,
Bogenberger J,
Skrine JD,
Knudten A,
Chen J,
Trail G,
Sleeman L,
Cole S,
Grampp G,
Hunt P:
Megakaryocyte growth and development factor. Analyses of in vitro effects on human megakaryopoiesis and endogenous serum levels during chemotherapy-induced thrombocytopenia.
J Clin Invest
95:2973,
1995
20. Nichol JL: Serum levels of thrombopoietin in health and disease,
in Kuter DJ, Hunt P, Sheridan W, Zucker-Franklin D (eds):
Thrombopoiesis and Thrombopoietins. Totowa, NJ, Humana, 1997, p 359
21.
Fielder PJ,
Hass P,
Nagel M,
Stefanich E,
Widmer R,
Bennett GL,
Keller G-A,
de Sauvage FJ,
Eaton D:
Human platelets as a model for the binding and degradation of thrombopoietin.
Blood
89:2782,
1997[Abstract/Free Full Text]
22. (abstr, suppl 1)
Li J,
Xia Y,
Kuter DJ:
Analysis of the thrombopoietin receptor (MPL) on platelets from normal and essential thrombocythemic (ET) patients.
Blood
88:545a,
1996
23.
Tomer A,
Hanson SR,
Harker LA:
Autologous platelet kinetics in patients with severe thrombocytopenia: Discrimination between disorders of production and destruction.
J Lab Clin Med
118:546,
1991[Medline]
[Order article via Infotrieve]
24. Paulus JM: Platelet Kinetics: Radioisotopic, Cytological,
Mathematical and Clinical Aspects. Amsterdam, The Netherlands, North-Holland, 1971
25.
Savage B,
McFadden PR,
Hanson SR,
Harker LA:
The relation of platelet density to platelet age: Survival of low and high-density 111Indium-labeled platelets in baboons.
Blood
68:386,
1986[Abstract/Free Full Text]
26.
Harker LA,
Finch CA:
Thrombokinetics in man.
J Clin Invest
48:963,
1969
27. Shapiro HM: Practical Flow Cytometry. New York, NY, Wiley-Liss,
1995
28.
Tomer A,
Harker LA,
Burstein SA:
Flow cytometric analysis of normal human megakaryocytes.
Blood
71:1244,
1988[Abstract/Free Full Text]
29.
Tomer A,
Friese P,
Conklin R,
Bales W,
Archer L,
Harker LA,
Burstein SA:
Flow cytometric analysis of megakaryocytes from patients with abnormal platelet counts.
Blood
74:594,
1989[Abstract/Free Full Text]
30. Tomer A, Harker LA: Thrombocytopoiesis, in Anderson KC, Ness PM
(eds): Scientific Basis of Transfusion Medicine. Philadelphia, PA, WB
Saunders, 1994, p 40
31.
Tomer A,
Scharf RE,
McMillan R,
Ruggeri ZM,
Harker LA:
Bernard-Soulier syndrome: Quantitative characterization of megakaryocytes and platelets by flow cytometric and platelet kinetic measurements.
Eur J Haematol
52:193,
1994[Medline]
[Order article via Infotrieve]
32.
Finch CA,
Harker LA,
Cook JD:
Kinetics of the formed elements of human blood.
Blood
50:699,
1977[Free Full Text]
33.
Slichter SJ,
Harker LA:
Thrombocytopenia mechanisms and management of defects in platelet production.
Clin Haematol
7:523,
1978[Medline]
[Order article via Infotrieve]
34. Debili N, Cramer E, Wendling F, Vainchenker W: In vitro effects
of Mpl ligand on human hemopoietic progenitor cells, in Kuter DJ, Hunt
P, Sheridan W, Zucker-Franklin D (eds): Thrombopoiesis and
Thrombopoietins. Totowa, NJ, Human, 1997, p 217
35.
Bailar JC,
Mosteller F:
Guidelines for statistical reporting in articles for medical journals.
Ann Intern Med
108:266,
1988
36.
Hanson SR,
Slichter SJ:
Platelet kinetics in patients with bone marrow hypoplasia: Evidence for a fixed platelet requirement.
Blood
66:1105,
1985[Abstract/Free Full Text]
37. George JN, El-Harake MA, Aster RH: Thrombocytopenia due to
enhanced platelet destruction by immunologic mechanisms, in Beutler E,
Lichtman MA, Coller BS, Kipps TJ (eds): Hematology. New York, NY,
McGraw-Hill, 1995, p 1315
38.
Hymes KB,
Greene JB,
Karpatkin S:
The effect of azidothymidine on HIV-related thrombocytopenia.
N Engl J Med
318:516,
1988[Medline]
[Order article via Infotrieve]
39.
Zucker-Franklin DA,
Cao YZ:
Megakaryocytes of human immunodeficiency virus-infected individuals express viral RNA.
Proc Natl Acad Sci USA
86:5595,
1989[Abstract/Free Full Text]
40.
Louche F,
Bettaieb A,
Henri A,
Ocksenhendler E,
Farcet J-P,
Bierling P,
Seligmann M,
Vainchenker W:
Infection of megakaryocytes by human immunodeficiency virus in seropositive patients with immune thrombocytopenic purpura.
Blood
78:1697,
1991[Abstract/Free Full Text]
41.
Zucker-Franklin D,
Termin CS,
Cooper MD:
Structural changes in the megakaryocytes of patients infected with the human immunodeficiency virus (HIV-1).
Am J Pathol
134:1295,
1989[Abstract]
42.
Zauli G,
Catani L,
Gibellini D,
Re MC,
Vianelli N,
Colangell V,
Celeghini C,
Capitani S,
LaPlaca M:
Impaired survival of bone marrow GPIIb/IIIa+ megakaryocytic cells as an additional pathogenetic mechanism of HIV-1-related thrombocytopenia.
Br J Haematol
92:711,
1996[Medline]
[Order article via Infotrieve]
43.
Bierling P,
Bettaieb A,
Oksenhendler E:
Human immunodeficiency virus-related immune thrombocytopenia.
Semin Thromb Hemost
21:68,
1995[Medline]
[Order article via Infotrieve]
44.
Finazzi G,
Mannucci PM,
Lazzarin A,
Gringeri A,
Arici C,
Ciaci D,
Terzi R,
Barbui T:
Low incidence of bleeding from HIV-related thrombocytopenia in drug addicts and hemophiliacs: Implications for therapeutic strategies.
Eur J Haematol
45:82,
1990[Medline]
[Order article via Infotrieve]
45.
Harker LA,
Marzec UM,
Kelly AB,
Cheung E,
Tomer A,
Nichol JL,
Hanson SR,
Stead RB:
Prevention of thrombocytopenia and neutropenia in a nonhuman primate model of marrow suppressive chemotherapy by combining pegylated recombinant human megakaryocyte growth and development factor and recombinant human granulocyte colony-stimulating factor.
Blood
89:155,
1997[Abstract/Free Full Text]
46.
Koyanagi Y,
O'Brien WA,
Zhao JQ,
Golde DW,
Gasson JC,
Chen ISY:
Cytokines alter production of HIV-1 from primary mononuclear phagocytes.
Science
241:1673,
1988[Abstract/Free Full Text]
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Abstract
Introduction
Abstract
