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Blood, Vol. 96 No. 1 (July 1), 2000:
pp. 188-194
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Morphological analysis of microparticle generation in
heparin-induced thrombocytopenia
Mary Hughes,
Catherine P. M. Hayward,
Theodore E. Warkentin,
Peter Horsewood,
Katherine A. Chorneyko, and
John G. Kelton
From the Departments of Medicine and Pathology, Faculty of Health
Sciences, McMaster University Medical Center, and The
Canadian Blood Services, Hamilton, Ontario, Canada.
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Abstract |
Heparin-induced thrombocytopenia (HIT) with thrombosis is a serious
complication of heparin use. HIT sera can generate platelet-derived microparticles, which are produced in a heparin-dependent manner and
are hypothesized to be important initial pathological participants because they promote vascular occlusion. To date, microparticles have
been studied using flow cytometric techniques. However, it is uncertain
whether the small-sized material seen in flow cytometric studies
represents true platelet microparticles shed from activated platelets
or whether they are platelets that have contracted after releasing
their internal components. This report describes a morphological investigation of platelet-derived microparticles in HIT using, among
other techniques, confocal, scanning electron, and transmission electron microscopy. Following incubation with HIT sera, the existence of small membrane-bound vesicles in the milieu of activated platelets was demonstrated. A population of microparticles, expressing
platelet-specific glycoproteins, was separated from platelets by
centrifugation over a sucrose layer. These microparticles had identical
flow cytometric profiles, size heterogeneity, and GPIb
and GPIIb/IIIa staining intensity as the microparticle population in
unfractionated samples. When microparticles were generated in situ and
fixed onto grids, they were demonstrated to be distinct membrane-bound
vesicles that originated near the platelet body and terminal ends of
pseudopods on activated platelets. These microparticles appeared to be
generated by localized swelling, budding, and release. Collectively,
these morphological studies document the existence of true
microparticles in platelets activated by HIT sera. The microparticles
may play an important role in the pathogenesis of HIT.
(Blood. 2000;96:188-194)
© 2000 by The American Society of Hematology.
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Introduction |
Heparin-induced thrombocytopenia (HIT), one of the most
common immune-mediated adverse drug reactions, occurs in 1%-3% of patients receiving therapeutic doses of heparin.1,2 Unlike other immunological drug-induced reactions, many patients with HIT
experience serious thrombotic morbidity including venous thrombosis, arterial thrombosis, and disseminated intravascular coagulation (DIC).1-3 Studies of the pathogenesis of this prothrombotic
condition demonstrated that circulating heparin-dependent antibodies
bind to a complex of heparin and platelet factor 4 (PF4).4-8 Several years ago, we demonstrated that the
binding of heparin-dependent immunoglobulin G (IgG) to platelet
Fc RII receptors leads to platelet activation9,10 and the generation of platelet-derived
microparticles.11 These microparticles were produced in a
heparin-dependent fashion and were shown to have procoagulant
activity.11,12 Although microparticles are thought to be
initial participants in venoocclusive events, their mechanism of
formation and confirmation of existence are still a matter of controversy.
Presently, flow cytometry is the most frequently reported technique
used to study platelet-derived microparticles, and it has been used by
our group to develop a diagnostic test for heparin-induced thrombocytopenia.13 However, some investigators have raised questions about the analysis of flow cytometry experiments. Studies by
Bode et al14 showed that the light scatter distribution of platelets is broad and that it is difficult to identify clearly where
the population of intact platelets ends and the population of smaller
particles begins. This observation of a continuum of particle size
rather than 2 distinct platelet and microparticle populations
questioned the nature of microparticles and their degree of
heterogeneity. Studies by Matzdorff et al15 demonstrated that counting microparticles becomes unreliable when platelet counts
drop to a low number. This was thought to be explained by the fact that
saturating amounts of antibodies may become unspecifically adsorbed to
other particles or may form antibody complexes. The observations that
an antibody surplus leads to antibody complexes and that these
complexes can interfere with platelet and microparticle counting have
also been reported by other groups.16,17 Finally, George et
al18 demonstrated that microparticle preparations derived
from washed activated platelets contained a heterogeneous array of
membrane fragments, vesicles, and granules. These observations suggested that morphological documentation of microparticles in heparin-induced thrombocytopenia was necessary for the confirmation of
their existence.
In previous studies, immunoassays and flow cytometry studies
demonstrated procoagulant properties of microparticles but failed to
provide information on their structural origin or mechanism of
generation. In this study we conducted a morphological analysis of
platelet-derived microparticles to document the existence of microparticles in heparin-induced thrombocytopenia. Several different techniques were used including flow cytometry, confocal microscopy, transmission electron microscopy (TEM), and scanning electron microscopy (SEM) to provide a network of support for the existence of
true microparticles in heparin-induced thrombocytopenia. Through a
novel approach of activating platelets directly on EM grids with
heparin-induced thrombocytopenia sera, we visually documented the
structural origin of microparticles in situ. Finally, microparticles from platelet reactions were isolated by centrifugation over a sucrose layer. We were able to demonstrate that microparticles are physically distinct from platelets and have scatter plot displays, size heterogeneity, and staining intensity similar to that of microparticles in unfractionated platelet reaction mixtures.
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Patients, materials, and methods |
Patient samples
This study was approved by the University and Hospital Ethics Review
Committee at McMaster University Medical Center, Hamilton, Ontario,
Canada. Patient and control samples were collected
according to the university-approved Ethics Review Committee
recommendations, and all entrants provided signed consent.
Studies were performed using sera from patients with HIT who met the
clinical criteria for diagnosis as described previously.9 In all patients, the diagnosis of HIT was confirmed using the 14C-serotonin platelet release assay.9,19
Control sera were obtained from patients who tested negative for HIT
using the 14C-serotonin platelet release assay and from
healthy aspirin-free volunteers. All samples were heat-inactivated for
30 minutes at 56°C, centrifuged at 8000g for 10 minutes,
and frozen at  70°C until use.
Materials
All materials were purchased from Sigma Chemical Co (St Louis, MO)
unless otherwise stated. Where applicable, brand names are noted in parentheses.
Antibodies
Flow cytometry studies were performed using the platelet-specific
monoclonal antibody (mAb) anti-GPIb
(TW-1).20 This antibody was conjugated directly with
fluorescein isothiocyanate (FITC). Confocal microscopy studies were
performed using the platelet-specific mAbs anti-GPIb
(TW-1) and anti-GPIIb/IIIa (Raj-1).20 The secondary
antibody used in these studies was a Texas Red-conjugated (TR-conjugated) goat antimouse IgG antibody (Jackson Immuno-Research Laboratories, West Grove, PA).
Platelet 14C-serotonin release assay
Diagnostic testing for HIT was performed using the
14C-serotonin release assay as previously
described.9,19 All tests were performed in triplicate.
Isolation of platelets
Whole blood was collected from healthy aspirin-free volunteers and
mixed with acid citrate dextrose (ACD, 6:1, vol/vol [pH 4.5])
containing 1 mmol/L theophylline and 1 µg/mL prostaglandin E1. Platelets were isolated by differential centrifugation
at 160g for 20 minutes and washed once in calcium-
and albumin-free Tyrode's solution (pH 6.2) containing apyrase.
For microparticle generation studies, washed platelets were
resuspended in albumin-free Tyrode's solution containing calcium
and magnesium (pH 7.4).
Preparation of platelets
In 12 × 75-mm round-bottom polystyrene tubes
(Becton Dickinson, Lincoln Park, NJ), 0.7 mL washed platelets
(300 × 109 cells per L) were added to 0.2 mL test
serum and 0.1 mL buffer, 0.1-0.3 U/mL final concentration of heparin
(Leo Laboratories, Ajax, Ontario, Canada), 0.1-1.0 U/mL final
concentration of thrombin,3 or 10 µm calcium ionophore
A23187.4 Following a 1-hour incubation, without stirring or
shaking, at room temperature, the reaction was stopped by adding an
equal volume of 1% paraformaldehyde (PFA) (wt/vol) in
phosphate-buffered saline (PBS)(pH 7.4) for 1 hour at room temperature.
For flow cytometry studies, samples were diluted 1:2 with filtered 0.22 µm PBS (pH 7.4) containing 0.2% glycine (wt/vol). A 100-µL sample
of PFA-fixed platelets was incubated with 10 µL FITC-labeled
anti-GPIb (20 µg/mL) for 1 hour at 37°C. Following incubation, samples were further diluted with 0.25-mL filtered PBS (pH
7.4) and kept in the dark until fluorescence activated cell sorter
(FACS) analysis (FACScan; Becton Dickinson, San Jose, CA).
For confocal microscopy studies, PFA-fixed samples were washed with
PBS/glycine buffer (pH 7.4), centrifuged at 2000g for 15 minutes, and resuspended in 3% bovine serum albumin (BSA)/PBS blocking
buffer (pH 7.4) containing 50 µg/mL normal mouse IgG. A 0.5-mL sample
of PFA-fixed platelets was incubated with anti-GPIb (1/100) or anti-GPIIb/IIIa (1/100) for 2 hours at
37°C. The platelets were washed once with blocking buffer and
incubated with TR-conjugated goat antimouse IgG secondary antibody
(1/100). Following a 2-hour incubation at 37°C, the platelets were
washed, spread onto glass coverslips, and analyzed using confocal
microscopy. For all confocal microscopy studies, samples were kept in
the dark until analysis.
Flow cytometry
Platelet-derived microparticles were identified using a FACScan.
FITC fluorescence was detected using a 530-nm bandpass filter. Data for
forward light angle scatter (FCS), side angle scatter (SSC), and FITC
fluorescence were obtained with gain settings in the logarithmic mode.
For each sample, 5000 events were acquired. Microparticles were
distinguished from platelets on the basis of their characteristic flow
cytometric profile of FCS versus FITC fluorescence. Analysis of the
fluorescence histograms (counts vs fluorescence) was used to quantitate
platelet microparticles, which were defined as
GPIb-positive events that exhibited less fluorescence
than 95%-99% of nonactivated FITC GPIb-labeled platelets. This setting gave background microparticle levels of less
than 6% in control samples.
Confocal microscopy
Platelets and platelet-derived microparticles were identified using
the platelet-specific mAbs, anti-GPIb and
anti-GPIIb/IIIa, and a TR-conjugated goat antimouse IgG secondary
antibody. Confocal microscopy was performed with a Universal Confocal
Laser Scan Research Microscopy System (Carl Zeiss,
Oberkochen, Germany), objective magnification × 100, and
individual excitation lasers and filters for the TR fluorochrome.
Controls included platelets incubated with and without
antibody, normal mouse IgG, or primary antibody with an irrelevant
fluorescent secondary antibody. Acquired images were imported into
Micrografx Picture Publisher (version 7, Micrografx Inc, Richardson, TX).
Electron microscopy
Using 12 × 75-mm round-bottom polystyrene tubes (Becton
Dickinson), 0.7 mL washed platelets (resuspended in albumin-free
Tyrode's solution containing calcium and magnesium; pH 7.4) at a
concentration of 300 × 109 cells per L were added
to 0.2 mL heat-treated test serum and 0.1-mL buffer or heparin at a
final concentration of 0.1-0.3 U/mL. Following a 1-hour incubation,
without stirring or shaking, at room temperature, the reaction was
stopped by adding an equal volume of 1% glutaraldehyde (wt/vol) in 0.1 mol/L phosphate buffer (pH 7.2) for 1 hour at room
temperature. Samples were pelleted at 2000g for 15 minutes,
embedded in glycolmethacrylate, and processed for routine TEM. Thin
platelet sections were cut on a Reichert-Jung Ultracut ultramicrotome
(Leica AG, Vienna, Austria), contrast-stained with uranyl acetate and
lead citrate, and analyzed under a JEOL 1200EX Transmission Electron
Microscope (Tokyo, Japan).
The washed platelets were resuspended in albumin-free Tyrode's
solution containing calcium and magnesium (pH 7.4) and incubated with a
heat-treated test serum and buffer or heparin at a final concentration
of 0.1-0.3 U/mL. After incubation for 30 minutes at room temperature,
the samples were fixed with 2% glutaraldehyde (wt/vol) in phosphate
buffer (pH 7.2). A drop of the platelet suspension was allowed to
settle on a poly-L-lysine-coated glass coverslip. Samples were
postfixed in 1% osmium tetroxide, dehydrated in graded alcohol, and
dried by the critical-point method. The dried samples were
sputter-coated with gold and observed under a JEOL 1200EX Scanning
Electron Microscope.
A 5-µL sample of washed platelets was then resuspended in
albumin-free Tyrode's solution containing calcium and magnesium (pH
7.4). Using a pipette, the platelets were gently put onto a
BSA-precoated formvar grid and allowed to settle
undisturbed for 2 minutes. A 3-µL sample of test serum and 2 µL
buffer,1 0.1-0.3 U/mL final concentration of
heparin,2 0.1-1.0 U/mL final concentration of
thrombin,3 or 10 µL calcium ionophore A231874
were then gently put onto the grid with a pipette. Following a 5- to
20-minute incubation at room temperature, the reaction was stopped by
the addition of 2% glutaraldehyde (wt/vol) in phosphate buffer (pH
7.2). Samples were negatively stained with 2% phosphotungstic acid and
analyzed under a JEOL 1200EX Transmission Electron Microscope.
Density centrifugation
Microparticles were isolated from activated platelets by sedimenting
platelet reactions across a sucrose layer according to Pasquet et
al.21,22 This procedure is a modification of the procedure
used in binding experiments to separate platelet-bound cells from
unbound ligands.23 Briefly, PFA-fixed
platelet reactions were centrifuged at 1000g for 10 minutes
to pellet the bulk of the platelets. The supernatant was layered onto 5 mL 27% sucrose (wt/vol), prepared in assay buffer, and centrifuged at
2000g for 10 minutes. The residual platelets sedimented
through the sucrose, and the upper phase, containing the
microparticles, was harvested for flow cytometry and confocal
microscopy analysis.
For flow cytometry studies, samples were diluted 1:2 with filtered PBS
(pH 7.4) containing 0.2% glycine (wt/vol). A 100-µL sample was
incubated with 10-µL FITC-labeled anti-GPIb at a
concentration of 20 µg/mL for 1 hour at 37°C. Following
incubation, the samples were further diluted with 0.25 mL filtered PBS
(pH 7.4) and analyzed by FACS analysis.
For confocal microscopy studies, PFA-fixed samples were washed with
PBS/glycine buffer (pH 7.4), centrifuged at 15 000g for 15 minutes, and resuspended in 3% BSA/PBS blocking buffer (pH 7.4)
containing normal mouse IgG at a concentration of 50 µg/mL. A 0.5-mL
sample was incubated with anti-GPIIb/IIIa (1/100) or anti-GPIb (1/100) for 2 hours at 37°C. Samples
were washed once with blocking buffer and incubated with a
TR-conjugated goat antimouse IgG secondary antibody (1/100). Following
a 2-hour incubation at 37°C, samples were washed and spread
onto glass cover slips. The samples were kept in the dark until
confocal microscopy analysis.
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Results |
Flow cytometry was used to detect platelet-derived
microparticles following incubation of normal washed platelets with
patient sera in the presence or absence of heparin. Microparticles were distinguished from platelets on the basis of size and relative fluorescence of an FITC-conjugated antibody to platelet membrane GPIb (Figure 1). Briefly,
the FL1/FSC (fluorescence of FITC anti-GPIb/forward lightscatter) representation of
platelets activated with HIT sera (n = 3) showed 2 populations: (1) a
major population, corresponding to platelets, and (2) a smaller second population, corresponding to the microparticle fraction defined in
previous studies.13 This second population was absent in platelets incubated with control sera (n = 3) or heparin alone. Microparticle generation by thrombin and calcium ionophore produced a
population of particles similar to that observed for HIT sera. Based on
fluorescence histograms (data not shown), the percentage of
microparticles generated from control sera was less than 5% in
comparison with 40% for HIT sera, 45% for thrombin, and 47% for
calcium ionophore.
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| Fig 1.
Flow cytometric analysis of platelets activated with HIT
sera, thrombin, and calcium ionophore.
Platelets (p) and microparticles (mp) were identified using
fluorescence (FL1, FITC anti-GPIb ) (y-axis) and size (FSC) (x-axis)
characteristics. Control platelets included; (A) platelets incubated in
buffer alone; (B) platelets incubated with patient serum, which tested
negative for HIT, in the presence of 0.1 U/ml heparin; and (C)
platelets incubated with HIT serum with no heparin added. (D) Platelets
were incubated with HIT serum in the presence of 0.1 U/ml heparin. (E)
As a positive control, platelets were also incubated with 1 U/mL
thrombin or (D) 10 µm calcium ionophore A23187. Microparticles (MP)
were generated with heparin-induced thrombocytopenia serum, thrombin,
and calcium ionophore and not with the control serum. The percent of
microparticles (the percent of fluorescent events in the microparticle
gate) generated by HIT serum was 40%; thrombin, 45%; and calcium
ionophore, 47%. The results of a representative experiment are
shown.
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Although flow cytometry demonstrated that significant levels of
microparticles were generated from platelets incubated with HIT sera,
it was not clear whether microparticles were a population of compressed
smaller platelets or true microparticles. To resolve these issues, we
used several additional techniques including confocal microscopy, TEM,
and SEM to visually analyze the morphology of microparticles generated
by HIT sera.
Confocal microscopy studies
In these studies, microparticles were distinguished from platelets
on the basis of size. Platelet microparticles were defined as
GPIIb/IIIa+ particles, which were less than 0.1 to 1.0 µm
in diameter. Platelet reactions with HIT sera in the presence of
heparin demonstrated numerous brightly stained particles surrounding
homogeneously stained platelets (Figure 2).
This "starry sky pattern" was also observed in platelet reactions
with thrombin and calcium ionophore. In comparison, very few stained
particles were observed in platelet reactions with control sera.
Confocal analysis of the microparticles generated by HIT sera,
thrombin, or calcium ionophore did not demonstrate apparent differences
in their size heterogeneity or the absolute numbers of microparticles
generated. Furthermore, there was no apparent difference in staining
intensity of their particles using antibodies for GPIb
or GPIIb/IIIa. Although these studies suggested that platelet
microparticles were generated by activating platelets with HIT sera,
electron microscopy was used to further investigate the morphology of
the microparticles and the mechanism of their generation.
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| Fig 2.
Confocal microscopy images of microparticles generated by
HIT serum, thrombin, and calcium ionophore.
Platelets and microparticles were identified using a platelet-specific
primary antibody (mAb anti-GPIIb/IIIa) and a fluorescent secondary
antibody (TR-conjugated goat antimouse). (A) Control platelets were
incubated with patient serum, which tested negative for HIT in the
presence of 0.1 U/mL heparin, (B) heparin alone, and (C) HIT serum in
the absence of heparin. (D) Platelets were incubated with HIT serum in
the presence of 0.1 U/mL heparin. As a positive control, (E) platelets
were also incubated with 1 U/mL thrombin or (F) 10 µm calcium
ionophore A23187. Platelet reactions with HIT serum in the presence of
heparin demonstrated numerous brightly stained particles surrounding
homogeneously stained platelets. This "starry sky pattern" was
also observed in platelet reactions with thrombin and calcium
ionophore. In comparison, very few stained particles were observed in
platelet reactions with control serum or heparin alone. The results of
a representative experiment are shown.
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Electron microscopy studies
TEM was used to study the ultrastructure of platelets incubated with
HIT sera. Figure 3 shows representative TEM
pictures of platelets incubated with control serum or platelets
incubated with HIT serum in the presence of heparin. Platelets from
control reactions, where there was no stimulus for platelet activation, were discoid in shape. This morphology is characteristic of resting platelets. In comparison, platelets activated by HIT sera displayed numerous pseudopodia and loss of their resting discoid shape. Separate
from the pseudopods, there appeared to be some discrete small particles
that were located adjacent to platelets and pseudopodia. Numerous
pseudopodia demonstrated a region of bulging at their terminal end or
along their body, suggesting these structures might be the origins of
microparticles. Areas of bulging were observed on both pseudopods and
the central platelet body. To address the issue that microparticles
might correspond to transverse sections of pseudopods in these studies,
using SEM, we further investigated the morphology of platelets
activated with HIT sera. In these studies, the entire platelet was
viewed, and cross-sectioning was not performed.
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| Fig 3.
Representative transmission electron micrographs of
resting platelets and platelets incubated with HIT serum.
Resting platelets or platelets incubated with HIT serum in the presence
of 0.1 U/mL heparin were fixed with 0.5% glutaraldehyde and embedded
in glycolmethacrylate. (A) The morphology of a representative normal
resting platelet. Alpha granules (a), mitochondria (m), and channels of
the open-cannalicular system (ocs) can be seen throughout the platelet.
The platelets appear round or discoid in shape, and there is an absence
of pseudopodia. (B-F) The morphology of representative platelets
incubated with HIT serum in the presence of heparin. Platelets are
activated with a centralized clustering of alpha granules (a) and
mitochondria (m). (B) Pseudopodia (psd) can be seen extending from the
platelet body, and several distinct membrane-bound structures
resembling small vesicles (v) are observed near the platelet. Numerous
pseudopodia demonstrated a region of bulging (E) at the terminal end of
the pseudopod or (C) along the body of the pseudopod.
Frequently, several areas of bulging were observed on (D) the same
pseudopod and (C) the platelet body itself. (F) In these platelets,
microparticles appeared to be released by the budding of pseudopods.
(Original magnification (A, B) × 15 000; (C, D) × 25 000;
and (E, F) × 50 000.)
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SEM studies (Figure 4) demonstrated that
platelets incubated with control sera or heparin alone (not shown)
maintained a discoid shape. These morphological observations were
consistent with those of resting platelets observed in TEM studies.
Platelets activated with HIT sera demonstrated morphological changes
consistent with those observed in cross-sectioned platelets including
absence of a discoid form; presence of pseudopodia; and
presence of microparticles, in which no linkage to a platelet body or
pseudopod could be discerned. These same morphological changes were
also observed in platelets activated with thrombin and calcium
ionophore (data not shown).
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| Fig 4.
Representative scanning electron micrographs of resting
platelets and platelets activated with HIT serum in the presence of
heparin.
Resting platelets or platelets incubated with HIT serum in the presence
of 0.1 U/mL heparin were fixed with 2% glutaraldehyde and processed
for SEM. (A) The morphology of a representative normal resting
platelet. Resting platelets were observed to generally maintain a
discoid form. (B) The morphology of a representative platelet incubated
with HIT serum. These platelets demonstrated several morphological
changes including absence of a discoid form,1 presence of
pseudopodia,2 and presence of microparticles near the ends
of pseudopodia3 (indicated by arrows). (C) Microparticles
that are clearly distinct and separate from the platelet body localize
near the terminal end of a pseudopod (indicated by arrow).
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In all of the previously described methods, some washing steps were
required as part of the platelet processing protocol. To address the
issue that some microparticles may have been lost in these washing
steps, we analyzed negatively stained platelets, which were activated
and fixed directly onto electron microscopy grids. In this technique,
washing steps were not used. Platelets incubated with control sera
(Figure 5) or therapeutic concentrations of
heparin alone (data not shown) appeared round or discoid in shape,
which is consistent with a resting state. When platelets were activated
directly on formvar-coated grids with HIT sera, formed microparticles
were observed. These platelets demonstrated morphological changes that
were consistent with those observed in TEM and SEM studies. Frequently,
these platelets were observed with pseudopodia extending from the
platelet body and with numerous microparticles surrounding the
pseudopods and platelet body. These microparticles ranged in size from
less than 0.1 to 1.0 µm in diameter and appeared as discrete
membrane-bound particles. Microparticles were observed to be localized
near the terminal ends and body of pseudopods, often near points of
bulging or swelling.
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| Fig 5.
Electron micrographs of negatively stained platelets
activated in situ with HIT sera.
Platelets were allowed to settle on BSA-coated formvar grids and then
incubated with patient serum or platelet agonists. (A) Control
platelets were incubated with patient serum, which tested negative for
HIT, or heparin alone (data not shown). (B) Platelets were inoculated
with HIT serum in the presence of 0.1 µ/mL heparin. As a positive
control, platelets were also incubated with (C) 1 U/mL thrombin or (D)
10 µm calcium ionophore A23187. Platelets were then fixed with 2%
glutaraldehyde and negatively stained with 2% phosphotungstic acid.
(A) Control platelets demonstrated a round or discoid shape,
which is characteristic of resting platelets. (B) Platelets incubated
with HIT serum demonstrated numerous microparticles surrounding the
platelet body. Frequently, these platelets were observed with
pseudopodia extending from the platelet body. Microparticles ranged in
size from less than 0.1 to 1.0 µm in diameter and appeared as
discrete membrane-bound particles. Platelets incubated with (C)
thrombin and (D) calcium ionophore demonstrated a similar platelet
morphology, with microparticles surrounding the platelet body.
(Original magnification (A-E) × 13 000.) (E) These particles
were observed to be near the terminal ends and body of the pseudopods,
and numerous points of bulging or swelling along the body and terminal
ends of the pseudopodia were observed.
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Platelet reactions with thrombin and calcium ionophore demonstrated a
similar phenomenon, with numerous microparticles surrounding the
platelet body. The apparent morphology or size of microparticles did
not appear to differ, whether microparticles were generated from HIT
sera, thrombin, or calcium ionophore. Furthermore, some microparticles
were observed to be clustered together at points, suggesting that
microparticles may be integrated into aggregates upon release.
Microparticle isolation studies
We have described a simple procedure to isolate microparticles from
platelets by sedimenting the latter across a sucrose layer. These
studies were performed to further confirm that microparticles are a
distinct population and not connected to activated platelets. When
microparticle-rich fractions were isolated from platelet reactions
incubated with HIT sera, FACS analysis confirmed that the supernatant
contained very few contaminating platelets. Furthermore, isolated
microparticles and microparticles in unfractionated reactions had
identical physical properties: scatter plot displays (Figure 6), size heterogeneity (Figure
7), and GPIb and GPII/IIIa staining intensity (Figure 7). The observation that very few
microparticles were isolated from platelets incubated with control
serum or heparin alone demonstrated that the sucrose
isolation procedure did not generate artefactual microparticle
formation during the centrifugation step. These studies demonstrated
that microparticles were physically distinct from platelets, as
evidenced by their ability to be isolated from platelet reaction
mixtures.
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| Fig 6.
Flow cytometric analysis of isolated microparticles from
platelets activated with HIT sera, thrombin, and calcium ionophore.
Very few microparticles were observed in control
reactions, in which platelets were incubated with patient serum that
tested negative for (E) heparin-induced thrombocytopenia or heparin
alone (data not shown). Microparticles (lower row) were identified
using fluorescence (FL1, FITC anti-GPIb) (y-axis) and size (FSC)
(x-axis) characteristics. Microparticles isolated from platelets
incubated with (F) heparin-induced thrombocytopenia serum, (G)
thrombin, and (H) calcium ionophore demonstrated similar light scatter
profiles as microparticles in unfractionated platelet reactions (upper
row). The results of a representative experiment are shown.
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| Fig 7.
Confocal microscopy images of isolated microparticles
from platelets activated with HIT sera, thrombin, and calcium
ionophore.
Microparticles were identified using platelet-specific primary antibody
(mAb anti-GPIb and anti-GPIIb/IIIa) and a fluorescent secondary
antibody (TR-conjugated goat antimouse). (A-D) Isolated microparticles
following centrifugation of the platelet reaction over a sucrose layer.
Very few microparticles were isolated from control reactions, in which
platelets were incubated with patient serum that tested
negative for (A) heparin-induced thrombocytopenia or heparin alone
(data not shown). Microparticles isolated from platelets
incubated with (B) heparin-induced thrombocytopenia serum, (C)
thrombin, and (D) ionophore were less than 0.1 to 1.0 µm in
diameter and demonstrated a similar degree of size heterogenosity
and staining intensity as microparticles in unfractionated platelet
reactions (not shown). The results of a representative experiment
are shown.
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Discussion |
Several years ago, we demonstrated that the binding of
heparin-dependent IgG to platelet FcRII receptors lead to platelet activation9,10 and the generation of platelet-derived
microparticles.11 These microparticles were produced in a
heparin-dependent fashion and were shown to have procoagulant activity,
as demonstrated by coagulation12 and amidolytic
assays.11 Using flow cytometry, increased levels of
circulating microparticles have also been observed in patients with
acute HIT.11 The observation of small numbers of
microparticles in the plasma of normal human blood14,24,25 and in increased amounts in HIT has led to their diagnostic
measurement.13 Although these microparticles are thought to
be distinct from those of activated platelets, their true existence has
not been morphologically confirmed. In this study, we document for the first time that true microparticles are generated from HIT sera and
that this microparticle population is separate and distinct from that
of activated platelets.
Several of the experimental conditions and techniques used in this
study provide important morphological evidence that true microparticles
are generated when platelets are activated by HIT sera. Using negative
staining, confocal microscopy, TEM, and SEM, we documented consistent
morphological observations. First, when platelets were activated with
HIT sera, they lost their discoid shape, and pseudopodia were observed.
Second, numerous sites of swelling were observed on both the platelet
body and pseudopodia, and small discrete membrane-bound particles were
frequently observed near these sites. Using SEM and TEM on
unfractionated platelet reaction mixtures, we were able to confirm that
a generation of microparticles are not attached to the platelet body or
to pseudopodia. The microparticles generated by HIT sera ranged in size
from less than 0.1 to 1.0 µm in diameter. These microparticles could
be physically isolated from platelet reaction mixtures by
centrifugation over a sucrose gradient, and they were indistinguishable
from the population of microparticles in unfractionated platelet
reaction mixtures in their size, membrane glycoprotein expression, and physical properties.
Based on the morphological observations associated with microparticle
generation, we hypothesize that microparticle shedding in HIT involves
(1) formation of localized points of swelling on the platelet body,
pseudopod body, or terminal pseudopod tips; (2) further development of
these points of swelling into well-defined buds; and (3) transformation
of buds into microparticles and concomitant release.
The clinical significance of microparticles in HIT has not been
established. There is now evidence to suggest that the incidence of HIT
depends on the clinical context of the patients receiving heparin
therapy and the duration of heparin exposure.26
This incidence appears to be greater in older patients
with underlying hemostatic activation such as inflammation, orthopedic
trauma, or cardiopulmonary bypass surgery.26 In this
context, the presence of activated platelets appears to favor the
development of HIT.26 In our study we noted that
microparticles generated from HIT sera, thrombin, or calcium ionophore
were quite similar, irrespective of the agonist. These results suggest
that the process of microparticle formation may involve a similar
mechanism irregardless of the agonist. Recent studies suggest that
agonist-induced microparticle formation is closely related to the
elevation of intracellular calcium, which affects the reorganization of
the platelet cytoskeletal architecture.27-29 It is unclear
whether microparticles in HIT are primarily markers of platelet
activation or whether they serve a more significant physiologic role.
It is unclear whether HIT-induced microparticles or microparticles
observed in other thrombotic conditions, such as transient ischemic
attacks,30 pulmonary embolism,31 thrombotic
thrombocytopenic purpura,32 or DIC,33 have
similar functional properties. We observed that microparticles
generated by HIT sera, thrombin, or calcium ionophore express similar
amounts of GPIb and GP IIb/IIIa. It is possible that the
microparticle membrane characteristics may not vary with respect to
specific inducers of microparticle formation.
Our study describes the ultrastructure of microparticle production in
platelets incubated with HIT sera. Using electron microscopy, these
microparticles appear to bud from pseudopodia and/or regions of the
platelet membrane following platelet activation, and they are distinct
and separate from activated platelets. The observations that
microparticles are no longer bound to activated platelets, carry
intense procoagulant activity, and have a smaller circulating size
suggest that they may be important promoters of thrombotic events in
HIT. Our study provides important morphologic clues as to the nature of
microparticle production in platelets. These results may
contribute to the understanding of thrombotic complications that
characterize heparin-induced thrombocytopenia.
| |
Acknowledgment |
We wish to express thanks to M. Kalina for technical assistance with
the TEM and SEM studies.
| |
Footnotes |
Submitted January 6, 2000; accepted February 28, 2000.
Supported by grants from the Heart and Stroke
Foundation of Ontario (J.G.K., grant no. T3390) (C.P.M.H., Research
Scholar) and the Medical Research Council of Canada (J.G.K., grant no. MT-7150), Ontario, Canada, and a Bayer/ Canadian Red Cross Society Research Studentship (M.H.).
Reprints: John G. Kelton, HSC 3W10, McMaster University
Medical Center, 1200 Main St West, Hamilton, Ontario, Canada L8N
3Z5; e-mail: keltonj{at}fhs.mcmaster.ca.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction