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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 96 No. 1 (July 1), 2000:
pp. 182-187
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Heparin-induced thrombocytopenia: new evidence for the dynamic
binding of purified anti-PF4-heparin antibodies to platelets and
the resultant platelet activation
Peter M. Newman and
Beng H. Chong
From the Centre for Thrombosis and Vascular Research, Prince of
Wales Hospital, Sydney, New South Wales, Australia.
 |
Abstract |
Immune heparin-induced thrombocytopenia (HIT) is associated with
antibodies directed against a complex of platelet factor 4 (PF4) and
heparin. We were able to affinity purify anti-PF4-heparin IgG (HIT
IgG) from the plasma of 2 patients with HIT. Under conditions that were
more physiological and sensitive than those in previous studies, we
observed that this HIT IgG caused platelet aggregation on the addition
of heparin. Platelets activated with HIT IgG increased their release
and surface expression of PF4. We quantitated, for the first time, the
binding of affinity-purified HIT iodine 125-IgG to platelets as they
activated in a plasma milieu. Binding of the HIT IgG was dependent on
heparin and required some degree of platelet activation. Blocking the
platelet Fc RII with the monoclonal antibody IV.3 did not prevent HIT
IgG binding to activated platelets. We concluded that anti-PF4-heparin
IgG is the component in these HIT plasmas that induces platelet
aggregation. The Fab region of HIT IgG binds to PF4-heparin on the
surface of activated platelets. We propose that only then does the Fc
portion of the bound IgG further activate the same or adjacent
platelets through the Fc receptor. Our data support a dynamic model of
platelet activation in which released PF4 enhances further antibody
binding and more release.
(Blood. 2000;96:182-187)
© 2000 by The American Society of Hematology.
| |
Introduction |
Immune heparin-induced thrombocytopenia (HIT) is a
potentially serious complication of heparin therapy.1,2
Patient plasma usually contains antibodies that aggregate platelets in
the presence of heparin.3,4 In addition, antibodies
directed against complexes of platelet factor 4 (PF4) and heparin are
associated with this disease.5-7 However, HIT plasma that
aggregates platelets does not always contain anti-PF4-heparin
antibodies and vice versa.6,8,9 Understanding how HIT
antibodies interact with platelets and whether the anti-PF4-heparin
antibodies bind platelets and cause aggregation is important for
understanding the pathophysiology of HIT. Unfortunately, conflicting
models have been proposed to describe the interaction of HIT antibodies
with platelets, and none of the previous studies directly investigated
the binding of affinity-purified anti-PF4-heparin IgG to platelets.
Visentin et al10 report that, with flow cytometry, IgG from
some patients with HIT bound to resting, washed platelets. Blocking the
platelet Fc receptor (FcRII) with the monoclonal antibody IV.3
abolished antibody binding, which implied that only the Fc portion of
HIT IgG interacts with platelets. Visentin et al10 and
others2,11,12 contend that HIT IgG forms an immune complex with circulating PF4-heparin and that the complex binds to the platelet FcRII through the Fc portion of the IgG and causes platelet activation.
This model is disputed by Horne and Alkins,13 who
investigated the binding of iodinated total IgG from patients with HIT to washed platelets. Antibody binding was reliably observed only with thrombin-activated platelets and was dependent on the addition of
approximately equimolar concentrations of PF4 and heparin. In contrast
to the findings of Visentin et al,10 they observed that Fc
receptors were not important for the binding of HIT antibodies. Specifically, HIT antibodies bound to platelets in the presence of
IV.3. Horne and Alkins13 and others5,14-16
support a model in which HIT IgG binds by its Fab region to
PF4-heparin complexes already on the platelet surface.
These studies by Visentin et al10 and Horne and
Alkins13 are valuable, but both investigated the binding of
HIT antibodies to platelets under static conditions in which the washed
platelets were either fully activated or held quiescent by
prostaglandin E1. There was no opportunity for secreted
platelet proteins to modulate antibody binding and no information
describing how binding changed during activation. We have studied the
binding of HIT IgG to platelets under more physiological conditions
using affinity-purified anti-PF4-heparin IgG from HIT plasma. This IgG
provides much greater sensitivity and is a more clearly defined
antibody than the total IgG or plasma used by others.10,13
Importantly, platelet aggregation was induced by the affinity-purified
HIT IgG, and antibody binding was studied in a plasma milieu with
platelets exposed to shear forces. Our data also help to resolve the
role played by the platelet FcRII in HIT, which previously had only
been investigated in artificial experiments.
| |
Materials and methods |
Purification of HIT IgG
Plasma was collected with informed consent from healthy
individuals and from 2 patients with HIT (patient 1 and patient 2). The
affinity purification of HIT anti-PF4-heparin IgG (HIT IgG) and a
clinical summary of these patients has been previously
described.17 Briefly, patients 1 and 2 were 72- and
67-year-old women. Thrombocytopenia developed in each of them after
approximately 1 week of heparin therapy, and findings on
14C-serotonin release and platelet aggregation assays were
positive. Other causes of thrombocytopenia were clinically excluded.
HIT plasma was passed through a heparin-PF4-agarose column, and bound
antibodies were eluted with 0.13 to 1.5 mol/L NaCl gradient. IgG was
further purified on a protein G Sepharose column. Normal IgG, which did
not bind PF4-heparin, was purified from the pooled plasma of 3 healthy
persons by affinity for protein G Sepharose. The molecular weights of
the HIT and normal IgG were characterized under native conditions by
size exclusion high-performance liquid chromatography using a Zorbax
G-450 column (Hewlett Packard, Palo Alto, CA) and an aqueous solvent of
0.2 mol/L NaCl, 0.1 mol/L phosphate, pH 7.0, running at 1 mL/minute.
The IgG was predialyzed against the running buffer and was detected at
210 nm. The IgG eluted as a single peak with an average molecular
weight of 157 kd when compared with gel filtration molecular weight
standards (Bio-Rad Laboratories, Hercules, CA). No larger IgG
aggregates were present. IgG was iodinated with Na 125I
using iodogen (Pierce, Rockford, IL).
IgG binding during platelet aggregation
The methods for platelet preparation and platelet
aggregometry are based on those previously described.4
Platelet-rich plasma (PRP) was prepared by centrifuging citrated blood
at 170g for 15 minutes. Plasma was obtained by
centrifuging the remaining blood at 1600g for 15 minutes. The
concentration of platelets was determined with an automated blood cell
counter (Sysmex NE-8000, Kobe, Japan). Platelet aggregometry was
performed in silicone-coated glass tubes at 37°C by monitoring the
increase in optical transmittance of the platelet suspension with a
ChronoLog (Chrono-Log, Havertown, PA) aggregometer. Aggregation was
recorded once a second on a personal computer with an 8-bit
analog-to-digital converter (Pocket Sampler; Jaycar Electronics,
Sydney, Australia).
The aggregation reaction involved stirring PRP (300 × 106 platelets/mL) with HIT IgG (12 µg/mL = 80 nmol/L), 125I-IgG tracer ( 0.07
µg/mL, 13 kBq/mL), in 650 µL plasma. Aggregation was
induced by the addition of heparin (0.5 U/mL) or collagen (4µg/mL).
The aggregation reaction was sampled by removing, in duplicate, 80 µL
platelets with a wide-mouth siliconized pipette tip. These samples were
layered onto a 800 µL cushion of 17% sucrose18 in
phosphate-buffered saline (PBS) within a microfuge tube then centrifuged at 11,000g for 2 minutes. The supernatant (free
counts) was carefully removed from the top down, and the pellet (bound counts) were briefly rinsed with 200 µL PBS without resuspension. The
bottom of the tube, containing the pellet, was cut off, and the
radioactivity level was determined with a -counter. The amount of
bound IgG was determined by multiplying the proportion of total counts
bound by the amount of IgG added per platelet.
PF4 expression and release during platelet activation
In a siliconized glass aggregometer tube, 650 µL citrated
PRP (300 × 106 platelets/mL) was stirred at 37°C
with heparin plus HIT IgG, collagen plus heparin, collagen alone, or
heparin alone. Heparin was used at 0.5 U/mL, collagen was used at 4 µg/mL, and affinity-purified anti-PF4-heparin IgG from HIT patient 2 was used at 12 µg/mL. Aggregation was only observed when platelets
were activated with HIT IgG, collagen plus heparin, or collagen alone.
Activation was arrested after 8 to 9 minutes by adding 65 µL ETP and
chilling on ice (ETP consists of 107 mmol/L EDTA, 12 mmol/L
theophylline, and 2.8 µmol/L prostaglandin E1). Platelets
were pelleted by centrifugation (3500g, 2 minutes) in the
siliconized tube. The plasma supernatant was passed though a 0.2 µm
filter to remove residual platelets19 and was stored at
 20°C before the PF4 concentration was measured.
To measure PF4 expression, the platelet pellets were resuspended in 220 µL of a solution containing 2% bovine serum albumin (BSA), 10% ETP,
50 µg/mL IV.3, and PBS. Ninety-microliter platelets were incubated on
ice in microfuge tubes for 10 minutes with an equal volume of iodinated
sheep anti-PF4 IgG (affinity-purified polyclonal; Cedarlane
Laboratories, Hornby, Ontario, Canada) or sheep total IgG (purified
from sheep serum by affinity for protein G Sepharose). Free
125I-IgG was removed from the platelets by direct
centrifugation (9400g, 2 minutes) and 2 washes with PBS. The
bottom of the microfuge tube, containing the pellet, was cut off, and
the radioactivity level was determined with a -counter. Sodium
dodecyl sulfate-polyacrylamide gel electrophoresis indicated that
neither sheep IgG preparation contained PF4.
PF4 assay
A competition radioimmunoassay was developed to measure the PF4
concentration in plasma derived from activated and inactivated platelets (see above). Microtiter wells that could be individually separated (MaxiSorp-BreakApart; Nunc, Roskilde, Denmark) were coated
with affinity-purified sheep anti-PF4 IgG and blocked with BSA.
Dilutions of test plasma and purified PF47 standards (0-2 µg/mL) were prepared in a solution containing 1% BSA, 0.38%
trisodium citrate, 10% ETP, and PBS-Tween. When the test plasma
contained 0.5 U/mL heparin, the same heparin concentration was
maintained throughout dilution of the test and standard PF4 solutions.
Standard or unknown PF4 solution (110 µL) was equilibrated for 30 minutes with an equal volume of 125I-PF4 solution (0.2
µg/mL, 4 KBq/mL) containing polybrene (50 µg/mL). One hundred
microliters of PF4 mixture was incubated, in duplicate, for 15 minutes
in the wells coated with anti-PF4 IgG. Wells were washed 3 times with
PBS-Tween and then broken apart, and radioactivity was measured in a
-counter. The PF4 concentration was determined by comparing counts
with the appropriate standard curve, depending on the presence or
absence of 0.5 U/mL heparin. The standard curve with heparin was
shifted by 0.25 µg/mL to the right of the curve without heparin. The
most sensitive region of the PF4 standard curve was 0.05 to 1 µg/mL
PF4, and samples were diluted to fall within this range. Polybrene was required in this assay to prevent heparin from abolishing the binding
between PF4 and antibody.
| |
Results |
HIT IgG binding during platelet aggregation
We investigated the binding of affinity-purified HIT
125I-IgG to platelets at various time points (A, B, C, and
D) during platelet aggregation induced by the antibody and heparin
(Figure 1). Before heparin was added
to the suspension of PRP and HIT IgG (time point A), the platelets
remained nonaggregated, and negligible binding of HIT IgG
was detected. The addition of heparin triggered platelet aggregation.
The beginning of aggregation (time point B) was associated with low but
detectable binding of HIT IgG. At time point C, when platelets were
approximately 50% aggregated, there was approximately half-maximal HIT
IgG binding. The greatest IgG binding was observed once platelets were
fully aggregated (time point D). The nonspecific binding of normal
125I-IgG remained low throughout, indicating minimal
entrapment of IgG during aggregation. These data suggest that some
degree of platelet activation is required for HIT IgG binding to
platelets.
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| Fig 1.
Binding of affinity-purified anti-PF4-heparin HIT IgG to
platelets during heparin-induced platelet aggregation.
Platelet-rich plasma (300 × 106 platelets/mL) was
mixed with HIT IgG (12 µg/mL) and 125I-IgG tracer in a
platelet aggregometer at 37°C for 12 minutes before heparin (0.5 U/mL) was added. The reaction was sampled 2 minutes before heparin
(time point A), either at the start (time point B) or in the middle
(time point C) of aggregation, and when platelets were fully aggregated
(time point D). Samples were centrifuged through a 17% sucrose cushion
to separate platelets from unbound IgG. Specific binding of patient 1 ( ) and patient 2 ( ) HIT IgG was measured by using HIT
125I-IgG tracer, whereas nonspecific binding (open symbols)
was determined with normal 125I-IgG instead. The gray trace
shows a typical aggregation profile that progresses from unaggregated
to fully aggregated on an arbitrary scale. IgG bound is
the mean ± SE of 3 to 8 experiments.
|
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Role of platelet activation and platelet FcRII
The role of the FcRII in the binding of HIT IgG to
platelets was investigated by blocking the FcRII with the monoclonal antibody IV.3. As expected, the presence of IV.3 prevented HIT IgG and
heparin from inducing platelet aggregation, and no binding of HIT
125I-IgG to these platelets was detected. However, after
collagen was added to activate the platelets, the HIT
125I-IgG did bind (Figure 2).
Thus, blocking the FcRII did not prevent binding of HIT IgG if the
platelets could be activated by another pathway. This indicates that
IgG binding to activated platelets must have occurred by the Fab region
of HIT IgG, probably binding to PF4-heparin immobilized on the
platelet surface. The same conclusion was reached when HIT
anti-PF4-heparin F(ab')2 bound to platelets aggregated with collagen plus heparin (Figure
3). The action of IV.3 appears to be that
it precludes HIT IgG from activating platelets. This prevents the
release of PF4 from platelet  -granules and minimizes the expression
of PF4-heparin on the platelet surface so there is little binding of
HIT IgG.
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| Fig 2.
Effect of blocking platelet Fc receptor on the binding of
HIT IgG during platelet aggregation.
Platelet-rich plasma (300 × 106 platelets/mL) was
mixed with the monoclonal antibody IV.3 (anti-FcRII, 50 µg/mL), HIT IgG (12 µg/mL), and 125I-IgG tracer in a
platelet aggregometer for 12 minutes before heparin (0.5 U/mL)
was added. Aggregation was induced by the addition of collagen (4 µg/mL) 12 minutes after heparin was added. The reaction was
sampled 2 minutes before heparin, 10 minutes after heparin (when
full aggregation would have occurred without IV.3), and when the
collagen-induced aggregation was complete. Samples were centrifuged
through a 17% sucrose cushion to separate platelets from unbound
IgG. Specific binding of patient 1 () and patient 2 () was
quantitated by the inclusion of the same HIT 125I-IgG
tracer, whereas nonspecific binding ( ) was measured by the
binding of normal 125I-IgG. The gray trace shows a typical
aggregation profile. IgG bound is the mean of 2 experiments.
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| Fig 3.
Binding of HIT F(ab')2 to
platelets.
Platelet-rich plasma (300 × 106 platelets/mL) was
mixed with HIT or normal F(ab')2 (8 µg/mL) and
125I-F(ab')2 tracer in a platelet
aggregometer for 12 minutes before heparin (0.5 U/mL) plus collagen (4 µg/mL) was added to induce aggregation. Platelets were sampled 2 minutes before heparin plus collagen and 5 minutes after heparin plus
collagen and were centrifuged through a 17% sucrose cushion to
separate platelets from unbound F(ab')2. Symbols
indicate the specific binding of patient 1 () and patient 2 ()
F(ab')2 and nonspecific binding of Normal
F(ab')2 ( ). The gray trace shows a typical
aggregation profile. F(ab')2 bound is the mean of 2 experiments.
|
|
Specificity of affinity-purified HIT IgG
To confirm that the HIT IgG indeed recognized PF4, we blocked the
binding of HIT IgG to activated platelets by preincubating the
platelets with affinity-purified sheep anti-PF4 IgG. The binding of HIT
125I-IgG tracer (without unlabeled HIT IgG) was
increasingly inhibited with higher concentrations of anti-PF4 (Figure
4). At 48 µg/mL anti-PF4, the binding of
HIT IgG from patients 1 and 2 was reduced to one quarter and one
eighth, respectively, of the binding without anti-PF4.
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| Fig 4.
Inhibition of HIT IgG binding to platelets by anti-PF4.
Platelets that were activated by collagen plus heparin were resuspended
in PBS containing 2% BSA, 6 mg/mL human IgG, 10% ETP, and 50 µg/mL
IV.3. Platelets (60 × 106) were blocked for 30 minutes with various concentrations of affinity-purified sheep anti-PF4
IgG before HIT 125I-IgG tracer was added for 10 minutes.
All incubations were performed at 0°C to minimize further platelet
activation. After centrifugation and 2 washes with PBS, free
125I-IgG was removed. Binding of HIT IgG from patient 1 () and patient 2 () is expressed as a percentage of the total
radioactivity added because the IgG concentration of the
125I-IgG tracer alone cannot be accurately measured.
Background binding of normal 125I-IgG was only 0.05%.
|
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Release of PF4 from platelets
To show that PF4 is released during platelet activation, we
quantitated the PF4 released from platelets during incubation of PRP
with various agonists (Figure 5). The
plasma from unstimulated platelets, anticoagulated with citrate,
contained 0.19 µg/mL PF4. When platelets were aggregated by the
addition of either HIT IgG plus heparin, collagen alone, or collagen
plus heparin, the plasma contained approximately 5 µg/mL PF4.
Treatment of platelets with heparin alone released a lesser amount of
PF4, and the resultant plasma contained 0.82 µg/mL PF4. The released
PF4 may be absorbed onto the platelet surface, resulting in elevated
surface expression of the protein. PF4 release by HIT IgG without
heparin was not investigated because IgG alone does not trigger
platelet activation.
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| Fig 5.
Release of PF4 after platelet activation.
Either 650 µL citrated platelet-rich plasma (300 × 106 platelets/mL) was not activated or it was
activated with heparin and HIT IgG, collagen and heparin together,
collagen alone, or heparin alone after being stirred in an
aggregometer. Activation was arrested when ETP was added and the plasma
was chilled on ice. Platelets were removed by centrifugation (for use
in the step described in Figure 6), and the plasma PF4 concentration
was measured by competition radioimmunoassay. Bars show the mean
values of 3 experiments.
|
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Increased expression of PF4 on the platelet surface
after activation
The relative surface expression of PF4 on activated and
nonactivated platelets was measured by the binding of sheep anti-PF4 125I-IgG (Figure 6).
Unstimulated citrated platelets displayed little specific binding of
anti-PF4 antibodies. Aggregation of platelets with either HIT IgG plus
heparin, collagen alone, or collagen plus heparin resulted in a marked
increase in PF4 expression. Platelets treated with heparin alone had
only a modest increase in surface PF4 compared with unstimulated
platelets.
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| Fig 6.
Expression of PF4 on the platelet surface after
activation.
Citrated PRP either was not activated or it was activated with heparin
and HIT IgG, collagen and heparin together, collagen alone, or heparin
alone. Activation was arrested after ETP was added and the plasma was
chilled on ice. Plasma was removed by centrifugation (for use in the
step described in Figure 5). Surface expression of PF4 was determined
by the binding of sheep anti-PF4 125I-IgG (dark bars). Open
bars represent the nonspecific binding of nonimmune sheep IgG. Graph
illustrates the percentage anti-PF4 125I-IgG bound to
platelets as a proportion of the total radioactivity added and shows
the mean of 2 experiments.
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These data support the notion that the surfaces of activated platelets
are covered in PF4 that binds heparin and HIT IgG. The bound IgG
activates other platelets through FcRII, which initiates a chain
reaction that leads to a rapid increase in platelet aggregation and HIT
IgG binding.
Role of heparin
Figure 7 demonstrates that
heparin is required to facilitate the binding of HIT IgG to aggregated
platelets. Platelets aggregated with collagen alone did not bind HIT
IgG. Binding only occurred when heparin was included with the collagen.
This supports the concept that PF4 must form a complex with heparin
before it is recognized by HIT IgG.
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| Fig 7.
Heparin dependence in the binding of HIT IgG to
collagen-aggregated platelets.
Platelet-rich plasma (300 × 106 platelets/mL) was
mixed with HIT or normal IgG (12 µg/mL) and 125I-IgG
tracer in a platelet aggregometer for 12 minutes before collagen (4 µg/mL) ± heparin (0.5 U/mL) was added. Platelets were sampled 2 minutes before collagen, 4 minutes after collagen (when platelets were
consistently fully aggregated), and 10 minutes after collagen by
centrifugation through 17% sucrose. Solid symbols indicate the
specific binding of patient 1 () and patient 2 () IgG and
nonspecific binding of normal IgG () in the presence of heparin.
Corresponding open symbols represent binding without heparin. Gray
trace shows a typical aggregation profile. Values are the means of 2 experiments.
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The amount of IgG bound to platelets was double (Figure 7) the amount
when FcRII was blocked by IV.3 (Figure 2). This probably reflects
differences in the degree of platelet activation between the 2 figures
and not a component of Fc binding. In Figure 7 platelets were activated
by both collagen and HIT IgG and so probably expressed more
PF4-heparin antigen than did Figure 2, in which activation by HIT IgG
was prevented by IV.3. Support for this view comes from our observation
that platelets activated by HIT IgG plus heparin bound more anti-PF4
IgG than those activated by collagen plus heparin (Figure 6).
| |
Discussion |
In this study we demonstrated that HIT IgG, affinity
purified on heparin-PF4-agarose, induces heparin-dependent
platelet aggregation (Figure 1). This finding extends that of
Greinacher et al,20 who showed that HIT antibodies,
isolated by affinity for endothelial cells, could bind PF4-heparin on
enzyme-linkedimmunosorbent assay and activate platelets. However,
our data contribute the strongest evidence that the anti-PF4-heparin
antibody is the heparin-dependent platelet-aggregating factor,
known to be present in the plasma of patients with HIT, and they
imply that the anti-PF4-heparin antibody may cause
in vivo platelet activation and, consequently, thromboembolism.
However, this mechanism may not necessarily reflect the pathophysiology
in all patients with HIT.
We provide, for the first time, fundamental data on the binding of
affinity-purified HIT anti-PF4-heparin IgG to platelets during
platelet aggregation. We used concentrations of
platelets,21 heparin,22,23 and specific
anti-PF4-heparin IgG17 that were achievable in vivo.
Significantly, no exogenous PF4 was added, and PF4 released from
the platelets was available to modulate further antibody binding to the
platelets. Our findings are particularly significant because the
platelet aggregation was induced by the HIT IgG itself rather than by
thrombin. The use of affinity-purified antibody provided high sensitivity.
Figure 1 indicates that before the addition of heparin, there was
little or no binding of HIT IgG to platelets. We only detected that HIT
IgG bound to platelets when heparin was added to the platelets and
aggregation began. The HIT IgG binding then increased sharply until
platelets were fully aggregated. There is clearly a close correlation
between the degree of aggregation and the amount of HIT IgG bound. This
confirms that platelet aggregation by HIT IgG is a dynamic process by
which activation promotes further antibody binding. We speculate that a
very low level of HIT IgG binding to the platelets occurred before aggregation.
We confirmed that PF4 is strongly implicated in forming the platelet
antigen recognized by HIT IgG because the binding of HIT IgG to
activated platelets was inhibited by the preincubation of platelets
with sheep anti-PF4 IgG (Figure 4). Furthermore, the activation of
platelets by HIT IgG plus heparin, or by collagen, triggered a
substantial increased in the expression of PF4 on the platelet membrane
(Figure 6). Of note, incubating platelets with heparin
alone was sufficient to elevate modestly the surface expression of PF4.
Many reports demonstrate that HIT antibodies optimally bind to
PF4-heparin in a narrow range of PF4:heparin ratios. In our hands,
this ratio corresponded to at least 20 µg PF4/U heparin, regardless of whether the PF4-heparin was immobilized on enzyme-linked immunosorbent assay wells or was free in solution. We have demonstrated that the ratio of PF4:heparin is perhaps not as important in a more physiological and sensitive system. We added 0.5 U/mL heparin to
platelet-rich plasma, but the PF4 was derived solely from that already
present in the plasma or the platelets, or both. The plasma PF4
concentration increased from 0.19 µg/mL in citrated platelet-rich plasma to 5.6 µg/mL when platelets were fully
aggregated by HIT IgG (Figure 5). Consequently, heparin was
always in excess, but the increase in PF4 concentration would have
moved the PF4:heparin ratio toward the optimum. It is likely that
this contributed to the increased binding of HIT IgG during platelet aggregation.
Previously, we and others5,10,17,20 observed that HIT IgG
binds weakly to PF4 alone, immobilized into microtiter wells in the
absence of heparin. Therefore, we investigated the importance of
heparin in the binding of HIT IgG to platelet-surface PF4. We observed
that binding of anti-PF4-heparin IgG to collagen-aggregated platelets
only occurred in the presence of heparin (Figure 7), despite the fact
that platelets aggregated by collagen alone expressed significant
amounts of PF4 on their surfaces (Figure 6). We concluded that PF4 on
the platelet surface may not be modified in the same way as PF4
adsorbed into microtiter wells. Instead heparin is required to
facilitate HIT antibody binding, perhaps by inducing a conformational
change within PF4.
Aster24 indicates that understanding the orientation of IgG
on the platelet surface is important. If the Fab region of IgG can bind
platelets, then anti-PF4-heparin antibodies of IgA or IgM classes may
opsonize platelets for destruction in vivo, and this may explain why
HIT can develop in patients with only IgA or IgM anti-PF4-heparin
antibodies.25 Figure 2 indicates that, as
expected,26,27 IV.3 prevented HIT IgG from aggregating
platelets in the presence of heparin, and these nonaggregated platelets did not bind HIT 125I-IgG. Platelet aggregation was then
induced by the addition of collagen, which acts through a mechanism
independent of FcRII. Once aggregated, the platelets bound HIT IgG
despite the presence of IV.3. Similarly, F(ab')2
fragments of HIT IgG also bound to platelets aggregated by
collagen and heparin (Figure 3). This implies that the binding of HIT
IgG to platelets occurs when the Fab portion of IgG recognizes
PF4-heparin already on the platelet surface. Consequently, HIT
IgG binding is dependent on platelet activation needed to increase PF4
expression on the platelet surfaceand not on the availability of
FcRII. The effect of IV.3 is simply to prevent HIT IgG
from activating platelets through the Fc receptor.
We present definitive evidence that anti-PF4-heparin IgG from patients
with HIT can cause heparin-dependent platelet aggregation. For the
first time, we also report the dynamic binding of HIT IgG to platelets
as the IgG (plus heparin) induces platelet aggregation under conditions
that are as close as possible to those found in vivo. Our data support
a mechanism of platelet activation by HIT IgG that is summarized in
Figure 8. Initially, on the addition of
heparin, tiny amounts of PF4-heparin complexes form on the platelet
surfaced. The Fab portion of HIT IgG binds to this antigen, and the Fc
region of the bound HIT IgG cross-links FcRII on the same or
adjacent platelets. This triggers platelet activation and
degranulation. The PF4 that is released binds more heparin and forms
more antigen on platelets. Thus, positive feedback accelerates platelet
activation until all the platelets are fully aggregated.
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| Fig 8.
Dynamic model of platelet activation by HIT IgG.
(1) Complexes of PF4-heparin form on the surfaces of weakly activated
platelets. (2) HIT IgG binds to the platelet-bound PF4-heparin through
the Fab region. (3) The Fc portion of the bound IgG cross-links
FcRII on the same (not illustrated) or adjacent platelets and
produces strong platelet activation. (4) PF4, released from the
activated platelets, forms more complexes with heparin on platelets and
promotes antibody binding. In this model, blocking FcRII with IV.3
would still permit the Fab part of IgG to bind platelets, but IV.3
would inhibit platelet activation.
|
|
These data on the dynamic binding of HIT IgG to platelets in the
presence of heparin provide further insights into the pathophysiology of heparin-induced thrombocytopenia and thrombosis.
| |
Acknowledgments |
We wish to thank Irene Bate and Johanne Lonie (Gradipore Ltd.)
for performing size exclusion high-performance liquid chromatography on
the IgG.
| |
Footnotes |
Submitted July 27, 1999; accepted February 18, 2000.
Supported by a grant from the National Health and Medical Research
Council (Australia) and an NSW Research and Development Infrastructure
grant. P.N. is an Australian Postgraduate Award Scholar.
Reprints: Beng H. Chong, Department of Haematology, Prince of
Wales Hospital, Randwick, New South Wales, 2031, Australia; e-mail:
b.h.chong{at}unsw.edu.au.
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.
| |
References |
1.
Chong BH.
Heparin-induced thrombocytopenia.
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Top
Abstract
Introduction