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Blood, Vol. 91 No. 2 (January 15), 1998:
pp. 549-554
Adenosine Diphosphate (ADP) and ADP Receptor Play a Major Role in
Platelet Activation/Aggregation Induced by Sera From Heparin-Induced
Thrombocytopenia Patients
By
János Polgár,
Petra Eichler,
Andreas Greinacher, and
Kenneth J. Clemetson
From the Theodor Kocher Institute, University of Berne, Berne,
Switzerland and the Institute for Immunology and Transfusion Medicine,
Ernst-Moritz-Arndt-University, Greifswald, Germany.
 |
ABSTRACT |
The molecular basis for heparin-induced thrombocytopenia (HIT), a
relatively common complication of heparin therapy, is not yet fully
understood. We found that pretreatment of platelets with AR-C66096
(formerly FPL 66096), a specific platelet adenosine diphosphate (ADP)
receptor antagonist, at a concentration of 100 to 200 nmol/L that
blocked ADP-dependent platelet aggregation, resulted in complete loss
of platelet aggregation responses to HIT sera. AR-C66096 also totally
inhibited HIT serum-induced dense granule release, as judged by
measurement of adenosine triphosphate (ATP) release. Apyrase, added to
platelets at a concentration that had only minor effects on
thrombin- or arachidonic acid-induced aggregation, also blocked
completely HIT serum-induced platelet aggregation. Furthermore,
AR-C66096 inhibited platelet aggregation and ATP release induced by
cross-linking Fc RIIA with specific antibodies. These data show that
released ADP and the platelet ADP receptor play a pivotal role in HIT
serum-induced platelet activation/aggregation. The thromboxane receptor
inhibitor, Daltroban, had no effect on HIT serum-induced platelet
activation whereas GPIIb-IIIa antagonists blocked platelet aggregation
but had only a moderate effect on HIT serum-induced dense granule
release. Pretreatment of platelets with chondroitinases
but not with heparinases resulted in concentration dependent inhibition
of HIT serum-induced platelet aggregation. These novel data relating to
the mechanism of platelet activation induced by HIT sera suggest that
the possibility should be examined that ADP receptor antagonists or
compounds that inhibit ADP release may be effective as therapeutic
agents for the prevention or treatment of complications associated with heparin therapy.
| |
INTRODUCTION |
HEPARIN-INDUCED thrombocytopenia (HIT) is
the most frequent therapeutic drug-induced immune mediated
complication.1,2 Thromboembolism as a consequence of
heparin administration was first described almost 40 years
ago.3 Recently, several important aspects of the
pathogenesis of HIT have been resolved. There is general agreement that
in the majority of cases the antigen in HIT is formed by multimolecular
complexes of heparin and platelet factor 4 (PF4)4-7 and
that immune complexes formed from heparin-PF4 and the resulting
antibodies induce an Fc-mediated platelet activation.8-11 Accordingly, cross-linking of the platelet Fc receptor,
FcRIIA,12 has a crucial role in platelet activation
induced by HIT antibodies. Recent reports suggested that a functional
dimorphism of FcRIIA, arginine, or histidine at position 131, which
affects the avidity of the receptor for Fc binding, might be
responsible for disease susceptibility.13,14 However, in
their recent study Arepally et al15 found no statistically
significant difference in the prevalence of each FcRIIA isoform
between HIT patients with or without thrombosis and controls or between
HIT patients with thrombosis or thrombocytopenia alone. This suggests
that FcRIIA dimorphism is not responsible for disease susceptibility
and the question of what kind of factors are responsible for different
clinical manifestations in patients with HIT is still open. To
investigate this very important question, a detailed knowledge of the
mechanism of platelet activation induced by HIT antibodies is
essential. Although several aspects of HIT antibodies-induced platelet
activation have already been resolved, there is still no general
agreement about basic questions such as whether immune complexes
containing heparin, PF4, and IgG are formed in the fluid phase and then
bind to platelet Fc receptors or if heparin-PF4 complexes on the
platelet surface are recognized and bound by HIT antibodies; two
distinct models with different consequences.16
One approach to finding factors that may be important in the mechanism
of platelet activation induced by HIT antibodies is to examine whether
a particular pretreatment of platelets with receptor antagonists or
(where these are not available, by specific degradation) affects the
platelet activating capacity of HIT antibodies. Some novel findings
obtained by this method are described here that may contribute to a
better understanding of platelet activation/aggregation mechanisms
involved in HIT.
| |
MATERIALS AND METHODS |
Materials.
ADP receptor inhibitor AR-C66096
(2-propylthio-D- ,-difluoromethylene ATP, trisodium salt, formerly
FPL 66096) was a kind gift from Mr R.G. Humphries, Astra Charnwood
(Loughborough, UK). Apyrase (Type III), chondroitinase ABC (EC
4.2.2.4.), heparinase I (EC 4.2.2.7.), heparinase III (EC 4.2.2.8.),
luciferin-luciferase, heparin (sodium salt, from porcine intestinal
mucosa), and antimouse IgG, Fc-specific F(ab )2 were
from Sigma Chemical Co (St Louis, MO). Anti-FcRIIA monoclonal
antibody (MoAb) (IV.3) was from Medarex Inc (Annandale, NJ). RGDS
tetrapeptide was from Bachem AG (Bubendorf, Switzerland). The low
molecular mass peptidomimetic GPIIb-IIIa antagonist Ro44-9883 was a
kind gift from Dr Beat Steiner, Hoffmann-La Roche (Basle, Switzerland).
Low molecular mass thrombin inhibitor LY288570 was a kind gift from Dr
Joseph A. Jakubowski, Lilly Research Laboratories (Indianapolis, IN).
Daltroban (Boehringer Mannheim, Mannheim, Germany) was a kind gift from
Dr H. Patscheke, Klinikum Karlsruhe, Germany. PF4 was purified as
previously described,17 with heparin-Sepharose affinity
chromatography.
Sera and platelets.
Sera from 11 patients with HIT were examined. HIT was verified in these
patients, as previously described.18 Informed consent was
obtained from each patient. All studies were conducted according to the
principles expressed in the Declaration of Helsinki. Human platelets
were isolated from buffy coats, less than 20 hours after blood
collection, obtained from the Central Laboratory of the Swiss Red Cross
Blood Transfusion Service. To one buffy coat was added 30 mL of 100 mmol/L citrate, pH 6.5. Platelet rich plasma and the platelet pellet
were isolated by successive centrifugation steps. Platelets were washed
twice with Tyrode's buffer and were finally resuspended in 20 mmol/L
Hepes, 140 mmol/L NaCl, 4.5 mmol/L KCl, 5.5 mmol/L glucose, pH 7.4. The
platelet count was adjusted to 400 to 800 × 109/L.
Samples were kept at room temperature until used for aggregation studies. Because of the well known but poorly understood donor-related platelet variability in platelet aggregation tests with HIT sera, platelets were isolated in parallel from six buffy coats each day. The
platelet suspension that aggregated most strongly with 3 HIT sera
chosen randomly from the 11 HIT sera examined in this study was used
for further experiments that day.
Platelet aggregation.
Platelet aggregation was monitored by light transmission with a
Labintec (Monpellier, France) aggregometer with continuous stirring at 1300 rpm at 37°C. Platelets were preincubated with 2 mmol/L CaCl2 at 37°C for 10 minutes (without stirring)
before starting the measurement by adding the samples for analysis. For standardization of PF4 supply and facilitating generation of
heparin-PF4-complexes, 10 µg/mL PF4 and 0.5 units/mL heparin were
added before adding HIT serum as an agonist. One vol HIT serum
containing 1 µg/mL low molecular mass thrombin inhibitor, LY288570,
was added to 20 vol of platelet suspension.
Measurement of platelet ATP release.
Determination of ATP by a luciferin/luciferase method was performed as
previously described.19 Briefly, 1, 2, and 5 minutes after
adding the agonists HIT serum or thrombin to activate the platelets,
the reaction was terminated by mixing 5 vol of platelet suspension with
1 vol of ice-cold 600 mmol/L formaldehyde, 60 mmol/L EDTA, pH 7.4. The
formaldehyde-fixed aliquots were kept on ice for 5 minutes before
centrifuging in an Eppendorf centrifuge at 6000 rpm for 3 minutes at
room temperature. The supernatants were analyzed for ATP. Luminescence
measurements were performed with a homemade luminometer incorporating a
model 1109 Photon Counter (Princeton Applied Research, Princeton, NJ).
Pretreatment of platelets with chondroitinases ABC and heparinases.
Platelets were incubated with various concentrations of enzymes, in the
presence or absence of 2 mmol/L CaCl2, in Eppendorf tubes
in a 37°C water bath. The samples were gently mixed every 5 minutes. After 30 minutes incubation the samples were transferred to
the aggregometer cuvettes and platelet aggregation was performed as
described above. Those samples that were treated with enzymes in the
absence of CaCl2 were incubated with 2 mmol/L
CaCl2 for 5 minutes before adding the agonists, thrombin,
arachidonic acid, or HIT sera.
| |
RESULTS |
Characteristics of HIT sera examined.
Sera from 11 HIT Type II patients were examined. Summary of clinical
status of the patients as well as laboratory test results are shown in
Table 1. After preliminary results with
randomly selected HIT sera we included sera from patients showing
different clinical manifestations of HIT for further studies. Thus,
patients no. 3 and 6 had thrombocytopenia only; no. 7 and 9 had
thromboembolic complications without a major decrease in platelet
count; no. 1, 4, and 5 had thrombocytopenia and venous thromboembolic
complications and no. 2 and 11 had thrombocytopenia as well as venous
and arterial thrombosis.
Preliminary experiments showed that in the presence of 2 mmol/L
CaCl2, 10 µg/mL PF4, and 0.5 U/mL heparin, 1 vol HIT
serum added to 20 vol of washed platelet suspension induced clearly detectable platelet aggregation in 5 minutes. Preincubation of platelets with 2 µg/mL anti-FcRIIA MoAb, IV.3, completely blocked the platelet aggregating effect of all HIT sera examined.
HIT serum-induced dense granule release was not prevented by
inhibiting GPIIb-IIIa.
It has been shown previously that
14C-serotonin release induced by HIT sera were
similar when normal platelets and platelets from a patient with
Glanzmann's thrombasthenia were used.20 RGDS, 500 µmol/L, or the low molecular mass peptidomimetic Ro44-9880, 1 µmol/L, which blocks the fibrinogen binding site on GPIIb-IIIa, inhibited HIT serum-induced and thrombin-induced aggregation by more
than 90%, however, they inhibited ATP release by only 30% to 40%
when HIT sera (no. 1 to 3; n=2), and 10% when 1 nmol/L thrombin was
used as an agonist (data not shown). These data strongly suggest a
GPIIb-IIIa-independent mechanism for dense granule release in HIT
serum-induced platelet aggregation.
ADP receptor inhibitor inhibits HIT serum-induced platelet
aggregation and dense granule release.
Pretreatment of platelets with the ADP receptor inhibitor AR-C66096
inhibited HIT serum-induced platelet aggregation in a concentration
dependent way (Fig 1). The thromboxane
receptor inhibitor Daltroban, added to platelets at a concentration
that completely blocked arachidonic acid (AA)-induced aggregation, had
no effect on HIT serum-induced aggregation (Fig 1).
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| Fig 1.
Concentration dependent inhibition of HIT serum-induced
platelet aggregation by the ADP receptor antagonist AR-C66096.
Platelets, 500 × 109/L, were preincubated with various
concentrations of AR-C66096 or thromboxane receptor inhibitor Daltroban
at 37°C for 2 minutes before stimulation with HIT serum no. 1 or
100 nmol/L AA. HIT serum no. 1: Mean % aggregation with control buffer
was 55 ± 6%. AA: Mean % aggregation with control buffer was 53 ± 4% (mean ± SD, n = 3). HIT sera no. 2 and 3 and platelet
suspensions from donors other than in the case of HIT serum no.1 gave
similar results.
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A direct relationship was found between ATP release and platelet
aggregation when the effects of different HIT sera were examined with
the same platelet suspension over 5 minutes incubation time (Table 2). HIT sera 9 to 11 did not induce
either ATP release or platelet aggregation. Sera 6 to 8 induced
moderate to low ATP release and moderate to weak aggregation. HIT sera
1 to 5 were the most potent platelet agonists determined either by
measurement of platelet aggregation or ATP release. Table 2 shows that
the presence of ADP receptor inhibitor blocked completely HIT
serum-induced platelet aggregation as well as ATP release (except with
serum No. 7) whereas it had only little effect on thrombin-induced
aggregation and ATP release. Control sera obtained from healthy
volunteers did not induce platelet aggregation or ATP release under the
same conditions as used with HIT sera. A platelet suspension from
another donor gave similar results to those shown in Table 2 (data not shown).
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Table 2.
Platelet Aggregation and ATP Release Induced by HIT
Sera, Control Sera, and Thrombin in the Presence and Absence of ADP
Receptor Inhibitor
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If released ADP plays a major role in HIT serum-induced platelet
aggregation, apyrase, which breaks down ADP, should inhibit the
process. Apyrase, 5 U/mL, inhibited HIT serum-induced platelet aggregation completely, whereas this concentration of apyrase had only
minor effects on thrombin- and AA-induced aggregation (not shown).
Figure 2 shows a comparison of time
dependent platelet aggregation and ATP release induced by HIT sera and
thrombin. HIT sera no. 3 and 4 induced approximately the same
aggregation response and a similar ATP release at 5 minutes. However,
there was a significant difference in the degree of aggregation and ATP
release between the two sera during the first 2 minutes after adding
them to platelets.
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| Fig 2.
Comparison of the time course of platelet aggregation and
ATP release induced by HIT sera no. 3 (A) and 4 (B) and 1 nmol/L thrombin (C). Agonists were added at time points indicated by arrows.
Ten µg/mL PF4 and 0.5 U/mL heparin was added 1 minute before adding
HIT sera as agonists. One, 2, and 5 minutes after adding the agonists
aliquots were taken for ATP measurement. Arrows and numbers indicate
released ATP (nmol/1010 platelets). The aggregation curves
and ATP release data are representative of the results from three
experiments.
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ADP receptor inhibitor inhibits platelet activation/aggregation and
dense granule release induced by cross-linking FcRIIA with specific
antibodies.
ADP receptor inhibitor inhibited HIT serum-induced platelet
aggregation, a process thought to involve FcRIIA cross-linking, encouraged us to examine the effect of the ADP receptor inhibitor on
platelet aggregation induced by cross-linking FcRIIA. After preincubation of platelets in the presence and absence of 200 nmol/L
AR-C66096, FcRIIA cross-linking was induced by successive addition
of anti-FcRIIA MoAb, IV.3, 2 µg/mL, and antimouse IgG, Fc specific
F(ab)2, 2 to 20 µg/mL. The platelet aggregation
response depended on the concentration of the cross-linker
F(ab)2, reaching maximal aggregation at 10 µg/mL
cross-linker. Low and moderate aggregation responses, up to 30% to
40% aggregation, were completely abolished by pretreatment of
platelets with 200 nmol/L AR-C66096 whereas strong responses (58 ± 4 % aggregation, n = 3) were inhibited 92 ± 3 % (percentage
inhibition ± SD, n = 3, not shown). Similarly, pretreatment of
platelets with 200 nmol/L AR-C66096 blocked or inhibited ATP-release
more than 80% when the original aggregation response in the absence of
AR-C66096 was moderate or strong, respectively (not shown).
Pretreatment of platelets with chondroitinases inhibits HIT
serum-induced platelet aggregation.
Preincubation of platelets with chondroitinase ABC resulted in
concentration dependent inhibition of HIT serum-induced platelet aggregation (Fig 3). Platelets preincubated
with chondroitinases ABC aggregated normally with thrombin or AA (not
shown). Strong aggregation responses (such as that shown in Fig 3A)
induced by HIT sera were not blocked completely by pretreatment of
platelets with chondroitinases ABC, up to 1 U/mL for 30 minutes. Weak
aggregation responses, induced by adding that amount of HIT sera that
resulted in 10% to 20% platelet aggregation, were abolished
completely by pretreatment of platelets with chondroitinases ABC (not
shown). The inhibitory effect of chondroitinases ABC pretreatment
was independent of the presence or absence of CaCl2 during
the incubation with the enzyme. Heparinase I and III (Sigma; up to 20 U/mL and 7 U/mL, respectively) did not inhibit HIT serum-induced
platelet aggregation (not shown). These results strongly suggest the
involvement of a chondroitin sulfate proteoglycan in HIT serum-induced
platelet activation.
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| Fig 3.
Pretreatment of platelets with chondroitinase ABC
inhibits HIT serum-induced platelet aggregation. Platelets, 800 × 109/L, were preincubated with 0 (A), 0.2 (B), 0.5 (C), and
1 (D) µ/mL chondroitinase ABC at 37°C for 30 minutes before
stimulation with HIT sera. Agonists were added at time points indicated
by arrows. A total of 10 µg/mL PF4 and 0.5 U/mL heparin was added 1 minute before adding HIT sera as agonists. The aggregation curves are
representative of the results from three experiments with HIT sera 1, 3, and 4 and platelets from three different donors.
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| |
DISCUSSION |
Sera from HIT patients contain specific antibodies developed against
multimolecular complexes of heparin and PF4.4-7 These antibodies induce platelet activation and aggregation via cross-linking of FcRIIA.8-11 Dense granule release is an integral part
of HIT serum-induced platelet effects and serotonin or ATP release
assays are often used for laboratory diagnosis of HIT.21,22
The platelet membrane receptor GPIIb-IIIa has a well documented role in
both platelet aggregation and the release reaction.23 When
we examined the action of GPIIb-IIIa antagonists on platelet effects
induced by HIT sera, GPIIb-IIIa antagonists were found to prevent HIT serum-induced platelet aggregation, as expected. However, they inhibited HIT serum-induced dense granule release only moderately as
judged by ATP release. This finding (which corroborates previous results that normal platelets and Glanzmann's thrombasthenia platelets lacking GPIIb-IIIa showed similar 14C-serotonin release
induced by HIT sera20) encouraged us to look for potential
platelet agonists/mechanisms, which are capable of inducing
GPIIb-IIIa-independent dense granule release, and may be involved in
early events of platelet activation with HIT sera. The first candidate
was thromboxane A2 supposedly generated as a result of
early activation of phospholipase C and arachidonic acid release.
However, the thromboxane receptor inhibitor Daltroban had no effect on
HIT serum-induced platelet aggregation although it clearly inhibited
AA-induced platelet aggregation.
Next, we examined the possibility that ADP, released in small amounts
from the dense granules as a result of HIT antibodies binding to and
activating platelets, could have an important role in further
activation and aggregation of platelets as in the case of
collagen.24 The ADP receptor on platelets is thought to be a member of the P2Y nucleotide receptor family of G
protein-coupled, seven transmembrane domain receptors.25,26
The availability of a newly developed specific ADP receptor antagonist,
AR-C66096,27,28 which is effective in vitro, made it
possible to investigate the role of ADP and the ADP receptor in HIT
serum-induced platelet activation/aggregation. Pretreatment of
platelets with AR-C66096, at a concentration of 100 to 200 nmol/L that
blocked ADP-dependent platelet aggregation, resulted in complete loss
of platelet aggregation response to HIT sera. In addition, and more
unexpected, HIT serum-induced dense granule release was also blocked by
AR-C66096. These data clearly show that ADP release is the major factor
responsible for HIT serum-induced platelet aggregation. This was
supported by the finding that apyrase, added to platelets at a
concentration that had only minor effects on thrombin- or AA-induced
aggregation, blocked completely HIT serum-induced platelet aggregation.
In all 11 HIT sera examined, the presence of antiheparin-PF4 IgG was
shown by enzyme-linked immunosorbent assay (ELISA) and none of these
sera-induced platelet aggregation or dense granule release in platelets
was preincubated with anti-FcRIIA MoAb, IV.3. This shows that
antiheparin-PF4 IgG-induced FcRIIA cross-linking is involved in the
platelet effect of HIT sera examined here, in agreement with earlier
reports.8-11
It was also found that AR-C66096 inhibited FcRIIA
cross-linking-induced platelet aggregation and ATP-release. A recent
report by Cattaneo et al29 suggests the existence of an
aggregation-independent and ADP receptor-dependent mechanism by which
released ADP potentiates platelet secretion. Their finding that
deficiency of ADP binding sites on platelets was associated with a
secretion defect is consistent with our results that dense granule
release was inhibited by the ADP receptor antagonist AR-C66096.
The recent report by Horne et al30 provided the first
direct evidence for platelet binding of IgG as well as
F(ab)2 from patients with HIT. It was also shown in
this study that neither addition of a 200-fold excess of normal IgG or
inclusion of anti-FcRIIA MoAb, IV.3, suppressed the binding. This
suggests that HIT-IgG binding to platelets occurs via heparin-PF4
rather than FcRIIA. However, Suh et al31 reported that
IV.3 completely blocked the binding to platelets of immune complexes
consisting of PF4, heparin, and immunopurified HIT IgG antibodies. So,
there is no general agreement about whether immune complexes are formed
in the plasma and then bind to platelets via Fc receptors or whether
heparin-PF4 complexes associated with the platelet surface are
recognized by HIT antibodies. We show that preincubation of platelets
with chondroitinase ABC resulted in decreased reactivity to HIT sera. Since the first report,32 there is now increasing evidence
that PF4 associates with platelet chondroitin sulfate
proteoglycans.33-35 Although PF4 has an apparent Kd of 30 nmol/L for heparin whereas for chondroitin sulfate it is 265 nmol/L,34 a ninefold difference, this may not represent the
situation in platelets. On the one hand, in a platelet proteoglycan the
glycosaminoglycan chains are likely to be concentrated along a short
peptide sequence of repeating serine and glycine residues that would
increase the affinity to PF4 compared to free chondroitin sulfate; and
on the other hand the affinity of heparin for PF4 might be decreased in
the HIT antibody-heparin-PF4 complex compared to free heparin. Thus,
the effect of the chondroitinases could be due to destruction of a
platelet surface proteoglycan containing multiple chondroitin sulfate
chains that may have a role in docking and aligning the heparin-PF4(-HIT antibody) complexes on platelets and therefore facilitating FcRIIA clustering. That the chondroitinases do not prevent the platelet activation completely in reponse to higher doses
of HIT sera may be due to the complexity of these proteoglycans and the
glycosaminoglycan linkages. Such an effect of platelet membrane
proteoglycans would not be unique because there is now general
agreement that the affinity of fibroblast growth factors for their
receptors is increased in the presence of proteoglycans and that this
is probably of physiological significance.36
The data presented here suggest that enhanced susceptibility of
platelets to ADP, or other differences enhancing granule release, perhaps based on elevated expression or variations in the structure of
the ADP receptor on platelets or its signaling pathways, may be
considered as predisposing factors for development of heparin-induced complications. The fact that ADP plays a major role in platelet activation/aggregation induced by antibodies to heparin-PF4 complexes suggests that ADP receptor antagonists inhibiting ADP release from
dense granules might be effective as therapeutic agents for prevention
or treatment of heparin-induced immune complications.
| |
FOOTNOTES |
Supported in part by a grant from the Swiss National Science Foundation
(31-42336.94 to K.J.C.) and in part by the Deutsche Forschungsgemeinschaft (Grant No.1096/2-2).
Address reprint requests to Kenneth J. Clemetson, PhD, Theodor Kocher
Institute, University of Berne, Freiestrasse 1, CH-3012 Berne,
Switzerland.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely
to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Corinne Birbaum for technical assistance. We are grateful to
the Central Laboratory of the Swiss Red Cross Blood Transfusion Service
(Berne, Switzerland) for the supply of buffy coats.
| |
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Abstract
Introduction
Abstract