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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 5 (September 1), 1998:
pp. 1526-1531
Heparin-Induced Thrombocytopenia: New Insights Into the Impact of
the Fc RIIa-R-H131 Polymorphism
By
Lena E. Carlsson,
Sentot Santoso,
Gudrun Baurichter,
Hartmut Kroll,
Stephanie Papenberg,
Petra Eichler,
Nomdo A.C. Westerdaal,
Volker Kiefel,
Jan G.J. van de Winkel, and
Andreas Greinacher
From the Department of Immunology and Transfusion Medicine,
Ernst-Moritz-Arndt-University, Greifswald, Germany; the Department of
Clinical Immunology and Transfusion Medicine, Justus-Liebig-University,
Giessen, Germany; the Department of Immunology and Medarex Europe,
University Hospital Utrecht, Utrecht, The Netherlands; and the
Department of Immunology and Transfusion Medicine, University of
Leipzig, Leipzig, Germany.
 |
ABSTRACT |
Heparin-induced thrombocytopenia (HIT), a severe complication of
heparin treatment, can be associated with new thrombotic complications.
HIT antibodies activate platelets via the platelet Fc -receptor
(FcRIIa), which carries a functionally relevant polymorphism
(FcRIIa-R-H131). The effect of this polymorphism on the clinical
manifestations of HIT is controversial. We determined prospectively the
FcRIIa-R-H131 genotypes in 389 HIT patients, in 351 patients with
thrombocytopenia or thrombosis due to causes other than HIT and without
detectable HIT antibodies, and in 256 healthy blood donors. For this
purpose, a novel nested sequence-specific primer-polymerase chain
reaction (SSP-PCR) was developed. FcRIIa-R/R131 was
found to be overrepresented in the HIT patients (27%) compared with
the control groups (non-HIT patients [21%] and blood donors [20%]). In a subgroup of 122 well-characterized HIT patients, the
genotype distribution in patients presenting with thrombocytopenia only
was compared with that of patients who developed thromboembolic complications. The frequency of FcRIIa-R/R131 among patients with
thrombotic events was significantly elevated (37% v 17%; P = .036). Our results indicate that genotype distribution
can be correlated to the clinical outcome of patients with HIT. We speculate that the reduced clearance of immune complexes in patients with the FcRIIa-R/R131 allotype causes prolonged activation of endothelial cells and platelets, thus increasing the risk for thrombotic complications.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
HEPARIN-INDUCED thrombocytopenia (HIT) is
the most common drug-induced immune thrombocytopenia. Heparin treatment
can induce antibodies that recognize a complex of heparin and platelet
proteins, in most cases platelet factor 4 (PF4).1-5 These
immune complexes activate platelets and possibly also endothelial
cells.2,4,6 Paradoxically, HIT patients are at high risk of
developing new venous or arterial thromboembolic vessel occlusions in
response to the anticoagulant, heparin. It is now widely accepted that there is a discrepancy between the number of patients who develop HIT
antibodies and the number of patients who develop clinical symptoms of
HIT (ie, a decrease in platelet counts of >50% on or after day 5 of
heparin treatment and/or new thromboembolic complications [TECs]).5,7-9 However, why some
HIT patients develop thrombocytopenia only and others develop
concomitant TECs remains controversial. In clinically symptomatic
patients, platelet activation10 and generation of platelet
microparticles11 seem to be important triggers for the
development of TECs. Both are mediated by the platelet
FcRIIa,12-14 which, after cross-linking by the
heparin/PF4-HIT antibody immune complexes, initiates platelet activation.
The FcRIIa is the only IgG Fc-receptor present on platelets and the
gene encoding the receptor is polymorphic. A point mutation (G507A)
causes an amino acid exchange, Arg (R)-His (H) at position 131.15,16 FcRIIa-R/R131 interacts well with mouse IgG1,
and was originally named high responder (HR), whereas FcRIIa-H/H131 binds mouse IgG1 poorly and was called low responder
(LR).16-18 The opposite affinities are noted for human IgG;
FcRIIa-H/H131 is the only Fc receptor effectively interacting
with IgG2, as shown with leukocytes.19,20
Currently, there is a debate about whether the allotype of FcRIIa is
correlated with the development of HIT and the clinical outcome of
affected patients. Several studies addressing the distribution of the
FcRIIa-R-H131 polymorphism have been published
recently.21-24 However, these studies have been performed
with relatively low numbers of patients and their results have been
discrepant. In this study, we prospectively determined the
FcRIIa-R-H131 polymorphism of 389 HIT patients and compared their
genotype distribution with that of patients with thrombocytopenia or
thrombosis who did not have HIT antibodies and with healthy blood
donors. Additionally, we performed a subanalysis to assess the
occurrence of the FcRIIa-R-H131 polymorphism in HIT patients with
isolated thrombocytopenia and in HIT patients presenting with TECs
during heparin administration.
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MATERIALS AND METHODS |
Patients and controls.
Patients (n = 389) with clinical symptoms of HIT and heparin-dependent
antibodies as determined by the heparin-induced platelet activation
(HIPA) test25,26 were investigated. In the HIPA test,
heparin-dependent antibodies activate platelets via the FcRIIa;
thus, only patients with FcRIIa-reactive HIT antibodies were
included in the study. Patients without detectable HIT antibodies whom
had either thrombocytopenia due to causes other than HIT (eg, leukemia,
autoimmune thrombocytopenia, or sepsis) or a thrombotic event that was
unrelated to heparin treatment (n = 351) and unrelated healthy blood
donors (n = 256) served as control groups. All patients and controls were of Caucasian ethnicity. The patient groups originate from the whole of Germany, with 50% of the blood donors originating from the Northern part of Germany and 50% from the central part of
Germany. Data for 122 of the 389 patients with acute HIT were obtained
in a prospective treatment trial (unpublished data). Clinical data regarding these patients had been evaluated by an independent investigator and cross-checked with the patient file in an
audit program. A subanalysis of these well-characterized patients was
performed to determine the correlation between the FcRIIa-R-H131
genotypes and the manifestation of TECs in HIT.
HIPA test.
Platelet-rich plasma (PRP) was prepared from citrated blood (1.6 vol
adenine-citrate-dextrose [ACD] and 8.4 vol blood) from normal blood
donors (with 10 medication-free days). PRP was acidified by the
addition of 111 µL/mL ACD, and 5 U/mL apyrase (grade III; Sigma,
Munich, Germany) were added. After centrifugation (7 minutes at
650g), the supernatant was discarded and platelets were
carefully resuspended in 5 mL washing buffer (tyrode buffer containing
2.5 U/mL apyrase and 1.0 U/mL hirudin [Pentapharm, Basel,
Switzerland], adjusted to pH 6.3 with HCl). The platelets were
incubated in sealed tubes (15 minutes at 37°C), centrifuged (7 minutes at 650g), and resuspended in 1 mL suspension buffer
(tyrode buffer containing 0.002 mol/L MgCl2 and 0.002 mol/L
CaCl2, adjusted to pH 7.2 with HCl). The suspension was
adjusted to 300,000 to 400,000 platelets/µL and incubated in a sealed
tube (45 minutes at 37°C) before use. Heat-inactivated (56°C
for 30 minutes) patient serum (20 µL) was dispensed in a microtiter
plate (Greiner, Nürtingen, Germany). For intra-assay negative
control, parallel samples are mixed with a high concentration of
heparin (final concentration, 100 U/mL). This procedure disrupts
heparin-PF4 complexes and detects antibodies that bind independently of
the heparin concentration. Platelet suspension (75 µL) is added to
all samples and, finally, the low concentration heparin (final
concentration, 0.2 U/mL) is added to allow PF4/heparin complex
formation. The microtiter plate was incubated (45 minutes at room
temperature) on a magnetic stirrer (1,000 rpm) with two
steel spheres (2 mm diameter; SKF, Schweinfurth, Germany). The
transparency of the suspension was assessed using an indirect light
source every 5 minutes. The patient sera was considered positive for
HIT antibodies if the suspension became transparent due to platelet
aggregation with 0.2 U/mL heparin but not with 100 U/mL heparin.
Primer design.
To genotype the FcRIIa-R-H131 polymorphism, a new nested
sequence-specific primer-polymerase chain reaction
(SSP-PCR) method was developed. After the first PCR
amplification, using primer pair P52 and P63,27 the product
obtained was reamplified using sequence-specific primers. The
sequence-specific sense primers P5G (specific for G507) and P4A
(specific for A507) are located at the polymorphic site on exon 4, and
a common antisense primer, P13, is located on intron 4 (Fig 1). Both exonic primers were constructed based on the published FcRIIa cDNA
sequence.28 To increase the specificity, two mismatch bases
(T instead of C) were introduced at positions 502 and 504 in both
primers. Because no data on the intronic sequences of FcRIIa were
available, we determined the nucleotide sequence of intron 4 by PCR
using genomic DNA. After 30 amplification cycles with primers P63 and
P52, a 1,000-bp product was isolated. Sequence analysis of this product identified an 800-bp intron with conserved donor and acceptor splice
junctions. Based on this nucleotide sequence, we designed the antisense
intronic primer, P13, located 118 bp downstream from exon 4, for the
second-round PCR (Fig 1). A 440-bp fragment from the C-reactive protein
(CRP) gene was used as an internal positive control.29,30
All primers were purchased from Eurogentec (Seraing, Belgium).
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| Fig 1.
Schematic illustration of the localization of the primers
for the nested SSP-PCR and representative results of FcRIIa genotype
of three individuals: homozygous FcRIIa-R/R131 (R/R), heterozygous
FcRIIa-R/H131 (R/H), and homozygous FcRIIa-H/H131 (H/H). For the
FcRIIa-specific amplification, primers P63 and P52 were
used.27 For the allele-specific amplification, primers P5G
(for the FcRIIa-R131 allele) and P4A (for the FcRIIa-H131 allele)
were combined with a common antisense primer, P13. The 440-bp
amplification product of CRP was used as an internal control. The
278-bp fragment represents the allele-specific amplification product
from the SSP-PCR of FcRIIa. Amplification with P13 and P5G (lane 1)
and with P13 and P4A (lane 2) shows a homozygous FcRIIa-R/R131
individual. Amplification with P13 and P5G (lane 3) and with P13 and
P4A (lane 4) shows a heterozygous FcRIIa-R/H131 individual.
Amplification with P13 and P5G (lane 5) and with P13 and P4A (lane 6)
shows a homozygous FcRIIa-H/H131 individual. Lane 7 contains a
molecular weight standard (100 bp).
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Nested SSP-PCR.
DNA was isolated from EDTA-anticoagulated peripheral blood using QIAAmp
blood kits (Qiagen, Hilden, Germany). One hundred nanograms of genomic
DNA was added to 100 µL reaction mixes containing 10 mmol/L Tris (pH
8.0), 50 mmol/L KCl, 2.75 mmol/L MgCl2, 0.25 mmol/L of each
dNTP, 100 µg/mL bovine serum albumin (BSA), 0.1 µmol/L each of P63
(5 -CAA GCC TCT GGT CAA GGT C) and P52 (5-GAA GAG CTG CCC
ATG CTG) primers, 20 nmol/L each of CRP I and CRP II primers, and 1 U
of AmpliTaq (Perkin Elmer, Vaterstetten, Germany). PCR conditions were
as follows: 1 cycle at 95°C for 5 minutes, 55°C for 5 minutes,
and 72°C for 5 minutes. This was followed by 35 cycles of 95°C
for 1 minute, 55°C for 1 minute, and 72°C for 2 minutes, ending
with an extension step at 72°C for 10 minutes. From this reaction,
1 µL was reamplified in the SSP-PCR using primers P13 (5-CTA
GCA GCT CAC CAC TCC TC) and P5G (5-GAA AAT CCC AGA AAT TTT
TCC G) or P4A (5-GAA AAT CCC AGA AAT TTT TCC A). The allele-specific bases are in bold type
and the inserted mismatch bases in the SSPs are underlined. The PCR conditions were as follows: 95°C for 5 minutes followed by 30 cycles of 95°C for 15 seconds, 58°C for 30 seconds, and
72°C for 30 seconds, with an extension step at 72°C for 10 minutes. The PCR products were analyzed by electrophoresis on 1.5%
agarose gels stained with ethidium bromide.
Nucleotide sequencing.
To analyze the polymorphic region, PCR products amplified by primers
P63 and P52 were purified by Gene Clean (Dianova, Hamburg, Germany) and
sequenced directly with primer P63 using Sequenase 2.0, as recommended
by the manufacturer (Amersham, Braunschweig, Germany).
Method validation.
To assess intra-assay reproducibility, EDTA-anticoagulated blood was
obtained from 80 patients at two different time points. In a blinded
manner, these 160 samples were typed for FcRIIa polymorphism. To
validate the inter-assay reproducibility, leukocytes from genotyped
donors were phenotyped for the FcRIIa polymorphism using monoclonal
antibodies (MoAbs) in flow cytometric analysis (fluorescence-activated
cell sorting [FACS]; n = 340) and
3H-thymidine incorporation in a T-cell proliferation assay
(n = 272).
Phenotypic analysis of FcRIIa allotypes.
FACS scan analysis was performed according to standard methodology,
using MoAb IV.3 (mIgG2b; Medarex, Annandale, NJ) that recognizes
monomorphic epitopes on FcRIIa and MoAb 41H16 (mIgG2a; kindly
provided by Dr B.M. Longenecker, University of Alberta, Edmonton,
Alberta, Canada) that reacts preferentially with
FcRIIa-R131.20 The anti-CD3-induced T-cell mitogenesis
assays were performed as described by Tax et al,17
including an extra reaction with human IgG2 anti-CD3 to allow
discrimination between FcRIIa-R/H131 and
FcRIIa-R/R131.20
Statistics.
Relative frequencies of the FcRIIa genotypes were compared using
 2 statistics for contingency tables with 2 × 3 fields.31 P values were calculated with the SPSS
PC+ statistical package (SPSS Inc, Chicago, IL).
| |
RESULTS |
Nested SSP-PCR.
Results of the SSP analyses for three representative individuals are
shown in Fig 1. Amplification of genomic DNA from donor 1 resulted in a
278-bp specific product with primer P5G, but not with primer P4A. In
contrast, with DNA from donor 3, this product could be amplified only
with primer P4A. Both primers, P5G and P4A, amplified the 278-bp
product from donor 2. In all reactions, the 440-bp internal control
fragment of the CRP gene was present. These results indicate that
donors 1, 2, and 3 represent homozygous FcRIIa-R/R131, heterozygous
FcRIIa-R/H131, and homozygous FcRIIa-H/H131, respectively.
Method validation.
Results of the nested SSP-PCR were validated by direct sequencing of
the polymorphic region; no discrepancies were found (data not shown).
Double sample processing (n = 80), FACS analysis using MoAbs
recognizing FcRIIa-R/H131 and FcRIIa-R/R131 (n = 340), and
anti-CD3-induced T-cell mitogenesis (n = 272) demonstrated the
intra-assay and inter-assay reproducibility to be 100%.
Genotype distribution.
The genotype distributions and allele frequencies of the
FcRIIa-R-H131 polymorphism are presented in
Table 1. There were no significant
differences in the genotype distribution between the two control groups
(ie, non-HIT patients with thrombocytopenia or thrombosis and healthy
blood donors; P = .45). However, in HIT patients, the
FcRIIa-R/R131 genotype was overrepresented and the FcRIIa-H/H131
genotype was underrepresented when compared with non-HIT control
patients (P < .001) and with healthy blood donors (P = .024). Approximately 50% of subjects in all three groups were
FcRIIa-R/H131 heterozygous.
Correlation between FcRIIa-R-H131 genotypes and manifestation of
TECs.
In the subanalysis of 122 well-characterized patients, 68 patients
(56%) developed TECs during heparin administration and 54 patients
(44%) presented with isolated thrombocytopenia. In HIT patients who
developed a TEC during heparin treatment, the FcRIIa-R/R131 genotype
was overrepresented and the FcRIIa-R/H131 and FcRIIa-H/H
genotypes were underrepresented compared with HIT patients presenting
with thrombocytopenia only; (P = .036; Table 2).
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Table 2.
Distribution of FcRIIa Genotypes and Allele
Frequencies in a Subanalysis of 122 HIT Patients With
Thrombocytopenia Only or With Thromboembolic Events During Heparin
Administration
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DISCUSSION |
HIT antibodies are known to interact with platelets via the FcRIIa.
The FcRIIa carries a polymorphic site at position 131 (R-H) that
affects its capacity to interact with immune complexes. In our
investigation, the FcRIIa-R/R131 genotype was overrepresented and
FcRIIa-H/H131 was underrepresented in HIT patients compared with
non-HIT patients and healthy blood donors. Furthermore, a subanalysis
with well-characterized HIT patients indicated a correlation between
genotype and clinical manifestations of HIT. We found a significantly
higher frequency of FcRIIa-R/R131 in HIT patients who developed TECs
than in patients presenting with isolated thrombocytopenia (P = .036; Table 2).
Four previous studies of the relationship between the FcRIIa-R-H131
polymorphism and the development of HIT have been reported (Table 3).21-24 In three of
these studies, FcRIIa-H/H131 was found to be overrepresented in HIT
patients.21,22,24 In the remaining study, no differences in
the distribution of FcRIIa-R-H131 genotypes were
detected.23 It may be that the discrepancy between FcRIIa genotype distribution in the present study and that of earlier studies is due to the inclusion of a different proportion of
HIT patients with TECs. In three of the earlier studies, no data are
given regarding the percentage of patients who developed HIT-related
thrombocytopenia or HIT-related TECs.21,22,24 However, in
the remaining study,23 23 of 36 patients had TECs and no
significant differences in the FcRIIa genotype distribution were
found. The disparity in patient sample sizes between our study and
earlier studies might also contribute to differences in results; ie,
our study includes many HIT patients relative to the small number of
HIT patients included in previous trials.
It is possible that the differing distributions of FcRIIa allotypes
in HIT patients reflect normal variations in the FcRIIa gene
frequency, which are found not only among populations of different
ethnic origins,27,32 but also within the Caucasian population, where the gene distribution ranges from 18% to 32% for
FcRIIa-R/R131 and from 19% to 36% for
FcRIIa-H/H131.22,33 However, this variability is not
likely to affect our findings, because all of our patients and controls
originate from the same population.
Our results lead us to speculate that, in HIT patients with the
FcRIIa-H/H131 allotype, uptake of antibody-coated platelets and
PF4/heparin antibody complexes is enhanced. Thus, these patients are
more likely to present with thrombocytopenia; whereas, in patients with
the FcRIIa-R/R131 allotype, the immune complexes are less
efficiently removed from the circulation and might, therefore, cause
prolonged immune-complex-dependent activation of platelets and
endothelial cells, leading to TECs. Indeed, there is increasing evidence that the FcRIIa-R131 allele is a risk factor for the manifestation of immune-complex-mediated diseases.34 The
impaired phagocytosis of immune complexes seen in patients with
systemic lupus erythematosus (SLE) can be correlated to the presence of the R131 allele.35 SLE patients with the FcRIIa-R/R131
allotype who remove the circulating immune complexes less efficiently
develop lupus nephritis at a higher rate than do patients with the
FcRIIa-H/H131 allotype.36,37 Furthermore, the FcRIIa
polymorphism has an impact on the susceptibility to bacterial
infections. Phagocytosis of IgG2-opsonized bacteria is less
effective in individuals with the FcRIIa-R/R131
allotype,38 and these patients exhibit a higher
susceptibility to infections by encapsulated bacteria than patients
with the FcRIIa-H/H131 allotype.39,40 Accordingly, a low
incidence of infections with encapsulated bacteria has been noted in
the Japanese population,41 where the FcRIIa-H/H131 allotype predominates.27,32 It is interesting to note that reports of HIT in Japanese patients are also very rare.
Because individuals with FcRIIa-H/H131 effectively clear immune
complexes, this allotype might be regarded as a protective factor
against HIT-related thrombosis. Although there is evidence that
platelets expressing the FcRIIa-R/R131 phenotype interact more
strongly with HIT antibodies in vitro than platelets with the
FcRIIa-H/H131 phenotype do,22 this need not contradict our hypothesis. Functional in vitro assays with isolated platelets cannot show interactions among cells of the reticulo-endothelial system, cells from the immune system, and platelets and cannot demonstrate the capacity to remove platelets and immune complexes from
the circulation.
In two of the previous studies, the IgG subclass of the HIT antibodies
was assessed.23,24 Both studies reported that the majority
of HIT antibodies belonged to the IgG1 subclass. The FcRIIa polymorphism is primarily responsible for binding differences in antibodies of the IgG2 and IgG3
subclasses20; however, this polymorphism also seems to
influence the interaction with immune complexes of the IgG1
subclass.24
Like the present study, all four of the earlier studies used functional
assays to determine the presence or absence of HIT antibodies. We chose
this diagnostic technique because functional tests are based on the
activation of platelets via the FcRIIa receptor, and because we were
studying FcRIIa polymorphism, only patients with antibodies that
reacted with FcRIIa were of interest. Because the HIPA test is a
functional assay that has been shown to have similar sensitivity and
specificity as compared with the 14C-serotonin release
assay,26 we do not see a major discrepancy to the
laboratory methods reported in previous studies on FcRIIa-R-H131 polymorphism in HIT patients. However, it is possible that HIT patients
not identified in this study might have been identified using a
PF4/heparin enzyme-linked immunosorbent assay (ELISA).
Together, our findings and those of other published studies suggest
that, although the polymorphism may not be the major risk factor for
clinical manifestation of HIT, once HIT develops, patients with the
FcRIIa-R/R131 genotype might be at a higher risk of developing new
TECs.
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FOOTNOTES |
Submitted October 15, 1997;
accepted April 28, 1998.
Supported by the Deutsche Forschungsgemeinschaft Gr 1096/2-2.
Address reprint requests to Andreas Greinacher, MD, Department of
Immunology and Transfusion Medicine, Ernst-Moritz-Arndt-University, Sauerbruchstr., D-17487 Greifswald, Germany; e-mail:
greinach{at}rz.uni-greifswald.de.
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 |
The technical assistance of C. Blumentritt and A. Raether is highly
appreciated, and the pre-PCR work by Dr K. Olbrich is gratefully
acknowledged. We thank S. Owens for editorial help with the
language.
| |
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Abstract
Introduction
Abstract