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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 8 (April 15), 1998:
pp. 2810-2817
Recurrent Arterial Thrombosis Linked to Autoimmune
Antibodies Enhancing von Willebrand Factor Binding to Platelets and
Inducing Fc RII Receptor-Mediated Platelet Activation
By
Marc F. Hoylaerts,
Chantal Thys,
Jef Arnout, and
Jos Vermylen
From the Center for Molecular and Vascular Biology, Katholieke
Universtiteit Leuven, Leuven, Belgium.
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ABSTRACT |
A patient with a history of recurrent late fetal loss associated
with multiple placental infarcts and cerebrovascular ischemia at the
age of 36, followed a year later by a myocardial infarction, was
referred for further investigation. Coronary angiography was normal.
Antinuclear factor, lupus anticoagulant, anticardiolipin antibodies,
and other thrombophilia parameters were negative, but there was
moderate hyperthyroidism with positive thyroid peroxidase antibodies.
Platelet numbers and von Willebrand factor (vWF) were normal. Her
platelets showed spontaneous aggregation that disappeared with aspirin
intake. However, aggregation still was induced by low levels of
ristocetin (0.3 to 0.5 mg/mL). The low-dose ristocetin aggregation in
patient platelet-rich plasma (PRP) was completely blocked by
neutralizing antiglycoprotein Ib (GPIb) and anti-vWF antibodies. The
monoclonal anti-Fc RII receptor antibody IV.3 inhibited partly, which
suggests that PRP aggregation by low-dose ristocetin was elicited by
vWF-immunoglobulin (Ig) complexes. Upon addition to washed human
platelets, with vWF (10 µg/mL), purified patient Igs dose-dependently
enhanced ristocetin (0.15 mg/mL)-induced aggregation between 0 and 500 µg/mL, an effect that disappeared again above 1 mg/mL. Aggregation
was dependent on the vWF concentration and was blocked by IV.3 or
neutralizing anti-GPIb or anti-vWF antibodies. The spontaneous
aggregation of normal platelets resuspended in patient plasma could be
inhibited totally by IV.3 and partially by neutralizing anti-GPIb or
anti-vWF antibodies. Perfusion with normal anticoagulated blood,
enriched with 10% of control or patient plasma, over surfaces coated
with vWF showed increased platelet adhesion and activation in the
presence of patient antibodies. Treatment of the patient with the
antithyroid drug thiamazol and temporary corticosteroids, aspirin, and
ticlopidine did not correct the platelet hypersensitivity to
ristocetin. These observations suggest that some autoantibodies to vWF
may both enhance vWF binding to platelets and cause platelet activation through binding to the FcRII receptor, and thereby may be
responsible for a new form of antibody-mediated thrombosis.
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INTRODUCTION |
RECURRENT FETAL LOSS and arterial
thrombotic events in a young nonatherosclerotic female suggest a
thrombophilic state.1 Several recent reports indeed link
fetal loss to congenital deficiencies of factor V Leiden2
or to hyperhomocysteinemia.3 Furthermore, fetal loss and
arterial thrombosis can be linked to circulating antibodies. The
prototype of this is the antiphospholipid syndrome.4-6 Antibody-mediated arterial thrombosis is also observed in the heparin-induced thrombocytopenia and thrombosis
syndrome.7-9 The pathogenetic mechanism of the latter has
recently been established. The antibodies bind to heparin-platelet
factor 4 complexes on the platelet surface, and subsequently activate
the platelet by interaction of their Fc portion with the platelet
FcRII receptor.10 Recent work has suggested that, also
in the antiphospholipid syndrome, antibody reacts with
phospholipid-binding proteins on minimally activated cells, and that
further cell activation leading to thrombosis is
Fc-dependent.11,12
In this report, we describe a young female with circulating autoimmune
antibodies. Although the existence of anti-von Willebrand factor (vWF)
antibodies could not be confirmed by direct evidence, these antibodies
appeared to react with vWF while maintaining normal vWF plasma levels.
Unexpectedly, this patient had no bleeding tendency, but on the
contrary had a history of thrombosis. Her platelet studies were
suggestive of increased sensitivity to vWF-mediated platelet
activation, a finding related to the patient's antibodies. This report
provides the first evidence for the existence of autoimmune anti-vWF
antibodies that are thrombogenic in humans. Since we found this
prothrombotic tendency to be associated with FcRII receptor-mediated
platelet activation, this case bears a strict similarity to the
aforementioned examples of antibody-mediated thrombosis, such as the
antiphospholipid syndrome and heparin-induced thrombocytopenia.
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MATERIALS AND METHODS |
Patient description.
A 37-year-old woman was referred to our hospital for further
investigation of a history of thrombosis. At the age of 22 and 23 years, both pregnancies of this patient resulted in intrauterine death
at 26 and 28 weeks, respectively, with evidence of fetal growth
retardation and pathologically documented multiple placental infarcts
on both occasions. At the age of 36, she developed an ischemic
cerebrovascular accident, confirmed by computer tomography and magnetic
resonance imaging. At the age of 37, she developed a myocardial
infarction; both arterial thrombotic episodes were preceded by flu-like
symptoms: moderate fever, arthralgia, and headache. Echocardiography
showed hypokinesia of the interventricular septum, but no valvular
abnormalities; she was not taking oral contraceptives. Angiography
performed 1 month after the myocardial infarction showed normal
coronary arteries. Antinuclear factor, lupus anticoagulant,
anticardiolipin antibodies, and other thrombophilia parameters
(antithrombin, protein C, protein S, activated protein C resistance,
and hyperhomocysteinemia) were negative. The patient had moderate
hyperthyroidism, with positive thyroid peroxidase antibodies. Her
platelet-rich plasma (PRP) manifested spontaneous platelet aggregation,
which disappeared with aspirin intake; however, aggregation with
low-dose ristocetin (0.5 mg/mL) persisted (Table 1). Ristocetin cofactor activity was 100%
at the start of the observation, but decreased to 50% over less than 6 months. Factor VIII activity, on the contrary, remained normal
throughout the study (Table 1). Treatment with thiamazol (antithyroid
drug), temporary corticosteroids, aspirin, and ticlopidine failed to correct the hypersensitivity to ristocetin, but the patient is currently doing well.
Isolation procedures.
Cryoprecipitate was made from normal and patient plasma following
thawing overnight at 4°C. vWF was isolated from cryoprecipitate by
reprecipitation and bentonite-mediated fibrinogen
depletion, followed by gel filtration on a Sepharose 4B-CL column
(2.6 × 95 cm, Pharmacia, Uppsala, Sweden) as
described.13 On reducing sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the isolated
vWF showed a single band. Monoclonal antibodies were isolated from
ascites by protein A-chromatography. Human immunoglobulins (Igs) were
also purified by protein A-chromatography, taking care to collect
acid-eluted antibodies in 1 mmol/L Tris-HCl buffer, pH 8. To maximally
avoid Ig aggregation during antibody preparation, patient Igs were also
isolated via ion-exchange chromatography on a mono-Q Sepharose column,
equilibrated in 20 mmol/L Tris-HCl buffer, pH 8.5. Bound Igs were
eluted from this column via a linear NaCl gradient (0 to 250 mmol/L),
pooled, dialyzed against phosphate-buffered saline (PBS), and
concentrated via reverse osmosis against polyethylene glycol with an
average Mr of 30,000.
Platelet aggregation studies.
Platelet aggregations were performed on a dual-channel
Chrono-Log Aggregometer (Chronolog Corp, Havertown, PA).
PRP was prepared from normal or patient blood collected on 0.38 mol/L
citrate, by centrifugation at 150g for 10 minutes. Washed
platelets were prepared by collecting blood from normal donors on 10%
(vol/vol) of acid-citrate-dextrose (ACD) solution pH 6.2 (93 mmol/L
Na3-citrate, 7 mmol/L citric acid, 0.14 mmol/L dextrose)
and by centrifugation at 150g for 20 minutes. The PRP was then
mixed with an equal volume of ACD and platelets were pelleted by
centrifugation at 1,000g for 10 minutes. Platelets were
carefully resuspended in Hanks balanced salt solution (HBSS), pH 7.4 (0.44 mmol/L KH2PO4, 0.34 mmol/L
Na2HPO4, 136 mmol/L NaCl, 5.4 mmol/L KCl, and
5.6 mmol/L D-glucose), plus a one-third volume fraction of
ACD and platelets were washed again via centrifugation at
1,000g for 10 minutes. Platelets were then resuspended either
in plasma or plasma mixtures, or in HBSS and mixed with vWF (0 to 10 µg/mL) or cryoprecipitate, in which the vWF concentration was
adjusted to 10 µg/mL, and aggregation was induced by ristocetin.
Neutralizing murine monoclonal antibodies, such as the anti-vWF
monoclonal antibody AJvW-2 (Ajinomoto, Yokohama, Japan) up to
20 µg/mL,14 an antiglycoprotein Ib (GPIb) monoclonal antibody (G19H10, raised in our laboratory) up to 20 µg/mL, or the
anti-FcRII receptor monoclonal antibody IV.3 (Medarex,
Annandale, NJ) up to 10 µg/mL, were added to PRP or to washed
platelets before induction of aggregation by ristocetin. Spontaneous
platelet aggregation was studied in an ELVI 840 dual-channel
aggregometer (Pabisch, Brussels, Belgium) in citrated PRP at 1,000 rpm.
Following resuspension of washed normal platelets in patient plasma,
the spontaneous aggregation was investigated at 1,500 rpm in the
presence of the indicated concentrations of G19H10, AJvW-2, and IV.3.
Flow cytometry analysis of Ig binding.
Washed normal platelets were resuspended in either normal plasma or
patient plasma and stirred in an aggregometer at 1,000 rpm in the
absence or the presence of 0.5 mg/mL ristocetin. After 2 minutes, 5 µL PRP was removed and fixed by the addition of 45 µL 1% formol in
PBS for 1 hour, following which the remaining formaldehyde was
neutralized by the addition of 50 µL 1-mol/L Tris-HCl buffer, pH 8. Platelet-associated human Igs were then revealed via addition of 400 µL of a 100-fold diluted fluorescein isothiocyanate (FITC)-labeled
goat antihuman Ig antiserum (Becton-Dickinson, San José, CA) and
acquisition on a FACS Calibur flow cytometer.
Shear stress-dependent perfusion studies.
Blood (blood group O) anticoagulated with low-Mr heparin (2 IU/mL) was recirculated at 1,300 s 1 through
parallel-plate flow chambers with a height of 0.4 mm, in the presence
of added normal or patient plasma (5% to 10%). Perfusions were
performed for 2 minutes over thermonox coverslips coated overnight with
calf-skin collagen (1 mg/mL in 50 mmol/L acetic acid) or vWF (25 µg/mL in PBS). Coverslips were removed from the perfusion chamber,
rinsed in HEPES buffer, and fixed with 1% glutaraldehyde in PBS before
staining with May-Grünwald-Giemsa as described
previously.15 To minimize nonspecific platelet activation
due to the recirculation of the anticoagulated blood, monoperfusion
experiments were also performed through rectangular glass capillaries
(0.2 × 2 mm internal diameters; Vitro Dynamics, Rockaway, NJ) at a
shear rate equal to 1,000 s1. These ethanol-rinsed glass
capillaries had been coated overnight at 4°C with calf-skin collagen
or vWF as described earlier. The glass surface was blocked via
perfusion with 1% bovine serum albumin dissolved in HEPES-buffered
saline (HBS: 10 mmol/L HEPES, 150 mmol/L NaCl, pH 7.5) for 10 minutes.
Blood was then aspirated through these capillaries by means of a
Harvard Apparatus (Holliston, MA) 22 pump at a constant reverse-flow
rate of 0.75 mL/min for 2 minutes. The capillaries were then perfused
with HBS for 10 minutes and stained using May-Grünwald (10-minute
perfusion)-Giemsa (1-hour perfusion), all by aspiration through the
capillaries. Following final rinsing with HBS and fixation with
methanol (10-minute perfusion), the capillaries were dried.
Quantitation of platelet adhesion was done by image analysis of the
surface covered using en face light microscopy (Dialux 20 EB, E; Leitz,
Wetzlar, Germany) at a low magnification (×40) and the TCL-Image
image-processing software (Multihouse TSI, Amsterdam, The Netherlands).
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RESULTS |
Spontaneous and ristocetin-induced patient platelet aggregation.
Before treatment, stirring of the patient's PRP induced spontaneous
platelet aggregation, with this aggregation reaching a plateau after 40 minutes (Fig 1). Platelet activation by
stirring responded well to high-dose aspirin: 4 days after the daily
intake of 160 mg, aggregation was reduced, and had disappeared almost entirely after 4 additional days of 1,000 mg/d (Fig 1). At the start of
the study, patient PRP aggregations were normal when induced by ADP,
collagen, the thromboxane A2 analog U46619, and arachidonic
acid (AA). In view of the treatment with aspirin, the AA-induced
platelet aggregation was abolished entirely after 7 weeks. Initially, a
full-blown aggregation was observed with ristocetin concentrations as
low as 0.5 mg/mL, but was somewhat tempered following aspirin intake
for 7 weeks, albeit the slope of the initial agglutination step
remained unaltered (Table 1 and Fig 2A).The low-dose ristocetin aggregation remained strong throughout the
entire follow-up period (Table 1), even at concentrations of ristocetin
as low as 0.3 mg/mL (Fig 2B) and despite the fact that the ristocetin
cofactor activity, ie, vWF antigen levels, decreased to
approximately 50% at the end of the observation period (Table
1).
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| Fig 1.
Spontaneous platelet aggregation in patient PRP. Stirring
induced aggregation, initially (1), after intake for 4 days of 160 mg
aspirin per day (2), and after an additional 4 days of 1,000 mg aspirin
per day (3).
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| Fig 2.
Ristocetin-induced patient platelet aggregation. (A)
Aggregation of patient's PRP by 0.5 mg/mL ristocetin before (1) and
after daily intake of 1,000 mg aspirin for 7 weeks (2). (B) Intensity of aggregation response as a function of ristocetin concentration during aggregation of normal ( ) and patient ( ) PRP, 7 weeks after
daily intake of 1,000 mg aspirin.
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FcRII participation in the aggregation process.
Platelet aggregation induced by 0.3 and 0.5 mg/mL ristocetin was
completely blocked by the neutralizing anti-vWF antibody AJvW-2 and by
the neutralizing anti-GPIb antibody G19H10 (Fig 3), with both monoclonal antibodies being
specific inhibitors of the vWF-mediated platelet activation. The
anti-FcRII receptor antibody IV.3, although it had no effect on the
ristocetin-induced agglutination of normal PRP, surprisingly reduced
the weak aggregation induced by 0.3 mg/mL ristocetin and had a partial
effect on the stronger aggregation observed with 0.5 mg/mL ristocetin
(Fig 3B). Aggregations of patient PRP induced by 0.5 mg/mL ristocetin
and performed in the presence of the GPIIb/IIIa antagonist G4120 also reduced the second wave of the aggregation curve, confirming that the
addition of 0.5 mg/mL ristocetin to PRP induced both an initial agglutination (first wave), followed by a subsequent platelet activation step and platelet aggregation (second wave).
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| Fig 3.
Inhibition of ristocetin-induced patient PRP aggregation.
Amplitude of aggregation of PRP by (A) 0.3 and (B) 0.5 mg/mL ristocetin in the absence ("none") and in the presence of 10 µg/mL of the inhibitory anti-vWF monoclonal antibody AJvW-2, of 14 µg/mL of the
inhibitory anti-GPIb monoclonal antibody G19H10, and of 10 µg/mL of
the anti-FcRII receptor monoclonal antibody IV.3.
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Stimulation of normal platelet aggregation by patient plasma.
To exclude that the patient's platelets were responsible for the
observed sensitivity to vWF, washed platelets were isolated from normal
donors and resuspended in either normal or patient plasma. Ristocetin
induced a dose-dependent agglutination/aggregation reaction in the
reconstituted normal PRP (Fig 4A),comparable to that in normal PRP. In the reconstituted mixture of
platelets and patient plasma, a normal response to AA was noted, but
the increased sensitivity toward ristocetin could be confirmed. The partial inhibition by IV.3 of the platelet aggregation induced by 0.5 mg/mL of ristocetin could also be reproduced using normal platelets in
combination with patient plasma (Fig 4B). When washed platelets were
resuspended in mixtures of normal and patient plasma, maximal
aggregation by 0.5 mg/mL of ristocetin was observed with 25% to 50%
of patient plasma (Fig 5).
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| Fig 4.
Resuspension of normal platelets in patient plasma.
Aggregation of washed normal platelets resuspended in (A) normal and
(B) patient plasma with 1 mmol/L AA (1) or with 1.2 (2), 0.9 (3), or
0.5 (4) mg/mL ristocetin, and inhibition by 10 µg/mL IV.3 of the
aggregation induced in patient plasma by 0.5 mg/mL ristocetin (5).
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| Fig 5.
Platelet aggregation as a function of proportion of
patient plasma. Amplitude for the ristocetin (0.5 mg/mL)-induced
aggregations of normal washed platelets resuspended in normal plasma
containing the indicated proportions of patient plasma (0  1).
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Platelet aggregations with patient Igs.
To evaluate the involvement of patient Igs, ristocetin-induced
aggregation was studied using washed platelets, purified normal vWF,
and protein A-purified Igs isolated from patient or normal plasma. At
0.15 mg/mL ristocetin, no agglutination developed, but the addition of
purified patient Igs induced a dose-dependent increase of platelet
activation (not shown). Optimal stimulation was observed between 0.25 and 1 mg/mL of added antibody, but at 2 mg/mL of antibody, virtually no
aggregation was observed, suggestive of a bell-shaped dose-response
curve. Control aggregations performed in the presence of Igs isolated
from different batches of normal plasma would yield considerably weaker
stimulation, although occasionally some aggregation was found,
indicative of weak Ig aggregate-induced platelet activation. However,
with vWF concentrations less than 5 µg/mL, aggregations gradually
became weaker.
In an effort to avoid Ig aggregate-induced platelet activation, patient
Igs were isolated via ion-exchange chromatography and platelet
aggregation studies were performed with these antibodies. As shown in
Fig 6, at 0.5 mg/mL, these antibodies
enhanced the weak aggregation of washed platelets induced by 0.17 mg/mL
ristocetin in the presence of vWF. Furthermore, this aggregation could
be inhibited almost completely by the anti-vWF monoclonal antibody AJvW-2, the anti-GPIb monoclonal antibody G19H10, and the anti-FcRII receptor antibody IV.3. The double involvement of vWF and Igs in the
aggregation process was further confirmed by the lack of aggregation in
the absence of ristocetin or of vWF. The low residual aggregation
observed after 10 minutes in the absence of ristocetin is compatible
with the formation of vWF-Ig complexes with some affinity for the
platelet Fc receptor. However, the lack of platelet aggregation in the
absence of vWF excludes Ig aggregates as a cause of platelet
activation. When the preincubation time of platelets, vWF, and antibody
was prolonged from 0 to 10 minutes, a clear enhancement of the initial
platelet response to ristocetin was observed (not shown), also
compatible with immune complex formation between vWF and Igs.
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| Fig 6.
Immune complex formation as the trigger for aggregation.
Amplitude of the aggregation at minute 10 of washed normal platelets in
the absence ( ) or presence (+) of ristocetin (0.17 mg/mL), vWF (10 µg/mL), ion-exchange chromatography-isolated patient Igs (0.5 mg/mL), the anti-vWF antibody AJvW-2 (20 µg/mL), the anti-GPIb antibody G19H10 (20 µg/mL), or the anti-FcRII antibody IV.3 (10 µg/mL), as indicated. Platelet mixtures were preincubated for 10 minutes before initiation of aggregation by ristocetin and/or stirring.
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When normal platelets were resuspended in patient plasma and stirred at
1,500 rpm, a mild spontaneous aggregation could be induced, with
approximately 40% aggregation after 45 minutes. In the presence of
IV.3, this aggregation was blocked entirely, but the slope of the
aggregation was also reduced by 62% by the anti-GPIb antibody G19H10
and by 78% by the anti-vWF antibody AJvW-2. These experiments confirm
that the weak aggregation observed after 10 minutes, shown in Fig 6 for
mixtures of vWF and patient Igs, is caused by the simultaneous binding
to platelets of vWF and Igs, even in the absence of further mediators
of vWF binding. The presence on the platelet membrane surface of such
complexes was further investigated via flow cytometry. Stirring washed
platelets resuspended in patient plasma for 2 minutes reduced single
platelet numbers to 27% of initial values. In the presence of 0.5 mg/mL ristocetin, this number dropped further to 19%. Resuspension in normal plasma and stirring, on the contrary, reduced single platelet numbers only to 52% and 53%, respectively, in agreement with a weaker
platelet activation. In addition, whereas the median antihuman Ig
antibody-bound fluorescence for the remaining single platelets in
normal plasma did not change in the absence (relative value, 28) and
the presence (relative value, 26) of ristocetin, the median fluorescence for the single platelets resuspended in patient plasma (27 in the absence of ristocetin) increased to 36 following stirring in the
presence of ristocetin. These data indicate that the patient vWF
binding induced by 0.5 mg/mL ristocetin is accompanied by binding of
patient Igs to the platelet.
Cryoprecipitate-dependent aggregations.
Cryoprecipitate was made from normal and patient plasma and adjusted to
10 µg vWF/mL. The cryoprecipitate from the patient was much more
capable of supporting low-dose ristocetin-induced aggregation of washed
platelets than normal cryoprecipitate, and this effect was inhibited by
IV.3. Because both the patient vWF antigen levels and ristocetin
cofactor activity were normal at the start of the study, these findings
suggested that the patient cryoprecipitate was enriched in vWF-IgG
immune complexes, supporting aggregation.
Increase of shear stress-induced platelet aggregation by patient
antibodies.
Reperfusion experiments under shear stress, more representative of
physiologic vWF-dependent platelet activation than ristocetin, were
performed in low-Mr heparin anticoagulated blood with
normal or patient plasma added to 5% and 10% of the volume. There was increased platelet adhesion to a collagen surface in the presence of
5% but not 10% patient plasma; platelet adhesion to vWF was enhanced
in the presence of 10%, but not yet at 5% patient plasma (Table
2).
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Table 2.
Platelet Surface Coverage (±SD) Following
Reperfusion for 2 Minutes of Normal Low-Mr Heparin
Anticoagulated Blood, Supplemented With Normal or Patient Plasma, Over
Coverslips Coated With Calf-Skin Collagen or Human vWF
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To investigate these observations further, monoperfusions of
anticoagulated blood were performed at 1,000 s1 through
glass capillaries coated with collagen or vWF. These monoperfusions at
moderately high shear forces through rectangular capillaries coated
with calf-skin collagen resulted in the adhesion of single platelets
and of small to moderately sized platelet aggregates (Fig
7a) that contained up to 20 platelets, as evaluated by
area analysis. In this setup, substitution of 5% normal plasma by 5%
patient plasma resulted in the formation of larger platelet aggregates
(Fig 7b), without increase in the degree of adhesion (normal plasma,
18.3% ± 1.8%; patient plasma, 20% ± 5.1%). Although monoperfusions performed over vWF yielded a less homogeneous platelet adhesion, substitution of 10% normal plasma by patient plasma resulted
in an increase of the degree of platelet adhesion from 7.9% ± 0.6%
to 14.1% ± 2.6% (Fig 7c and d, P < .05), but no
significant differences in the size of the aggregates could be detected
(~50% of platelets adhered either as single platelets or as very
small aggregates consisting of two to three platelets only).
Substitution of 20% normal plasma with patient plasma no longer showed
any difference either in the degree of adhesion or in the size of the
aggregates formed.
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| Fig 7.
Platelet adhesion during perfusion over collagen and vWF.
Platelet adherence after perfusion for 2 minutes of anticoagulated normal blood through glass capillaries coated with collagen (a, b) or
vWF (c, d), in the absence (a, c) or presence of 5% (b) or 10% (d)
patient plasma.
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DISCUSSION |
The patient reported in this study was referred to our hospital
following a history of recurrent fetal loss and arterial thrombosis. The usual risk factors associated with fetal loss or thrombosis such as
activated protein C resistance, antiphospholipid antibodies, and
hyperhomocystinemia could all be excluded. However, the patient's platelets aggregated spontaneously on stirring; spontaneous aggregation has previously been linked to thrombocythemia,16 which was
not present in this patient; it disappeared with aspirin, although initially only when given at high doses.
Platelet aggregation by low ristocetin concentrations persisted
throughout aspirin treatment; mutations in vWF enhancing the binding of
vWF to its receptor on the platelet, the GPIb/IX/V complex, and giving
rise to von Willebrand disease (vWD) type IIb,17,18 could
be excluded as a basis for the observed phenomenon, because the patient
had no bleeding tendency and had a normal ristocetin cofactor, as well
as a normal platelet count; furthermore, the enhanced aggregation was
reproduced in a purified system that contained normal, not patient,
vWF. Alternatively, mutations in GPIb itself, equally giving rise to
enhanced vWF binding and a platelet type vWD,19 were also
excluded, since low-dose ristocetin-induced aggregation could be
reproduced with normal platelets when resuspended in patient plasma.
Evidence is provided that vWF-antibody complexes may explain the
observed low-dose ristocetin-induced aggregations. Aggregations performed in patient plasma could be inhibited completely by
neutralizing antibodies reactive with vWF or with GPIb and could be
blocked partially by the monoclonal antibody IV.3. By binding to the
platelet FcRII receptor, this monoclonal antibody prevents
Fc-mediated interactions and signal transduction leading to platelet
activation.20 The finding that IV.3 primarily blocked the
second phase of the low-dose ristocetin-induced aggregation curves is
compatible with this interpretation. Normal plasma enhanced the
response to patient plasma, according to a bell-shaped curve, a finding
that is compatible with optimization of immune complex formation as a
consequence of plasma mixing. The data with patient cryoprecipitate and
normal washed platelets further support the concept of vWF-antibody
complexes being responsible for enhanced response to low-dose
ristocetin.
The interpretation of results obtained with Igs isolated via acid
elution was somewhat more difficult, since the isolated Igs in some
batches tended to aggregate and promote aggregation by themselves.
However, ion-exchange isolated patient antibodies supported the
aggregation of washed platelets only in the presence of vWF and low
concentrations of ristocetin. Furthermore, aggregations were dependent
on the concentration of added vWF and could almost completely be
inhibited by the vWF neutralizing antibody AJvW-2, the GPIb
neutralizing antibody G19H10, or the Fc receptor neutralizing antibody
IV.3.
It appears that the patient's autoimmune antibodies facilitate vWF
binding to platelets. This occurs to some extent even in the absence of
further mediators of vWF binding, a finding that may explain the
spontaneous aggregation also observed following resuspension of normal
platelets in patient plasma. The inhibition by IV.3 of the spontaneous
aggregation confirms the involvement of immune complexes; in addition,
the substantial inhibition by G19H10 and AJvW-2 confirms that
aggregation not only depends on the binding of vWF-Ig complexes to the
FcRII receptor, but that it involves vWF-GPIb-dependent
interactions. We have observed previously that a murine monoclonal
anti-vWF antibody is indeed capable of enhancing the binding of vWF to
its receptor on the platelet,21 a finding that may explain
why in the present study antibody binding to vWF facilitates vWF
binding to GPIb. Once the vWF-antibody complexes have become bound to
platelets, the Fc part of the Ig would then interact with the FcRII
receptor and activate the platelet, as outlined in Fig
8. Alternatively, antibody bound to vWF may
first interact with the Fc receptor, following which the binding of vWF
to GPIb will be facilitated, as evidenced by the faint and slow
aggregation of washed platelets observed in the absence of ristocetin.
Finally, as recently proposed,22 a quaternary complex is
formed between platelet receptors, vWF, and antibodies, stabilized by
multiple interactions, and in which platelet activation occurs
primarily via the Fc receptor (Fig 8). During the "spontaneous"
platelet activation, stirring in the presence of antibody alone would
then suffice to prompt binding of vWF-Ig complexes, followed by
subsequent antibody-mediated platelet activation, which can be
suppressed by aspirin.
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| Fig 8.
Representation of antibody mediated activation of
platelets via autoimmune antibodies against vWF: antibodies stabilize
vWF bound to GPIb on the platelet surface via Fc-mediated interactions and induce FcRII receptor-mediated platelet activation.
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The anti-vWF antibodies would have low affinity, which would not allow
the direct demonstration of immune complex formation by enzyme-linked
immunosorbent assay (ELISA), Western blotting, or surface plasmon
resonance interaction studies. This low affinity is compatible with the
aforementioned model in which both vWF and antibody need to be
stabilized via secondary interactions on platelet receptors to form a
more stable quaternary complex. This interpretation would support our
results obtained via flow cytometry, the only approach that enabled us
to show that increased vWF binding was associated with human Ig binding
to the platelet. The low affinity is compatible with the normal plasma
vWF levels found in the patient, at least at the beginning of the
follow-up period, and also would explain her higher platelet numbers in comparison to other situations of immune-mediated thrombocytopenia. Shortly after the onset of treatment, platelet numbers tend to increase, in parallel with an improved prevention of Fc-mediated platelet activation. These findings are compatible with a diminished platelet consumption following initiation of treatment. In view of the
continued sensitivity to low-dose ristocetin-induced aggregation and
the persistence of anti-vWF antibodies, higher platelet numbers in turn
may have an impact on the clearance of vWF via platelet-bound immune
complexes, explaining the small drop in vWF antigen levels and
ristocetin cofactor activity toward the end of the follow-up period.
Thrombosis would be facilitated by optimal proportions of antigen and
antibody, as was demonstrated by varying the antigen/antibody ratios
during the ristocetin-induced platelet aggregation studies. In this
regard, it is remarkable that both episodes of arterial thrombosis were
immediately preceded by flu-like symptoms, possibly suggesting
circulating immune complexes at that moment. At moderate shear stress,
the antibody could be expected to somewhat stimulate platelet adhesion
to vessel wall-exposed vWF, as was observed during the perfusion
experiments over collagen and vWF-coated surfaces. Furthermore, it
could also be expected to promote platelet thrombus growth. Within
limited dose ranges, this was observed.
These observations suggest the existence of a new type of
antibody-mediated thrombosis, characterized by the production of autoantibodies to vWF, in which the platelet FcRII receptor plays a
central role both by stabilizing platelet-bound vWF-Ig complexes and by
participating in platelet activation. This type of thrombosis bears a
high mechanistic similarity to other types of antibody-mediated thrombosis, such as heparin-induced thrombocytopenia and perhaps the
antiphospholipid syndrome.
| |
FOOTNOTES |
Submitted July 24, 1997;
accepted November 21, 1997.
Supported by Research Grant No. 3.0030.90 from the Belgian Fonds Voor
Wetenschappelük Onderzoek-Vlaanderen and by Interuniversitaire Attractiepool Grant No. P4/34.
Presented in abstract form at the Twenty-Sixth ISTH Congress, Florence,
Italy, June 1997 (Thromb Haemost p. 271, 1997 [abstr 1107]).
Address reprint requests to Marc F. Hoylaerts, PhD, Center for
Molecular and Vascular Biology, Katholieke Universtiteit Leuven, Campus
Gasthuisberg, O&N, Herestraat 49, B-3000 Leuven, Belgium.
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 M. Vanrusselt for performing the initial platelet aggregations
reported in this study, and I. Vreys for doing the perfusion studies.
J. Vermylen is holder of the "Dr J. Choay Chair in Haemostasis
Research."
| |
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Abstract
Introduction
Abstract