HEMATOPATHOLOGY AND COAGULATION MEDICINE
Original Article
The Rapid Differentiation of Type lib
von Willebrand's Disease from
Platelet-Type (Pseudo-) von Willebrand's
Disease by the "Neutral" Monoclonal
Antibody Binding Assay
JOHN PAUL SCOTT, M.D., 12 AND ROBERT R. MONTGOMERY, M.D.1
The differentiation of type lib von Willebrand's disease from
other variants of von Willebrand's disease, especially platelettype (pseudo-) von Willebrand's disease, poses a significant clinical problem because, although they are similar in the clinical
and diagnostic laboratory settings, the therapy of type lib von
Willebrand's disease is different from the therapy of platelettype von Willebrand's disease. This discrimination has required
cumbersome assays using fresh platelet-rich plasma that often
yielded equivocal results. Because it was shown by other researchers that type lib von Willebrand factor binds to normal
platelets with increased avidity at low concentrations of ristocetin,
it was reasoned that von Willebrand factor from patients with
type lib von Willebrand's disease would also bind to formalinfixed washed platelets at low concentrations of ristocetin. Using
the radiolabeled "neutral" monoclonal antibody AVW1 to label
plasma von Willebrand factor, the binding of von Willebrand
factor to formalin-fixed washed platelets was studied as a function
of ristocetin concentration. These studies demonstrated that the
I25
I-AVW1 von Willebrand factor from 13 patients with type
lib von Willebrand's disease binds to formalin-fixed washed
platelets at significantly lower concentrations of ristocetin than
plasma von Willebrand factor from 18 normal individuals, 3 patients with platelet-type von Willebrand's disease and 8 patients
with other variant forms of von Willebrand's disease. This radiolabeled "neutral" monoclonal antibody technique provides a
rapid, simple method for the differentiation on frozen plasma
samples of type lib von Willebrand's disease from platelet-type
and other variants of von Willebrand's disease. (Key words: von
Willebrand's disease; Type lib; Platelet-type; AVW1 factor;
"Neutral" monoclonal antibody; Differentiation) Am J Clin Pathol 1991;96:723-728
brand's disease is caused by an abnormality of the platelet
membrane that leads to increased affinity of the membrane for structurally normal high-molecular-weight
multimers of vWf.7"9 The differentiation between type lib
and platelet-type von Willebrand's disease previously required assays using fresh platelet-rich plasma, which often
failed to distinguish clearly between the two entities. Using the radiolabeled "neutral" monoclonal antibody
AVW1, 1 0 " we compared the binding of plasma vWf from
patients with type lib von Willebrand's disease with the
binding of similarly labeled plasma vWf from normal individuals, from patients with platelet-type von Willebrand's disease, and from patients with other variants of
From 'The Blood Center of Southeastern Wisconsin and the
department ofPathology, The Medical College of Wisconsin. Milwaukee.
von Willebrand's disease as a function of ristocetin conWisconsin.
centration.
Received January 29, 1991; received revised manuscript and accepted
for publication May 20, 1991.
MATERIALS AND METHODS
Supported in part by grants 88-GA-27 and 89-GA-30 from the American Heart Association, Wisconsin affiliate, by a grant from the Research
Foundation of The Blood Center of Southeastern Wisconsin, and by Patient Population
grants R01-HL33721-06 and P01-H244612-01 from the National Institutes of Health.
Patients with von Willebrand's disease were studied in
Address reprint requests to Dr. Scott: The Blood Center of Southeastern
Wisconsin, 1701 W. Wisconsin Avenue, Milwaukee, Wisconsin 53233. the Hemostasis Reference Laboratory of The Blood Center
von Willebrand factor (vWf) is a plasma and platelet protein that functions as an adhesive protein, linking damaged subendothelium to the platelet glycoprotein Ib-IX
complex, and also as the carrier protein for factor VIII.1,2
The clinical syndrome called von Willebrand's disease
results from quantitative or qualitative abnormalities of
vWf.3 In type lib von Willebrand's disease, a structural
abnormality of the vWf leads to increased binding of the
high-molecular-weight multimers of vWf to a structurally
normal platelet membrane.3"6 Platelet-type von Wille-
723
724
HEMATOPATHOLOGY AND COAGULATION MEDICINE
Article
of Southeastern Wisconsin using techniques that have
been described previously.12"16 Plasma from each patient
was assayed for vWf antigen, vWf ristocetin co-factor activity, multimeric analysis of vWf using sodium dodecyl
sulfate-0.65 agarose gel electrophoresis, and factor VIII
coagulant activity. When possible, platelet-rich plasma
also was studied to detect aggregation by low concentrations of ristocetin (0.3-0.6 mg/mL) for each patient with
abnormal vWf multimers, i.e., loss of the high-molecularweight multimers of vWf. Platelet-rich plasma that demonstrated increased sensitivity to low-dose ristocetin also
was studied to determine whether normal plasma or cryoprecipitate would induce aggregation of patient plateletrich plasma. Frozen plasma samples sent to the Hemostasis Reference Laboratory of The Blood Center of
Southeastern Wisconsin also were included in this study
when data were available regarding the vWf antigen, vWf
ristocetin co-factor activity, factor VIII coagulant activity,
vWf multimeric analysis, and ristocetin-induced platelet
aggregation of platelet-rich plasma. Four well-characterized plasma samples were supplied by Dr. Zavierio Ruggeri and one sample from a patient with platelet-type von
Willebrand disease was supplied by Dr. Jonathan Miller.
Reagents
Ristocetin was purchased from H Lundbeck A/S (Copenhagen, Denmark) and reconstituted in 0.9% normal
saline at a concentration of 30 mg/mL. The monoclonal
antibody AVWl was developed in our laboratory. AVWl
binds to vWf and does not block the binding of vWf to
either thrombin-stimulated washed platelets or to formalin-fixed washed platelets in the presence of ristocetin
and therefore has been referred to as a "neutral" monoclonal antibody." AVWl binds to all multimers of vWf.10
Formalin-fixed washed platelets were prepared from type
0 + platelet concentrate, as previously described.12 Sodium l25Iodide was purchased from Amersham (Arlington
Heights, IL). Iodo-gen was obtained from Pierce Chemical
Corp. (Rockford, IL). Agarose was obtained from FMC
(Rockland, ME). Other reagents were purchased from
Sigma Chemical Company (St. Louis, MO).
Measurement of the Binding of Plasma
von Willebrand Factor to Formalin-Fixed
Washed Platelets
To study vWf in the plasma milieu, the "neutral"
monoclonal antibody AVWl was labeled first with
125
Iodine using Iodo-gen.17 To trace label plasma vWf, a
small volume of 125I-AVW1 was added to the test plasma
so a 60-ML sample possessed approximately 50,000
counts per minute. 10 Ristocetin stock solution (30 mg/
AJ.C.P. •
mL) was diluted sequentially at the time of each assay so
a 20-fiL sample, when added to the reaction mixture,
would yield a final ristocetin concentration of 0.15, 0.3,
0.6, and 1.2 mg/mL, respectively. To perform the assay,
a 20-jiL sample of each ristocetin solution was added to
400 /xL of formalin-fixed washed platelets, 300,000 per
microliter, and allowed to incubate for 30 minutes at room
temperature. A 60-jiL sample of 125I-AVW1-labeled
plasma was then added, mixed, and allowed to incubate
for 30 minutes at room temperature. The suspension was
then centrifuged at 10,000# for 10 minutes at room temperature in a Beckman Microfuge (Beckman Industries,
Palo Alto, CA). The supernatant was aspirated gently and
the radioactivity levels in the pellet and the supernatant
were counted separately. Each determination was duplicated. On the date of each determination, a ristocetin doseresponse curve for pooled normal plasma also was performed. To correct for the effect of variable activity of the
radiolabel, data were normalized by dividing the percentage of 125I-AVW1 vWf bound at each ristocetin concentration by the percentage of I25I-AVW1 vWF bound
of normal pooled plasma at 1.2 mg/mL ristocetin to yield
the percentage of maximal vWf bound. Severe von Willebrand's disease plasma was used as a control to demonstrate the specificity of the binding of 125I-AVW1 for
plasma vWf. Assays for plasma 125I-AVW1 vWf were performed in a blinded fashion by a research technologist on
numbered samples in a laboratory physically separate
from the Hemostasis Reference Laboratory.
RESULTS
As depicted in Figure 1, our studies demonstrate minimal platelet binding of plasma vWf from 18 normal individuals at ristocetin concentrations of 0.15 and 0.3 mg/
mL. Measurable binding of 125I-AVW1 normal plasma
vWf to formalin-fixed washed platelets begins at 0.6 mg/
mL ristocetin. We then examined the binding of l25 IAVW1-labeled plasma from seven patients with type lib
von Willebrand's disease diagnosed in our laboratory by
studies of fresh plasma and platelet-rich plasma. Each type
lib plasma demonstrated increased binding of the radiolabeled plasma vWf to formalin-fixed washed platelets at
0.15 and/or 0.3 mg/mL ristocetin when compared to normal plasma. At 0.15 mg/mL ristocetin, the plasma vWf
from two of seven patients with type lib von Willebrand's
disease had binding to fixed washed platelets that fell
within the range obtained from the 18 normal individuals.
However, at 0.30 mg/mL ristocetin, there was a striking
increase in the amount of labeled plasma vWf bound to
formalin-fixed washed platelets from the patients with type
lib von Willebrand's disease (range, 27-51% of maximal
vWf bound) when compared to the values obtained from
ember 1991
SCOTT AND MONTGOMERY
Differentiation of Type lib and Platelet-type von WiUebrand's Disease
A — A
Normal plasma
•
TYPE lib vWD
120
o
z
• — •
100-1
'
0.0
0.2
725
TypellbvWD
Platelet-type vWD
100
3
O
m
80
5
>
_i
<
?
60
X
<
5
40
<
20
, i
0.0
8
0.2
0.4
0.6
0.8
1.0
1.2
RISTOCETIN, mg/ml
120n
A — A Normal plasma
Type I vWD
• — •
Type lla vWD
TypellbvWD
A —
120'
o
z
0:4
(Mi
0.6
RISTOCETIN, mg/ml
1.0
1.2
A Normal plasma
* Shipped lib Plasma
100'
o
80'
CO
60'
<
40
5
X
20
<
s
,-A-A'
0.4
0.6
0.8
RISTOCETIN, mg/ml
0.0
1.2
0.2
0.4
0.6
0.8
1.0
1.2
RISTOCETIN, mg/ml
FIG. 1 (upper left). This figure depicts the mean binding of I25I-AVW1-labeled plasma von Willebrand factor from 18 healthy individuals to formalinfixed washed platelets in comparison to the binding of I251-AVW1 -labeled plasma von Willebrand factor from seven individuals shown in our
laboratory to have type lib von WiUebrand's disease. Error bars represent one standard deviation.
FlG. 2 (upper right). This figure contrasts the ristocetin-induced binding of ,25I-AVW1-labeled plasma von Willebrand factor from three patients
with platelet-type von WiUebrand's disease with the mean binding of labeled von Willebrand factor from seven patients with type lib von WiUebrand's
disease to formalin-fixed washed platelets as a dose response to ristocetin.
FIG. 3 (lower left). The ristocetin-induced binding of '"I-AVW1 -labeled plasma von Willebrand factor to formalin-fixed washed platelets from three
patients with type I and five patients with type Ha von WiUebrand's disease is shown and contrasted with the mean binding of plasma von Willebrand
factor from 18 normal donors and with the mean binding of labeled von Willebrand factor from seven individuals with type lib von WiUebrand's
disease.
FIG. 4 (lower right). The binding of l25 I-AVWl-labeled plasma von Willebrand factor to formalin-fixed washed platelets from five individuals found
to have type lib von WiUebrand's disease on frozen, shipped plasma samples as a function of ristocetin dose is depicted in this figure in comparison
to the mean binding of 18 healthy individuals.
the 18 normal volunteers (range, 2.9-8.6% of maximal
vWf bound). There was variation in the range of values
obtained from these patient plasma samples; however,
when stimulated with 0.3 mg/mL ristocetin, the mean
value for 125I-AVW1 vWf binding to formalin-fixed platelets of the seven type lib patients was six times the mean
value for the 18 normal individuals studied.
In sharp contrast, plasma from three patients with
platelet-type von WiUebrand's disease demonstrated low
or normal binding of plasma vWf to formalin-fixed
washed platelets. Data contrasting the findings from the
patients with type lib and platelet-type von WiUebrand's
disease are presented in Figure 2.
We also studied the plasma from eight patients with
Vol. 96 • No. 6
726
HEMATOPATHOLOGY AND COAGULATION MEDICINE
Article
other variants of von Willebrand's disease (three type I
and five type Ha). In none of these cases did we demonstrate increased binding of I25I-AVW1-labeled plasma
vWf at 0.15 or 0.3 mg/mL ristocetin. The data for the
patients with other variants of von Willebrand's disease
are contrasted with the mean values obtained for the patients with type lib von Willebrand's disease and with the
mean values for the control individuals in Figure 3. We
then studied the ability of this assay to diagnose type lib
von Willebrand's disease on shipped frozen specimens.
In five patients, the clinical diagnosis of type lib von
Willebrand's disease was confirmed on the basis of markedly increased binding of 125I-AVW1-labeled plasma vWf
at 0.15 or 0.3 mg/mL ristocetin, as depicted in Figure 4.
After the initial studies outlined in this report, we subsequently had the opportunity to analyze the binding of
I25
I-AVW1-labeled plasma vWf from 26 additional previously undiagnosed cases with untyped von Willebrand's
disease undergoing evaluation. The plasma from 11 of
these individuals had clearly increased binding of 125 lAVW1 vWf at 0.3 mg/mL ristocetin and the remaining
15 individuals had normal results. When reviewed, the
clinical history and other laboratory parameters were
compatible with a diagnosis of type lib von Willebrand's
disease in those patients with increased binding.
Previously we observed slightly increased aggregation
of platelet-rich plasma after the addition of cryoprecipitate
in the index case that triggered this investigation. Using
the "neutral" monoclonal binding assay, we demonstrated
that an increased amount of the patient's plasma vWf
bound to normal formalin-fixed washed platelets at 0.15
and 0.3 mg/mL. These results were confirmed by showing
that the patient's washed platelets bound normal amounts
of labeled vWf at low doses of ristocetin, whereas her labeled plasma vWf demonstrated increased binding to
normal washed platelets at low concentrations of ristocetin.
DISCUSSION
Although multiple variants of von Willebrand's disease have been described using sophisticated laboratory
techniques, the most challenging and clinically significant
differentiation is the identification of type lib von Willebrand's disease and differentiation of it from platelet-type
(pseudo-) von Willebrand's disease. In type lib von Willebrand's disease, the abnormal vWf molecule demonstrates
increased avidity toward platelet glycoprotein lb and triggers platelet aggregation.1819 The precise nature of the
molecular defect inducing this abnormality is unclear; although now that the purported sites on vWf that bind to
glycoprotein lb have been identified,20 it is probable that
A.J.C.P. •
there will be a rapid improvement in our understanding
of the structural changes that result in type lib von Willebrand's disease. Platelet-type von Willebrand's disease
results from an abnormality of the platelet membrane
glycoprotein Ib/IX complex, in which there is an increased
avidity of the platelet membrane for the high-molecularweight multimers of normal plasma vWf.21,22 The binding
area on glycoprotein lb for vWf recently was localized to
the amino terminus of glycoprotein Iba, 23 and investigations into the molecular structure of GPIb in patients
with platelet-type von Willebrand's disease probably will
clarify the fundamental nature of this disorder. The increased avidity of the platelet membrane for vWf triggers
platelet aggregation and selective removal of the highmolecular-weight multimers of vWf from the plasma.
In the clinical laboratory, the findings for type lib and
platelet-type von Willebrand's disease are similar in that
there is evidence of increased agglutination of plateletrich plasma by low doses (0.3-0.6 mg/mL) of ristocetin.
Multimeric analysis shows loss of the high-molecularweight multimers of plasma vWf. However, the plateletrich plasma of patients with platelet-type von Willebrand's
disease undergoes aggregation when exposed to high-molecular-weight multimers of vWf from normal plasma or
cryoprecipitate, whereas there is no aggregation when the
platelets from a patient with type lib von Willebrand's
disease are stimulated with normal plasma vWf. In our
laboratory, these studies sometimes have been equivocal
and they are inconvenient because they require fresh
platelet-rich plasma. Fresh washed platelets would be an
ideal tool to study the binding of the patient's vWf to the
platelet membrane; however, these also require fresh
platelet-rich plasma as a starting material, and washing
platelets is an onerous, labor-intensive procedure.
In light of our understanding of the pathophysiologic
difference of type lib von Willebrand's syndrome from
other variants of von Willebrand's syndrome, we reasoned
that the plasma vWf from these individuals with type lib
von Willebrand's disease would demonstrate increased
binding to formalin-fixed washed platelets at low doses
of ristocetin, whereas vWf from other variants of
von Willebrand's disease should not bind at low doses of
ristocetin. In our laboratory, we previously generated the
monoclonal antibody AVWI, which binds readily to all
multimeric species of vWf and does not inhibit the binding
of vWf to formalin-fixed or fresh washed platelets in the
presence of ristocetin. Previously we developed the "neutral" monoclonal antibody binding assay for the study of
vWf binding to platelets in the plasma milieu and now
used this technique to measure the binding of vWf from
patients with type lib von Willebrand's disease to formalin-fixed washed platelets in the presence of ristocetin.
1991
SCOTT AND MONTGOMERY
Differentiation of Type lib and Platelet-type von Willebrand's Disease
To confirm that AVWl-labeled vWf binds to glycoprotein
lb, we demonstrated that the ristocetin-induced binding
of I25I-AVW1 vWf from patients with type lib von Willebrand's disease is blocked by the monoclonal antibodies
AVW3 and AP-1 (data not presented). AVW3 binds to
vWf and inhibits ristocetin-induced platelet agglutination.
AP-1 binds to platelet GPIb and also inhibits ristocetininduced platelet agglutination.
When the "neutral" monoclonal antibody binding assay was applied to the study of clinical samples, the plasma
from 18 normal individuals demonstrated minimal binding of labeled vWf at 0.15 and 0.3 mg/mL ristocetin. The
plasma vWf from eight patients with type I or type Ha
von Willebrand's disease also demonstrated minimal
binding at these concentrations. More significantly, the
plasma from three patients with platelet-type (pseudo) von
Willebrand syndrome also demonstrated minimal binding
of plasma vWf to formalin-fixed washed platelets at 0.15
and 0.3 mg/mL ristocetin. In marked contrast, the plasma
vWf from seven local patients with type lib von Willebrand's disease and from five frozen, shipped samples
demonstrated increased binding of plasma vWf to formalin-fixed washed platelets at 0.15 and 0.3 mg/mL ristocetin, thus providing a ready differentiation of type lib
from other variant forms of von Willebrand's syndrome.
The difference in binding was particularly striking at 0.30
mg/mL ristocetin. The clinical history and laboratory data
from the patients whose shipped samples were analyzed
were compatible with a diagnosis of type lib von Willebrand's disease. In a subsequently studied larger series of
patients with von Willebrand's disease, the "neutral"
monoclonal antibody binding assay was again readily able
to differentiate lib from other variants of von Willebrand's
disease.
The differentiation of type lib from platelet-type von
Willebrand's disease is crucial because the therapy of these
variants is different. Patients with type lib von Willebrand's disease should be treated for bleeding episodes
with cryoprecipitate or a vWf concentrate containing the
high-molecular-weight multimersofvWf. l-desamino-8D-arginine vasopressin (DDAVP) is contraindicated in
patients with both type lib and platelet-type von Willebrand's disease because it frequently induces thrombocytopenia.24"26 In platelet-type von Willebrand's disease,
treatment for bleeding episodes requires the administration of platelet concentrates and not vWf because these
patients appear to synthesize a normal vWf molecule.
The "neutral" monoclonal antibody technique provides
rapid plasma assay for the differentiation of type lib from
other variant forms of von Willebrand's syndrome. This
is a marked improvement over currently available assays
because it does not require the use of platelet-rich plasma
727
but rather can be performed on frozen samples that can
be shipped to a central reference laboratory. In our laboratory, the "neutral" monoclonal antibody binding
technique yields reproducible results within 2 hours and
allows the rapid diagnosis and differentiation of type lib
von Willebrand's disease in situations requiring prompt
therapeutic intervention.
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