614 Blood. Vol 64, No 3 (September), 1984: pp 614-62 1 Studies of the Pathophysiology of Acquired von Willebrand’s Disease in Seven Patients With Lymphoproliferative Disorders or Benign Monoclonal Gammopathies By P.M. Mannucci, R. Lombardi, R. Bader, M.H. Horellou, G. Finazzi, C. Besana, J. Conard, and M. Samama In seven patients with acquired von Willebrand’s disease (AvWD) associated with lymphoproliferative disorders or benign monoclonal gammopathies. the platelet contents of von Willebrand factor antigen and ristocetin cofactor (vWF:Ag and vWF:RiCof, respectively) were normal. All the multimers of vWF:Ag could be seen in the 1.6% SDS-agarose gel electrophoresis patterns of plasma and platelet lysates. Infusion of 1 -deamino-8-D-arginine vaso- pressin (DDAVP) augmented plasma levels of vWF:Ag and vWF:RiCof of all patients and corrected prolonged bleeding times (BT). However. compared with patients with congen- ital vWD type I and comparable degrees of baseline abnor- A SYN DROM E RESEM BLING the congenital bleeding disorder, von Willebrand’s disease (vWD), sometimes occurs in patients without family history or previous symptoms of abnormal bleeding. The syndrome, usually called acquired von Wille- brand’s disease (AvWD), is strikingly similar to the congenital disease in terms of laboratory findings, being characterized by a prolonged bleeding time (BT) and low plasma levels of factor VIII-von Willebrand factor (FVIII-vWF) measurements.’ FVIII-vWF is a macromolecular complex of two related, but nonidenti- cal, proteins: factor VIII, which can be measured as coagulant activity ( FV I I I :C) or antigen ( FVI I I:CAg), and von Willebrand factor, measured either immuno- logically (vWF:Ag) or as ristocetin cofactor activity (vWF:RiCof).’ Our review of 36 cases reported in full in the literature indicates that AvWD occurs most often in association with autoimmune or lymphoproliferative disorders, with or without associated monoclonal gam- mopathies. Even though this association strongly mdi- cates that AvWD has an immunologic basis, there are cases in which the underlying disease (solid tumors, angiodysplasias, etc) has no obvious relationship to any From the Angelo Bianchi Bonomi Hemophilia and Thrombosis Centre and Third Institute ofClinical Medicine, Maggiore Hospital and University of Milano. Italy: the Central Hematology Labora- tory, Hotel Dieu Hospital. Paris; and the Institute of Medical Pathology, San Raffaele Hospital and University of Milano. Supported in part by a grant from Consiglio Nazionale delle Ricerche, Progetto Finalizzato ‘Ingegneria Genetica, “ Sottopro- getto Basi Molecolari delle Malattie Genetiche (No. 83.0/0.11.5/). Submitted Oct 5, 1983; accepted March 29, 1984. Address reprint requests to Dr P.M. Mannucci, Via Pace 9. Milano. Italy. (C) /984 by Grune & Stratton. Inc. 0006-497l/84/6403-0005$03.00/0 malities treated in the same way. vWF:Ag and vWF:RiCof were increased less and cleared more rapidly from plasma and the BT remained normal for a shorter period of time. These studies provide evidence that these AvWD patients have qualitatively normal vWF in plasma. but at lower concentrations. that vWF in platelets is normal both quali- tatively and quantitatively. and that cellular vWF can be rapidly released into plasma by DDAVP to correct the hemostatic abnormalities. However. vWF is removed rap- idly from plasma. making the correction more transient than in congenital vWD type I. abnormality of the immune system. Three general mechanisms to explain the pathogenesis of AvWD have been proposed. The first implies that antibodies either inactivate the biologic activities of FVIII- vWF,2 bind to the complex without affecting the active sites, or induce rapid clearance of the complex from the circulation through the formation of immuno- complexes.’2”3 The second proposes that AvWD is the result of the selective absorption of FVIII-vWF to abnormal lymphocytic clones or malignant cells, lead- ing to low plasma levels.’’9 Finally, the third states that there is defective synthesis of FVIII-vWF in cellular compartments (megakaryocytes, endothelial cells) and/or there is defective release into plasma. We decided to apply three new approaches to investigating the problem of the pathogenesis of AvWD in seven patients with AvWD associated with lymphoprolifera- tive disorders or benign monoclonal gammopathies. The first was the measurement of vWF (as vWF:Ag and vWF:RiCof) in platelets, which are easily accessi- ble cells of megakaryocytic origin from which the vWF content of cellular compartments can be evaluated.20’2’ The second was the assessment of the plasma release and clearance of FVIII-vWF by monitoring the changes of vWF:Ag, vWF:RiCof, and FVIII:C after infusion of I-deamino-8-D-arginine vasopressin (DDAVP), a drug that probably releases FVIII-vWF from cellular com- partments.22 The third was the electrophoretic analysis of the structure of FVIII-vWF, which consists of a multimeric series of oligomers of a protomer composed of a variable number of identical subunits,2325 in plasma and platelets. MATERIALS AND METHODS Patients The clinical details for the patients are listed in Table 1. The most typical features were the onset of mildly to moderately severe For personal use only. on October 3, 2017. by guest www.bloodjournal.org From
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Observed values are expressed as percentages of pooled normal plasma incubated under the same conditions as the mixtures, set arbitrarily at 100%
(medians of three different experiments). Expected values were calculated from the values of normal and patient plasmas incubated and assayed
separately. Percentage differences between observed and expected values were also calculated.
Means and 95% confidence limits (between parentheses) for nine normal subjects. Percentage differences were not significantly different from those
for patients.
tMeans and 95% confidence limits (between parentheses) for seven AvWD patients. Percentage differences were not significantly different from zeronor from the control values.
616 MANNUCCI ET AL
Table 2. Baseline Labors tory Findings of Seven Patients With Acquired von Willebr and’s Disease
are given in Table 2. Patients 1 , 2, and 3 were the most
severely affected patients, with plasma vWF:Ag (EIA
method) and vWF:RiCof below the lower limits ofdetection of the methods (6 U/dL). Very low concen-
trations of vWF:Ag, however, could be measured by
the IRMA method, which gave dose-response curves
parallel to those of normal plasma and maximal anti-
body-binding capacity like that of normal plasma. The
BT was prolonged, but less than would have beenexpected from the severity of the plasma FVIII-vWFdeficits. In the remaining four patients (No. 4 to 7), all
the plasma FVIII-vWF measurements were less low
than in the first three patients. The BT of patients 4 to
6 were normal.
Inhibitors of FVIIJ-vWF
In AvWD plasma heated to destroy FVIII:C, therewas no evidence of inhibitory activity against FVIII:C
(data not shown). After prolonged incubation of nor-
mal and AvWD plasmas, observed values of residual
vWF:Ag and vWF:RiCof did not significantly differfrom the values expected from the values of plasmas
incubated separately (nor from the values of the con-
trol plasmas), indicating that no significant inactiva-
tion of vWF:Ag and vWF:RiCof was induced in nor-
mal plasma by incubation with AvWD plasma (Table3).
Platelet FVJII-vWF Measurements
Platelet vWF:Ag and vWF:RiCof concentrations
were normal in all patients except No. 5, who had low
borderline values (Fig 1). This patient has myeloma
and was anemic, and hence, it was difficult to avoid
contamination with red cells in washed platelets and
lysates. Such contamination is likely to have led to a
higher protein content and thus to underestimation of
platelet vWF:Ag and vWF:RiCof.
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Fig 1 . Platelet von Willebrand factor antigen (vWF:Ag) mea-
sured with IRMA (squares) and ristocetin cofactor (vWF:RiCof)(triangles) in nine healthy subjects (normals). seven patients withcongenital von Willebrand’s disease (CvWD. closed symbols). andseven patients with acquired von Willebrand’s disease (AvWD,open symbols). No significant difference between groups for eachmeasurement was found with a one-way analysis of variance.
vWF:Ag IRMA (#{149}).vWF: RiCof (A).
A
ACQUIRED VON WILLEBRAND’S DISEASE 617
FVIII-vWF Multimeric Pattern
All the multimers present in normal plasma could be
seen in all patients (Fig 2), with the pattern similar to
that for normal plasma and plasma from patients with
the “classical” form of congenital vWD (type I). The
relative intensities of the bands were decreased in
rough proportion to the plasma concentrations of
FVIII-vWF measurements. Larger multimers than
Fig 2. Autoradiograph pattern of factor VIll-von Wille-brand factor (FVlll-vWF) electrophoresed in 1 .6% agarose inthe presence of sodium dodecyl sulfate and detected byreaction with ‘2�l-labeled affinity-purified antibody. Thearrow indicates the origin of the running gel. and the anode isat the botton of the gel. (A) From left to right: normal plasma;plasmas from AvWD patients 4. 5. 6. 2, and 3; platelet lysatesfrom a normal subject and AvWD patient 3. (B) From left toright: plasmas from patient 1 . normal control. and patient 7.
4
were present in plasma were seen in the platelet lysates
of all the patients tested. A representative example
(patient 3) is shown in Fig 2.
DDA VP Studies
In order to evaluate the magnitude and time courseof FVIII:C, vWF:Ag, and vWF:RiCof changes in
AvWD patients after DDAVP, we have chosen to use
as a control group seven patients with congenital vWD
(CvWD) type I treated with DDAVP in the same
manner (Table 4). The rationale for this choice was
that these patients had the following features in com-
mon with the AvWD patients: low plasma levels of
FVIII:C, vWF:Ag, and vWF:RiCof (Table 4); normalplatelet vWF:Ag and vWF:RiCof (Fig 1); and intact
multimeric structure in plasma and platelets. Bleeding
times, however, were prolonged in all patients. Figure
3 compares the extent and time course of plasma
changes in these measurements after DDAVP in the
two groups. In both, there was a marked increase of
FVIII:C, vWF:Ag, and vWF:RiCof over baseline val-
ues. The mean increase of vWF:Ag and vWF:RiCof
for AvWD patients, however, was slightly less
(P < .05) than for CvWD immediately after infusion.
In AvWD, there was also a more rapid return of
vWF:Ag and vWF:RiCof to the baseline after the end
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Table 5. Half-Disappearance Times of Factor VIII Coagulant
Activity (FVlll:C). von Willebrand Factor Antigen (vWF:Ag). and
Ristocetin Cofactor (vWF:RiCof) in Patients With Congenital and
Acquired von Willebrand’s Disease
Fig 3. Quantitative changes of FVlll-vWF factor (measured asFVlll:C, vWF:RiCof. and vWF:Ag) at various time intervals (hours)after DDAVP infusion in seven patients with CvWD (solid lines)and seven patients with AvWD (dotted lines). Results areexpressed as mean changes ( ± SD) over baseline values taken as 1..-. CvWD, #{149}---#{149}AvWD.
Acquired vWD (N - 7)
Congenital vWD (N - 7)
‘Means ± SEM.
tP < .05 v CvWD patients.�P < .01 vCvWD patients.
618 MANNUCCI ET AL
Table 4. Baseline Lab oratory Findi ngs for Seven “Control” Patients With Congenital von Will ebrand’s Dise ase Type I
Fig 4. (A) Autoradiograph pattern of plasma FVlll-vWF mul-timers of CvWD patient 3 before (time 0) and at various timesafter DDAVP infusion. N shows a normal plasma pattern. (B)Autoradiograph pattern of plasma FVlll-vWF multimers of AvWDpatient 3 before (time 0) and after DDAVP. N shows a normalplasma pattern.
4
4
A
N
N
VWD TYPE I0’ 15’ 30’ 60’ 2h
AVWD
0’ 15’ 30’ 60’ 2h
4h
4h
AvWD-defective release of FVIII-vWF from cellular
compartments or increased plasma clearance-were
evaluated by comparing the effects of DDAVP in
AvWD and CvWD patients, on the assumption thatthis agent should amplify the physiologic mechanisms
for releasing FVIII-vWF from cellular compartments
and clearing it from plasma. In AvWD, FVIII:C,
vWF:Ag, and vWF:RiCof markedly increased soon
after the infusion, and larger multimers, possibly
released from cellular compartments,22 appeared tran-
siently in the circulation. This behavior was similar to
that of patients with CvWD type I, but differed in that
the increase was of lesser magnitude and more short-
lived, at least for vWF:Ag and vWF:RiCof. A more
rapid plasma clearance of FVIII-vWF could probably
explain both the lower peak values and the shorter
half-disappearance times of vWF:Ag and vWF:RiCof,
even though an additional role of defective release
from cells cannot be excluded by our data. Rapid
plasma clearance might be due to at least three causes:
(1) specific autoantibodies that inactivate FVIII-vWF;
(2) specific autoantibodies that bind FVIII-vWF but
are not directed against the so-called FVII1-vWF
active sites; and (3) nonspecific antibodies that form
circulating immunocomplexes with FVIII-vWF and
favor its clearance by Fc-bearing cells of the reticu-
loendothelial system.’2”3 Unlike some previous stud-
ies,2�”3#{176}but in agreement with many others,’2�9’29’3133
our patients’ plasmas did not inactivate FVIII:C,vWF:Ag, and vWF:RiCof in normal plasma in vitro,
Table 6. Bleeding Times Before and After DDAVP in Patients
With Congenital and Acquired von Willebrand’s Disease
PatientNo. Diagnosis
Bleeding Times (mm)
Baseline
30 mm
Postinfusion
4 h
Postinfusion
1
2
3
4
5
6
7
8
9
10
11
12
13
14
AvWD
AvWD
AvWD
AvWD
AvWD
AvWD
AvWD
CvWD
CvWD
CvWD
CvWD
CvWD
CvWD
CvWD
10
11
14
5
5
6
8
19
12
13
11
12
13
14
4
6
7
ND
ND
ND
ND
10
6
4
5
6
7
8
11
10
13
ND
ND
ND
ND
8
7
5
4
7
5
5
ND, not done because baseline bleeding times were normal or
borderline.
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brand’s disease in a patient with Wilm’s tumor. J Pediatr 95:997,30. Mazurier C, Parquet-Gernez A, Descamps i, Bauters F,1979
Goudemand M: Acquired von Willebrand’s syndrome in the courseof Waldenstr#{246}m disease. Thromb Haemostas 44:1 15, 1980 � Clough V. MacFarlane IA, O’Connor i, Wood JK: Acquired
von Willebrand syndrome and Ehlers-Danhos syndrome presenting3 1 . Rosborough TK, Swaim WR: Acquired von Wilhebrand’s with gastrointestinal bleeding. Scand i Haematol 22:305, 1979
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PM Mannucci, R Lombardi, R Bader, MH Horellou, G Finazzi, C Besana, J Conard and M Samama gammopathiesseven patients with lymphoproliferative disorders or benign monoclonal Studies of the pathophysiology of acquired von Willebrand's disease in
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