THROMBOSIS RESEARCH 65; 343-351,1992 0049-3848/92 $5.00 + .OO Printed in the USA. Copyright (c) 1992 Pergamon Press Ltd. All rights reserved.
A NEW
0F VAN WILLEBRAND DISEASE (TYPE II I) WITH A NORMAL DEGREE OF PROTEOLYTIC CLEAVAGE OF VON WILLEBRAND FACTOR
VARIANT
G. Castaman, F. Rodeghiero,
A. Lattuada and P.M. Mannucci
From the Division of Hematology and Hemophilia and Thrombosis Hospital, Vicenza; A. Bianchi Bonomi Center, San Bortolo Hemophilia and Thrombosis Center and Institute of Internal Medicine, IRCCS Maggiore Hospital and University, Milan, Italy (Received
10.10.1991;
accepted
in revised form 29.11.1991
by Editor S. Coccheri)
ABSTRACT A variant of type II von Willebrand disease (vWd) is described in a young woman and her mother with severe lifelong histories. On bleeding electrophoresis with low-resolution agarose gels the the proband lacked large and plasma of multimers of von Willebrand factor intermediate-size (vWF) but the platelet multimeric structure was normal. On high-resolution gels, smaller multimers could be resolved into a broader central band and four satellite bands, which were much fainter than in normal plasma. In the proband plasma, the relative concentrations of proteolytic fragments of the vWF subunit were within the normal laboratory range. Since this variant of vWd appears to differ from those reported hitherto, the designation of type II I is proposed.
INTRODUCTION Von Willebrand's disease (vWd) is a heterogeneous bleeding disorder caused by the decrease and/or abnormality of von Willebrand factor (vWF) (l), resulting in an impaired primary hemostasis, reflected by a prolonged skin bleeding time, and in defective blood coagulation due to the secondary decrease of factor VIII. On the basis of the multimeric analysis of vWF, several main types of the disease have been described (1). In particular, among type II vWD, Key words: von Willebrand factor VIII
disease, von Willebrand
343
factor,
344
A NEW VARIANT
OF VW DISEASE
Vol. 65, No. 3
characterized by the lack of large vWF multimers in plasma, eight phenotypes have been identified and called types II A to II H (2-8). In this paper, we describe a previously unreported type II variant.
PATIENTS AND METHODS Case ReDort The proband was 18 years old when she was first referred to the Hematology Division of Vicenza for evaluation of a severe lifelong bleeding history. Since her early childhood, she had suffered from severe epistaxis and on two instances she was transfused with packed red cells. Iron therapy was needed to correct iron deficiency secondary to menorrhagia. Pregnancy at age 23 was uneventful, with ldeamino-8-D-arginine vasopressin (DDAVP) infused immediately after delivery and 24 hours later. DDAVP was also repeatedly administered to control severe epistaxis. Epistaxis and severe menorrhagia were reported by her mother, who had required transfusion of packed red cells when she developed severe bleeding after a dental extraction. Life-threatening bleeding occurred after each of her two deliveries, requiring several units of packed red cells and whole blood. The father and the sister of the proband are asymptomatic. Methods Blood was collected into 3.8 % sodium citrate and centrifuged at 1,500 g for 15 min. In experiments carried out to evaluate the degree of proteolytic cleavage of vWF in vivo (6), 5 mM EDTA, 6 mM N-ethylmaleimide and 1 mM leupeptin (Sigma, St. Louis, USA) were added to citrate to avoid in vitro proteolytic degradation of vWF after venepuncture. The bleedins time (BT) was measured with the Simplate II device (General Diagnostics, Morris Plains, USA), making VIII two vertical incisions on the forearm. Factor coauulant activity (VIII:C) was assayed in plasma by a onestage method (9) and von Willebrand factor antisen (vWF:Ag) by two-site ELISA, using a polyclonal antiserum (Stago, Asnieres, France) (10). Ristocetin cofactor activity (RiCof) was measured in plasma using formalin-fixed platelets, as previously described (9,lO). Ristocetin-induced nlatelet atyryrecation (RIPA) was evaluated by adding increasing amounts of ristocetin to platelet-rich plasma, and measuring the extent of aggregation 3 minutes later. The multimeric structure of vWF was analysed by sodium dodecyl sulphate (SDS) agarose gel electrophoresis in low (0.8% or 0.9% low-gelling temperature agarose), inJ;;mediate high agarose) temperature low-gelling (1.4% resolution (2% low-gelling temperature agarose) gel systems, as previously described (6,ll). In normal plasma, low resolution gels resolve each smaller multimer as a single band; intermediate resolution gels, into one broader band and two subbands (triplet); high resolution gels, into five bands, two migrating above and two below a broader and denser
Vol. 65, No. 3
A NEW VARIANT
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345
central band. The degree of proteolvtic cleavase of plasma vWF was analysed (after immunoaffinity purification of vWF, reduction and SDS-polyacrylamide gel electrophoresis) by immunoblotting with a panel of anti-human vWF monoclonal antibodies (kindly provided by Dr. Z.M. Ruggeri, S~s$pps Clinic and Research Foundation, La Jolla, USA) and I-rabbit anti-mouse IgG antibodies (5). Concentrations of proteolytic fragments relative to that of the intact vWF subunit were calculated by measuring in a gamma scintillation counter the radioactivity of the bands excised from the nitrocellulose blot (5,ll). Platelet vWF was measured after platelet separation on Ficoll-Hypaque gradients (12). Lysis was obtained adding l/40 volumes of 20,% Triton X-100. The supernatants were snapfrozen at -80 C.
TABLE
Family
I. Clinical
member
Bleeding
and
laboratory
history
data
Bleeding
time
(min)
Proband*
Mother*
for
the
proposita
and
VIII:C
vWF:Ag
(IU/dL)
(IU/dL)
her
family
members
RiCof
RiCof/vWF:Ag
(IlJ/dL)
ratio
RIPA (q/ml)
Epistaxis,
menorrhagia
16-
> 20
16-35
6-25
< 3-5
0.21-0.39
1.6-1.9
Epistaxis,
menorrhagia,
1%
> 20
12-25
10-29
< 3-6
0.26-0.41
1.7-1.9
post-partum bleeding
bleeding.
after
tooth
extraction
Father
None
6
100
105
104
0.99
1.0
Sister
NOlX
5
76
66
72
1.06
1.0
55-150
50-146
Normal
(0
52-164
0.71-1.16
0.76-1.15
_________--___________-----________--_---_-____------_-_-_-------__------____-----____-_----------
*
Data
represent
the
range
of
5 separate
determinations
RESULTS
The main clinical and laboratory data are summarized in Table 1. The proband and her mother had correspondingly low levels of VIII:C and vWF:Ag in plasma, with lower or unmeasurable RiCof. Accordingly, RiCof/vWF:Ag ratios (measured on 5 different occasions) were low, ranging from 0.21 to 0.41 (range in 40 normals: 0.71 + 1.16), as usually observed in patients with dysfunctional vWF. Greater than normal concentrations of ristocetin were needed to elicit aggregation. Normal values were obtained in the father.
A NEW VARIANT OF VW DISEASE
346
Fig.
1
Vol. 65, No. 3
vWF multimers for proband plasma (PPLA), mother plasma (PPLT), (MPLA), normal plasma (NPLA), proband platelets normal platelets (NPLT). SDS gel electrophoresis in a lowresolution gel (0.9% low-gelling temperature agarose). The arrow at the top indicates the interface between stacking and running gel.
On lOW-reSOlUtiOn CfelS, multimeric analysis of vWF of the proband and her mother indicated that multimers of larger and intermediate size were missing from plasma and that each smaller multimer was thinner than in normal plasma (Ficr. 1). Normal patterns were observed in the father and sister of the proband (not shown). Platelet vWF had a normal multimeric structure in the proband (Ph. 1) and her mother (not shown). Intermediateresolution gels of proband plasma (Fis. 2) and her mother indicated that the triplet structure present in normal plasma was replaced by a single central band, with no visible subbands. A normal triplet structure was present in the plasma from the father and the sister (not shown). In normal plasma, hishresolution gels resolved smaller multimers into five bands, but in proband plasma the four satellite bands of the broader central band were thinner and fainter than in normal plasma (E'iu. 3), even after prolonged exposure of the autoradiographs to achieve intensity of the bands comparable to that seen in normal plasma. The multimeric structure of platelet vWF was identical to that of normal platelets (Fis. 3) (for platelet vWF, each smaller multimer is made of a doublet). The content of platelet vWF in the proband and her mother
Vol. 65. No. 3
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347
was normal (vWF:A 8:0.45 and 0.75 IV/lo' platelets, normal rang5 0.21-0.59 IU/lO platelets; RiCof* 0.34 and 0.68 IU/lO platelets, normal range 0.27-1.02 IU/109 platelets). Analysis of proteolvtic cleavacre of the vWF subunit in the plasma of the proband demonstrated the presence of the same 189-, 176- and 140-kd fragments present in normal plasma. No new fragment was seen, nor was there any evidence of uncleaved provWF (not shown). The intact 225-kd subunit comprised between 82.8% and 86% of the total vWF in the proband (range of three determinations) (normal, 74% to 86%); the 189-kd fragment, between 2.0% and 2.5% (normal, 1.9% to 5.8%); the 176-kd fragment, between 8% and 11% (normal, 5.5% to 12.6%); the 140 -kd fragment, between 3.4% and 5% (normal, 2% to 7.4%). Hence, the relative concentrations of all fragments were within the normal laboratory range.
Fig. 2 SDS gel electrophoresis in an intermediateresolution gel (1.4% low-gelling temperature agarose). Marker bars denote the pattern triplet in normal plasma P denotes (N). proband plasma, with no visible triplet structure. The arrow at the top indicates the interface between and stacking running gel.
-
348
A NEW VARIANT OF VW DISEASE
PLT N
P
Vol. 65. No. 3
PLA
Fig. 3 SDS gel electrophoresis in a high-resolution gel (2% low-gelling temperature agarose). Plasma (PLA) and platelet (PLT) vWF for a normal subject (N) and proband (P). The arrow at the top indicates the interface between stacking and running gel.
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DISCUSSION The diagnosis of type II vWd was made in this young woman with a severe life-long bleeding history on the basis of a prolonged BT,low VIII/vWF measurements and lack of large vWF multimers (Fiq. 1). Intermediate-resolution gels indicated that the triplet structure of each smaller multimer was replaced by a single central band (Fis. 2). Highresolution gels showed that for each smaller multimer the four satellite bands of the central band were much less prominent than in normal plasma (Fis. 3), even after prolonged exposure of the autoradiographs. Platelet vWF content and multimeric pattern were normal (Fis. 1 and 3). The pattern observed in this case is unique among reported type II subtypes (2-8). Unlike this case, types II by quantitative and/or D, II G and II H are characterized qualitative abnormalities of platelet vWF (larger and intermediate multimers are missing) (4,7,8). The abnormality of plasma vWF multimeric pattern peculiar of type II E, i.e., a single central band seen on intermediate-resolution gels, is quite distinct from that of this case, i.e., a central band with fainter adjacent bands; moreover, the degree of proteolysis of the vWF subunit was normal in our case, whereas it was low in II E vWd (5). Type II B is easily distinguishable from this subtype (in spite of the fact that in both the platelet multimeric structure is intact), because in type II B RIPA is elicited at much lower ristocetin concentrations and thrombocytopenia occurs after DDAVP (13,14),but not in our patient. This subtype has some similarities with types II A, II C large vWF multimers are usually and II F. Even though defective in plasma and platelets of type II A patients, Weiss et al (15) reported that in some of them (called IIA-3) the multimeric structure of platelet vWF was intact, contrasting with defects of large multimers in plasma, as in our proband. On the other hand, her normal concentrations of proteolytic fragments of the vWF subunit represent a clear differentiation from the type II A phenotype, which is characterized by increased concentrations of the fragments (5). For both II C and this, the triplet structure is missing and replaced by a single band on intermediate resolution gels II C, however, is also characterized by (3). Type abnormalities of the multimeric structure of platelet vWF; and by recessive transmission (3), in contrast with the dominant pattern of inheritance of this variant. Both type II F and this case are characterized by an alteration of the multimeric pattern of plasma vWF, in contrast with a normal platelet pattern (6). Despite these similarities, however, our case differs from type II F for several aspects. In II F, there is an abnormal mobility of satellite bands of plasma vWF, seen on high-resolution gels (6), whereas in our proband the corresponding bands had a mobility similar to normal plasma. The transmission of the defect is dominant in our family, whereas it appears to be recessive in type II F (none of the parents of the type II F proband had a bleeding
350
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history and the one who could be examined had normal vWF measurements and BT) (6). The mechanism underlying this abnormality of vWF is unknown. In some type II plasmas proteolysis of the vWF subunit is higher than for normal plasma, whereas in others proteolysis is lower (5). In our case, there was a normal degree of proteolysis, a previously unreported finding in type II vWD. In accordance with the currently used phenotypic terminology, we propose that this variant of von Willebrand disease is designated type II I.
This work was supported in part by CNR, Progetto Finalizzato Ingegneria Genetica (PMM), grant HL-41135 from National Institutes of Health (PMM), and by the Health Department of the Veneto Region (FR).
REFERENCES 1.
RUGGERI, Z.M. and ZIMMERMAN, T.S. von Willebrand factor and von Willebrand disease. Blood, 70, 895-907, 1987.
2. RUGGERI, Z.M. and ZIMMERMAN, T.S. Variant von Willebrand disease. Characterization of two subtypes by analysis of multimeric composition of factor VIII/van Willebrand factor in plasma and platelets. J. Clin. Invest., 65,318-324, 1980. 3. RUGGERI, Z.M., NILSSON, I.M., LOMBARDI, R., HOLMBERG, L., and ZIMMERMAN, T.S. Aberrant multimeric structure of von Willebrand factor in a new variant of von Willebrand's disease (type II C). J. Clin. Invest., 70, 1124-1127, 1982. 4. KINOSHITA, S., HARRISON, J., LAZERSON, J. and ABILDGAARD, C.F. A new variant of dominant type II von Willebrand's disease with aberrant multimeric pattern of factor VIIIrelated antigen. Blood, 63, 1369-1372, 1984. 5. ZIMMERMAN, T.S., DENT, J.A., RUGGERI, Z.M. and NANNINI L.H. Subunit composition of plasma von Willebrand factor. Cleavage is present in normal individuals, increased in II A and II B von Willebrand disease, but minimal in variants with aberrant structure of individual multimers (type II C, II D, II E). J. Clin. Invest., 77, 947-951, 1986. 6. MANNUCCI, P.M., LOMBARDI, R., FEDERICI, A-B., DENT, J.A., T.S. and RUGGERI, Z.M. A new variant of type II ZIMMERMAN, von Willebrand disease with aberrant multimeric structure of plasma but not platelet von Willebrand factor. Blood, 68,269-274, 1986.
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7.
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GRALNICK, H.R., WILLIAMS, S.B., McKEONN, P., MAISONNEUVE, P ., JENNEAU, C. and SULTAN, Y. A variant of type II von Willebrand disease with abnormal triplet structure and discordant effects of protease inhibitors on plasma and platelet von Willebrand factor structure. Am. J. Iiematol., 24,259-266,
1987.
8.
FEDERICI, A.B., MANNUCCI, P.M., LOMBARDI, R., LATTUADA, A ., COLIBRETTTI, M.L., DENT, J.A. and ZIMMERMAN, T.S. Type II H von Willebrand disease: new structural abnormality of plasma and platelet von Willebrand factor in a patient with prolonged bleeding time and borderline levels of ristocetin cofactor activity. Am. J. Hematol., 32,287-293, 1989.
9.
RODEGHIERO,
F., CASTAMAN, G. and
investigation of the prevalence disease. Blood, 69,454-459, 1987. 10.
of von Willebrand's
RODEGHIERO, F., CASTAMAN, G. and TOSETTO, A. von Willebrand factor antigen is less sensitive than ristocetin cofactor for the diagnosis of type I von Willebrand disease Results based on an epidemiological investigation. Thromb. Haemostas.,
11.
DINI, E. Epidemiological
MANNUCCI, LATTUADA,
64,349-352,
1990.
P.M., LOMBARDI, R., CASTAMAN, G., DENT, J-A., ZIMMERMAN, T.S. von A., RODEGHIERO, F. and
Willebrand disease "Vicenza" su ranormal) von Willebrand $1 , 69-70I 1988 .
with larger-than -normal factor multimers. Blood,
12.
RODEGHIERO, F., CASTAMAN, G., TOSETTO, A., LATTUADA, A. Platelet von Willebrand factor assay and MANNUCCI, P.M. Results using two methods for platelet lysis. Thromb. Res , 59,259-267, 1990.
13.
RUGGERI, Z.M., PARETI, F.I., MANNUCCI, P.M., CIAVARELLA, N. Heightened interaction between and ZIMMERMAN, T.S.
platelets and factor VIII/van Willebrand factor in a new subtype of von Willebrand's disease. N. Enal. J. Med., 302,1047-1051,
1980.
14.
HOLMBERG,L., NILSSON, I.M., BORGE, L., GUNNARSON, M. and SJORIN, E. Platelet aggregation induced by 1-desamino-8-Darginine vasopressin (DDAVP) in type IIB von Willebrand's disease. N. Enul. J. Med., 309,816-821, 1983.
15.
WEISS, H.J., PIETU, G., RABINOWITZ, R., GIRMA, J.P., ROGERS, J. and NEYER, D. Heterogenous abnormalities in the multimeric structure, antigenic properties and plasmaplatelet content of factor VIII/van Willebrand factor in subtypes of classic (type I) and variant (type II A ) von Willebrand's disease. J. Lab. Clin. Med., 101,411425,1983.
A NEW
0F VAN WILLEBRAND DISEASE (TYPE II I) WITH A NORMAL DEGREE OF PROTEOLYTIC CLEAVAGE OF VON WILLEBRAND FACTOR
VARIANT
G. Castaman, F. Rodeghiero,
A. Lattuada and P.M. Mannucci
From the Division of Hematology and Hemophilia and Thrombosis Hospital, Vicenza; A. Bianchi Bonomi Center, San Bortolo Hemophilia and Thrombosis Center and Institute of Internal Medicine, IRCCS Maggiore Hospital and University, Milan, Italy (Received
10.10.1991;
accepted
in revised form 29.11.1991
by Editor S. Coccheri)
ABSTRACT A variant of type II von Willebrand disease (vWd) is described in a young woman and her mother with severe lifelong histories. On bleeding electrophoresis with low-resolution agarose gels the the proband lacked large and plasma of multimers of von Willebrand factor intermediate-size (vWF) but the platelet multimeric structure was normal. On high-resolution gels, smaller multimers could be resolved into a broader central band and four satellite bands, which were much fainter than in normal plasma. In the proband plasma, the relative concentrations of proteolytic fragments of the vWF subunit were within the normal laboratory range. Since this variant of vWd appears to differ from those reported hitherto, the designation of type II I is proposed.
INTRODUCTION Von Willebrand's disease (vWd) is a heterogeneous bleeding disorder caused by the decrease and/or abnormality of von Willebrand factor (vWF) (l), resulting in an impaired primary hemostasis, reflected by a prolonged skin bleeding time, and in defective blood coagulation due to the secondary decrease of factor VIII. On the basis of the multimeric analysis of vWF, several main types of the disease have been described (1). In particular, among type II vWD, Key words: von Willebrand factor VIII
disease, von Willebrand
343
factor,
344
A NEW VARIANT
OF VW DISEASE
Vol. 65, No. 3
characterized by the lack of large vWF multimers in plasma, eight phenotypes have been identified and called types II A to II H (2-8). In this paper, we describe a previously unreported type II variant.
PATIENTS AND METHODS Case ReDort The proband was 18 years old when she was first referred to the Hematology Division of Vicenza for evaluation of a severe lifelong bleeding history. Since her early childhood, she had suffered from severe epistaxis and on two instances she was transfused with packed red cells. Iron therapy was needed to correct iron deficiency secondary to menorrhagia. Pregnancy at age 23 was uneventful, with ldeamino-8-D-arginine vasopressin (DDAVP) infused immediately after delivery and 24 hours later. DDAVP was also repeatedly administered to control severe epistaxis. Epistaxis and severe menorrhagia were reported by her mother, who had required transfusion of packed red cells when she developed severe bleeding after a dental extraction. Life-threatening bleeding occurred after each of her two deliveries, requiring several units of packed red cells and whole blood. The father and the sister of the proband are asymptomatic. Methods Blood was collected into 3.8 % sodium citrate and centrifuged at 1,500 g for 15 min. In experiments carried out to evaluate the degree of proteolytic cleavage of vWF in vivo (6), 5 mM EDTA, 6 mM N-ethylmaleimide and 1 mM leupeptin (Sigma, St. Louis, USA) were added to citrate to avoid in vitro proteolytic degradation of vWF after venepuncture. The bleedins time (BT) was measured with the Simplate II device (General Diagnostics, Morris Plains, USA), making VIII two vertical incisions on the forearm. Factor coauulant activity (VIII:C) was assayed in plasma by a onestage method (9) and von Willebrand factor antisen (vWF:Ag) by two-site ELISA, using a polyclonal antiserum (Stago, Asnieres, France) (10). Ristocetin cofactor activity (RiCof) was measured in plasma using formalin-fixed platelets, as previously described (9,lO). Ristocetin-induced nlatelet atyryrecation (RIPA) was evaluated by adding increasing amounts of ristocetin to platelet-rich plasma, and measuring the extent of aggregation 3 minutes later. The multimeric structure of vWF was analysed by sodium dodecyl sulphate (SDS) agarose gel electrophoresis in low (0.8% or 0.9% low-gelling temperature agarose), inJ;;mediate high agarose) temperature low-gelling (1.4% resolution (2% low-gelling temperature agarose) gel systems, as previously described (6,ll). In normal plasma, low resolution gels resolve each smaller multimer as a single band; intermediate resolution gels, into one broader band and two subbands (triplet); high resolution gels, into five bands, two migrating above and two below a broader and denser
Vol. 65, No. 3
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345
central band. The degree of proteolvtic cleavase of plasma vWF was analysed (after immunoaffinity purification of vWF, reduction and SDS-polyacrylamide gel electrophoresis) by immunoblotting with a panel of anti-human vWF monoclonal antibodies (kindly provided by Dr. Z.M. Ruggeri, S~s$pps Clinic and Research Foundation, La Jolla, USA) and I-rabbit anti-mouse IgG antibodies (5). Concentrations of proteolytic fragments relative to that of the intact vWF subunit were calculated by measuring in a gamma scintillation counter the radioactivity of the bands excised from the nitrocellulose blot (5,ll). Platelet vWF was measured after platelet separation on Ficoll-Hypaque gradients (12). Lysis was obtained adding l/40 volumes of 20,% Triton X-100. The supernatants were snapfrozen at -80 C.
TABLE
Family
I. Clinical
member
Bleeding
and
laboratory
history
data
Bleeding
time
(min)
Proband*
Mother*
for
the
proposita
and
VIII:C
vWF:Ag
(IU/dL)
(IU/dL)
her
family
members
RiCof
RiCof/vWF:Ag
(IlJ/dL)
ratio
RIPA (q/ml)
Epistaxis,
menorrhagia
16-
> 20
16-35
6-25
< 3-5
0.21-0.39
1.6-1.9
Epistaxis,
menorrhagia,
1%
> 20
12-25
10-29
< 3-6
0.26-0.41
1.7-1.9
post-partum bleeding
bleeding.
after
tooth
extraction
Father
None
6
100
105
104
0.99
1.0
Sister
NOlX
5
76
66
72
1.06
1.0
55-150
50-146
Normal
(0
52-164
0.71-1.16
0.76-1.15
_________--___________-----________--_---_-____------_-_-_-------__------____-----____-_----------
*
Data
represent
the
range
of
5 separate
determinations
RESULTS
The main clinical and laboratory data are summarized in Table 1. The proband and her mother had correspondingly low levels of VIII:C and vWF:Ag in plasma, with lower or unmeasurable RiCof. Accordingly, RiCof/vWF:Ag ratios (measured on 5 different occasions) were low, ranging from 0.21 to 0.41 (range in 40 normals: 0.71 + 1.16), as usually observed in patients with dysfunctional vWF. Greater than normal concentrations of ristocetin were needed to elicit aggregation. Normal values were obtained in the father.
A NEW VARIANT OF VW DISEASE
346
Fig.
1
Vol. 65, No. 3
vWF multimers for proband plasma (PPLA), mother plasma (PPLT), (MPLA), normal plasma (NPLA), proband platelets normal platelets (NPLT). SDS gel electrophoresis in a lowresolution gel (0.9% low-gelling temperature agarose). The arrow at the top indicates the interface between stacking and running gel.
On lOW-reSOlUtiOn CfelS, multimeric analysis of vWF of the proband and her mother indicated that multimers of larger and intermediate size were missing from plasma and that each smaller multimer was thinner than in normal plasma (Ficr. 1). Normal patterns were observed in the father and sister of the proband (not shown). Platelet vWF had a normal multimeric structure in the proband (Ph. 1) and her mother (not shown). Intermediateresolution gels of proband plasma (Fis. 2) and her mother indicated that the triplet structure present in normal plasma was replaced by a single central band, with no visible subbands. A normal triplet structure was present in the plasma from the father and the sister (not shown). In normal plasma, hishresolution gels resolved smaller multimers into five bands, but in proband plasma the four satellite bands of the broader central band were thinner and fainter than in normal plasma (E'iu. 3), even after prolonged exposure of the autoradiographs to achieve intensity of the bands comparable to that seen in normal plasma. The multimeric structure of platelet vWF was identical to that of normal platelets (Fis. 3) (for platelet vWF, each smaller multimer is made of a doublet). The content of platelet vWF in the proband and her mother
Vol. 65. No. 3
A NEW VARIANT
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was normal (vWF:A 8:0.45 and 0.75 IV/lo' platelets, normal rang5 0.21-0.59 IU/lO platelets; RiCof* 0.34 and 0.68 IU/lO platelets, normal range 0.27-1.02 IU/109 platelets). Analysis of proteolvtic cleavacre of the vWF subunit in the plasma of the proband demonstrated the presence of the same 189-, 176- and 140-kd fragments present in normal plasma. No new fragment was seen, nor was there any evidence of uncleaved provWF (not shown). The intact 225-kd subunit comprised between 82.8% and 86% of the total vWF in the proband (range of three determinations) (normal, 74% to 86%); the 189-kd fragment, between 2.0% and 2.5% (normal, 1.9% to 5.8%); the 176-kd fragment, between 8% and 11% (normal, 5.5% to 12.6%); the 140 -kd fragment, between 3.4% and 5% (normal, 2% to 7.4%). Hence, the relative concentrations of all fragments were within the normal laboratory range.
Fig. 2 SDS gel electrophoresis in an intermediateresolution gel (1.4% low-gelling temperature agarose). Marker bars denote the pattern triplet in normal plasma P denotes (N). proband plasma, with no visible triplet structure. The arrow at the top indicates the interface between and stacking running gel.
-
348
A NEW VARIANT OF VW DISEASE
PLT N
P
Vol. 65. No. 3
PLA
Fig. 3 SDS gel electrophoresis in a high-resolution gel (2% low-gelling temperature agarose). Plasma (PLA) and platelet (PLT) vWF for a normal subject (N) and proband (P). The arrow at the top indicates the interface between stacking and running gel.
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DISCUSSION The diagnosis of type II vWd was made in this young woman with a severe life-long bleeding history on the basis of a prolonged BT,low VIII/vWF measurements and lack of large vWF multimers (Fiq. 1). Intermediate-resolution gels indicated that the triplet structure of each smaller multimer was replaced by a single central band (Fis. 2). Highresolution gels showed that for each smaller multimer the four satellite bands of the central band were much less prominent than in normal plasma (Fis. 3), even after prolonged exposure of the autoradiographs. Platelet vWF content and multimeric pattern were normal (Fis. 1 and 3). The pattern observed in this case is unique among reported type II subtypes (2-8). Unlike this case, types II by quantitative and/or D, II G and II H are characterized qualitative abnormalities of platelet vWF (larger and intermediate multimers are missing) (4,7,8). The abnormality of plasma vWF multimeric pattern peculiar of type II E, i.e., a single central band seen on intermediate-resolution gels, is quite distinct from that of this case, i.e., a central band with fainter adjacent bands; moreover, the degree of proteolysis of the vWF subunit was normal in our case, whereas it was low in II E vWd (5). Type II B is easily distinguishable from this subtype (in spite of the fact that in both the platelet multimeric structure is intact), because in type II B RIPA is elicited at much lower ristocetin concentrations and thrombocytopenia occurs after DDAVP (13,14),but not in our patient. This subtype has some similarities with types II A, II C large vWF multimers are usually and II F. Even though defective in plasma and platelets of type II A patients, Weiss et al (15) reported that in some of them (called IIA-3) the multimeric structure of platelet vWF was intact, contrasting with defects of large multimers in plasma, as in our proband. On the other hand, her normal concentrations of proteolytic fragments of the vWF subunit represent a clear differentiation from the type II A phenotype, which is characterized by increased concentrations of the fragments (5). For both II C and this, the triplet structure is missing and replaced by a single band on intermediate resolution gels II C, however, is also characterized by (3). Type abnormalities of the multimeric structure of platelet vWF; and by recessive transmission (3), in contrast with the dominant pattern of inheritance of this variant. Both type II F and this case are characterized by an alteration of the multimeric pattern of plasma vWF, in contrast with a normal platelet pattern (6). Despite these similarities, however, our case differs from type II F for several aspects. In II F, there is an abnormal mobility of satellite bands of plasma vWF, seen on high-resolution gels (6), whereas in our proband the corresponding bands had a mobility similar to normal plasma. The transmission of the defect is dominant in our family, whereas it appears to be recessive in type II F (none of the parents of the type II F proband had a bleeding
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history and the one who could be examined had normal vWF measurements and BT) (6). The mechanism underlying this abnormality of vWF is unknown. In some type II plasmas proteolysis of the vWF subunit is higher than for normal plasma, whereas in others proteolysis is lower (5). In our case, there was a normal degree of proteolysis, a previously unreported finding in type II vWD. In accordance with the currently used phenotypic terminology, we propose that this variant of von Willebrand disease is designated type II I.
This work was supported in part by CNR, Progetto Finalizzato Ingegneria Genetica (PMM), grant HL-41135 from National Institutes of Health (PMM), and by the Health Department of the Veneto Region (FR).
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