American Journal of Hematology 40:20-27 (1992)

Further Evidence for Recessive Inheritance of Von Willebrand Disease With Abnormal Binding of Von Willebrand Factor to Factor Vlll M.F. Lopez-Fernandez, M.J. Blanco-Lopez, M.P. CastiAeira, and J. Batlle Servicio de Hematologia y Hemoterapia, Hospital Juan Canalejo-Teresa Herrera, La Corufia, Spain

A new family with a bleeding diathesis and FVlll deficiency secondary to abnormal binding of von Willebrand factor (vWF) to factor Vlll (FVIII) is described. Two propositi of this family, an 18-year-old male and a 33-year-old female, both with a history of epistaxis, bruising, bleeding from the gums, epistaxis, hemarthrosis, and hematoma, were analyzed. Also additional members of the same family with no bleeding history were also studied. The propositi showed normal vWF activities, low FVlll activity: one of them had been diagnosed as having hemophilia A and the other was a hemophilia A carrier. Both showed a very poor response to treatment with FVlll concentrates and desmopressin (DDAVP) but a good clinical response to cryoprecipitate. APTT was prolonged and no inhibitory activity was noticeable in their plasmas. Thirty-five units per kilogram body weight of Hemofil M was infused to both propositi and FVlll reached basal level within 60 minutes of the infusion. No FVlll response at all was observed in the female after intravenous DDAVP administration. However, the male who received the infusion of 35 U/kg body weight of Humate-P achieved a normal FVlll level that was maintained for 12 hours. Multimeric analysis of vWF was normal in all the members studied. Von Willebrand factor domain for FVlll binding was assayed in the two propositi and in six other members of the same family by using a non-isotopic and sensitive method, a modification of the one previously described, using the Hemofil M concentrate as exogenous FVIII. The data obtained showed that both propositi had similar binding to that observed by using plasma of a patient with severe von Willebrand disease. Furthermore, five siblings had a decreased binding of vWF to FVIII, when compared with plasma from normal individuals or patients with hemophilia A. We also observed that, for screening purpose, the ratio of bound FVlll/immobilized vWF (at saturation of the anti-vWF and offering of 1 U/ml of exogenous FVIII) distinguished two levels of abnormality (normal range 0.7rk1.15, propositi 0.0040.007, and remaining members affected 0.25-0.42). The most probable explanation is that the propositi are homozygous or double heterozygous, the other five siblings affected being heterozygous for a recessive vWF defect. This more accessible assay presented here may be of help in routine analysis for diagnosing this type of von Willebrand disease, which has important implications for therapy and genetic counseling. 6 1992 Wiley-Liss, Inc.

Key words: desmopressin, FVlll concentrate, bleeding diathesis

INTRODUCTION

Von Willebrand disease (vWD) results from quantitative and/or qualitative abnormalities of von Willebrand factor (vWF). Different qualitative defects have been found in vWD: Phenotypic alterations of multimeric structure of vWF and a functional abnormality, for example the enhanced interaction of vWF with platelet receptors in the presence of low concentrations of ristocetin [1,2]. Recently, a new disfunctional form of vWD was reported in which vWF fails to bind properly to normal

C? 1992 Wiley-Liss, Inc.

factor VlII procoagulant protein (FVIII) [3-51. This new form of vWD (the “Normandy variant”) may be diagReceived for publication April 2 3 . 199 I; accepted October 16, 1991. Address reprint requests to Javier Batlle. M.D.. Servicio de Hernatologia y Hemoterapia, Hospital Teresa Herrera, Carretera del Pasaje sin, 15006 La Coruiia. Spain. This work wa\ wpported in part by grants #90/0832. 9013229, and 9 110372 (FlSss, INSALUD. Spain)

Defective Binding of vWF to FVlll

nosed incorrectly as hemophilia A , since a low level of FVIlI can be the only alteration detected during routine analysis [ 5 ] .Furthermore, patients with this defect seem to respond poorly to commercial FVIII concentrates. Some initial evidence for a possible recessive inheritance of this abnormality has been reported [ 5 ] . We describe here a new family, from Spain, with two propositi, one of them diagnosed previously as having hemophilia A and the other a hemophilia A carrier. In both a defective binding of vWF to normal FVIIl has been demonstrated by using a sensitive non-isotopic method, simpler than those previously described [3.4]. Five siblings of the same family also had a partial binding defect, despite showing normal levels of FVIII in their plasmas, which would strongly suggest a heterozygous expression of a recessive form of vWD. MATERIALS AND METHODS

Both patients, six additional members of the same faniily, and the normal controls included in this study were niedication-free for at least 10 days before the nieasurement of skin bleeding time and/or before drawing venous blood (with the exception of the mother, 1-2, who was under treatment for hypertension). For comparison plasmas from a patient with severe von Willebrand disease (vWF:Ag < 0.01 U/dL), from two patients with the recently described type ID vWD [6], and from 26 patients with moderate or severe hemophilia A were included in this study. 1 -Deamino-8-D-arginine-vasopressin (DDAVP, Minirin, Ferring Laboratories, Malmo, Sweden) was infused to the propositi at a dose of 0.4 pg/kg. Thirty-five units per kilogram body weight (b.w.) of Heniofil M (Hyland Baxter, Glendale, CA) was infused to both propositi for FVIII recovery studies; 35 U/kg b.w. of Humate-P (Behringwerke, Marburg, Germany) was infused to one of the two propositi before dental extraction. For each kind of infusion blood samples were obtained before and at various times after the infusion. Reagents

Proteinase inhibitors included leupeptin, EDTA, N-ethylmaleimide (NEM), aprotinin, and phenylmethylsulfonylfluoride (PMSF) (Sigma Chemical Co., St. Louis, MO). Electrophoresis-pure reagents were acquired from Bio-Rad (Richmond, CAI. Agarose HGT(P) and Seaplaque from Seakem (Marine Colloids, FL) were used. Factor VlIl deficient plasma was obtained from Baxter Dade (Miami, FL), and the ainidolytic kits for measurement of VIII:C were from KabiVitruni (Coatest factor VIII, Stockholm). Protein G Sepharose 4 (MAb Trap, fast flow) was from Pharmacia (Uppsala, Sweden). Polystyrene 96-well microtitration plates were obtained from Dynatech Laboratories Inc. (Hamburg, Germany). A monoclonal antibody specific for vWF which bound all

21

the vWF multimers, previously described [7], was kindly provided by Ruggeri and Zimmerman. Human serum albumin was from lnstituto Berna (Madrid, Spain). Rabbit antiniouse immunoglobulins was from Dakopatts A/S (Denmark). A rabbit antihuman vWF Alkaline Phosphatase conjugate was from Serotec Ltd. (Oxford, UK). Hemofil M (specific activity before albumin addition: 2,0004,000 U/mg of protein) was used as a exogenous FVIII. Bovine serum albumin and p-nitrophenyl phosphate disodium in alkaline buffer solution were from Sigma (Sigma Chemical Co., St. Louis, MO). All other reagents were of the highest grade available. Methods

The methods used for blood collection and preparation of platelet-rich plasma (PRP) and platelet-poor plasma (PPP), as well as for the determination of ristocetininduced platelet agglutination (RIPA) in PRP were as previously described [8]. Plasma with proteinase inhibitors was obtained in the presence of 1 mM leupeptin, 14 mM NEM, and 5 mM EDTA [9]. Platelets were washed free of plasma constituents and then lysed following previously published procedures [ 10. I I ] . The activity of von Willebrand factor antigen (vWF:Ag) by Laurel1 technique, ristocetin cofactor (vWF:RCo). and RIPA were assayed as explained elsewhere [9]. Bleeding time (BT) was measured by using Simplate I1 (General Diagnostics, Anaheim, CA). Factor VIII procoagulant activity (VII1:C) was measured in plasma samples by a one stage method based on the partial thromboplastin time [ 121. The results of the assays were expressed in units per milliliter, using a standard plasma pooled from 30 healthy donors. For VII1:C the normal pool was calibrated in international units against the International Standard for FVIII and vWF, obtained by courtesy of Dr. Barrowcliffe (National Institute of Biological Standard and Control, Hampstead. UK). SDS-agarose electrophoresis (high and low resolution agarose gels) for analysis of vWF multimers was performed as per our previous publication [9]. Binding of exogenous FVIII to immobilized plasma vWF. It was assayed as described by Nishino et al. 131 but with some modifications. The IgG fraction of the monoclonal anti vWF, obtained by affinity chromatography using MAb Trap from ascitic fluid, was coated onto polystyrene well microtitration plates at a concentration of 5-6 pg/mL, in 0.1 M Na carbonate buffer (pH 9.3) overnight, at room temperature. The wells were washed and incubated with 3% bovine serum albumin at 37°C for 1 hour to block the remaining plastic active sites. Afterward, different plasma samples diluted in 3% human albumin in pH 7.2 PBS were added in duplicate to two different microtitration plates and then incubated at 37°C for 1 hr. After washing, endogenous FVlIl was eluted by incubation with 0.25 M CaCI,. Then, any possible ex-

22

Lopez-Fernandez et al.

cess of anti vWF monoclonal antibody was blocked with 0.43 mg/niL rabbit antimouse immunoglobulins in PBS at 37°C for 30 min, in order to avoid a direct binding of purified FVIII throughout the small amounts of vWF present in the Hemofil M . One set was assayed for vWF antigen (vWF:Ag) by incubating with rabbit anti human vWF:Ag alkaline phosphatase conjugate, and after washing, it was developed with p-nitrophenyl phosphate disodium in alkaline buffer solution ( 1 mg/mL) and the O.D. 405 was measured in an Easy Reader EAR 400 SLTLabinstruments (Salzburg, Austria). One unit of Hemofil M in PBS with 5%’human serum albumin (in one type of experiment), or different dilutions of the same product (0.25. 0.5, and 1 Uiml) (in the second type of experiment), were offered to the second set of microtitration plates. After incubation at 37°C for 1 hr, the wells were washed and the FVIII activity was measured as described by Nishino et al. (31. Exponential lines for test plasmas and normal pool were calculated (according to the equation y = a + blnx; x = immobilized vWF and y bound FVIII) for each individual experiment and statistical analysis was carried out by using the Student’s t test as well as the Mann-Whitney U test. Finally, the ratio bound FVIW immobilized vWF was calculated at saturation of the monoclonal anti-vWF (usually when 115 plasma dilution was used for normal vWF concentration). The normal range of this ratio was obtained by analyzing plasma from 33 normal individuals. The assay for vWF antigen did not show any significative difference between the behavior of vWF:Ag from normal individuals and that from the propositi, thus suggesting a normal immobilization to the monoclonal, as well as a normal detection by the rabbit antihuman vWF:Ag alkaline phosphatase conjugate used in this assay. Most of the FVllI bound to the vWF was eluded after incubation with 0.25 M CaCI?.

0

I 1 (0.38)

2 (0.43)

1 (0.25)

NORMAL (Range):

(0.70- 1.15)

Fig. 1. Pedigree of this family. The propositi are 11-2 and 11-3. The ratio bound FVlll/immobilized vWF (at saturation of monoclonal anti vWF antibody and offering of 1 U/mL of Hemophil M) is indicated in parentheses. Both propositi are hornozygous or doubly heterozygous and the five siblings are heterozygous for the vWF abnormality or abnormalities. 11-1 is normal. A characteristic autosomal recessive inheritance pattern is seen in this family.

RESULTS

The pedigree of this family is shown in Figure I . The two propositi were a 33-year-old female (11-2) and an 18-year-old inale (11-3), with a very long bleeding history characterized by episodes of epistaxis, bruising, prolonged hemorrhage following dental extractions, as well as many heniarthrosis and hematoma. She had also had postdelivery bleeding. They referred a poor clinical response to commercial FVIII concentrates and no response at all to desrnopressin (DDAVP). Patient 11-2 underwent surgery after cryoprecipitate infusion without showing abnormal bleeding. The remaining members of this family had no bleeding history. Consanguinity was not dis-

TABLE 1. Clinical Data of the Patients

Patient 1- I 1-2

11- 1

11-2 11-3 11-4 11-5 111- I

Normal range

Platelet count (X 109L)

APTT (sec)

Bleeding time (min)

VI1I:C (U/dL)

vWF:Ag (UldL)

vWF:RCo (U/dL)

250 I68 252 204 315 295 215 235 150-300

31 32 28 (53-74)a (62-72)a 32 32.5 28 28-33

10.5 12 7 (6-9)* (6-7)a 6.5 6 n.th