COAGULATION AND TRANSFUSION MEDICINE Original Article The Evaluation of Factor VIII Binding Activity of von Willebrand Factor by Means of an ELISA Method Significance and Practical Implications ALESSANDRA CASONATO, PhD, ELENA PONTARA, PhD, PATRIZIA ZERBINATI, PhD, ALESSANDRO ZUCCHETTO, MD, AND ANTONIO GIROLAMI, MD Downloaded from https://academic.oup.com/ajcp/article/109/3/347/1758043 by guest on 28 September 2021 One of the functions of von Willebrand factor (vWF) is to serve as chromogenic assay and our ELISA. A patient who was homozy­ a carrier of clotting factor VIII (FVIII). Deficiency of this function gous for the R53W mutation and had no FVIII binding capacity results in the von Willebrand disease (vWD) variant type 2N, according to the chromogenic method showed undetectable FVIII which resembles hemophilia A. We describe a new sandwich binding by ELISA. The remaining two patients, one who was enzyme-linked immunosorbent assay (ELISA) to study the abil­ homozygous for the R91Q mutation and one with compound het­ ity of vWF to bind exogenous recombinant FVIII (rFVIII), in erozygosity for the R91Q and R53W mutations, showed which anti-vWF-coated plates are incubated with plasma vWF, markedly decreased FVIII binding by the chromogenic method followed by exogenous FVIII and a peroxidase-coupled anti- and yielded ELISA values ranging from 4 to 8 U/dL. Therefore, FVIII antibody. Dose-response curves obtained using normal although the two methods produce qualitatively similar results, plasma vWF and purified normal vWF revealed a hyperbolic rela­ the ELISA method offers the advantage of allowing quantifica­ tionship between the optical density and the vWF concentration. tion of the FVIII binding function. FVIII binding was also ana­ The assay allows the quantification of FVIII binding with values lyzed in 20 patients with type 1 vWD; we found a decrease of expressed in U/dL; 100 U/dL was the amount present in normal FVIII binding that was proportionate to the decrease in vWF lev­ plasma. The sensitivity and specificity of the method are demon­ els, showing a normal FVIII binding activity/vWF molecule ratio. strated by its ability to measure binding levels as low as 1 to 2 We define the binding activity measured by this assay as U/dL and the fact that no FVIII binding was observed using vWF:FVIII binding activity and propose its use in the functional plasma known to contain less than 1 U/dL vWF. To verify the analysis of vWF. (Key words: Factor VIII; von Willebrand factor; accuracy of the assay, three patients with type 2N vWD with char­ FVIII binding activity; von Willebrand disease; Type 2N vWD) acterized vWF gene mutations were studied using an existing Am J Clin Pathol 1998;109:347-352. Von Willebrand factor (vWF) is a high-molecular- includes vWF abnormalities characterized by defec­ weight glycoprotein that fulfills a crucial role in tive functions independent of multimer representa­ hemostasis by promoting platelet adhesion at the site tion. This group includes type 2N vWD, a variant of vascular injury1 and by serving as a carrier for clot­ previously called type Normandy vWD. Type 2N ting factor VIII (FVIII) in the plasma,2 thereby pro­ vWD is characterized by defective binding of vWF to tecting FVIII from proteolytic degradation.3'4 FVIII9-10 that is associated with normal platelet adhe­ Deficiency of vWF is responsible for the most fre­ sion. The phenotypic expression is characterized by quent inherited bleeding disorder, von Willebrand levels of FVIII that are substantially lower than the disease (vWD),5 in which quantitative defects (type 1 levels of vWF Although the condition is similar to and type 3) and molecular abnormalities (type 2) mild hemophilia A, its inheritance is not X-linked; have been described.6'7 According to the most recent nevertheless, misdiagnosis of hemophilia A has been proposed classification of vWD,8 the type 2 group described in type 2N vWD.11'12 Point mutations resulting in the amino acid substitutions Argl9Trp, Thr28Met, Arg53Trp, His54Gln and Arg91Gln have 13 16 From the University of Padua Medical School, Institute of Medical been reported in type 2N vWD. " Semeiotics, Padua, Italy. The FVIII-vWF interaction has been studied in This work was supported by grants from Ministero Universita' vitro using a chromogenic assay that estimates the Ricerca Scientifica e Tecnologica, Rome, Italy, and from Veneto exogenous FVIII binding to vWF immunoisolated Region, Venice, Italy. from plasma.4,5'8 We describe a new quantitative Manuscript received March 7,1997; revision accepted June 26,1997. Address reprint requests to Dr Casonato: Istituto di Semeiotica method that measures the binding of FVIII to Medica, Via Ospedale Civile 105, Padova, Italia. immobilized vWF. 347 348 COAGULATION AND TRANSFUSION MEDICINE Original Article MATERIALS AND METHODS RESULTS Patients and healthy volunteers were studied. Demonstration of the Specific Binding Informed consent was obtained in accordance with of Recombinant FVIII to Immobilized vWF the declaration of Helsinki. Fifty healthy volunteers (30 women and 20 men), 21 to 65 years of age, who A sandwich immunoassay was developed to had not taken medication for at least 2 weeks before quantify the capability of plasma vWF to bind to the study served as controls. Nineteen patients with exogenous rFVIII. As described in the "Materials and type 1 subtype platelet-normal, 1 patient with type 1 Methods" section, the assay used microtiter plates platelet-low, and 1 patient with type 3 vWD were coated with anti-vWF antibody, which were incu­ Downloaded from https://academic.oup.com/ajcp/article/109/3/347/1758043 by guest on 28 September 2021 studied. Blood was drawn from the antecubital vein bated with plasma samples followed by rFVIII and and anticoagulated using 3.8% sodium citrate (1:10 horseradish peroxidase-conjugated anti-FVIII anti­ vol/vol). Platelet-poor plasma was prepared and body. Figure 1 shows the dose-response curves bleeding time was measured as previously obtained with serial dilutions of purified vWF and described.17 Assays measuring vWF ristocetin cofac- vWF present in NP at a constant concentration of tor activity (vWF:RCo), vWF antigen (vWF:Ag), and exogenous rFVIII (1.0 U/mL). Results showed that FVIII activity (FVIILC) were previously reported.18-20 the binding of rFVIII was a function of the amount of The vWF was purified from human cryoprecipitates according to the method of De Marco and Shapiro.21 Binding Assay of Recombinant FVIII to Immobilized vWF Purified normal von Willebrand factor Microtiter wells in 96-well plates were coated 1,500 Normal pooled plasma overnight at 4°C with 200 uL of polyclonal anti-vWF Type 3 von Willebrand disease antibody (Dako, Amsterdam, the Netherlands), 1.5 ug/mL in 0.05 mol/L sodium carbonate-bicarbonate buffer, pH 9.6. After extensive washing, the wells were incubated with phosphate-buffered saline (PBS) en 1,000 containing 2% bovine serum albumin (BSA) for 1 c hour at room temperature. The following steps were D then performed at room temperature. After washing, 200 uL of selected plasma dilutions, prepared in PBS 8 containing 0.05% polysorbate (Tween) and 2% BSA, were incubated in the wells for 1 hour. Endogenous FVIII was then removed from the immobilized vWF by incubation with buffered 0.4 mol/L calcium chlo­ ride for 30 minutes. After washing selected dilutions of recombinant FVIII (rFVIII; Baxter, Deerfield, 111), prepared in a PBS-Tween-BSA mixture were added and incubated for different times. After washing, the 1:100 1:400 1:800 1:1,600 bound rFVIII molecule was detected by incubation with horseradish peroxidase-conjugated polyclonal Plasma Dilution anti-FVIII antibodies (Enzymes Research, South Bend, FIG 1. Binding of recombinant factor VIII (rFVIII) to immobilized von Ind). The color was developed by addition of ortho- Willebrand factor (vWF) from normal pooled plasma (NP), purified normal vWF, and type 3 von Willebrand disease (vWD) plasma. The phenylenediamine dihydrochloride. The values were dose-response curves were obtained by incubating immobilized vWF plotted against plasma vWF dilutions and were with a constant amount of rFVIII (1 U/mL) for 30 minutes at 22°C. expressed as U/dL, with 100 U/dL the value The bound rFVIII was detected and quantified using horseradish per­ obtained with a 1:25 dilution of normal pooled oxidase-conjugated anti-FVIII antibody. The color was developed by plasma (NP). The ratio of FVIII binding activity to addition of o-phenylenediamine dihydrochloride. The NP was serially diluted starting from 1:25, and purified normal vWF was serially vWF:Ag was also calculated to normalize the binding diluted starting at a concentration of 0.4 |ig/mL, which corresponds to function to the vWF levels. the amount of vWF present in a 1:25 dilution of NP. AJCP • March 1W8 CASONATO ET AL 349 FVIII Binding to v\ Evaluated by ELISA immobilized vWF molecule and that the decrease in Binding of FVIII to Plasma vWFFrom Healthy FVIII binding had a hyperbolic relationship to the Persons and Patients With Type 1 vWD decrease in vWF. The specificity of rFVIII binding to vWF was demonstrated by the dose-response curve Fifty healthy subjects were studied to define a nor­ obtained with normal purified vWF, which showed a mal range of FVIII-vWF binding; three dilutions (1:25, relationship similar to that obtained using NP at sim­ 1:50, 1:100) were performed for each sample. ilar concentrations (see Fig 1). As a further test for Moreover, vWF:Ag was also evaluated by using an possible nonspecific binding of rFVIII to plasma pro­ ELISA method that uses the same coated anti-vWF teins other than vWF, plasma from a patient with antibody at a similar concentration.