Mapping and functional studies of two alloantibodies developed in patientswith type 3 von Willebrand disease

Inhibitors against von Willebrand factor (vWF) developed in two unrelated m ultitransfused patients (patients 1 and 2) with severe (type 3) von Willebr and disease (vWD) were analyzed. Both inhibitors were identified as antibod ies of the IgG class by ELISA using immobilized purified VWF and either ser um or purified Ig from the patients. Typing, mapping and functional studies of both antibodies revealed significantly distinct properties. Patient 1 a ntibody contained all subclasses of IgG (1, 2. 3 and 4) whereas antibody fr om patient was a mixture of only IgG1 and 1. By ELISA using a series of imm obilized purified proteolytic fragments of VWF, patient 1 antibody mainly b ound to fragment SpIII and, to a lower extent, to fragments SpII and SpI: i t poorly bound to P34 and the 39/34 kDa fragment. In contrast, patient 2 an tibody only bound to fragments corresponding to the N-terminal portion of V WF but failed to bind to SpII. Functional studies were performed by testing the capacity of each antibody to inhibit VWF binding to its various ligand s. Both antibodies blocked VWF binding to Factor VIII (FVIII), fibrillar ty pe III collagen, bitiscetin and the subsequent induced binding to GPIb. Pat ient 1 antibody also blocked VWF binding to platelet GPIb when induced by r istocetin. However it failed to block VWF binding to GPIb when induced by b otrocetin as well as the binding of botrocetin itself to VWF. Our data thus suggest that this inhibitor does not recognize the GPIb-binding site on VW F bur the sites of VWF involved in its interaction with ristocetin. In cont rast, we observed that patient 2 antibody blocked vWF binding to platelet G PIb induced by either agonist as well as VWF binding to botrocetin. Finally , the effect of the antibodies was tested on vWF binding to GPIIb/IlIa. As expected from the mapping experiments, only Ige from patient 1 blocked the interaction while IgG from patient 2 had no effect. In conclusion, we have shown that two multitransfused patients with type 3 vWD have developed allo antibodies with similar propel ties to those of polyclonal antibodies but w ith distinct effects on the functions of vWF.