LOCALIZATION OF A FACTOR VIII BINDING DOMAIN ON THE N-TERMINAL PORTION (FRAGMENT SpIII) OF VON WILLEBRAND FACTOR

Abstract

A new domain has been identified on the von Willebrand Factor (vWF) subunit. vWF binds to platelet glycoproteins GPIb and GPIIb/IIIa as well as to collagen and corresponding domains have been isolated. vWF also binds to Factor VIII (F.VIII). We show here that the corresponding domain is located on the N-terminal portion of the vWF subunit (residues 1 to 1,365). For this purpose, F.VIII was tested for its ability to bind to purified vWF degradation fragments obtained by digestion with S.aureus V-8 protease, ie a dimeric N-terminal fragment of 320 kd (SpIII) and a dimeric C-terminal fragment of 220 kd (SpII). Human F.VIII was purified from cryoprecipitate by immunoadsorption of F.VIII/vWF onto a monoclonal antibody (MAb) to vWF coupled to Sepharose, followed by elution using 0.25 M CaCl2. The F.VIII preparation contained 100 U/ml VIII:C and less than 0.001 XJ/ml vWFAg. The binding assay was performed using polystyrene tubes coated with 2 ug/ml of purified vWF, SpIII or SpII. Coated albumin, fibrinogen or fibronectin were used as controls. Purified F.VIII (0.1 to 2 U/ml VIII:C) was incubated in the coated tubes for 1 h at 37°C. Following washing, bound F.VIII was estimated in situ by one-stage clotting and chromogenic assays. Immunoradiometric assay with 125 I-MAbs to SpII or SpIII demonstrated that the amount of coated protein remained constant throughout the experiments. F.VIII bound in a dose-dependent manner to coated vWF and SpIII but not to SpII. Binding was specific for F.VIII as demonstrated by inhibition experiments. Bound F.VIII could be removed with 0.25 M CaCl2 and its coagulant activity inhibited by a MAb or an oligoclonal (homologous) antibody neutralising VIII:C. Binding of F.VIII to coated vWF or SpIII was also inhibited in a dose-dependent way by vWF or SpIII. In contrast, addition of SpII had no effect upon the binding. Binding of F.VIII to SpIII was confirmed using MAbs to vWF. Among 28 MAbs which bound SpIII and had no anti VIII:C activity, 12 inhibited binding of F.VIII whereas the others had no effect. Among the latter, 3 MAbs blocked binding of vWF or SpIII to GPIb and 6 MAbs inhibited binding of vWF or SpIII to collagen. Ten MAbs to SpII had no inhibitory effect upon binding of F.VIII. These results indicate that a F.VIII binding domain of vWF is located in the N-terminal portion of vWF (residues 1 to 1,365) and that it is distinct from the GPIb and collagen binding domains. The 12 MAbs to SpIII which block binding of F.VIII to vWF or SpIII should allow the precise localization of this new domain.