FACTOR VIII INTERACTIONS WITH PLATELET VON WILLEBRAND FACTOR

Abstract

In plasma, factor VIII circulates in a non-covalent complex with von Willebrand factor. This complex is an important component in hemostasis and thrombosis. A second pool of von Willebrand factor stored in the platelet alpha granules is not exposed to or complexed with factor VIII. Platelet von Willebrand factor was purified from normal human platelet lysates and its role in the factor VIII/von Willebrand factor interaction was examined using a solid phase assay system. The multimeric composition of purified platelet von Willebrand factor determined by sodium dodecyl sulfate-agarose gel electrophoresis was comparable to normal platelet lysate preparations. No factor VIII activity or antigen was associated with the platelet von Willebrand factor. Purified multimeric platelet von Willebrand factor was directly immobilized in microtiter wells and the factor VIII activity was determined using a chromogenic assay specific for factor VIII. In contrast, when recombinant factor VIII was added to platelet von Willebrand factor coated wells, it bound in a specific, dose-dependent, and saturable manner. Dissociation of recombinant factor VIII from the factor VIII/von Willebrand factor complex was demonstrated in the presence of 0.25M CaCL₂, or 20mM EDTA, or 1.5M NaCl. Recombinant factor VIII was unable to bind to reduced and alkylated platelet von Willebrand factor. Two monoclonal antibodies and two synthetic peptide sequences known to inhibit the factor VIII interaction with plasma von Willebrand factor were used to further characterize factor VIII binding to platelet von Willebrand factor. Incubation of recombinant factor VIII with the anti-factor VIII light chain monoclonal antibody 60-B inhibited subsequent binding of recombinant factor VIII to platelet von Willebrand factor in a dose-dependent manner. The binding site for this antibody has been localized between amino acid residues Lys-1674 to Glu-1684 of the factor VIII light chain. A pentadecapeptide was constructed spanning amino acid residues Val-1670 to Glu-1684 (VEMKKEDFDIYDEDE) of the factor VIII light chain. This peptide did not inhibit the binding of recombinant factor VIII to immobilized platelet von Willebrand factor; however, when this peptide was included in an incubation mixture containing the antibody 60-B and recombinant factor VIII, 60-B inhibition of recombinant factor VIII binding was diminished in a dose-dependent manner. A monoclonal anti-von Willebrand factor antibody, W5-6A, was used to investigate the factor VIII binding site on platelet von Willebrand factor. This binding site has been localized to amino acid residues Thr-78 to Thr-96 on the amino-terminus of the plasma von Willebrand factor subunit. In a competitive binding assay, W5-6A inhibited binding of recombinant factor VIII to platelet von Willebrand factor in a concentration-dependent manner. A second peptide was synthesized corresponding to amino acid residues Ala-74 to Trp-93 (APGETVKIGCNTCVCRDRKW) on the amino-terminus of the plasma von Willebrand factor subunit. It also inhibited the binding of recombinant factor VIII to platelet von Willebrand factor in a concentration-dependent manner. These findings demonstrate that platelet von Willebrand factor interacts with factor VIII in a manner similar to that previously described for plasma von Willebrand factor and may suggest another potential role for platelet von Willebrand factor in hemostasis and thrombosis.