Binding of Factor VIII to von Willebrand Factor Is Enabled by Cleavage of the von Willebrand Factor Propeptide and Enhanced by Formation of Disulfide-Linked Multimers

Abstract

von Willebrand factor (vWF) is a multimeric adhesive glycoprotein with one factor VIII binding site/subunit. Prior reports suggest that posttranslational modifications of vWF, including formation of N-terminal intersubunit disulfide bonds and subsequent cleavage of the propeptide, influence availability and/or affinity of factor VIII binding sites. We found that deletion of the vWF propeptide produced a dimeric vWF molecule lacking N-terminal intersubunit disulfide bonds. This molecule bound fluorescein-labeled factor VIII with sixfold lower affinity than multimeric vWF in an equilibrium flow cytometry assay (approximate KDs, 5 nmol/L v 0.9 nmol/L). Coexpression of propeptide-deleted vWF with the vWF propeptide in trans yielded multimeric vWF that displayed increased affinity for factor VIII. Insertion of an alanine residue at the N-terminus of the mature vWF subunit destroyed binding to factor VIII, indicating that the native mature N-terminus is required for factor VIII binding. The requirement for vWF propeptide cleavage was shown by (1) a point mutation of the vWF propeptide cleavage site yielding pro-vWF that was defective in factor VIII binding and (2) correlation between efficiency of intracellular propeptide cleavage and factor VIII binding. Furthermore, in a cell-free system, addition of the propeptide-cleaving enzyme PACE/furin enabled factor VIII binding in parallel with propeptide cleavage. Our results indicate that high-affinity factor VIII binding sites are located on N-terminal disulfide-linked vWF subunits from which the propeptide has been cleaved.

Identifying Carriers of Type 2N von Willebrand Disease

“…Other type 2N forms appear to have a compound heterozygosity for 2N mutations and a VWF type 1 allele or silent allele (18,19). Moreover, a new category includes patients with a persistence of propeptide of VWF, that interferes with the FVIII binding capacity of VWF (12,20,21). VWF:FVIIIB assay, the specific test for diagnosing type 2N VWD, explores in vitro the capacity of a patient's VWF to bind exogenous FVIII, after the endogenous FVIII has been removed.…”

Why All the Fury over Furin?

“…Furin has >400 predicted protein substrates featuring the Arg-X-Arg/Lys-Arg↓-X sequence motif, including extracellular matrix proteins such as ADAM metalloproteinases, , bone morphogenetic protein 1 (BMP-1), collagen, fibrillin, and integrins. − Furin can process several signaling peptides, hormones, and growth factors such as endothelin 1, , growth hormone releasing hormone, insulin-like growth factor 1, natriuretic peptide precursors, , platelet-derived growth factor polypeptides, transforming growth factor beta (TGF-β), , parathyroid hormone, and vascular endothelial growth factors C and D. , The substrates also include serum proteins, such as albumin, coagulation factors IX and X, complement component 3, and von Willebrand factor, transmembrane receptors, such as insulin receptor, met proto-oncogene (hepatocyte growth factor receptor), notch 1 receptor, and low density lipoprotein related receptor, the membrane bound nonvoltage-gated sodium channel alpha ENaC-α, and others . Furin’s proteolytic processing of these substrates has been linked to multiple normal physiological and pathogenic processes, such as viral infection, bacterial toxin activation, cancer, − neurodegenerative disorders, diabetes, and atherosclerosis.…”

Factor VIII and von Willebrand factor interaction: biological, clinical and therapeutic importance

“…The FVIII binds to VWF within the first 272 residues of the mature N-terminal region of the VWF polypeptide (DÕ and D3 domains within the corresponding to residues 763-1035) [35][36][37][38][39]. Cleavage of the propeptide from the mature polypeptide is required for FVIII binding, however the prior involvement of the propeptide in mature VWF processing increases the subsequent affinity of VWF for FVIII by approximately 10-fold [40,41].…”