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.

Circumventing furin enhances factor VIII biological activity and ameliorates bleeding phenotypes in hemophilia models

“…Furin processing is critical for hFVIIa and hFIX biological activity. The deleterious effect of furin processing upon FVIII secretion is the converse of other clotting factors in which furin activity is necessary for efficient secretion of biologically functional proteins (13,(15)(16)(17). As shown in Figure 3 with the known supportive role of furin processing of FIX (15) and FVIIa 37, confirms the specific inhibition of furin by A1AT-PDX in these experimental models.…”

“…Furin is a serine protease responsible for the intracellular cleavage and processing of myriad proteins that contribute to health as well as to neoplastic, autoimmune, inflammatory, and infectious diseases (13,14). Cleavage by furin is also required for the biological activity of a number of proteins needed for hemostasis including factor VII (FVII), FIX, protein C, protein S, and vWF (13,(15)(16)(17). Recombinant expression systems for commercial FIX products have relied on cotransfection with furin to enhance production of biologically active protein (18,19).…”

Structure and Function of a Recombinant von Willebrand Factor Drug Candidate

“…FVIII binding capacity was determined using a modified ELISA combined with a chromogenic assay (ECA) developed in-house. 26 A pre-formed complex of VWF and FVIII was transferred to a microtiter plate, where an antibody directed against human VWF was immobilized. Bound FVIII was determined with a commercially available FVIII chromogenic assay, and the FVIII binding capacity was calculated from a fitted reference curve constructed from a normal human plasma assumed to have 1 U of VWF:FVIIIB capacity/1 IU VWF:Ag.…”

von Willebrand Factor: Form for Function

“…43 This stabilization effect is achieved through the noncovalent interaction between circulating VWF and plasma FVIII, which results in a tightly bound complex (K D ¼ 0.2-0.9 nM). [44][45][46][47] High-molecular-weight and low-molecular-weight multimeric VWF have similar binding affinities for FVIII. 48 VWD patients characterized with loss of high-molecularweight VWF multimers (i.e., VWD types 2A and 2B) have a FVIII-to-VWF antigen (VWF:Ag) ratio (a measure of FVIII levels normalized to VWF levels in plasma) comparable with that of healthy controls.…”

“…[35][36][37] Similar to mature VWF, ΔPro begins the N-termini with S764 but exhibits a sixfold reduced affinity for FVIII, suggesting that propeptide processing of the D'D3 assemblies optimizes VWF for FVIII association. 47 Interestingly, although each molecule of mature VWF contains multiple copies of FVIII-binding sites that may be saturated in vitro, the stoichiometry is about 1 FVIII molecule to 50-100 subunits of VWF in vivo. 44,48 Although a detailed structure of VWF in complex with FVIII has yet to be solved, several lines of evidence point to the importance of the D'D3 assemblies in binding FVIII.…”