von Willebrand Factor (VWF) is a large, multimeric adhesive glycoprotein that is involved in the formation of a platelet plug after vascular injury. In addition, VWF functions as a carrier protein for clotting factor VIII (FVIII) which prevents rapid clearance of plasma FVIII. Decreased levels of VWF or defects in VWF function are found in patients with von Willebrand disease (VWD). Quantitative defects of VWF protein in type 1 and 3 VWD affect plasma levels of both VWF and FVIII, whereas qualitative defects in type 2 VWD result in abnormal binding of VWF to platelets such as in type 2A, 2B, and 2M, or to FVIII such as in type 2N. Previous studies have demonstrated that FVIII binds VWF, which dramatically accelerates the proteolytic cleavage of multimeric VWF by ADAMTS13 under mechanically-induced shear stresses. This rate-enhancing effect of FVIII on VWF proteolysis appears to depend on the ability of FVIII to bind VWF, as a FVIII variant lacking the A3 acidic region fails to exhibit the cofactor activity that accelerates VWF proteolysis. To determine whether reduced FVIII binding in VWF type 2N variants affects VWF proteolysis, we examined the proteolytic cleavage of recombinant VWF type 2N variants in the presence of FVIII (and lyophilized platelets) for variants with a moderate VWD phenotype (Arg854Gln and His817Gln) or severe VWD phenotype (Arg763Gly, Arg782Trp, Thr791Met, and Arg782Trp + His817Gln). Recombinant VWF type 2N variants (37.5 μg/ml or 150 nM) were incubated with ADAMTS13 (25 nM) in the absence and the presence of various concentrations of FVIII (0-40 nM) with or without lyophilized platelets (0-600×103/μl) under fluid shear stress. The proteolytic cleavage products (350 kDa) were determined by 5% SDS-PAGE and Western blot under denaturing but non-reducing conditions. We show that the proteolytic cleavage of VWF type 2N variants by ADAMTS13 under these conditions was variably reduced as compared that of wild type VWF. The reduction in the cleavage rate was proportional to the degree of reduction in VWF FVIII binding activity, which was assessed with a microtiter assay, with the least cleavage by ADAMTS13 of the variants with the lowest FVIII binding activity. This reduced cleavage of the type 2N variants was not correlated with the binding affinity between the type 2N variants and ADAMTS13 protease. These results provide further evidence that binding of FVIII to VWF, which may alter VWF conformation, is necessary to accelerate VWF proteolysis by ADAMTS13 under fluid shear stress. This variability in ADAMTS13 cleavage may contribute to the heterogeneity of bleeding phenotype of type 2N VWD variants. The bleeding phenotype may be modulated not only by plasma FVIII levels, but also the extent of VWF proteolysis by ADAMTS13 under physiological fluid shear stress.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.