Abstract
We previously demonstrated that coagulation factor VIII (FVIII) accelerates proteolytic cleavage of von Willebrand factor (VWF) by A disintegrin and metalloprotease with thrombospondin type 1 repeats (ADAMTS13) under fluid shear stress. In this study, the structural elements of FVIII required for the rate-enhancing effect and the biological relevance of this cofactor activity are determined using a murine model. An isolated light chain of human FVIII (hFVIII-LC) increases proteolytic cleavage of VWF by ADAMTS13 under shear in a concentration-dependent manner. The maximal rate-enhancing effect of hFVIII-LC is ∼8-fold, which is comparable with human full-length FVIII and B-domain deleted FVIII (hFVIII-BDD). The heavy chain (hFVIII-HC) and the light chain lacking the acidic (a3) region (hFVIII-LCΔa3) have no effect in accelerating VWF proteolysis by ADAMTS13 under the same conditions. Although recombinant hFVIII-HC and hFVIII-LCΔa3 do not detectably bind immobilized VWF, recombinant hFVIII-LC binds VWF with high affinity (K(D), ∼15 nM). Moreover, ultra-large VWF multimers accumulate in the plasma of fVIII(-/-) mice after hydrodynamic challenge but not in those reconstituted with either hFVIII-BDD or hFVIII-LC. These results suggest that the light chain of FVIII, which is not biologically active for clot formation, is sufficient for accelerating proteolytic cleavage of VWF by ADAMTS13 under fluid shear stress and (patho) physiological conditions. Our findings provide novel insight into the molecular mechanism of how FVIII regulates VWF homeostasis.
Highlights
The structural component of factor VIII (FVIII) required for regulating von Willebrand factor (VWF) proteolysis is not fully understood
Biochemical Characterization of Recombinant FVIII Variants—We previously found that full-length FVIII accelerates proteolytic cleavage of VWF by ADAMTS13 under fluid shear stress [19]
These results suggest that the acidic a3 region in the light chain of FVIII is required for accelerating VWF proteolysis by ADAMTS13 under shear stress
Summary
The structural component of FVIII required for regulating VWF proteolysis is not fully understood. Ultralarge VWF multimers accumulate in the plasma of fVIII؊/؊ mice after hydrodynamic challenge but not in those reconstituted with either hFVIII-BDD or hFVIII-LC These results suggest that the light chain of FVIII, which is not biologically active for clot formation, is sufficient for accelerating proteolytic cleavage of VWF by ADAMTS13 under fluid shear stress and (patho) physiological conditions. We show that an isolated light chain of FVIII, which is biologically inactive for clot formation, is sufficient for accelerating proteolytic cleavage of VWF by ADAMTS13 in vitro using a fluid shear-based assay and in vivo using fVIIIϪ/Ϫ mice expressing FVIII variants via a hydrodynamic approach This rate-enhancing effect by FVIII light chain depends on its high affinity binding with VWF; a light chain of FVIII lacking the a3 region and a heavy chain of FVIII, which do not bind. Our findings may shed more light on the structure-function relationship of FVIII in regulation of the VWF-ADAMTS13 axis, which helps in understanding the clinical heterogeneity of patients with severe hemophilia A
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