Structural Elements of FVIII Required for Accelerating Proteolytic Cleavage of Von Willebrand Factor by ADAMTS13.

Abstract 849 ADAMTS13 ( A D isintegrin A nd M etalloprotease with T hrombo S pondin type 1 repeats-13) cleaves von Willebrand factor (vWF) at the Tyr1605-Met1606 sessile bond. An inability to cleave ultra large vWF by ADAMTS13 on endothelial cell surface and in blood may result in disseminated microvascular thromboses seen in patients with thrombotic thrombocytopenic purpura. Recent study has demonstrated that human coagulation factor VIII (FVIII) can accelerate proteolytic cleavage of human vWF by ADAMTS13 under fluid shear stress ( Cao et al, PNAS, 2008, 105:7416-21 ). However, the structural elements of FVIII required for such rate enhancing effect are not known. Using recombinant technology, biochemical assays and an animal model, we were able to show that an isolated light chain of FVIII (FVIII-LC) was sufficient to accelerate proteolytic cleavage of vWF by ADAMTS13 under fluid shear stress in a concentration-dependent manner. At concentration of 5 nM, FVIII-LC increased the proteolytic cleavage product of VWF (350 kDa) by approximately 5-7 fold, similar to wild type FVIII and B-domain deleted FVIII-SQ. The concentration of FVIII-LC achieving the half maximal rate enhancing effect (IC50) was estimated to be ∼2.0 nM. In contrast, an isolated heavy chain of FVIII (FVIII-HC) or a FVIII-LC with an acidic region being deleted (FVIII-LCda3) did not exhibit significant rate enhancing effect under the same conditions. A microtiter binding assay demonstrated that the FVIII-LC bound vWF with a dissociation constant ( K D ) of ∼17 nM, whereas FVIII-HC and FVIII-LCda3 did not bind the immobilized vWF detectably. These results suggest that binding of FVIII-LC to vWF may be necessary and sufficient to increase the susceptibility of vWF to proteolytic cleavage by ADAMTS13 under fluid shear stress. To corroborate our biochemical findings, we performed animal studies by investigating plasma vWF antigen levels and multimer distribution using mini-agarose gel electrophoresis and Western blot. We showed that even at baseline plasma vWF antigen and high molecular weight (HMW) vWF multimers in fVIII knockout mice ( fVIII -/- ) (n=18) were modestly increased compared with those of wild-type mice (n=14). Upon challenge by a hydrodynamic injection of phosphate-buffered saline (2 ml in 5 seconds), plasma vWF antigen and HMW-vWF multimer were dramatically increased in the fVIII −/− mice (n=10) at 48-hour post-injection as a result of endothelial cell activatin and injury. Supprisingly, this dramatic increase in plasma vWF antigen and HMW-vWF multimers were abrogated in the fVIII −/− mice with the similar hydrodynamic injection of saline containing a plasmid encoding canine FVIII-SQ. At 48-h post injection, plasma canine FVIII-SQ ranged from 150% to 700% of normal canine FVIII activity based on a Coatest assay (n=20). A microtiter binding assay further showed that canine FVIII-SQ bound immobilized murine vWF with a dissociation constant ( K D ) of ∼1.7 nM. These findings provide further evidence that FVIII may be a cofactor that regulates proteolytic processing of vWF by ADAMTS13 under both physiological and pathological conditions. The cofactor activity of FVIII depends on high affinity interactions between the FVIII light chain and vWF under fluid shear stress. Disclosures: No relevant conflicts of interest to declare.