4 Engineered FVIII variants without clotting activity accelerate proteolytic cleavage of VWF by ADAMTS13 under shear: Implication for a potential novel therapy of TTP

Background Previous studies have demonstrated that factor VIII (FVIII) can bind von Willebrand factor (VWF) and accelerate its proteolytic cleavage by ADAMTS13 under mechanical shear. Plasma derived FVIII and recombinant FVIII concentrates are shown to reducing circulating ultra large VWF multimers in wild-type mice and in patients with hereditary thrombotic thrombocytopenic purpura (TTP). These results suggest that FVIII may be useful for treatment of TTP. However, an increased plasma level of FVIII may be a risk factor for venous thrombosis that limits its therapeutic use. We aim to reengineer FVIII to create FVIII variant that exhibits no clotting activity but may retain its binding to VWF that enhances ADAMTS13-dependent proteolysis of VWF proteolysis by ADAMTS13 under shear. Methods We used site-directed mutagenesis, recombinant protein expression and purification, shear-dependent cleavage assay and one stage clotting assay to fully characterize two FVIII variants (R372Q and R1689C). Results Purified recombinant FVIII-SQ and its variant exhibited molecular sizes of 165 kDa (single chain), 93 kDa (heavy chain), and 80 kDa (light chain) on a denatured and reduced gel in the absence of thrombin activation. Thrombin cleaved both heavy and light chains at R372, R740, and R1689 to generate 3 small fragments in FVIII-SQ. However, thrombin did not cleave at R372 or R1689 in the R372Q or R1689C variant as expected. Furthermore, no specific clotting activity was detected in R372Q and R1689C variants by one-stage clotting assay. Most importantly, recombinant R372Q and R1689C variants were able to accelerate the proteolytic cleavage of recombinant multimeric VWF by ADAMTS13 under shear in a concentration-dependent manner with the maximal increase of 3 to 6 folds. Conclusions Reengineered FVIII variants without clotting activity may be further explored for therapy of TTP and perhaps other arterial thrombosis through binding VWF and accelerating its proteolysis by ADAMTS13 under shear. The therapeutic efficacy may be accomplished without the increased risk of potential venous thrombosis.