Summary. Background: The multidomain metalloprotease ADAMTS13 regulates the size of von Willebrand factor (VWF) multimers upon their release from endothelial cells. How the different domains in ADAMTS13 control VWF proteolysis in vivo remains largely unidentified. Methods: Seven C-terminally truncated murine ADAMTS13 (mADAMTS13) mutants were constructed and characterized in vitro. Their ability to cleave VWF strings in vivo was studied in the ADAMTS13 )/) mouse. Results: Murine MDTCS (devoid of T2-8 and CUB domains) retained full enzyme activity in vitro towards FRETS-VWF73 and the C-terminal T6-8 (del(T6-CUB)) and CUB domains (delCUB) are dispensable under these assay conditions. In addition, mADAMTS13 fragments without the spacer domain (MDT and M) had reduced catalytic efficiencies. Our results hence indicate that similar domains in murine and human ADAMTS13 are required for activity in vitro, supporting the use of mouse models to study ADAMTS13 function in vivo. Interestingly, using intravital microscopy we show that removal of the CUB domains abolishes proteolysis of platelet-decorated VWF strings in vivo.In addition, whereas MDTCS is fully active in vivo, partial (del(T6-CUB)) or complete (delCUB) addition of the T2-8 domains gradually attenuates its activity. Conclusions: Our data demonstrate that the ADAMTS13 CUB and T2-8 domains influence proteolysis of platelet-decorated VWF strings in vivo.

The monoclonal antibody 6B4 has a potent antithrombotic effect in nonhuman primates by binding to the flexible loop, also known as the ␤-switch region (amino acids 230 -242), of glycoprotein Ib␣ (GPIb␣). This interaction blocks, in high shear stress conditions, the specific interaction between GPIb␣ and von Willebrand factor suppressing platelet deposition to the damaged vessel wall, a key event in the pathogenesis of arterial thrombosis. To understand the interactions between this antibody and its antigen at the amino acid level, we here report the identification of the paratope and epitope in 6B4 and GPIb␣, respectively, by using computer modeling and site-directed mutagenesis. The docking programs ZDOCK (rigid body docking) and HADDOCK (flexible docking) were used to model the interaction of 6B4 with GPIb␣ and to delineate the respective paratope and epitope. 6B4 and GPIb␣ mutants were constructed and assayed for their capacity to bind GPIb␣ and 6B4, respectively. From these data, it is found that the paratope of 6B4 is mainly formed by five residues: Tyr

secretion and contributes to thrombotic thrombocytopenic purpura in mice. J Thromb Haemost 2015; 13: 283-92.Summary. Background: Congenital thrombotic thrombocytopenic purpura (TTP) is characterized by mutations in the ADAMTS13 gene, which either impair protein secretion or influence ADAMTS13 (A Disintegrin-like And Metalloprotease domain with ThromboSpondin type-1 motif, member 13) activity. Phenotypic consequences of these mutations have not yet been evaluated in animal models for TTP. Objectives: To identify the in vitro effect of a novel ADAMTS13 mutation and to investigate whether this mutation induces TTP in vivo. Methods: All 29 ADAMTS13 exons with exon-intron boundaries of a patient with pregnancy-onset TTP were sequenced. Wildtype and mutant ADAMTS13 proteins were both transiently and stably expressed in human embryonic kidney cells, and their activity was evaluated in vitro using fluorescence resonance energy transfer and flow assays. Molecular dynamics simulations were performed to study Ca 2+ stability. Adamts13 -/-mice were hydrodynamically injected with wild-type and mutant expression plasmids and triggered with recombinant human von Willebrand factor. Results: We identified a novel heterozygous c.559G>C mutation in exon 6 of the proposita's ADAM-TS13 gene. This mutation resulted in a p.Asp187His substitution (p.D187H), which was located in the high affinity Ca 2+ -binding site in the metalloprotease domain of ADAMTS13. The homozygous p.D187H mutation down-regulated ADAMTS13 activity in vitro. Impaired proteolytic activity was linked to unstable Ca 2+ binding as visualized using a molecular dynamics simulation. In addition, the p.D187H mutation affects protein secretion in vitro. In Adamts13 -/-mice, the homozygous p.D187H mutation reduced ADAMTS13 secretion and activity and contributed to TTP when these mice were triggered with recombinant human von Willebrand factor. Conclusions: Our data indicate that the p.D187H mutation impairs ADAMTS13 activity and secretion and is responsible for TTP onset in mice.