Abstract Number: OC 66.1
Meeting: ISTH 2021 Congress
Theme: Platelet Disorders, von Willebrand Disease and Thrombotic Microangiopathies » ADAMTS13 and TTP
Background: ADAMTS13 is a metalloprotease that regulates the platelet-capturing capacity of von Willebrand factor (VWF). Unlike other ADAMTS and coagulation proteases, ADAMTS13 has a long circulating half-life (days) as an active protease, suggesting that it is resistance to inhibitors of metalloproteases in blood.
Aims: Herein, we seek to understand the mechanism(s) by which ADAMTS13 is resistant to inhibition.
Methods: C-terminal domain truncations of ADAMTS13 and chimeras with ADAMTS5 were generated. Synthetic peptides of VWF were synthesized and site-directed mutagenesis (SDM) was used to disrupt unique features of metalloprotease domain (gatekeeper triad and calcium-binding loop). Alpha 2-macroglobulin, tissue inhibitors of metalloproteinases, and small molecule inhibitor (marimastat) were used as inhibitors, and tested using FRETS-VWF73 and Western blot.
Results: MDTCS, MD, and MD13/TCS5 constructs were resistant to all inhibitors, while MD5/TCS13 was inhibited. Therefore the closed conformation does not protect ADAMTS13 from inhibition. Instead, the MD region may play an important role in protecting ADAMTS13 from inhibition. Engagement of the disintegrin domain exosite using synthetic peptides was unable to sensitize MDTCS or MD to inhibition. Mutagenesis studies of the metalloprotease domain are on-going to examine the role of the ionic interaction between R193, D217, and D252 (gatekeeper triad), and the calcium-binding loop (R180-R193).
Conclusions: Our data suggests that features within the metalloprotease domain restrict access to the active site of ADAMTS13. VWF may be the only substrate that can unlock ADAMTS13, which may explain why ADAMTS13 displays no off-target proteolysis and is protected from inhibitors. Delineating the mechanisms of ADAMTS13 regulation may help improve diagnoses and treatment of thrombotic thrombocytopenic purpura and other cardiovascular diseases.
To cite this abstract in AMA style:
Singh K, Sohrabipour S, Madarati H, Sparring T, Kretz C. Investigating the Resistance of ADAMTS13 towards Protease Inhibitors [abstract]. Res Pract Thromb Haemost. 2021; 5 (Suppl 2). https://abstracts.isth.org/abstract/investigating-the-resistance-of-adamts13-towards-protease-inhibitors/. Accessed August 16, 2022.« Back to ISTH 2021 Congress
ISTH Congress Abstracts - https://abstracts.isth.org/abstract/investigating-the-resistance-of-adamts13-towards-protease-inhibitors/