P11. Abstract Title: Investigating the resistance of ADAMTS13 towards Alpha 2-Macroglobulin

Abstract

<h3>Background</h3> ADAMTS13 is a metalloprotease that regulates the platelet-capturing capacity of von Willebrand factor. ADAMTS13 is secreted in its active form and has a circulating half-life of 4-7 days despite the presence of relatively high concentrations of inhibitors known to target other members of the ADAMTS protease family. This suggests that ADAMTS13 is resistant to inhibition by natural protease inhibitors, such as Alpha 2-Macroglobin (A2M) and tissue inhibitors of metalloproteinases (TIMPs). ADAMTS13 is known to adopt a closed conformation whereby C-terminal CUB domains bind to the proximal SPACER domain. The impact of this closed form of ADAMTS13 on accessibilty of the active site is not currently known. <h3>Methods</h3> To better understand the mechanism of ADAMTS13 resistance to natural protease inhibitors, C-terminal deletion constructs of ADAMTS13 and chimeras between ADAMTS13/ADAMTS5 were generated. Resistance was tested by examining the activity of ADAMTS13 variants in the presence of A2M using FRETS-VWF73; and then confirmed by Western blot examining the presence of the cleaved form of A2M. <h3>Results</h3> Activity assay showed that both full length (FL) -ADAMTS13 and MDTCS were resistant towards A2M up to 24 hours. Further, FL-ADAMTS13 and MDTCS were unable to cleave A2M; and therefore, were not inhibited. The MD variant in the presence of A2M showed marked reduction in activity and cleaved A2M. Therefore, MD was inhibited by A2M. <h3>On-going Studies</h3> Additional studies with ADAMTS5 (MD5TCS13, MD5TCST1-CUB2(13) and MD13TCS5) are on-going and should shed additional light on this unique property of ADAMTS13. These experiments will be repeated using the 4 isoforms of TIMPs; which are direct inhibitors of the metalloprotease active site. <h3>Conclusion</h3> Based on our domain truncations of ADAMTS13, its resistance towards A2M is likely mediated by proximal domains closest to its active site. On-going and future studies will try to determine the exact mechanism of resistance.