ADAMTS13 resistance to protease inhibition / Background: ADAMTS13 is a metalloprotease that regulates the delicate balance between VWF multimeric length and its platelet capturing capacity. Unlike other ADAMTS and coagulation proteases, ADAMTS13 exhibits a prolonged half-life of several days as an active protease, suggesting that it is protected from inhibitors of metalloproteases in blood. Here, we investigate the mechanism by which ADAMTS13 is resistant to protease inhibition.
Methods: C-terminal domain truncations of ADAMTS13 (MDTCS and MD) and chimeras with ADAMTS5 (MD13/TCS5, M13/DTCS5, MD5/TCS13, and MD5(TCS-CUB13)) were generated. Metalloprotease domain segments from ADAMTS5 were swapped into MDTCS13 corresponding to the gatekeeper triad (R193, D217, and D252) (MDTCS-G), the variable loop (G236-S263) (MDTCS-V5), and the calcium-binding loop (R180-R193) (MDTCS-C5). MDTCS-GVC5 was generated to study these features simultaneously. Alpha 2-macrogloublin (A2M), tissue inhibitors of metalloproteinases (TIMPs), and small molecule inhibitor (Marimastat) were used as inhibitors, and tested using FRETS-VWF73 and Western blot.
Results: MDTCS, MD, MD13/TCS5, M13/DTCS5, MDTCS-G, MDTCS-V5, and MDTCS-C5 constructs were resistant to all inhibitors, whereas MD5/TCS13 was inhibited. The presence of the closed conformation attenuated MD5(TCS-CUB13) proteolysis by 50-fold, while displaying a slower rate of inhibition compared to MD5/TCS13. We report the kinetic parameters of the unique features of the metalloprotease domain (the gatekeeper triad, the variable loop, and the calcium-binding loop). Moreover, simultaneously swapping these features sensitized MDTCS-GVC5 to Marimastat.
Conclusion: Our findings reveal that the closed conformation confers global latency, while the metalloprotease domain confers local latency of ADAMTS13. The local latency is maintained by the flexibility of the variable loop and the calcium-binding loop, which fold across the active site cleft to restrict inhibitor and substrate access. Extensive engagement of exosites by VWF can readily displace these loops, thereby activating ADAMTS13 from its latent form. Altogether, we present novel insight into the mechanism by which ADAMTS13 is resistant to protease inhibition. / Thesis / Doctor of Philosophy (PhD) / Hemostasis is the body’s natural process to prevent bleeding and maintain blood flow. The ability of a blood protein, called VWF, to stop bleeding upon injury is regulated by the protein ADAMTS13. ADAMTS13 circulates in the blood for days, but its function cannot be stopped by inhibitors. Here, we investigate the mechanism by which ADAMTS13 is resistant to inhibition. We found that several structures of ADAMTS13, called domains and loops, protect it from inhibitors. Folding of the distal domains to the centre of ADAMTS13 partially protected ADAMTS13 from inhibitors. Further investigation revealed that two flexible loops close to the active site of ADAMTS13 were primarily responsible for protecting ADAMTS13 from inhibitors. We suggest that the flexibility of these loops guard against inhibition by folding across the active site. These results are important because advances have been made to use ADAMTS13 therapeutically in many clotting illnesses, such as strokes.
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/27448 |
| Date | January 2022 |
| Creators | Singh, Kanwal |
| Contributors | Kretz, Colin, Medical Sciences (Blood and Cardiovascular) |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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