ADAMTS13 is a metalloprotease that regulates the length, and thus, the platelet-capturing capacity of von Willebrand factor. The regulation of ADAMTS13 activity remains poorly understood. Numerous circulating proteases cleave ADAMTS13 in vitro, impairing its activity, but the physiological significance of this mechanism remains unknown. Two commonly cleaved regions within ADAMTS13 were identified and mutants were developed: two with one of each region mutated (T4L and T8L mutants), one with both regions mutated (T4L/T8L or “double” mutant), and one with an additional elastase site mutated (T4L/T8L + I380G). This work characterizes the mutants’ resistance to proteolysis and compares the activity of the double mutant to wild-type ADAMTS13 (WT). Each mutant and WT was incubated with purified coagulation and neutrophil proteases, activated neutrophils, or added to plasma before initiating coagulation with or without tissue plasminogen activator. Cleavage patterns were visualized with western blot. FRETS-VWF73 and microfluidic flow assays were used to compare WT and mutant activity. Coagulation proteases cleave both predicted sites within WT, and the double mutant exhibits near complete resistance to cleavage over 3 hours. Resistance to degradation by neutrophil proteases is prolonged in the double mutant, but additional cleavage sites are present. Elastase cleavage is prevented in the T4L/T8L + I380G mutant. In plasma, WT is degraded upon initiating coagulation and subsequent fibrinolysis, which is prevented in the double mutant. WT is also degraded in the presence of activated neutrophils, and the double and T4L/T8L + I380G mutants exhibit improved but incomplete resistance. Finally, the mutants exhibit similar activity to WT using FRETS-VWF73 and the microfluidic assay. This work validates the location of two protease-sensitive regions within ADAMTS13 and confirms the resistance of the double mutant to coagulation proteases in vitro. Future work will complete the activity analysis, and compare the mutants’ therapeutic efficacy to WT in vivo. / Thesis / Master of Science (MSc) / Current drugs used to dissolve blood clots can cause major bleeding. Therefore, safer treatments need to be developed. An important step in the clotting pathway is platelet accumulation in the injured vessel. Platelets stick to string-like protein, von Willebrand Factor (VWF), and ADAMTS13 is a protein that regulates this by cutting VWF strings. ADAMTS13 shows promise as a treatment for clots without causing bleeding, but it is unclear how its activity is controlled. ADAMTS13 can be degraded by other proteins, however the importance of this process in the body is unknown. This work characterizes a degradation-resistant ADAMTS13 mutant, which may be used to study whether ADAMTS13 degradation reduces its therapeutic effectiveness. The mutant has normal VWF-cutting activity, is resistant to degradation by clotting proteins, and is partially resistant to proteins released by neutrophils, an important immune cell in clotting. Future studies will investigate its effectiveness at treating clots in animals.
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/28685 |
| Date | January 2023 |
| Creators | DeYoung, Veronica A |
| Contributors | Kretz, Colin, Medical Sciences (Thrombosis & Haemostasis & Atherosclerosis) |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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