PB0315 - Characterizing Protease-Resistant ADAMTS13 Mutants

Background: ADAMTS13 is a metalloprotease that proteolytically regulates the length and thus, the platelet-binding capacity, of von Willebrand Factor. The regulation of ADAMTS13 activity is poorly understood, as it evades conventional regulatory mechanisms. Coagulation and neutrophil-derived proteases have been shown to cleave ADAMTS13 in vitro, but the significance of this degradation to its physiological regulation remains unknown. We hypothesize that ADAMTS13 is degraded by other proteases generated at sites of vessel injury, limiting its activity. Two common sites of cleavage on ADAMTS13 have been identified through in silico and in vitro analysis. Three ADAMTS13 variants have been developed: two with one of each site mutated, and one with both sites mutated. These mutants will be used to study the importance of proteolytic regulation of ADAMTS13.

Aims: To characterize protease-resistant mutants of ADAMTS13.

Methods: Each ADAMTS13 mutant and wild-type (WT-)ADAMTS13 were incubated with purified coagulation and neutrophil-derived proteases for 3 hours. Additionally, plasma was supplemented with WT-ADAMTS13 or the double mutant, and coagulation was initiated with tissue factor in the presence of tissue plasminogen activator. Cleavage patterns were visualized using western blotting.

Results: Coagulation proteases (plasmin, thrombin, kallikrein, and factor XIa) are capable of cleaving both predicted sites within WT-ADAMTS13. The double mutant exhibits near complete resistance to cleavage by these proteases over 3 hours. Resistance to degradation by neutrophil-derived proteases human proteinase 3, elastase, and cathepsin G is prolonged in the double mutant, but additional cleavage sites for are still present. Moreover, WT-ADAMTS13 is degraded in plasma upon initiating coagulation and subsequent fibrinolysis, and the double mutant exhibits complete resistance to this degradation.

Conclusion(s): These findings validate the location of two protease-sensitive regions within ADAMTS13 and confirms the resistance of an ADAMTS13 mutant to degradation in plasma upon initiating coagulation and fibrinolysis.