Proteolytic Inactivation of ADAMTS13 By Activated Factor XI
Authors: Garland Kathleen , McCarty Owen J.T. , Puy Cristina 
Published: October 2019

Abstract
Background: ADAMTS13, a plasma metalloprotease, is secreted into blood as an active enzyme that cleaves and inactivates von Willebrand factor (VWF), which binds collagen, facilitating platelet adhesion under vascular flow. Plasma ADAMTS13 has a molecular weight of 200 kDa, consisting of a metalloprotease (MET) domain, a disintegrin-like domain, a first thrombospondin type-1 repeat (TSP1) domain, a Cys-rich domain, and a spacer domain. Moreover, the C-terminal domain of ADAMTS13 contains an additional seven TSP1 repeats and two CUB domains. ADAMTS13 has been shown to adopt a natural folded conformation, allowing its CUB domains to interact with its spacer domain. This more closed conformation prohibits the functional exosite on the spacer domain from interacting with its proteolytic site on the A2 domain of VWF. In plasma, globular ADAMTS13 will associate with VWF via necessary binding of the CUB domains to the VWF D4CK fragment. Under shear stress or flow conditions, bound ADAMTS13 will unfold leading to exposure of the spacer domain exosite and ultimately increased ADAMTS13 proteolysis VWF. Without the CUB domains, ADAMTS13 does not proteolyze VWF under flow conditions. To date, it is still uncertain how ADAMTS13 activity is regulated, and what impact this has on the inactivation of VWF. The serine proteases thrombin, activated FX (FXa), and plasmin have been shown to cleave and inactivate ADAMTS13. Based on the fact that congenital factor XI deficiencies are associated with bleeding disorders and that elevated levels of FXI is an independent risk factor for deep vein thrombosis and ischemic stroke, we hypothesize that the serine protease activated FXI (FXIa) inactivates ADAMTS13 leading to platelet aggregation and thrombus formation.
Aim: To determine whether FXIa is able to cleave and inactivate ADAMTS13.
Methods and results: Recombinant ADAMTS13 (250 nM) was incubated with FXIa (50 nM) for selected times (0-3 hours) at 37oC before being separated by SDS-PAGE and analyzed by Coomassie blue staining, resulting in the disappearance of the ADAMTS13 band (~200 kDa) and the appearance of lower molecular weight bands under reducing conditions. The presence of aprotinin, which inhibits FXIa activity, blocked the degradation of ADAMTS13 by FXIa.
Samples were analyzed by western blot to determine the cleavage site using an anti-ADAMTS13 antibody, which specifically binds the two CUB domains, and an anti-ADAMTS13 antibody which specifically binds the MET domain. ADAMTS13 has been shown to be cleaved by serine proteases, such as plasmin, thrombin and FXa. We incubated ADAMTS13 (200nM) with equivalent concentrations of plasmin, thrombin, FXa, FXIa, FXIIa or Kallikrein at 37oC over a time interval of 0-3 hours. The addition of Ca++(5 mM) was necessary for proteolytic activity of thrombin and FXa. We observed that the ability of FXIa to cleave ADAMTS13 was found to be similar to the ability of thrombin to cleave ADAMTS13. Neither FXa, kallikrein, nor FXIIa appeared to cleave ADAMTS13.
The antibody against the MET domain detected a single broad band at approximately 150 kDa. When the samples were analyzed with the antibody specific for the two CUB domains, a single broad band at approximately 50 kDa was detected, suggesting that the proteolysis of ADAMTS13 by FXIa preferentially occurs near the start of the first CUB domain. Interestingly, it has been previously reported that the CUB domains are necessary for VWF strand cleavage under flow conditions.
It has been shown that the cleavage of a fluorescence-quenching substrate, FRETS-VWF73, by ADAMTS13 was enhanced after CUB1-2 domain removal. We observed that after the incubation of ADAMTS13 (30nM) with FXIa (30nM) at 37oC for 3 hours, the activity of ADAMTS13 was increased. Analysis of the samples by western blot using an anti-ADAMTS13 MET antibody confirmed the generation of the 150 kDa fragment.
Conclusion: Our study suggests a novel molecular link between the regulation of VWF activity and FXI through inactivation of ADAMTS13. The results suggest that the hemostatic role of FXIa may be attributed not only to activation of FIX but also through limiting ADAMTS13-mediated VWF inactivation. Our future studies are focused on determining the physiological role of the proteolytic removal of the CUB domains of ADAMTS13 by FXIa under flow conditions by measuring platelet aggregation.

Disclosures
No relevant conflicts of interest to declare.