Structure-function and regulation of ADAMTS-13 protease

Abstract

ADAMTS-13, a plasma reprolysin-like metalloprotease, cleaves von Willebrand factor (VWF). Severe deficiency of plasma ADAMTS-13 activity results in thrombotic thrombocytopenic purpura (TTP), while mild to moderate deficiencies of plasma ADAMTS-13 activity are emerging risk factors for developing myocardial and cerebral infarction, pre-eclampsia, and malignant malaria. Moreover, Adamts13(-/-) mice develop more severe inflammatory responses, leading to increased ischemia/perfusion injury and formation of atherosclerosis. Structure-function studies demonstrate that the N-terminal portion of ADAMTS-13 (MDTCS) is necessary and sufficient for proteolytic cleavage of VWF under various conditions and attenuation of arterial/venous thrombosis after oxidative injury. The more distal portion of ADAMTS-13 (TSP1 2-8 repeats and CUB domains) may function as a disulfide bond reductase to prevent an elongation of ultra-large VWF strings on activated endothelial cells and inhibit platelet adhesion/aggregation on collagen surface under flow. Remarkably, the proteolytic cleavage of VWF by ADAMTS-13 is accelerated by FVIII and platelets under fluid shear stress. A disruption of the interactions between FVIII (or platelet glycoprotein 1bα) and VWF dramatically impairs ADAMTS-13-dependent proteolysis of VWF in vitro and in vivo. These results suggest that FVIII and platelets may be physiological cofactors regulating VWF proteolysis. Finally, the structure-function and autoantibody mapping studies allow us to identify an ADAMTS-13 variant with increased specific activity but reduced inhibition by autoantibodies in patients with acquired TTP. Together, these findings provide novel insight into the mechanism of VWF proteolysis and tools for the therapy of acquired TTP and perhaps other arterial thrombotic disorders.

Keywords: ADAMTS13 protein; animals; arterial thrombosis; autoimmune diseases; factor VIII; human; thrombotic microangiopathies; von Willebrand factor (445-733).

Fig. 1. ADAMTS13 cleaves UL-VWF under various conditions

A. ADAMTS13 rapidly cleaves newly released UL-VWF or large VWF strings/bundles in the absence (A) and in the presence (B) of flow; ADAMTS13 also cleaves platelet-decorated UL-VWF or large VWF strings/bundles anchored on the endothelial cell surface, in solution, and within growing thrombi under fluid shear stress (C).

Fig. 2. Domain organisation and partial crystal structure of ADAMTS13

On the left, the domain organization of human mature ADAMTS13 is shown, which consists of a metalloprotease domain (M), a disintegrin-like domain (D), the first TSP1 repeat, a Cys-rich domain (C), and a spacer domain (S). In addition, the C-terminus contains 7 more TSP1 repeats (–8) and two CUB domains (C1 and C2). On the right, the surface and carton presentation of the crystal structure of ADAMTS13 disintegrin-like domain (Dis), first TSP1 repeat (TSP1), Cys-rich (Cys) and spacer domain (Spa) in addition to a modelled metalloprotease domain (based on the metalloprotease domains of ADAMTS4 and ADAMTS5).

Fig. 3. Zinc and calcium binding sites in the metalloprotease domain of ADAMTS13

A. The ADAMTS13 metalloprotease domain with an active site and three putative calcium-binding sites; B. Three histidine residues and one glutamic acid coordinate the zinc binding.

Fig. 4. Sequence alignment of the spacer domains of ADAMTS13 and other ADAMTS proteases

A. Domain organization of ADAMTS13; B. Sequence alignment of a partial spacer domain (Arg629-Lys681) from human (h), murine (m), and zebrafish (z) ADAMTS13 (A13), as well as a partial spacer domain of various ADAMTS family proteases (AD1–AD20). Boxed area in pink presents a region (exosite 3) that is highly conserved in the ADAMTS13-spacer domain but absent in the spacer domain in other ADAMTS proteases. C and D are the surface and ribbon representation of exosite 3, respectively.