Phylogenetic and functional analysis of ADAMTS13 identifies highly conserved domains essential for allosteric regulation

Abstract

The metalloprotease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats member 13) prevents microvascular thrombosis by cleaving von Willebrand factor (VWF) within platelet-rich thrombi, and cleavage depends on allosteric activation of ADAMTS13 by the substrate VWF. Human ADAMTS13 has a short propeptide, metalloprotease (M), disintegrin-like (D), thrombospondin-1 (T), Cys-rich (C), and spacer (S) domains (proximal domains), followed by 7 T and 2 CUB (complement components C1r and C1s, sea urchin protein Uegf, and bone morphogenetic protein-1) domains (distal domains). Distal domains inhibit the catalytic proximal domains; binding of distal T8-CUB domains to the VWF D4 domain relieves autoinhibition and promotes cleavage of the nearby VWF A2 domain. However, the role of specific ADAMTS13 distal domains in this allosteric mechanism is not established. Assays of plasma ADAMTS13 from 20 placental mammals, birds, and amphibians show that allosteric regulation is broadly conserved, and phylogenetic analysis of 264 vertebrates shows the long propeptide, T3, T4, T6, and T6a domains have been deleted several times in placental mammals, birds, and fish. Notably, pigeon ADAMTS13 has only 3 distal T domains but was activated normally by human VWF D4 and cleaved VWF multimers, preferentially under fluid shear stress. Human ADAMTS13 constructed to resemble pigeon ADAMTS13 retained normal allosteric regulation and shear-dependent cleavage of VWF. Thus, the T3-T6 domains of human ADAMTS13 are dispensable. Conversely, deletion of T7 or T8 abolished allosteric activation. For most species, some sequence changes in the VWF substrate can markedly increase the rate of cleavage, suggesting that ADAMTS13 and VWF have not evolved to be optimal enzyme-substrate pairs. These properties may reflect evolutionary pressure to balance the risk for VWF-dependent bleeding and thrombosis.

Domain-specific mechanical modulation of VWF–ADAMTS13 interaction

“…It is conceivable that the MP-TSP6–CUB interdomain interaction may also exhibit dynamic bond characteristics, allowing hemodynamic forces to modulate the conformational change of ADAMTS13 and facilitate the cleavage of VWF. CUB has been reported to bind the spacer domain, which masks the previously found binding exosites on the spacer domain (Gao et al , 2006; Akiyama et al , 2009; Muia et al , 2019; Zhu et al , 2019). Thus, the A1A2A3 interactions with ADAMTS13 and MP-TSP6 observed in this study may not involve the exosites on the spacer domain.…”

Crystal structure and substrate-induced activation of ADAMTS13

“…These hemostatic enzymes are all serine proteases, and, to the best of our knowledge, this description of ADAMTS family allostery involving conformational activation of the MP domain may provide mechanistic insight into other family members. Previous descriptions referring to ADAMTS13 allosteric activation have used this term to describe the opening of ADAMTS13 by dissociation of the CUB domains from the Spacer domain 10–12,37 , rather than more classical enzyme allostery associated with structural shifts in the protease domain. We propose that the allosteric activation of ADAMTS13 serves to spatially and temporally localize its proteolytic function and confer its high substrate specificity.…”

“…The crystal structure of the ADAMTS13 DTCS domains has been resolved (i.e., without the catalytic MP domain) 8 . The C-terminal domains consist of seven further TSP1 repeats and two CUB domains that fold back and interact with the central Spacer domain 9–12 .

Fig. 1Von Willebrand factor (VWF) and its exosite interactions with ADAMTS13.

…”

International Council for Standardization in Haematology (ICSH) recommendations for laboratory measurement of ADAMTS13

“…The other domains of ADAMTS13 play a role in the attachment and unravelling of VWF, while VWF itself induces conformational changes in ADAMTS13, making it fully active. [3][4][5][6][7] Thrombotic microangiopathies (TMA) are a group of disorders characterized by thrombocytopenia and microangiopathic haemolytic anaemia (MAHA) resulting in varying degrees of organ damage; they are rare, but potentially life-threatening unless they are recognized and treated rapidly. They include thrombotic thrombocytopenic purpura (TTP) and haemolytic uraemic syndrome (HUS), 8,9 but may also be secondary to cancer, viral infection (eg HIV), organ or bone marrow transplantation, pregnancy (preeclampsia/eclampsia or HELLP syndrome), severe hypertension, drugs, autoimmune disorders, sepsis and disseminated intravascular coagulation.…”

“…It should be noted that ADAMTS13 activity can also vary significantly due to other factors, for example changes in assay buffer conditions (pH, ionic strength and surfactants) can affect ADAMTS13 activity due to allosteric mechanisms. [3][4][5][6] EDTA plasma cannot be used for activity assays, as it irreversibly disrupts the quaternary structure of ADAMTS13, causing false low/absent activity. In cases of unexpected absence of ADAMTS13 activity, where results do not fit the clinical picture and to exclude pre-analytical variables such as incorrect sample type, a prothrombin time or activated partial thromboplastin time on the sample can be helpful as grossly prolonged times suggest EDTA contamination or serum.…”