Binding of ADAMTS13 to von Willebrand Factor

Abstract

ADAMTS13, a metalloprotease, cleaves von Willebrand factor (VWF) in plasma to generate smaller, less thrombogenic fragments. The interaction of von Willebrand factor with specific ADAMTS13 domains was characterized with a binding assay employing von Willebrand factor immobilized on a plastic surface. AD-AMTS13 binding was saturable and reversible. Equilibrium binding occurred within 2 h and the half-time for dissociation was ϳ4 h. Binding to von Willebrand factor was similar with either recombinant ADAMTS13 or normal plasma ADAMTS13; plasma from a patient who lacked ADAMTS13 activity showed no binding. The stoichiometry of binding was one ADAMTS13 per two von Willebrand factor monomers, and the K d was 14 nM. The ADAMTS13 metalloprotease and disintegrin domains did not bind VWF detectably. ADAMTS13 truncated after the first thrombospondin type 1 repeat bound VWF with a K d of 206 nM, whereas ADAMTS13 truncated after the spacer domain had a K d of 23 nM, which is comparable with that of full-length ADAMTS13. Truncation after the eighth thrombospondin type 1 repeat reduced the binding affinity by ϳ3-fold and truncation after the seventh thrombospondin type 1 repeat in addition to the CUB domains increased the affinity for von Willebrand factor by ϳ2-fold. Therefore, the spacer domain is required for ADAMTS13 binding to von Willebrand factor. The first thrombospondin repeat also affects binding, and the C-terminal thrombospondin type 1 and CUB domains of ADAMTS13 may modulate this interaction.von Willebrand factor (VWF) 1 is a large multimeric protein that promotes hemostasis by tethering platelets at sites of vascular injury (1). The largest multimers of VWF are most active in platelet interactions, and thrombosis can result if large multimers accumulate as a result of ADAMTS13 deficiency. VWF is secreted into plasma as ultra-large multimers that are proteolysed to smaller forms by the metalloprotease, ADAMTS13 (1-3). Deficiency of ADAMTS13 activity because of a congenital absence of the protein or because of the production of inhibitory anti-ADAMTS13 antibodies leads to thrombotic thrombocytopenic purpura (TTP) (4, 5). TTP is characterized by uncontrolled microvascular thrombosis that especially affects the cerebral and renal circulation (6).ADAMTS13 is a member of the ADAMTS family of metalloproteinases that are frequently involved in proteolysis of extracellular matrix proteins (7-11). The ADAMTS family is characterized by a modular domain structure consisting of a metalloproteinase domain, a disintegrin-like domain, a thrombospondin type 1 (TSP1) repeat, a cysteine-rich region, and a spacer domain (12). Most ADAMTS family members have a variable number of additional C-terminal TSP1 repeats. AD-AMTS13 contains seven additional TSP1 repeats and two CUB domains at its C terminus and is the most divergent member of the family (13-15).The CUB domains are unique to ADAMTS13 among the ADAMTS family members. CUB domains were first identified in the complement proteins C1r and C1s and have since been identified in ma...

Exosite interactions contribute to tension-induced cleavage of von Willebrand factor by the antithrombotic ADAMTS13 metalloprotease

“…ADAMTS13 binds with high affinity and cleaves either multimeric VWF or GST-VWF73, provided the spacer domain is present (12,23). Therefore, the structural requirements for ADAMTS13 to cleave small substrates like VWF73 are similar to those for cleaving multimeric VWF, which implies that interactions limited to the A2 domain are sufficient to determine the substrate specificity of ADAMTS13.…”

“…Nevertheless, VWF is the only known substrate of ADAMTS13 in plasma, even though ADAMTS13 is constitutively active (11). Furthermore, VWF is resistant to AD-AMTS13 until it is subjected to high fluid shear stress (7), adsorbed onto a surface (12), or treated with chaotropic agents such as urea (8) or guanidine hydrochloride (7).…”

stall Encodes an ADAMTS Metalloprotease and Interacts Genetically With Delta in Drosophila Ovarian Follicle Formation

“…The less severe truncation (stl pa49 ) would remove only the C-terminal spacer. The identical null phenotypes that are associated with both of these truncation alleles affirm the importance of the C-terminal spacer region, which mediates substrate specificity in other ADAMTS proteins (Flannery et al 2002;Zheng et al 2003;Majerus et al 2005). Finally, stl a16 was found to be the most polymorphic strain, and in this mutant we found several missense mutations.…”

“…First, several ADAMTSs undergo processing at the C terminus for regulated proteolytic activity, and the C-terminal spacer region has been shown to be important for substrate binding and the specificity of the enzyme's biological activity Majerus et al 2005). ADAMTS4 is cleaved extracellularly, thereby greatly increasing its aggrecanase activity (Flannery et al 2002;Gao et al 2002Gao et al , 2004, and ADAMTS1 loses a portion of its C terminus, thereby losing affinity for heparin (Rodriguez-Manzaneque et al 2000).…”

ADAMTS13, la protéase spécifique du clivage du facteur von Willebrand

“…de la molécule de VWF native [12]. Ainsi, seul le dépliement du VWF induit soit par les forces de cisaillement élevées du flux sanguin dans les microvaisseaux in vivo, soit par des agents dénaturants (urée, hydrochlorure de guanidine), ou son immobilisation sur une surface plastique [13] in vitro, permet l'exposition de ce pont peptidique et le rend donc accessible au clivage par ADAMTS13. De plus, cet accès est facilité par la liaison du VWF à certains ligands comme la P-sélectine (qui intervient dans la fixation du VWF à la surface des cellules endothéliales à la phase initiale de sa sécrétion dans le plasma [14]) ou la glycoprotéine Ib plaquettaire (qui permet la liaison du VWF aux plaquettes au sein du clou plaquettaire formé pour réparer une brèche vasculaire [15]).…”