scholarly journals The ADAMTS13 metalloprotease domain: roles of subsites in enzyme activity and specificity

Blood ◽  
2010 ◽  
Vol 116 (16) ◽  
pp. 3064-3072 ◽  
Author(s):  
Rens de Groot ◽  
David A. Lane ◽  
James T. B. Crawley
Keyword(s):  
Single Point ◽  
Minor Importance ◽  
Variable Regions ◽  
Cleavage Efficiency ◽  
Scissile Bond ◽  
A Minor ◽  
Von Willebrand ◽  
A2 Domain ◽  
Willebrand Factor

Abstract ADAMTS13 modulates von Willebrand factor (VWF) platelet-tethering function by proteolysis of the Tyr1605-Met1606 bond in the VWF A2 domain. To examine the role of the metalloprotease domain of ADAMTS13 in scissile bond specificity, we identified 3 variable regions (VR1, -2, and -3) in the ADAMTS family metalloprotease domain that flank the active site, which might be important for specificity. Eight composite sequence swaps (to residues in ADAMTS1 or ADAMTS2) and 18 single-point mutants were generated in these VRs and expressed. Swapping VR1 (E184-R193) of ADAMTS13 with that of ADAMTS1 or ADAMTS2 abolished/severely impaired ADAMTS13 function. Kinetic analysis of VR1 point mutants using VWF115 as a short substrate revealed reduced proteolytic function (kcat/Km reduced by 2- to 10-fold) as a result of D187A, R190A, and R193A substitutions. Analysis of VR2 (F216-V220) revealed a minor importance of this region. Mutants of VR3 (G236-A261) proteolysed wild-type VWF115 normally. However, using either short or full-length VWF substrates containing the P1′ M1606A mutation, we identified residues within VR3 (D252-P256) that influence P1′ amino acid specificity, we hypothesize, by shaping the S1′ pocket. It is concluded that 2 subsites, D187-R193 and D252-P256, in the metalloprotease domain play an important role in cleavage efficiency and site specificity.

Essential role of the disintegrin-like domain in ADAMTS13 function

Blood ◽  
2009 ◽  
Vol 113 (22) ◽  
pp. 5609-5616 ◽  
Author(s):  
Rens de Groot ◽  
Ajoy Bardhan ◽  
Nalisha Ramroop ◽  
David A. Lane ◽  
James T. B. Crawley
Keyword(s):  
Active Site ◽  
Catalytic Efficiency ◽  
Targeted Mutagenesis ◽  
Fold Reduction ◽  
Scissile Bond ◽  
And Function ◽  
Von Willebrand ◽  
A2 Domain ◽  
Willebrand Factor

ADAMTS13 is a highly specific multidomain plasma metalloprotease that regulates the multimeric size and function of von Willebrand factor (VWF) through cleavage at a single site in the VWF A2 domain. The precise role that the ADAMTS13 disintegrin-like domain plays in its function remains uncertain. Truncated ADAMTS13 variants suggested the importance of the disintegrin-like domain for both enzyme activity and specificity. Targeted mutagenesis of nonconserved regions (among ADAMTS family members) in the disintegrin-like domain identified 3 of 8 ADAMTS13 mutants (R349A, L350G, V352G) with reduced proteolytic activity. Kinetic analyses revealed a 5- to 20-fold reduction in catalytic efficiency of VWF115 (VWF residues 1554-1668) proteolysis by these mutants. These residues form a predicted exposed exosite on the surface of the disintegrin-like domain that lies approximately 26 Å from the active site. Kinetic analysis of VWF115 carrying the D1614A mutation suggested that Arg349 in the ADAMTS13 disintegrin-like domain interacts directly with Asp1614 in VWF A2. We hypothesize that this interaction assists in positioning the scissile bond within the active site of ADAMTS13 and therefore plays a major role in determining cleavage parameters (Km and kcat), as opposed to binding affinity (Kd) of ADAMTS13 for VWF, the latter being primarily determined by the spacer domain.

scholarly journals Extensive contacts between ADAMTS13 exosites and von Willebrand factor domain A2 contribute to substrate specificity

Blood ◽  
2008 ◽  
Vol 112 (5) ◽  
pp. 1713-1719 ◽  
Author(s):  
Weiqiang Gao ◽  
Patricia J. Anderson ◽  
J. Evan Sadler
Keyword(s):  
Von Willebrand Factor ◽  
Plasma Proteins ◽  
Scissile Bond ◽  
Substrate Cleavage ◽  
Α Helix ◽  
Substrate Binding Sites ◽  
Von Willebrand ◽  
A2 Domain ◽  
Willebrand Factor ◽  
Specific Distance

Abstract The metalloprotease ADAMTS13 efficiently cleaves only the Tyr1605-Met1606 bond in the central A2 domain of multimeric von Willebrand factor (VWF), even though VWF constitutes only 0.02% of plasma proteins. This remarkable specificity depends in part on binding of the noncatalytic ADAMTS13 spacer domain to the C-terminal α-helix of VWF domain A2. By kinetic analysis of recombinant ADAMTS13 constructs, we show that the first thrombospondin-1, Cys-rich, and spacer domains of ADAMTS13 interact with segments of VWF domain A2 between Gln1624 and Arg1668, and together these exosite interactions increase the rate of substrate cleavage by at least approximately 300-fold. Internal deletion of Gln1624-Arg1641 minimally affected the rate of cleavage, indicating that ADAMTS13 does not require a specific distance between the scissile bond and auxiliary substrate binding sites. Smaller deletions of the P2-P9 or the P4′-P18′ residues on either side of the Tyr1605-Met1606 bond abolished cleavage, indicating that the metalloprotease domain interacts with additional residues flanking the cleavage site. Thus, specific recognition of VWF depends on cooperative, modular contacts between several ADAMTS13 domains and discrete segments of VWF domain A2.

scholarly journals A novel binding site for ADAMTS13 constitutively exposed on the surface of globular VWF

Blood ◽  
2009 ◽  
Vol 114 (13) ◽  
pp. 2819-2828 ◽  
Author(s):  
Sara Zanardelli ◽  
Alain C. K. Chion ◽  
Evelyn Groot ◽  
Peter J. Lenting ◽  
Thomas A. J. McKinnon ◽  
...  
Keyword(s):  
Surface Plasmon Resonance ◽  
Binding Site ◽  
Surface Plasmon ◽  
Binding Assay ◽  
Initial Step ◽  
Static Conditions ◽  
Von Willebrand ◽  
A2 Domain ◽  
Willebrand Factor

AbstractADAMTS13 metalloprotease regulates the multimeric size of von Willebrand factor (VWF) by cleaving the Tyr1605-Met1606 bond in the VWF A2 domain. The mechanisms of VWF recognition by ADAMTS13 have yet to be fully resolved. Most studies have focused on the role of exosites within the VWF A2 domain, involved in interaction with the ADAMTS13 spacer domain. In the present study, we expressed different C-terminal domain VWF fragments and evaluated their binding to ADAMTS13 and its truncated mutants, MDTCS and del(TSP5-CUB). Using plate binding assay and surface plasmon resonance, we identified a novel ADAMTS13 binding site (KD ∼ 86 nM) in the region of VWF spanning residues 1874 to 2813, which includes the VWF D4 domain and that interacts with the C-terminal domains of ADAMTS13. We show that the interaction occurs even when VWF is in static conditions, assumed to be globular and where the VWF A2 domain is hidden. We demonstrate that C-terminal VWF fragments, as well as an antibody specifically directed toward the VWF D4 domain, inhibit VWF proteolysis by ADAMTS13 under shear conditions. We propose that this novel VWF C-terminal binding site may participate as the initial step of a multistep interaction ultimately leading to proteolysis of VWF by ADAMTS13.

scholarly journals The importance of vicinal cysteines, C1669 and C1670, for von Willebrand factor A2 domain function

Blood ◽  
2010 ◽  
Vol 115 (23) ◽  
pp. 4910-4913 ◽  
Author(s):  
Brenda M. Luken ◽  
Luke Y. N. Winn ◽  
Jonas Emsley ◽  
David A. Lane ◽  
James T. B. Crawley
Keyword(s):  
Crystal Structure ◽  
Disulfide Bond ◽  
Von Willebrand Factor ◽  
Thermal Unfolding ◽  
Von Willebrand ◽  
A2 Domain ◽  
Domain Stabilization ◽  
Willebrand Factor ◽  
Increased Susceptibility

Abstract The von Willebrand factor (VWF) A2 crystal structure has revealed the presence of a rare vicinal disulfide bond between C1669 and C1670, predicted to influence domain unfolding required for proteolysis by ADAMTS13. We prepared VWF A2 domain fragments with (A2-VicCC, residues 1473-1670) and without the vicinal disulfide bond (A2-ΔCC, residues 1473-1668). Compared with A2-ΔCC, A2-VicCC exhibited impaired proteolysis and also reduced binding to ADAMTS13. Circular dichroism studies revealed that A2-VicCC was more resistant to thermal unfolding than A2-ΔCC. Mutagenesis of C1669/C1670 in full-length VWF resulted in markedly increased susceptibility to cleavage by ADAMTS13, confirming the important role of the paired vicinal cysteines in VWF A2 domain stabilization.

scholarly journals Unraveling the scissile bond: how ADAMTS13 recognizes and cleaves von Willebrand factor

Blood ◽  
2011 ◽  
Vol 118 (12) ◽  
pp. 3212-3221 ◽  
Author(s):  
James T. B. Crawley ◽  
Rens de Groot ◽  
Yaozu Xiang ◽  
Brenda M. Luken ◽  
David A. Lane
Keyword(s):  
Von Willebrand Factor ◽  
Conformational Changes ◽  
Conformational Transformation ◽  
Vessel Injury ◽  
Scissile Bond ◽  
Von Willebrand ◽  
A2 Domain ◽  
Willebrand Factor ◽  
Natural Inhibitors ◽  
Multiple Interactions

Abstract von Willebrand factor (VWF) is a large adhesive glycoprotein with established functions in hemostasis. It serves as a carrier for factor VIII and acts as a vascular damage sensor by attracting platelets to sites of vessel injury. VWF size is important for this latter function, with larger multimers being more hemostatically active. Functional imbalance in multimer size can variously cause microvascular thrombosis or bleeding. The regulation of VWF multimeric size and platelet-tethering function is carried out by ADAMTS13, a plasma metalloprotease that is constitutively active. Unusually, protease activity of ADAMTS13 is controlled not by natural inhibitors but by conformational changes in its substrate, which are induced when VWF is subject to elevated rheologic shear forces. This transforms VWF from a globular to an elongated protein. This conformational transformation unfolds the VWF A2 domain and reveals cryptic exosites as well as the scissile bond. To enable VWF proteolysis, ADAMTS13 makes multiple interactions that bring the protease to the substrate and position it to engage with the cleavage site as this becomes exposed by shear. This article reviews recent literature on the interaction between these 2 multidomain proteins and provides a summary model to explain proteolytic regulation of VWF by ADAMTS13.

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

Blood ◽  
2019 ◽  
Vol 133 (17) ◽  
pp. 1899-1908 ◽  
Author(s):  
Joshua Muia ◽  
Jian Zhu ◽  
Suellen C. Greco ◽  
Karen Vanhoorelbeke ◽  
Garima Gupta ◽  
...  
Keyword(s):  
Fluid Shear Stress ◽  
Allosteric Regulation ◽  
Allosteric Activation ◽  
Complement Components ◽  
Enzyme Substrate ◽  
Morphogenetic Protein ◽  
Von Willebrand ◽  
A2 Domain ◽  
Willebrand Factor

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.

scholarly journals Role of calcium in regulating the intra- and extracellular cleavage of von Willebrand factor by the protease ADAMTS13

2017 ◽  
Vol 1 (23) ◽  
pp. 2063-2074 ◽  
Author(s):  
Shobhit Gogia ◽  
Anju Kelkar ◽  
Changjie Zhang ◽  
Kannayakanahalli M. Dayananda ◽  
Sriram Neelamegham
Keyword(s):  
Von Willebrand Factor ◽  
Calcium Binding ◽  
Key Points ◽  
Platelet Binding ◽  
Von Willebrand ◽  
A2 Domain ◽  
Willebrand Factor ◽  
Intracellular Proteolysis

Key Points VWF A2-domain intracellular proteolysis within ECs is enhanced upon disrupting calcium binding (eg, in VWD type 2A mutants). VWF string cleavage on ECs is calcium independent and is strongly dependent on platelet binding.

Von Willebrand disease and Weibel-Palade bodies

2010 ◽  
Vol 30 (03) ◽  
pp. 150-155 ◽  
Author(s):  
J. W. Wang ◽  
J. Eikenboom
Keyword(s):  
Platelet Adhesion ◽  
Coagulation Factor ◽  
Von Willebrand Disease ◽  
Inflammatory Factors ◽  
Limited Data ◽  
Storage Organelle ◽  
And Function ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryVon Willebrand factor (VWF) is a pivotal haemostatic protein mediating platelet adhesion to injured endothelium and carrying coagulation factor VIII (FVIII) in the circulation to protect it from premature clearance. Apart from the roles in haemostasis, VWF drives the formation of the endothelial cell specific Weibel-Palade bodies (WPBs), which serve as a regulated storage of VWF and other thrombotic and inflammatory factors. Defects in VWF could lead to the bleeding disorder von Willebrand disease (VWD).Extensive studies have shown that several mutations identified in VWD patients cause an intracellular retention of VWF. However, the effects of such mutations on the formation and function of its storage organelle are largely unknown. This review gives an overview on the role of VWF in WPB biogenesis and summarizes the limited data on the WPBs formed by VWD-causing mutant VWF.

Factor VIII Inhibitor Antibodies with C2 Domain Specificity Are Less inhibitory to Factor VIII Complexed with von Willebrand Factor

1996 ◽  
Vol 76 (05) ◽  
pp. 749-754 ◽  
Author(s):  
Suzuki Suzuki ◽  
Morio Arai ◽  
Kagehiro Amano ◽  
Kazuhiko Kagawa ◽  
Katsuyuki Fukutake
Keyword(s):  
Complex Formation ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Domain Specificity ◽  
Factor Viii Inhibitor ◽  
C2 Domain ◽  
Recombinant Fviii ◽  
Von Willebrand ◽  
A2 Domain ◽  
Willebrand Factor

SummaryIn order to clarify the potential role of von Willebrand factor (vWf) in attenuating the inactivation of factor VIII (fVIII) by those antibodies with C2 domain specificity, we investigated a panel of 14 human antibodies to fVIII. Immunoblotting analysis localized light chain (C2 domain) epitopes for four cases, heavy chain (A2 domain) epitopes in five cases, while the remaining five cases were both light and heavy chains. The inhibitor titer was considerably higher for Kogenate, a recombinant fVIII concentrate, than for Haemate P, a fVIII/vWf complex concentrate, in all inhibitor plasmas that had C2 domain specificity. In five inhibitor plasmas with A2 domain specificity and in five with both A2 and C2 domain specificities, Kogenate gave titers similar to or lower than those with Haemate P. The inhibitory effect of IgG of each inhibitor plasma was then compared with recombinant fVIII and its complex with vWf. When compared to the other 10 inhibitor IgGs, IgG concentration, which inhibited 50% of fVIII activity (IC50), was remarkably higher for the fVIII/vWf complex than for fVIII in all the inhibitor IgGs that had C2 domain reactivity. Competition of inhibitor IgG and vWf for fVIII binding was observed in an ELISA system. In 10 inhibitors that had C2 domain reactivity, the dose dependent inhibition of fVIII-vWf complex formation was observed, while, in the group of inhibitors with A2 domain specificity, there was no inhibition of the complex formation except one case. We conclude that a subset of fVIII inhibitors, those that bind to C2 domain determinants, are less inhibitory to fVIII when it is complexed with vWf that binds to overlapping region in the C2 domain.

Sign in / Sign up

Export Citation Format

Share Document