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 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.

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.

scholarly journals An autoantibody epitope comprising residues R660, Y661, and Y665 in the ADAMTS13 spacer domain identifies a binding site for the A2 domain of VWF

Blood ◽  
2010 ◽  
Vol 115 (8) ◽  
pp. 1640-1649 ◽  
Author(s):  
Wouter Pos ◽  
James T. B. Crawley ◽  
Rob Fijnheer ◽  
Jan Voorberg ◽  
David A. Lane ◽  
...  
Keyword(s):  
Polyclonal Antibodies ◽  
Catalytic Efficiency ◽  
Full Length ◽  
Human Monoclonal Antibodies ◽  
Thrombocytopenic Purpura ◽  
Domain Interactions ◽  
Von Willebrand ◽  
A2 Domain ◽  
Willebrand Factor ◽  
Reduced Activity

Abstract In the majority of patients with acquired thrombotic thrombocytopenic purpura (TTP), antibodies are directed toward the spacer domain of ADAMTS13. We have previously shown that region Y658-Y665 is involved. We now show that replacement of R660, Y661, or Y665 with alanine in ADAMTS13 reduced/abolished the binding of 2 previously isolated human monoclonal antibodies and polyclonal antibodies derived from plasma of 6 patients with acquired TTP. We investigated whether these residues also influenced cleavage of short von Willebrand factor (VWF) fragment substrate VWF115. An ADAMTS13 variant (R660A/Y661A/Y665A, ADAMTS13-RYY) showed a 12-fold reduced catalytic efficiency (kcat/Km) arising from greatly reduced (> 25-fold) binding, demonstrated by surface plasmon resonance. The influence of these residue changes on full-length VWF was determined with denaturing and flow assays. ADAMTS13-RYY had reduced activity in both, with proteolysis of VWF unaffected by autoantibody. Binding of ADAMTS13-RYY mutant to VWF was, however, similar to normal. Our results demonstrate that residues within Y658-Y665 of the ADAMTS13 spacer domain that are targeted by autoantibodies in TTP directly interact with a complementary exosite (E1660-R1668) within the VWF A2 domain. Residues R660, Y661, and Y665 are critical for proteolysis of short VWF substrates, but wider domain interactions also make important contributions to cleavage of full-length VWF.

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 METALLOPROTEASE ADAMTS-13

2019 ◽  
Vol 64 (4) ◽  
pp. 471-482
Author(s):  
A. V. Koloskov ◽  
A. A. Mangushlo
Keyword(s):  
Shear Stress ◽  
Coagulation System ◽  
Functional Roles ◽  
Closed Conformation ◽  
And Function ◽  
Available Information ◽  
Cryptic Epitopes ◽  
Von Willebrand ◽  
A2 Domain ◽  
Willebrand Factor

Introduction. The signifi cance of ADAMTS-13 extends beyond its key role in the pathogenesis of thrombotic thrombocytopenic purpura (TTP); there is evidence of a relationship between a decrease in the ADAMTS-13 activity and thrombotic events in acute myocardial infarction and ischemic stroke.Aim. To generalise available information on the structure and function of the metalloprotease ADAMTS-13.General findings. The biological function of ADAMTS-13 consists in the cleavage of ultra-large von Willebrand factor (vWF) multimers. The fact that its defi ciency causes the development of TTP provides a basis for understanding the function of vWF–cleaving protease. ADAMTS-13 has a domain structure. The functional roles of most ADAMTS-13 domains, as well as the key role of the ADAMTS-13-vWF interaction in the regulation of haemostasis, are defi ned. The conformational activation of ADAMTS-13 by vWF constitutes an important aspect of its function. After getting into the bloodstream, ultra-large vWF multimers quickly adopt a closed conformation, which becomes very resistant to ADAMTS-13 proteolysis in the absence of shear stress. Ultra-large plasma vWF multimers regain their sensitivity to ADAMTS-13 after being exposed to high fl uid shear stress, which unfolds the central vWF-A2 domain. The unfolding of a vWF molecule under shear stress conditions reveals previously hidden exosites in domain A2, which gradually increase the binding affi nity between ADAMTS-13 and vWF. The mechanism underlying the production of autoantibodies against ADAMTS-13 is unknown and requires further study. The masking of cryptic epitopes in the closed conformation of ADAMTS-13 prevents the formation of autoantibodies. Early antigen recognition of ADAMTS-13 occurs through surface-exposed epitopes in the C-terminal domains. More detailed information on the mechanisms underlying the interaction between ADAMTS-13 and the vWF can improve the understanding of mechanisms involved in the regulation of the coagulation system.Conflict of interest: the authors declare no confl ict of interest.Financial disclosure: the study had no sponsorship.

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.

Thrombocytopenia and platelet hypoaggregation induced by Bothrops asper snake venom

2005 ◽  
Vol 94 (07) ◽  
pp. 123-131 ◽  
Author(s):  
Mónica Soto ◽  
Teresa Escalante ◽  
Gilbert D. Loría ◽  
Raghuvir Arni ◽  
José María Gutiérrez ◽  
...  
Keyword(s):  
Degradation Products ◽  
Ex Vivo ◽  
Serine Proteinase ◽  
Α2 Macroglobulin ◽  
Hemorrhagic Activity ◽  
Lectin Family ◽  
And Function ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryThrombocytopenia and platelet dysfunction occur in patients bitten by Bothrops sp snakes in Latin America. An experimental model was developed in mice to study the effects of B. asper venom in platelet numbers and function. Intravenous administration of this venom induces rapid and prominent thrombocytopenia and ex vivo platelet hypoaggregation. The drop in platelet numbers was primarily due to aspercetin, a protein of the C-type lectin family which induces von Willebrand factor-mediated platelet aggregation/agglutination. In addition, the effect of class P-III hemorrhagic metalloproteinases on the microvessel wall also contributes to thrombocytopenia since jararhagin, a P-III metalloproteinase, reduced platelet counts. Hypoaggregation was associated with the action of procoagulant and defibrin(ogen)ating proteinases jararacussin-I (a thrombin-like serine proteinase) and basparin A (a prothrombin activating metalloproteinase). At the doses which induced hypoaggregation, these enzymes caused defibrin(ogen)ation, increments in fibrin(ogen) degradation products and D-dimer and prolongation of the bleeding time. Incubation of B. asper venom with batimastat and α2-macroglobulin abrogated the hypoaggregating activity, confirming the role of venom proteinases in this effect. Neither aspercetin nor the defibrin(ogen)ating and hypoaggregating components induced hemorrhage upon intravenous injection. However, aspercetin, but not the thrombin-like or the prothrombin-activating proteinases, potentiated the hemorrhagic activity of two hemorrhagic metalloproteinases in the lungs.

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.

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