scholarly journals A p.Arg127Gln variant in GPIbα LRR5 allosterically enhances affinity for VWF: a novel form of platelet-type VWD

2021 ◽  
Author(s):  
Loredana Bury ◽  
Emanuela Falcinelli ◽  
Haripriya Kuchi Bhotla ◽  
Anna Maria Mezzasoma ◽  
Giuseppe Guglielmini ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Conformational Dynamics ◽  
Von Willebrand Disease ◽  
High Affinity ◽  
Laboratory Test Results ◽  
Hydrogen Deuterium ◽  
Von Willebrand ◽  
The Impact ◽  
First Time ◽  
Willebrand Factor

Gain-of-function (GoF) variants in GP1BA cause platelet-type von Willebrand disease (PT-VWD), a rare inherited autosomal dominant bleeding disorder characterized by enhanced platelet GPIbα-von Willebrand factor (VWF) interaction and thrombocytopenia. To date, only 6 variants causing PT-VWD have been described, 5 in the C-terminal disulfide loop of the VWF-binding domain of GPIbα and 1 in the macroglycopeptide. GoF GP1BA variants generate a high affinity conformation of the C-terminal disulfide loop with a consequent allosteric conformational change on another region of GPIbα, the leucine-rich-repeat (LRR) domain. We identified a novel GP1BA variant (p.Arg127Gln) affecting the LRR5 domain of GPIbα in a boy with easy bruising and laboratory test results suggestive of PT-VWD. We thus aimed to investigate the impact of the p.Arg127Gln variant on GPIbα affinity for VWF and on GPIbα structure. CHO cells expressing p.Arg127Gln GPIbα showed increased binding of VWF induced by ristocetin and enhanced tethering on immobilized VWF as compared with cells expressing wild-type (WT) GPIbα. Surface plasmon resonance confirmed that p.Arg127Gln enhances the binding affinity of GPIbα for VWF. Hydrogen-deuterium exchange mass spectrometryshowed that p.Arg127Gln of LRR, while having little effect on the dynamics of the LRR locally, enhances the conformational dynamics of the GPIbα C-terminal disulfide loop structure. Our data demonstrate for the first time that GoF variants outside the GPIbα C-terminal disulfide loop may be pathogenic and that aminoacidic changes in the LRR may cause allosterically conformational changes in the C-terminal disulfide loop of GPIbα inducing a conformation with high affinity for VWF.

The D′ domain of von Willebrand factor requires the presence of the D3 domain for optimal factor VIII binding

2018 ◽  
Vol 475 (17) ◽  
pp. 2819-2830 ◽  
Author(s):  
Małgorzata A. Przeradzka ◽  
Henriet Meems ◽  
Carmen van der Zwaan ◽  
Eduard H.T.M. Ebberink ◽  
Maartje van den Biggelaar ◽  
...  
Keyword(s):  
Factor Viii ◽  
Binding Affinity ◽  
Von Willebrand Factor ◽  
Conformational Changes ◽  
Effective Interaction ◽  
Von Willebrand Disease ◽  
Binding Assays ◽  
Surface Plasmon Resonance Analysis ◽  
Von Willebrand ◽  
Willebrand Factor

The D′–D3 fragment of von Willebrand factor (VWF) can be divided into TIL′-E′-VWD3-C8_3-TIL3-E3 subdomains of which TIL′-E′-VWD3 comprises the main factor VIII (FVIII)-binding region. Yet, von Willebrand disease (VWD) Type 2 Normandy (2N) mutations, associated with impaired FVIII interaction, have been identified in C8_3-TIL3-E3. We now assessed the role of the VWF (sub)domains for FVIII binding using isolated D′, D3 and monomeric C-terminal subdomain truncation variants of D′–D3. Competitive binding assays and surface plasmon resonance analysis revealed that D′ requires the presence of D3 for effective interaction with FVIII. The isolated D3 domain, however, did not show any FVIII binding. Results indicated that the E3 subdomain is dispensable for FVIII binding. Subsequent deletion of the other subdomains from D3 resulted in a progressive decrease in FVIII-binding affinity. Chemical footprinting mass spectrometry suggested increased conformational changes at the N-terminal side of D3 upon subsequent subdomain deletions at the C-terminal side of the D3. A D′–D3 variant with a VWD type 2N mutation in VWD3 (D879N) or C8_3 (C1060R) also revealed conformational changes in D3, which were proportional to a decrease in FVIII-binding affinity. A D′–D3 variant with a putative VWD type 2N mutation in the E3 subdomain (C1225G) showed, however, normal binding. This implies that the designation VWD type 2N is incorrect for this variant. Results together imply that a structurally intact D3 in D′–D3 is indispensable for effective interaction between D′ and FVIII explaining why specific mutations in D3 can impair FVIII binding.

scholarly journals Platelet dysfunction in platelet-type von Willebrand disease due to the constitutive triggering of the Lyn-PECAM1 inhibitory pathway

Haematologica ◽  
2021 ◽  
Author(s):  
Loredana Bury ◽  
Emanuela Falcinelli ◽  
Anna Maria Mezzasoma ◽  
Giuseppe Guglielmini ◽  
Stefania Momi ◽  
...  
Keyword(s):  
Intracellular Signaling ◽  
Von Willebrand Disease ◽  
Bleeding Tendency ◽  
Platelet Dysfunction ◽  
Platelet Disorder ◽  
Granule Secretion ◽  
Inhibitory Signaling ◽  
Von Willebrand ◽  
First Time ◽  
Willebrand Factor

Platelet-type von Willebrand disease (PT-VWD) is an inherited platelet disorder characterized by macrothrombocytopenia and mucocutaneous bleeding, of variable severity, due to gain-of-function variants of GP1BA conferring to glycoprotein Ibα (GPIbα) enhanced affinity for von Willebrand factor (VWF). The bleeding tendency is conventionally attributed to thrombocytopenia and large VWF-multimers depletion. Some clues, however, suggest that platelet dysfunction may contribute to the bleeding phenotype but no information on its characteristics and causes are available. Aim of the present study was to characterize platelet dysfunction in PT-VWD and shed light on its mechanism. Platelets from a PT-VWD patient carrying the p.M239V variant and from PT-VWD mice carrying the p.G233V variant showed a remarkable platelet function defect, with impaired aggregation, defective granule secretion and reduced adhesion under static and flow conditions. VWF-binding to GPIbα is known to trigger intracellular signaling involving Src-family kinases (SFKs). We found that constitutive phosphorylation of the platelet SFK Lyn induces a negative-feedback loop downregulating platelet activation through phosphorylation of PECAM1 on Tyr686 and that this is triggered by the constitutive binding of VWF to GPIbα binding. These data show for the first time that the abnormal triggering of inhibitory signals mediated by Lyn and PECAM1 may lead to platelet dysfunction.In conclusion, our study unravels the mechanism of platelet dysfunction in PT-VWD caused by deranged inhibitory signaling triggered by the constitutive binding of VWF to GPIbα which may significantly contribute to the bleeding phenotype of these patients.

Impact of mutations in the von Willebrand factor A2 domain on ADAMTS13-dependent proteolysis

Blood ◽  
2006 ◽  
Vol 107 (6) ◽  
pp. 2339-2345 ◽  
Author(s):  
Wolf Achim Hassenpflug ◽  
Ulrich Budde ◽  
Tobias Obser ◽  
Dorothea Angerhaus ◽  
Elke Drewke ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Von Willebrand Factor ◽  
Von Willebrand Disease ◽  
Von Willebrand ◽  
A2 Domain ◽  
The Impact ◽  
Willebrand Factor ◽  
High Molecular Weight Multimers

Abstract Classical von Willebrand disease (VWD) type 2A, the most common qualitative defect of VWD, is caused by loss of high-molecular-weight multimers (HMWMs) of von Willebrand factor (VWF). Underlying mutations cluster in the A2 domain of VWF around its cleavage site for ADAMTS13. We investigated the impact of mutations commonly found in patients with VWD type 2A on ADAMTS13-dependent proteolysis of VWF. We used recombinant human ADAMTS13 (rhuADAMTS13) to digest recombinant full-length VWF and a VWF fragment spanning the VWF A1 through A3 domains, harboring 13 different VWD type 2A mutations (C1272S, G1505E, G1505R, S1506L, M1528V, R1569del, R1597W, V1607D, G1609R, I1628T, G1629E, G1631D, and E1638K). With the exception of G1505E and I1628T, all mutations in the VWF A2 domain increased specific proteolysis of VWF independent of the expression level. Proteolytic susceptibility of mutant VWF in vitro closely correlated with the in vivo phenotype in patients. The results imply that increased VWF susceptibility for ADAMTS13 is a constitutive property of classical VWD type 2A, thus explaining the pronounced proteolytic fragments and loss of HMWM seen in multimer analysis in patients.

The Impact of Mutations in the A2 Domain of Von Willebrand Factor on its Cleavage by ADAMTS13.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3666-3666
Author(s):  
Wolf A. Hassenpflug ◽  
Ulrich Budde ◽  
Tobias Obser ◽  
Dorothea Angerhaus ◽  
Elke Drewke ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Von Willebrand Disease ◽  
Full Length ◽  
Hemorrhagic Diathesis ◽  
Primary Hemostasis ◽  
Von Willebrand ◽  
A2 Domain ◽  
The Impact ◽  
Willebrand Factor ◽  
Group 1

Abstract Von Willebrand factor (VWF) plays an important role in primary hemostasis as it mediates platelet adhesion to the vessel wall and subsequent platelet aggregation at the site of vascular injury. Since the adhesive function of VWF depends on its multimer size, the loss of high molecular weight multimers (HMWM) results in hemorrhagic diathesis as seen in classical von Willebrand disease (VWD) type 2A, which represents the most common qualitative defect of VWD. The size distribution of VWF multimers in plasma is strongly influenced - if not regulated - by the specific VWF cleaving protease ADAMTS13 that cleaves VWF at the Y1605-M1606 bond in the A2 domain. Mutations in classical VWD type 2A cluster in the A2 domain and earlier studies suggest that some of the mutations make VWF more susceptible to ADAMTS13 dependent proteolysis (group 2) while others decrease the secretion of VWF HMWM (group 1). Our aim was to investigate the impact of VWF A2 domain mutations on ADAMTS13 dependent proteolysis of VWF. We used recombinant human ADAMTS13 (rhuADAMTS13) to digest recombinant full-length VWF and a fragment spanning the VWF A1-A2-A3 domains, harboring 13 different mutations that we found in patients with VWD type 2A. Proteolysis was monitored by VWF multimer analysis and by SDS-PAGE of the VWF A1-A2-A3 fragment. Cleavage of full-length VWF resulted in multimer patterns similar to that seen in plasma of patients with VWD type 2A, confirming the specifity of the reaction. Eleven VWF mutants (C1272S, G1505R, S1506L, M1528V, delR1569, R1597W, V1607D, G1609R, G1629E, G1631D, E1638K) showed less HMWM and more pronounced proteolytic fragments than wildtype (wt) VWF digested under the same conditions. Co-expression of the wt allele attenuated the proteolysis-permissive phenotype. The G1629E mutation resulted in highly increased proteolysis, suggesting an important role of this residue in the interaction between VWF and ADAMTS13. Surprisingly, G1505E and I1628T mutations failed to increase cleavage of the full-length VWF by rhuADAMTS13. However, when these mutations were introduced in the monomeric VWFA1-A2-A3 fragments they (like others) allowed cleavage of the Y1605-M1606 bond even under non-denaturing conditions, suggestive of an increased proteolytic susceptibility since wt VWF is only cleaved under denaturing conditions. The differences between the assays of full-length VWF and A1-A2-A3 domain fragment might be due to the lack of shear in our assay. This study provides direct evidence that in VWD type 2A, VWF with mutations in the A2 domain is subject to increased cleavage by ADAMTS13. This includes mutations previously designated as group 1 (G1505R, S1506L and V1607D) suggesting that increased susceptibility to ADAMTS13 is a more general property of VWF with A2 domain mutations. Therefore future therapies for patients with VWD type 2A might target VWF cleavage by ADAMTS13. RhuADAMTS13 and VWF constructs with mutations in the A2 domain are valuable tools to investigate VWF cleavage under varying conditions. Further work should address the question how shear influences ADAMTS13 dependent cleavage of VWF mutants.

scholarly journals Structure and nucleotide-induced conformational dynamics of the Chlorobium tepidum Roco protein

2019 ◽  
Vol 476 (1) ◽  
pp. 51-66 ◽  
Author(s):  
Egon Deyaert ◽  
Margaux Leemans ◽  
Ranjan Kumar Singh ◽  
Rodrigo Gallardo ◽  
Jan Steyaert ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Bound States ◽  
Conformational Dynamics ◽  
Bound State ◽  
Chlorobium Tepidum ◽  
Leucine Rich Repeat ◽  
Hydrogen Deuterium ◽  
Gtpase Cycle ◽  
Hdx Ms ◽  
First Time

Abstract The LRR (leucine-rich repeat)–Roc (Ras of complex proteins)–COR (C-terminal of Roc) domains are central to the action of nearly all Roco proteins, including the Parkinson's disease-associated protein LRRK2 (leucine-rich repeat kinase 2). We previously demonstrated that the Roco protein from Chlorobium tepidum (CtRoco) undergoes a dimer–monomer cycle during the GTPase reaction, with the protein being mainly dimeric in the nucleotide-free and GDP (guanosine-5′-diphosphate)-bound states and monomeric in the GTP (guanosine-5′-triphosphate)-bound state. Here, we report a crystal structure of CtRoco in the nucleotide-free state showing for the first time the arrangement of the LRR–Roc–COR. This structure reveals a compact dimeric arrangement and shows an unanticipated intimate interaction between the Roc GTPase domains in the dimer interface, involving residues from the P-loop, the switch II loop, the G4 region and a loop which we named the ‘Roc dimerization loop’. Hydrogen–deuterium exchange coupled to mass spectrometry (HDX-MS) is subsequently used to highlight structural alterations induced by individual steps along the GTPase cycle. The structure and HDX-MS data propose a pathway linking nucleotide binding to monomerization and relaying the conformational changes via the Roc switch II to the LRR and COR domains. Together, this work provides important new insights in the regulation of the Roco proteins.

scholarly journals Type 2M and Type 2A von Willebrand Disease: Similar but Different

2016 ◽  
Vol 42 (05) ◽  
pp. 483-497 ◽  
Author(s):  
Emmanuel Favaloro ◽  
Leonardo Pasalic ◽  
Jennifer Curnow
Keyword(s):  
Hemophilia A ◽  
Von Willebrand Disease ◽  
Test Results ◽  
Laboratory Test Results ◽  
Similarities And Differences ◽  
Treatment Responses ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Future Management

Analogous to the differentiation between hemophilia A and B, respectively, reflecting deficiency in factor VIII (FVIII) and FIX, and increasing being recognized as reflecting clinically different disorders, types 2A and 2M von Willebrand disease (VWD) can also be shown to express both similarities and differences in their prevalence, genetic defects, laboratory test results, clinical features, and treatment responses. In this narrative review, we explore these two “subtypes” of type 2 VWD, identifying parallels and dissimilarities in various aspects of their presentation to clinicians and to scientists/laboratories. This differential will become increasingly important as we strive to provide personalized approaches to future management of patients with VWD, particularly in the emerging landscape of recombinant von Willebrand factor.

Platelet Activation and Aggregation Induced by Recombinant von Willebrand Factors Reproducing Four Type 2B von Willebrand Disease Missense Mutations

1998 ◽  
Vol 79 (01) ◽  
pp. 211-216 ◽  
Author(s):  
Lysiane Hilbert ◽  
Claudine Mazurier ◽  
Christophe de Romeuf
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Von Willebrand Disease ◽  
Platelet Glycoprotein ◽  
Missense Mutations ◽  
Inhibit Platelet Aggregation ◽  
Wild Type ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryType 2B of von Willebrand disease (vWD) refers to qualitative variants with increased affinity of von Willebrand factor (vWF) for platelet glycoprotein Ib (GPIb). All the mutations responsible for type 2B vWD have been located in the A1 domain of vWF. In this study, various recombinant von Willebrand factors (rvWF) reproducing four type 2B vWD missense mutations were compared to wild-type rvWF (WT-rvWF) for their spontaneous binding to platelets and their capacity to induce platelet activation and aggregation. Our data show that the multimeric pattern of each mutated rvWF is similar to that of WT-rvWF but the extent of spontaneous binding and the capacity to induce platelet activation and aggregation are more important for the R543Q and V553M mutations than for the L697V and A698V mutations. Both the binding of mutated rvWFs to platelets and platelet aggregation induced by type 2B rvWFs are inhibited by monoclonal anti-GPIb and anti-vWF antibodies, inhibitors of vWF binding to platelets in the presence of ristocetin, as well as by aurin tricarboxylic acid. On the other hand, EDTA and a monoclonal antibody directed against GPIIb/IIIa only inhibit platelet aggregation. Furthermore, the incubation of type 2B rvWFs with platelets, under stirring conditions, results in the decrease in high molecular weight vWF multimers in solution, the extent of which appears correlated with that of plasma vWF from type 2B vWD patients harboring the corresponding missense mutation. This study supports that the binding of different mutated type 2B vWFs onto platelet GPIb induces various degrees of platelet activation and aggregation and thus suggests that the phenotypic heterogeneity of type 2B vWD may be related to the nature and/or location of the causative point mutation.

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.

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