A cluster of mutations in the D3 domain of von Willebrand factor correlates with a distinct subgroup of von Willebrand disease: type 2A/IIE

Blood ◽  
2010 ◽  
Vol 115 (23) ◽  
pp. 4894-4901 ◽  
Author(s):  
Reinhard Schneppenheim ◽  
Jan Jacques Michiels ◽  
Tobias Obser ◽  
Florian Oyen ◽  
Antje Pieconka ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Molecular Mechanisms ◽  
Intrinsic Property ◽  
Von Willebrand Disease ◽  
Phenotypic Characterization ◽  
Single Family ◽  
Distinct Subgroup ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Among the different phenotypes of von Willebrand disease (VWD) type 2A, we identified a particular subgroup with a high frequency of 29%, characterized by a relative decrease of large von Willebrand factor (VWF) multimers and decreased A Disintegrin And Metalloproteinase with ThromboSpondin type 1 motifs, member 13 (ADAMTS13)–mediated proteolysis previously described in a single family as VWD type IIE (VWD2A/IIE). Phenotype and genotype of 57 patients from 38 unrelated families displaying a particular multimer pattern resembling the original VWD2A/IIE were studied. Pathogenicity of candidate mutations was confirmed by expression studies and phenotypic characterization of recombinant mutants. Specific mutations were identified in all patients. Twenty-two different mutations, most of them affecting cysteine residues, 17 of them being novel, are clustering mainly in the VWF D3 domain and correlate with the VWD2A/IIE phenotype. An intracellular retention of most mutants and/or a defect of multimerization seem to be the main pathogenic molecular mechanisms. ADAMTS13 proteolysis of mutant VWF was not different from wild-type VWF in a static assay, suggesting that reduced in vivo proteolysis is not an intrinsic property of mutant VWF. Our study identified a distinct VWD subtype with a common molecular background which contributes significantly to the heterogeneous spectrum of VWD.

scholarly journals Molecular Characterization of the Von Willebrand Factor Type D Domain of Vitellogenin from Takifugu flavidus

Marine Drugs ◽  
2021 ◽  
Vol 19 (4) ◽  
pp. 181
Author(s):  
Kun Qiao ◽  
Caiyun Jiang ◽  
Min Xu ◽  
Bei Chen ◽  
Wenhui Qiu ◽  
...  
Keyword(s):  
Amino Acids ◽  
Von Willebrand Factor ◽  
Molecular Mechanisms ◽  
Protein Domain ◽  
Type D ◽  
Protein Toxin ◽  
Factor Type ◽  
Von Willebrand ◽  
Willebrand Factor

The von Willebrand factor type D (VWD) domain in vitellogenin has recently been found to bind tetrodotoxin. The way in which this protein domain associates with tetrodotoxin and participates in transporting tetrodotoxin in vivo remains unclear. A cDNA fragment of the vitellogenin gene containing the VWD domain from pufferfish (Takifugu flavidus) (TfVWD) was cloned. Using in silico structural and docking analyses of the predicted protein, we determined that key amino acids (namely, Val115, ASP116, Val117, and Lys122) in TfVWD mediate its binding to tetrodotoxin, which was supported by in vitro surface plasmon resonance analysis. Moreover, incubating recombinant rTfVWD together with tetrodotoxin attenuated its toxicity in vivo, further supporting protein–toxin binding and indicating associated toxicity-neutralizing effects. Finally, the expression profiling of TfVWD across different tissues and developmental stages indicated that its distribution patterns mirrored those of tetrodotoxin, suggesting that TfVWD may be involved in tetrodotoxin transport in pufferfish. For the first time, this study reveals the amino acids that mediate the binding of TfVWD to tetrodotoxin and provides a basis for further exploration of the molecular mechanisms underlying the enrichment and transfer of tetrodotoxin in pufferfish.

VON WILLEBRAND FACTOR (VWF) ANTIGENS IN URINE

1987 ◽  
Author(s):  
A M V Silveira ◽  
B Hessel ◽  
B Blombäck
Keyword(s):  
Monoclonal Antibodies ◽  
Von Willebrand Factor ◽  
High Performance ◽  
Molecular Size ◽  
Von Willebrand Disease ◽  
Enzyme Linked Immunosorbent Assay ◽  
Normal Urine ◽  
Von Willebrand ◽  
Willebrand Factor

Human urine was analyzed using a sensitive enzyme linked immunosorbent assay (ELISA) for von Willebrand factor (VWF) antigen. Urine of healthy persons contained VWF immunoreactivity. In the urine of a patient with severe von Willebrand disease, the VWF antigen was only detectable after intravenous infusion of VWF-Factor VIII concentrate. The VWF antigen in normal urine was analyzed by gel permeation high performance liquid chromatography (HPLC) and gel electrophoresis combined with immunoblotting. These analyses revealed three immunoreactive components of Mr 350 kDa, 60 kDa, and 20 kDa, respectively, the 60 kDa being the major component. Monoclonal antibodies of known specificity to VWF molecule were used in ELISA and immunoblotting to analyze urinary VWF. The three components reacted with an antibody to the central part of VWF, which is called fragment I, and contains the binding site for collagen. No significant immunoreac-tion was observed with monoclonal antibodies to the Nor C-terminal portions of VWF.VWF derivatives of molecular size similar to the largest urinary antigens were also observed in normal plasma. However, there is not an obvious relationship between these plasma forms and the products in urine since reduction of plasma and urine yields different products.These results indicate that VWF antigens excreted in normal urine are most likely fragments of VWF produced by limited degradation in vivo. This degradation preserves the central part of VWF molecule, the one which reacts with the antibody that blocks the binding of VWF to collagen.

scholarly journals Expressed full-length von Willebrand factor containing missense mutations linked to type IIB von Willebrand disease shows enhanced binding to platelets

Blood ◽  
1992 ◽  
Vol 79 (8) ◽  
pp. 2048-2055 ◽  
Author(s):  
PA Kroner ◽  
ML Kluessendorf ◽  
JP Scott ◽  
RR Montgomery
Keyword(s):  
Von Willebrand Factor ◽  
Messenger Rna ◽  
Von Willebrand Disease ◽  
Full Length ◽  
Platelet Glycoprotein ◽  
Missense Mutations ◽  
Mutant Proteins ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract von Willebrand disease (vWD) variant type IIB is an inherited bleeding disorder resulting from the spontaneous binding of defective von Willebrand factor (vWF) to platelets in vivo. To identify the molecular basis for type IIB vWD, we used reverse transcription and the polymerase chain reaction to examine the nucleotide sequence of the platelet glycoprotein (GP) Ib-binding domain encoded by the vWF messenger RNA in an affected family, and in an unrelated affected individual. We identified two different missense mutations linked with expression of type IIB vWD. These mutations, which lead to Pro574---- Leu and Val553----Met substitutions, respectively, were each introduced into the full-length vWF expression vector pvW198, and both wild-type (wt) and mutant vWF were transiently expressed in COS-7 cells. Binding assays showed that both mutant proteins showed significant non- ristocetin-dependent spontaneous binding to platelets, and that complete binding was induced by low concentrations of ristocetin that failed to induce platelet binding by wt vWF. The vWF/platelet interaction was inhibited by the anti-vWF monoclonal antibody (MoAb) AvW3, and the anti-GPIb MoAb AP1, which both block vWF binding to platelets. These results show that the identified missense mutations are the likely basis for the expression of type IIB vWD in these affected individuals.

Endothelial von Willebrand factor recruits platelets to atherosclerosis-prone sites in response to hypercholesterolemia

Blood ◽  
2002 ◽  
Vol 99 (12) ◽  
pp. 4486-4493 ◽  
Author(s):  
Gregor Theilmeier ◽  
Carine Michiels ◽  
Erik Spaepen ◽  
Ingrid Vreys ◽  
Désiré Collen ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Human Platelets ◽  
Platelet Glycoprotein ◽  
Perfusion Studies ◽  
Von Willebrand ◽  
Willebrand Factor

Platelets are thought to play a causal role during atherogenesis. Platelet-endothelial interactions in vivo and their molecular mechanisms under shear are, however, incompletely characterized. Here, an in vivo platelet homing assay was used in hypercholesterolemic rabbits to track platelet adhesion to plaque predilection sites. The role of platelet versus aortic endothelial cell (EC) activation was studied in an ex vivo flow chamber. Pathways of human platelet immobilization were detailed during in vitro perfusion studies. In rabbits, a 0.125% cholesterol diet induced no lesions within 3 months, but fatty streaks were found after 12 months. ECs at segmental arteries of 3- month rabbits expressed more von Willebrand factor (VWF) and recruited 5-fold more platelets than controls (P < .05, n = 5 and 4, respectively). The 3-month ostia had an increased likelihood to recruit platelets compared to control ostia (56% versus 18%, P < .0001, n = 89 and 63, respectively). Ex vivo, the adhesion of 3-month platelets to 3-month aortas was 8.4-fold increased compared to control studies (P < .01, n = 7 and 5, respectively). In vitro, endothelial VWF–platelet glycoprotein (GP) Ib and platelet P-selectin– endothelial P-selectin glycoprotein ligand 1 interactions accounted in combination for 83% of translocation and 90% of adhesion (P < .01, n = 4) of activated human platelets to activated human ECs. Platelet tethering was mainly mediated by platelet GPIbα, whereas platelet GPIIb/IIIa contributed 20% to arrest (P < .05). In conclusion, hypercholesterolemia primes platelets for recruitment via VWF, GPIbα, and P-selectin to lesion-prone sites, before lesions are detectable.

scholarly journals Clearance of von Willebrand factor

2008 ◽  
Vol 99 (02) ◽  
pp. 271-278 ◽  
Author(s):  
Olivier D. Christophe ◽  
Beatrijs D. Oortwijn ◽  
Peter J. Lenting ◽  
Cécile V. Denis
Keyword(s):  
Von Willebrand Factor ◽  
Half Life ◽  
Von Willebrand Disease ◽  
In Vivo Studies ◽  
Missense Mutations ◽  
Common Component ◽  
Starting Point ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryThe life cycle of von Willebrand factor (VWF) comprises a number of distinct steps, ranging from the controlled expression of theVWF gene in endothelial cells and megakaryocytes to the removal of VWF from the circulation. The various aspects of VWF clearance have been the objects of intense research in the last few years, stimulated by observations thatVWF clearance is a relatively common component of the pathogenesis of type 1 von Willebrand disease (VWD). Moreover, improving the survival of VWF is now considered as a viable therapeutic strategy to prolong the half-life of factor VIII in order to optimise treatment of haemophilia A. The present review aims to provide an overview of recent findings with regard to the molecular basis of VWF clearance. A number of parameters have been identified that influence VWF clearance, including its glycosylation profile and a number of VWF missense mutations. In addition, in-vivo studies have been used to identify cells that contribute to the catabolism of VWF, providing a starting point for the identification of receptors that mediate the cellular uptake ofVWF.Finally, we discuss recent data describing chemically modification of VWF as an approach to prolong the half-life of the VWF/FVIII complex.

The anti-von Willebrand factor aptamer ARC1779 increases von Willebrand factor levels and platelet counts in patients with type 2B von Willebrand disease

2012 ◽  
Vol 108 (08) ◽  
pp. 284-290 ◽  
Author(s):  
Petra Jilma-Stohlawetz ◽  
Paul Knöbl ◽  
James C. Gilbert ◽  
Bernd Jilma
Keyword(s):  
Von Willebrand Factor ◽  
Plasma Concentrations ◽  
Fold Increase ◽  
Von Willebrand Disease ◽  
Platelet Counts ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Ristocetin Cofactor

SummaryBlockade of hyperactive von Willebrand factor (VWF) by ARC1779 blunted the platelet drop induced by desmopressin in patients with type 2B von Willebrand disease (VWD). Thus, we hypothesised that ARC1779 may increase VWF levels and correct thrombocytopenia. Three thrombocytopenic patients suffering from type 2B VWD received a loading dose of 0.23 mg/kg ARC1779 followed by 4 μg/kg/min intravenously for 72 hours in a prospective clinical trial. ARC1779 was well tolerated and safe. Plasma concentrations of ARC1779 increased to 76 μg/ml (59–130) leading to an immediate decrease of free VWF A1 domains. VWF/FVIII levels increased as early as 12 h after start of infusion, peaked near the end of infusion, and returned to baseline at follow-up. VWF ristocetin cofactor activity (VWF:RCo) showed a median 10-fold increase 8 hours after end of infusion, while the median VWF-antigen and FVIII increase was less (5-fold and 4-fold, respectively). Most importantly inhibition of hyperactive VWF rapidly increased platelet counts from 40x109/l (38–58 x109/l) to a maximum of 146 x109/l (107–248 x109/l). In conclusion, ARC1779 markedly increases VWF/FVIII levels and most importantly improves or even corrects thrombocytopenia in VWD type 2B patients. This underscores the in vivo potency of ARC1779.

CALPAIN AND ELASTASE ARE NOT RESPONSIBLE FOR THE VON WILLEBRAND FACTOR FRAGMENTS IN NORMAL PLASMA AND IIA VON WILLEBRAND DISEASE

1987 ◽  
Author(s):  
S D Berkowitz ◽  
H Nozaki ◽  
K Titani ◽  
T Murachi ◽  
T S Zimmerman
Keyword(s):  
Von Willebrand Factor ◽  
Epitope Mapping ◽  
Human Leukocyte ◽  
Von Willebrand Disease ◽  
Neutral Protease ◽  
Leukocyte Elastase ◽  
Human Leukocyte Elastase ◽  
Von Willebrand ◽  
Willebrand Factor

Recent evidence suggests that proteolysis plays an important role in some forms of inherited and acquired von Willebrand disease (vWD). Using monoclonal epitope mapping, we have examined the proteolysis of the von Willebrand factor (vWF) subunit with platelet calcium activated neutral protease (CANP) and human leukocyte elastase and found that they are not responsible for the proteolytic cleavage sejen in normal individuals and IIA vWD. Previously we have shown that in vivo proteolysis of vWF is a normal event with a small but consistent proportion of plasma vWF being composed of 189, 176, and 140 kD fragments cleaved from the 225 kD subunit. In IIA vWD the proportion of cleaved vWF is increased. Because calcium activated neutral protease (CANP, calpain) and one or more enzymes released from polymorphonuclear leukocytes are known to proteolyze vWF in vitro with resultant loss of large multimers similar to that seen in IIA vWD, they have been suggested as being responsible for the proteolysis in vivo. We have now digested highly purified vWF with porcine CANP I and II and performed monoclonal epitope mapping on the resulting fragments. We found no difference in the size, location, and quantity of the fragments produced by calpain I versus calpain II. New fragments were detected of approximately 200, 170, 150, and 125 kD. There was no evidence for generation of the native fragments. Mapping of the 170 and 150 kD calpain-cleaved fragments revealed them to be from different parts of the molecule than the native 176 and 140 kD fragments. Digestion of vWF with human leukocyte elastase produced new fragments at 210/205, 190, 165, 145/140, and 130/125 kD. No generation of native fragments was detected. Monoclonal epitope mapping of the 190 and 145/140 kD elastase-cleaved bands proved that they come from opposite ends of the vWF molecule than the native 189 and 140 kD fragments, respectively. Therefore, CANP and human leukocyte elastase do not produce the proteolyzed fragments present in normal and IIA vWD and probably do not cause the loss of large multimers seen in that disorder.

Loss of cysteine 584 impairs the storage and release, but not the synthesis of von Willebrand factor

2014 ◽  
Vol 112 (12) ◽  
pp. 1159-1166 ◽  
Author(s):  
Viviana Daidone ◽  
Giovanni Barbon ◽  
Elena Pontara ◽  
Grazia Cattini ◽  
Lisa Gallinaro ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Von Willebrand Disease ◽  
Hek293 Cells ◽  
Loss Of Function ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryCysteines play a key part in von Willebrand factor (VWF) dimerisation and polymerisation, and their loss may severely affect VWF structure and function. We report on three patients with type 3 von Willebrand disease carrying the new c.1751G>T missense mutation that induces the substitution of cysteine 584 by phenylalanine (C584F), and the deletion of seven nucleotides in exon 7 (c.729_735del), producing a premature stop codon at position 454 (E244Lfs*211). VWF was almost undetectable in the patients’ plasma and platelets, while a single, poorly represented, oligomer emerged on plasma VWF multimer analysis. No post-DDAVP increase in VWF and factor VIII was observed. Expressing human recombinant C584F-VWF in HEK293T cells showed that C584F-VWF was synthesised and multimerised but not secreted – apart from the first oligomer, which was slightly represented in the conditioned medium, with a pattern similar to the patients’ plasma VWF. The in vitro expression of the E244Lfs*211–VWF revealed a defective synthesis of the mutated VWF, with a behavior typical of loss of function mutations. Cellular trafficking, investigated in HEK293 cells, indicated a normal C584F-VWF content in the endoplasmic reticulum and Golgi apparatus, confirming the synthesis and multimerisation of C584F-VWF. No pseudo-Weibel Palade bodies were demonstrable, however, suggesting that C584F mutation impairs the storage of C584F-VWF. These findings point to cysteine 584 having a role in the release of VWF and its targeting to pseudo-Weibel Palade bodies in vitro, as well as in its storage and release by endothelial cells in vivo.

scholarly journals Visualizing the von Willebrand factor/glycoprotein Ib-IX axis with a platelet-type von Willebrand disease mutation

Blood ◽  
2009 ◽  
Vol 114 (27) ◽  
pp. 5541-5546 ◽  
Author(s):  
Jose A. Guerrero ◽  
Mark Kyei ◽  
Susan Russell ◽  
Junling Liu ◽  
T. Kent Gartner ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Thrombus Formation ◽  
Von Willebrand Disease ◽  
Platelet Glycoprotein ◽  
Glycoprotein Ib ◽  
Ligand Interaction ◽  
Von Willebrand ◽  
Willebrand Factor

AbstractPlatelet-type von Willebrand disease (PT-VWD) is a bleeding disorder of the platelet glycoprotein Ib-IX/von Willebrand factor (VWF) axis caused by mutations in the glycoprotein Ib-IX receptor that lead to an increased affinity with VWF. In this report, platelets from a mouse expressing a mutation associated with PT-VWD have been visualized using state-of-the art image collection and processing. Confocal analysis revealed that VWF bound to the surface of single platelets and bridging micro-aggregates of platelets. Surface-bound VWF appears as a large, linear structure on the surface of 50% of the PT-VWD platelets. In vivo thrombus formation after chemical injury to the carotid artery revealed a severe impairment to occlusion as a consequence of the PT-VWD mutation. In vitro stimulation of PT-VWD platelets with adenosine diphosphate or thrombin demonstrates a significant block in their ability to bind fibrinogen. The impairment of in vivo thrombus formation and in vitro fibrinogen binding are more significant than might be expected from the observed platelet binding to VWF polymers over a small portion of the plasma membrane. Visualization of the receptor/ligand interaction and characterization of a severe antithrombotic phenotype provide a new understanding on the molecular basis of bleeding associated with the PT-VWD phenotype.

scholarly journals Endothelial von Willebrand factor regulates angiogenesis

Blood ◽  
2011 ◽  
Vol 117 (3) ◽  
pp. 1071-1080 ◽  
Author(s):  
Richard D. Starke ◽  
Francesco Ferraro ◽  
Koralia E. Paschalaki ◽  
Nicola H. Dryden ◽  
Thomas A. J. McKinnon ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Vascular Malformations ◽  
Integrin Αvβ3 ◽  
Von Willebrand Disease ◽  
Vegf Receptor ◽  
Therapeutic Implications ◽  
Von Willebrand ◽  
Willebrand Factor

AbstractThe regulation of blood vessel formation is of fundamental importance to many physiological processes, and angiogenesis is a major area for novel therapeutic approaches to diseases from ischemia to cancer. A poorly understood clinical manifestation of pathological angiogenesis is angiodysplasia, vascular malformations that cause severe gastrointestinal bleeding. Angiodysplasia can be associated with von Willebrand disease (VWD), the most common bleeding disorder in man. VWD is caused by a defect or deficiency in von Willebrand factor (VWF), a glycoprotein essential for normal hemostasis that is involved in inflammation. We hypothesized that VWF regulates angiogenesis. Inhibition of VWF expression by short interfering RNA (siRNA) in endothelial cells (ECs) caused increased in vitro angiogenesis and increased vascular endothelial growth factor (VEGF) receptor-2 (VEGFR-2)–dependent proliferation and migration, coupled to decreased integrin αvβ3 levels and increased angiopoietin (Ang)–2 release. ECs expanded from blood-derived endothelial progenitor cells of VWD patients confirmed these results. Finally, 2 different approaches, in situ and in vivo, showed increased vascularization in VWF-deficient mice. We therefore identify a new function of VWF in ECs, which confirms VWF as a protein with multiple vascular roles and defines a novel link between hemostasis and angiogenesis. These results may have important consequences for the management of VWD, with potential therapeutic implications for vascular diseases.

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