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1.
Cell ; 183(3): 717-729.e16, 2020 10 29.
Article in English | MEDLINE | ID: mdl-33031746

ABSTRACT

The respiratory and intestinal tracts are exposed to physical and biological hazards accompanying the intake of air and food. Likewise, the vasculature is threatened by inflammation and trauma. Mucin glycoproteins and the related von Willebrand factor guard the vulnerable cell layers in these diverse systems. Colon mucins additionally house and feed the gut microbiome. Here, we present an integrated structural analysis of the intestinal mucin MUC2. Our findings reveal the shared mechanism by which complex macromolecules responsible for blood clotting, mucociliary clearance, and the intestinal mucosal barrier form protective polymers and hydrogels. Specifically, cryo-electron microscopy and crystal structures show how disulfide-rich bridges and pH-tunable interfaces control successive assembly steps in the endoplasmic reticulum and Golgi apparatus. Remarkably, a densely O-glycosylated mucin domain performs an organizational role in MUC2. The mucin assembly mechanism and its adaptation for hemostasis provide the foundation for rational manipulation of barrier function and coagulation.


Subject(s)
Biopolymers/metabolism , Mucins/metabolism , von Willebrand Factor/metabolism , Amino Acid Sequence , Animals , Cryoelectron Microscopy , Disulfides/metabolism , Female , Glycosylation , HEK293 Cells , Humans , Hydrogen-Ion Concentration , Mice, Inbred C57BL , Models, Molecular , Mucins/chemistry , Mucins/ultrastructure , Peptides/chemistry , Protein Domains , Protein Multimerization , von Willebrand Factor/chemistry , von Willebrand Factor/ultrastructure
3.
J Mol Biol ; 432(2): 305-323, 2020 01 17.
Article in English | MEDLINE | ID: mdl-31628947

ABSTRACT

Von Willebrand factor (VWF), an exceptionally large multimeric plasma glycoprotein, functions to initiate coagulation by agglutinating platelets in the blood stream to sites of vascular injury. This primary hemostatic function is perturbed in type 2 dysfunctional subtypes of von Willebrand disease (VWD) by mutations that alter the structure and function of the platelet GPIbα adhesive VWF A1 domains. The resulting amino acid substitutions cause local disorder and misfold the native structure of the isolated platelet GPIbα-adhesive A1 domain of VWF in both gain-of-function (type 2B) and loss-of-function (type 2M) phenotypes. These structural effects have not been explicitly observed in A1 domains of VWF multimers native to blood plasma. New mass spectrometry strategies are applied to resolve the structural effects of 2B and 2M mutations in VWF to verify the presence of A1 domain structural disorder in multimeric VWF harboring type 2 VWD mutations. Limited trypsinolysis mass spectrometry (LTMS) and hydrogen-deuterium exchange mass spectrometry (HXMS) are applied to wild-type and VWD variants of the single A1, A2, and A3 domains, an A1A2A3 tridomain fragment of VWF, plasmin-cleaved dimers of VWF, multimeric recombinant VWF, and normal VWF plasma concentrates. Comparatively, these methods show that mutations known to misfold the isolated A1 domain increase the rate of trypsinolysis and the extent of hydrogen-deuterium exchange in local secondary structures of A1 within multimeric VWF. VWD mutation effects are localized to the A1 domain without appreciably affecting the structure and dynamics of other VWF domains. The intrinsic dynamics of A1 observed in recombinant fragments of VWF are conserved in plasma-derived VWF. These studies reveal that structural disorder does occur in VWD variants of the A1 domain within multimeric VWF and provides strong support for VWF misfolding as a result of some, but not all, type 2 VWD variants.


Subject(s)
Protein Structure, Secondary/genetics , Proteostasis Deficiencies/genetics , von Willebrand Disease, Type 2/genetics , von Willebrand Factor/genetics , Amino Acid Substitution , Blood Platelets/chemistry , Blood Platelets/metabolism , Gene Expression Regulation/genetics , HEK293 Cells , Humans , Loss of Function Mutation/genetics , Mass Spectrometry , Protein Domains/genetics , Protein Folding , Protein Multimerization/genetics , Proteostasis Deficiencies/blood , Proteostasis Deficiencies/pathology , von Willebrand Disease, Type 2/blood , von Willebrand Disease, Type 2/pathology , von Willebrand Factor/chemistry , von Willebrand Factor/ultrastructure
4.
Nat Commun ; 10(1): 3781, 2019 08 22.
Article in English | MEDLINE | ID: mdl-31439947

ABSTRACT

Platelet recruitment to sites of blood vessel damage is highly dependent upon von Willebrand factor (VWF). VWF platelet-tethering function is proteolytically regulated by the metalloprotease ADAMTS13. Proteolysis depends upon shear-induced conformational changes in VWF that reveal the A2 domain cleavage site. Multiple ADAMTS13 exosite interactions are involved in recognition of the unfolded A2 domain. Here we report through kinetic analyses that, in binding VWF, the ADAMTS13 cysteine-rich and spacer domain exosites bring enzyme and substrate into proximity. Thereafter, binding of the ADAMTS13 disintegrin-like domain exosite to VWF allosterically activates the adjacent metalloprotease domain to facilitate proteolysis. The crystal structure of the ADAMTS13 metalloprotease to spacer domains reveals that the metalloprotease domain exhibits a latent conformation in which the active-site cleft is occluded supporting the requirement for an allosteric change to enable accommodation of the substrate. Our data demonstrate that VWF functions as both the activating cofactor and substrate for ADAMTS13.


Subject(s)
ADAMTS13 Protein/metabolism , Protein Interaction Domains and Motifs/physiology , von Willebrand Factor/metabolism , ADAMTS13 Protein/ultrastructure , Allosteric Regulation/physiology , Crystallography, X-Ray , Models, Molecular , Protein Binding/physiology , Proteolysis , Recombinant Proteins/metabolism , Recombinant Proteins/ultrastructure , Substrate Specificity , von Willebrand Factor/ultrastructure
5.
PLoS One ; 14(1): e0210963, 2019.
Article in English | MEDLINE | ID: mdl-30645640

ABSTRACT

The formation of hemostatic plugs at sites of vascular injury crucially involves the multimeric glycoprotein von Willebrand factor (VWF). VWF multimers are linear chains of N-terminally linked dimers. The latter are formed from monomers via formation of the C-terminal disulfide bonds Cys2771-Cys2773', Cys2773-Cys2771', and Cys2811-Cys2811'. Mutations in VWF that impair multimerization can lead to subtype 2A of the bleeding disorder von Willebrand Disease (VWD). Commonly, the multimer size distribution of VWF is assessed by electrophoretic multimer analysis. Here, we present atomic force microscopy (AFM) imaging as a method to determine the size distribution of VWF variants by direct visualization at the single-molecule level. We first validated our approach by investigating recombinant wildtype VWF and a previously studied mutant (p.Cys1099Tyr) that impairs N-terminal multimerization. We obtained excellent quantitative agreement with results from earlier studies and with electrophoretic multimer analysis. We then imaged specific mutants that are known to exhibit disturbed C-terminal dimerization. For the mutants p.Cys2771Arg and p.Cys2773Arg, we found the majority of monomers (87 ± 5% and 73 ± 4%, respectively) not to be C-terminally dimerized. While these results confirm that Cys2771 and Cys2773 are crucial for dimerization, they additionally provide quantitative information on the mutants' different abilities to form alternative C-terminal disulfides for residual dimerization. We further mutated Cys2811 to Ala and found that only 23 ± 3% of monomers are not C-terminally dimerized, indicating that Cys2811 is structurally less important for dimerization. Furthermore, for mutants p.Cys2771Arg, p.Cys2773Arg, and p.Cys2811Ala we found 'even-numbered' non-native multimers, i.e. multimers with monomers attached on both termini; a multimer species that cannot be distinguished from native multimers by conventional multimer analysis. Summarizing, we demonstrate that AFM imaging can provide unique insights into VWF processing defects at the single-molecule level that cannot be gained from established methods of multimer analysis.


Subject(s)
Microscopy, Atomic Force/methods , Single Molecule Imaging/methods , von Willebrand Factor/chemistry , von Willebrand Factor/ultrastructure , Amino Acid Substitution , Cysteine/chemistry , Dimerization , HEK293 Cells , Humans , Models, Molecular , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutant Proteins/ultrastructure , Mutation, Missense , Particle Size , Protein Multimerization/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/ultrastructure , von Willebrand Diseases/blood , von Willebrand Diseases/genetics , von Willebrand Factor/genetics
6.
Anal Chem ; 87(20): 10299-305, 2015 Oct 20.
Article in English | MEDLINE | ID: mdl-26369694

ABSTRACT

Vital functions of mammals are only possible due to the behavior of blood to coagulate most efficiently in vessels with particularly high wall shear rates. This is caused by the functional changes of the von Willebrand Factor (VWF), which mediates coagulation of blood platelets (primary hemostasis) especially when it is stretched under shear stress. Our data show that shear stretching also affects other functions of VWF: Using a customized device to simulate shear conditions and to conserve the VWF molecules in their unstable, elongated conformation, we visualize at single molecule level by AFM that VWF is preferentially cleaved by the protease ADAMTS13 at higher shear rates. In contrast to this high shear-rate-selective behavior, VWF binds FVIII more effectively only below a critical shear rate of ∼30.000 s(-1), indicating that under harsh shear conditions FVIII is released from its carrier protein. This may be required to facilitate delivery of FVIII locally to promote secondary hemostasis.


Subject(s)
ADAM Proteins/chemistry , Factor VIII/chemistry , Microscopy, Atomic Force , von Willebrand Factor/chemistry , ADAM Proteins/metabolism , ADAM Proteins/ultrastructure , ADAMTS13 Protein , Factor VIII/metabolism , Factor VIII/ultrastructure , Humans , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Recombinant Proteins/ultrastructure , von Willebrand Factor/metabolism , von Willebrand Factor/ultrastructure
7.
J Thromb Haemost ; 13(9): 1699-708, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26178390

ABSTRACT

BACKGROUND: Under severe stenotic conditions, von Willebrand factor (VWF) multimerizes into large insoluble fibers at pathological shear rates. OBJECTIVE: Evaluate the mechanics and biology of VWF fibers without the confounding effects of endothelium or collagen. METHODS: Within a micropost-impingement microfluidic device, > 100-µm long VWF fibers multimerized on the post within 10 min using EDTA-treated platelet-free plasma (PFP) perfused at wall shear rates > 5000 s(-1) . RESULTS: von Willebrand factor fiber thickness increased to > 10 µm as a result of increasing the shear rate to 10,000 s(-1) . In a stress-strain test, fibrous VWF had an elastic modulus of ~50 MPa. The insoluble VWF fibers were non-amyloid because they rapidly dissolved in trypsin, plasmin or 2% SDS, but were resistant to 50 nm ADAMTS13 or 100 nm tissue plasminogen activator in plasma. Following fiber formation, perfusion of low corn trypsin inhibitor (CTI)-treated (4 µg mL(-1) ), recalcified citrated plasma at 1500 s(-1) caused fibrin formation on the VWF fibers, a result not observed with purified type 1 collagen or a naked micropost. During VWF fiber formation, contact pathway factors accumulated on VWF because the use of EDTA/D-Phe-Pro-Arg chloromethylketone (PPACK)/apixaban/high CTI-treated PFP during VWF fiber formation prevented the subsequent fibrin production from low-CTI, recalcified citrated PFP. VWF fibers displayed FXIIa-immunostaining. When PPACK-inhibited whole blood was perfused over VWF fibers, platelets rolled and arrested on the surface of VWF, but only displayed P-selectin if prevailing shear rates were pathological. Platelet arrest on VWF fibers was blocked with αIIb ß3 antagonist GR144053. CONCLUSIONS: We report VWF fiber-contact pathway crosstalk and mechanisms of thrombolytic resistance in hemodynamic settings of myocardial infarction.


Subject(s)
ADAM Proteins/pharmacology , Blood Coagulation/drug effects , Hemorheology , Platelet Activation/drug effects , Tissue Plasminogen Activator/pharmacology , von Willebrand Factor/chemistry , ADAMTS13 Protein , Biopolymers , Elastic Modulus , Fibrinolysin/pharmacology , Humans , In Vitro Techniques , Lab-On-A-Chip Devices , P-Selectin/blood , Piperazines/pharmacology , Piperidines/pharmacology , Platelet Adhesiveness , Platelet Glycoprotein GPIIb-IIIa Complex/antagonists & inhibitors , Protein Subunits , Solubility , von Willebrand Factor/physiology , von Willebrand Factor/ultrastructure
8.
Blood ; 126(8): 935-8, 2015 Aug 20.
Article in English | MEDLINE | ID: mdl-26065652

ABSTRACT

Association with the D'D3 domain of von Willebrand factor (VWF) stabilizes factor VIII (FVIII) in the circulation and maintains it at a level sufficient to prevent spontaneous bleeding. We used negative-stain electron microscopy (EM) to visualize complexes of FVIII with dimeric and monomeric forms of the D'D3 domain. The EM averages show that FVIII interacts with the D'D3 domain primarily through its C1 domain, with the C2 domain providing a secondary attachment site. Hydrogen-deuterium exchange mass spectrometry corroborated the importance of the C1 domain in D'D3 binding and implicates additional surface regions on FVIII in the interaction. Together, our results establish that the C1 domain is the major binding site on FVIII for VWF, reiterate the importance of the a3 acidic peptide in VWF binding, and suggest that the A3 and C2 domains play ancillary roles in this interaction.


Subject(s)
Factor VIII/chemistry , Factor VIII/metabolism , von Willebrand Factor/chemistry , von Willebrand Factor/metabolism , Binding Sites , Factor VIII/ultrastructure , HEK293 Cells , Humans , Mass Spectrometry , Microscopy, Electron , Protein Structure, Tertiary , von Willebrand Factor/ultrastructure
9.
J Atheroscler Thromb ; 22(10): 1091-9, 2015.
Article in English | MEDLINE | ID: mdl-25972025

ABSTRACT

AIM: von Willebrand factor (VWF) plays an important role in the regulation of hemostasis and thrombosis formation, particularly under a high shear rate. However, the adhesive force due to the molecular interaction between VWF and glycoprotein Ibα (GPIbα) has not been fully explored. Thus, we employed atomic force microscopy to directly measure the adhesive force between VWF and GPIbα. METHODS: We measured the adhesive force between VWF and GPIbα at the molecular level using an atomic force microscope (AFM). An AFM cantilever was coated with recombinant N-terminus VWF binding site of GPIbα, whereas a cover glass was coated with native VWF. RESULTS: The adhesive force at the molecular level was measured using an AFM. In the presence of 1 µg/mL VWF, the adhesion force was nearly 200 pN. As per the Gaussian fit analysis, the adhesive force of a single bond could have been 54 or 107 pN. CONCLUSION: Our consideration with the Gaussian fit analysis proposed that the adhesive force of a single bond could be 54 pN, which is very close to that obtained by optical tweezers (50 pN).


Subject(s)
Blood Platelets/metabolism , Hemostasis/physiology , Microscopy, Atomic Force/methods , Platelet Glycoprotein GPIb-IX Complex/chemistry , Thrombosis/blood , von Willebrand Factor/chemistry , Blood Platelets/ultrastructure , Humans , Molecular Structure , Platelet Glycoprotein GPIb-IX Complex/ultrastructure , Thrombosis/diagnosis , von Willebrand Factor/ultrastructure
10.
Blood ; 123(12): 1785-93, 2014 Mar 20.
Article in English | MEDLINE | ID: mdl-24394662

ABSTRACT

The C-terminal cystine knot (CK) (CTCK) domain in von Willebrand factor (VWF) mediates dimerization of proVWF in the endoplasmic reticulum and is essential for long multimers required for hemostatic function. The CTCK dimer crystal structure reveals highly elongated monomers with 2 ß-ribbons and 4 intra-chain disulfides, including 3 in the CK. Dimerization buries an extensive interface of 1500 Å(2) corresponding to 32% of the surface of each monomer and forms a super ß-sheet and 3 inter-chain disulfides. The shape, dimensions, and N-terminal connections of the crystal structure agree perfectly with previous electron microscopic images of VWF dimeric bouquets with the CTCK dimer forming a down-curved base. The dimer interface is suited to resist hydrodynamic force and disulfide reduction. CKs in each monomer flank the 3 inter-chain disulfides, and their presence in ß-structures with dense backbone hydrogen bonds creates a rigid, highly crosslinked interface. The structure reveals the basis for von Willebrand disease phenotypes and the fold and disulfide linkages for CTCK domains in diverse protein families involved in barrier function, eye and inner ear development, insect coagulation and innate immunity, axon guidance, and signaling in extracellular matrices.


Subject(s)
von Willebrand Factor/chemistry , Amino Acid Sequence , Crystallography, X-Ray , Humans , Microscopy, Electron , Models, Molecular , Molecular Sequence Data , Mutation , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/ultrastructure , Protein Interaction Domains and Motifs , Protein Multimerization , Protein Structure, Quaternary , Sequence Homology, Amino Acid , von Willebrand Factor/genetics , von Willebrand Factor/ultrastructure
11.
Colorectal Dis ; 14(12): 1500-6, 2012 Dec.
Article in English | MEDLINE | ID: mdl-22507880

ABSTRACT

AIM: Elevated levels of von Willebrand factor (VWF) are often observed in many diseases including colorectal cancer, but this finding is not definite. The aim of our study was to examine the change in VWF multimer distribution in patients with colorectal cancer. METHOD: We randomly selected nine patients from each of the four Union for International Cancer Control (UICC) stages of colon cancer. VWF antigen (VWF:Ag), VWF-cleaving protease ADAMTS-13 level and factor VIII activity (FVIII:C) were determined. The multimer distribution of VWF was visualized using electrophoretic multimer analysis. RESULTS: The VWF multimer structure was normal with no difference between the four UICC stages. There was no significant increase in VWF:Ag and FVIII:C levels in the more advanced UICC stages. There was no significant difference in the ADAMTS-13 level according to the UICC stage. CONCLUSION: There was no change in the VWF multimer distribution to indicate acquired von Willebrand disease.


Subject(s)
Carcinoma/blood , Carcinoma/pathology , Colorectal Neoplasms/blood , Colorectal Neoplasms/pathology , von Willebrand Factor/metabolism , von Willebrand Factor/ultrastructure , ABO Blood-Group System , ADAM Proteins/blood , ADAMTS13 Protein , Adult , Aged , Aged, 80 and over , Case-Control Studies , Factor VIII/metabolism , Female , Humans , Male , Middle Aged , Neoplasm Staging , Protein Multimerization , von Willebrand Factor/immunology
12.
Blood ; 120(2): 449-58, 2012 Jul 12.
Article in English | MEDLINE | ID: mdl-22490677

ABSTRACT

In the present study, we re-annotated von Willebrand factor (VWF), assigned its entire sequence to specific modules, and related these modules to structure using electron microscopy (EM). The D domains are assemblies of smaller modules visible as lobes in EM. Modules in the D-domain assemblies include von Willebrand D, 8-cysteine, trypsin inhibitor-like, E or fibronectin type 1-like domains, and a unique D4N module in D4. The D1-D2 prodomain shows 2 large connected assemblies, each containing smaller lobes. The previous B and C regions of VWF are re-annotated as 6 tandem von Willebrand C (VWC) and VWC-like domains. These 6 VWC domains correspond to 6 elongated domains that associate in pairs at acidic pH in the stem region of VWF dimeric bouquets. This correspondence is demonstrated by binding of integrin α(IIb)ß(3) to the fourth module seen in EM, VWC4, which bears the VWF Arg-Gly-Asp motif. The C-terminal cystine knot domain dimerizes end-to-end in a manner predicted by homology to TGF-ß and orients approximately perpendicular to the VWC domains in dimeric bouquets. Homologies of domains in VWF to domains in other proteins allow many disulfide bonds to be tentatively assigned, which may have functional implications.


Subject(s)
von Willebrand Factor/chemistry , von Willebrand Factor/genetics , Amino Acid Sequence , Binding Sites , Dimerization , Humans , Microscopy, Electron , Models, Molecular , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/genetics , Protein Structure, Quaternary , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Sequence Homology, Amino Acid , von Willebrand Factor/ultrastructure
13.
Thromb Haemost ; 104(3): 523-30, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20589314

ABSTRACT

Human plasma protein von Willebrand factor (VWF) is composed of a series of multimers with molecular weights ranging from 600 to 20,000 kDa or even more. Plasma-derived VWF (pdVWF) and recombinant VWF (rVWF) differ in that the ultra-large molecular weight multimer portion present in rVWF is usually missing in pdVWF due to partial cleavage of VWF by the plasma protease ADAMTS13. Here, tapping mode atomic force microscopy (TM-AFM) was used to visualise the shape and size of rVWF and pdVWF. The morphology of the variants of VWF was comparable, containing both globular and stretched domains. Mean chain lengths of the filaments and diameters of the core globular domains were determined and analysed on a statistical basis. About 72% of the pdVWF molecules and 70% of the rVWF molecules were 100-300 nm long. The portion of very long molecules (>300 nm) was only slightly greater in rVWF than in pdVWF (20% vs. 18%). The diameters of the globular core structures were in the range of 12 to 30 nm for both types of VWF. Inspection of a purified rVWF dimer revealed a similar range for the globular domain (14-32 nm). Finally, we demonstrate a dramatic conformational change for rVWF upon exposure to high shear stress, as has been reported for pdVWF. Our TM-AFM data show that the overall structure of rVWF is similar to that of pdVWF and that rVWF will extend its conformation under shear stress, which is required to exert its function in primary haemostasis.


Subject(s)
Microscopy, Atomic Force , von Willebrand Factor/ultrastructure , Electrophoresis, Agar Gel , Humans , Molecular Weight , Particle Size , Protein Conformation , Protein Multimerization , Protein Structure, Tertiary , Recombinant Proteins/ultrastructure , Stress, Mechanical , Structure-Activity Relationship , von Willebrand Factor/isolation & purification
14.
Biochem Biophys Res Commun ; 369(2): 507-12, 2008 May 02.
Article in English | MEDLINE | ID: mdl-18298947

ABSTRACT

The crucial role of the biopolymer "Von Willebrand factor" (VWF) in blood platelet binding is tightly regulated by the shear forces to which the protein is exposed in the blood flow. Under high-shear conditions, VWFs ability to immobilize blood platelets is strongly increased due to a change in conformation which at sufficient concentration is accompanied by the formation of ultra large VWF bundles (ULVWF). However, little is known about the dynamic and mechanical properties of such bundles. Combining a surface acoustic wave (SAW) based microfluidic reactor with an atomic force microscope (AFM) we were able to study the relaxation of stretched VWF bundles formed by hydrodynamic stress. We found that the dynamical response of the network is well characterized by stretched exponentials, indicating that the relaxation process proceeds through hopping events between a multitude of minima. This finding is in accordance with current ideas of VWF self-association. The longest relaxation time does not show a clear dependence on the length of the bundle, and is dominated by the internal conformations and effective friction within the bundle.


Subject(s)
Microfluidic Analytical Techniques/methods , Microscopy, Atomic Force/methods , Models, Chemical , Models, Molecular , von Willebrand Factor/chemistry , von Willebrand Factor/ultrastructure , Computer Simulation , Elasticity , Multiprotein Complexes/chemistry , Multiprotein Complexes/ultrastructure , Protein Conformation , Stress, Mechanical
15.
Thromb Res ; 119(6): 731-40, 2007.
Article in English | MEDLINE | ID: mdl-17010412

ABSTRACT

Adsorption of plasma proteins such as von Willebrand factor (vWF) on thrombogenic surfaces can induce conformational changes in tertiary structure so that the prothrombotic functional epitopes are exposed for interactions with platelets, resulting in platelet adhesion and thrombus formation. Thus, understanding platelet binding following changes in the structure of vWF is critical in understanding the mechanisms of thrombogenesis. The present study examined the accessibility of platelet binding epitopes within vWF adsorbed on two different thrombogenic surfaces, a hydrophobic synthetic surface and collagen VI coated substrates, under physiological buffer conditions using atomic force microscopy (AFM) in combination with immunogold labeling. Our results demonstrated that the glycoprotein Ib (GPIb) binding domain in vWF undergoes changes when adsorbed on collagen VI compared to vWF on a hydrophobic synthetic surface. This study provides a basis for a novel approach to understand the molecular mechanisms of surface-induced thrombosis by directly examining the structure-function relationships of plasma proteins involved in the thrombus formation.


Subject(s)
Blood Platelets/metabolism , Platelet Glycoprotein GPIb-IX Complex/metabolism , von Willebrand Factor/genetics , von Willebrand Factor/metabolism , Adsorption , Binding Sites , Collagen Type VI/chemistry , Epitopes , Humans , Immunohistochemistry , Microscopy, Atomic Force , Protein Structure, Tertiary , Silanes/chemistry , Structure-Activity Relationship , Surface Properties , Thrombosis/etiology , von Willebrand Factor/immunology , von Willebrand Factor/ultrastructure
16.
Blood ; 107(5): 1943-50, 2006 Mar 01.
Article in English | MEDLINE | ID: mdl-16293606

ABSTRACT

The metalloproteinase ADAMTS13 regulates the size of released von Willebrand factor (VWF) multimers bound to endothelial cells, but it is unknown whether it can cleave plasma VWF during thrombogenesis. To address this issue, we perfused blood over immobilized VWF and used videomicroscopy to visualize an activation-independent platelet aggregation process mediated by soluble VWF at shear rates greater than 10 000 s(-1). At normal Ca2+ concentration, platelets formed rolling as well as surface-attached clusters that grew larger during the first 5 minutes but then lost more than 70% of their mass by 10 minutes. In contrast, platelet clusters were stable in size when metal ions were chelated, anti-ADAMTS13 IgG were added, or washed blood cells were perfused with purified VWF but no plasma. In the latter case, addition of recombinant ADAMTS13 reduced platelet cluster size by more than 70%. Incubating ADAMTS13 with VWF before perfusion did not prevent the initial platelet clustering, indicating that the enzyme may act on platelet-bound VWF under shear stress. At the concentrations tested, ADAMTS13 had no effect on platelet aggregates formed upon blood perfusion over collagen fibrils. ADAMTS13, therefore, may regulate thrombus size preferentially when the cohesion between platelets depends on VWF binding induced by pathologically elevated shear stress.


Subject(s)
ADAM Proteins/metabolism , Blood Platelets/metabolism , Platelet Aggregation , Thrombosis/metabolism , von Willebrand Factor/metabolism , ADAMTS13 Protein , Calcium/metabolism , Calcium/pharmacology , Humans , Immunoglobulin G/pharmacology , Platelet Aggregation/drug effects , Stress, Mechanical , Time Factors , von Willebrand Factor/ultrastructure
17.
Blood ; 99(6): 2070-6, 2002 Mar 15.
Article in English | MEDLINE | ID: mdl-11877281

ABSTRACT

We have developed an immunogold von Willebrand factor (VWF) detection method that permits almost complete coverage of individual VWF molecules, and by this unequivocal localization and morphologic analysis of collagen-bound VWF by atomic force microscopy (AFM). Perfusion of gel filtration-purified VWF in parallel plate perfusion chambers over glass coverslips coated with calf skin collagen, followed by AFM imaging in air, enabled us to assess possible morphologic differences between VWF bound at low (0.07 N/m(2) = 0.7 dynes/cm(2)) and high (4.55 N/m(2) = 45.5 dynes/cm(2)) shear stresses. No significant differences in VWF morphology were found, the molecules were oriented almost randomly, and there were no clear signs of VWF "uncoiling" either at a high or at a low shear regime. After perfusing 1 microg/mL VWF for 5 minutes, surface coverage at high shear was almost twice the one seen at low shear, and some larger and more irregularly shaped VWF molecules could be seen at high shear. This difference disappeared, however, at 15 minutes of perfusion and was probably caused by diffusion kinetics. Moreover, the presence of 68 x 10(9)/L washed fixed platelets in the perfusate did not have any visible effect on VWF morphology at high versus low shear stress. These findings suggest that shear stress does not influence significantly the overall molecular morphology of VWF during its binding to collagen-coated surface and are consistent with a constitutively expressed affinity of collagen-bound VWF for glycoprotein Ib.


Subject(s)
Collagen Type I/metabolism , von Willebrand Factor/metabolism , Animals , Blood Platelets/metabolism , Cattle , Humans , Immunohistochemistry/methods , Microscopy, Atomic Force/methods , Perfusion , Platelet Adhesiveness , Protein Binding , Stress, Mechanical , von Willebrand Factor/chemistry , von Willebrand Factor/ultrastructure
18.
Bol. Acad. Nac. Med. B.Aires ; 79(2): 275-290, jul.-dic. 2001. tab
Article in Spanish | BINACIS | ID: bin-6812

ABSTRACT

La enfermedad de von Willebrand (VWD) se caracteriza por manifestaciones hemorrágicas de tipo muco-cutáneo, causadas por una deficiencia cuali o cuantitativa del factor von Willebrand, responsable de la interacción de las plaquetas entre sí y con superficies subendoteliales a través de receptores específicos. Se diagnosticó VWD en 1885 individuos, encontrando una prevalencia de mujeres del 60,5 por ciento. La mayoría de los pacientes (71,1 por ciento) eran mayores de 13 años al momento de la consulta. El 91 por ciento de los pacientes fue tipo 1; 3,1 por ciento tipo 2 A; 2,7 por ciento tipo intraplaquetario bajo; 1,8 por ciento tipo 2N; 1,3 por ciento tipo severo y 0,1 por ciento fenotipo combinado 2N+1. Los síntomas clínicos más frecuentes fueron: equimosis-hematomas y epistaxis y, entre mujeres mayores de 13 años, menometrorragia. Se halló 6,7 por ciento de pacientes sin historia personal ni familiar de hemorragia, que consultaron por tener estudios preoperatorios anormales. Niveles normales de FVIII se hallaron en 37,8 por ciento de los pacientes. La prevalencia de grupo 0 entre nuestros pacientes fue del 70,5 por ciento, con disminución significativa sólo en los valores de VWF:Ag con respecto de los no-0. No hubo correlación entre el grupo 0 y la presencia de síntomas clínicos, cantidad de sitios de sangrado, sangrado gineco-obstétrico ni requerimientos transfusionales. De 81 individuos pertenecientes a 8 familias seleccionadas con 8 o más miembros estudiados (4 con miembros con VWD tipo 1 y 4 con tipo 2A) confirmamos que hay mayor número de miembros afectados en las familias con VWD tipo 2A, sugiriendo mayor penetrancia de VWD en este subgrupo. En el grupo de individuos no diagnosticados, la presencia de miembros sintomáticos fue más frecuente entre las familias con VWD tipo 1. (AU)


Subject(s)
Humans , Male , Adolescent , Adult , Female , von Willebrand Factor/genetics , von Willebrand Factor/ultrastructure , von Willebrand Diseases/diagnosis , von Willebrand Diseases/physiopathology , von Willebrand Diseases/epidemiology , von Willebrand Diseases/complications , von Willebrand Diseases/genetics , Clinical Laboratory Techniques , Signs and Symptoms
19.
Blood ; 94(1): 172-8, 1999 Jul 01.
Article in English | MEDLINE | ID: mdl-10381510

ABSTRACT

Aggregation of blood platelets contributes to the arrest of bleeding at sites of vascular injury, but it can occlude atherosclerotic arteries and precipitate diseases such as myocardial infarction. The bonds that link platelets under flow conditions were identified using confocal videomicroscopy in real time. Glycoprotein (GP) Ibalpha and von Willebrand factor (vWF) acted in synergy with alphaIIbbeta3 and fibrinogen to sustain platelet accrual at the apex of thrombi where three-dimensional growth resulted in increasing shear rates. The specific function of distinct adhesion pathways in response to changing hemodynamic conditions helps to explain hemostatic and thrombotic processes.


Subject(s)
Blood Platelets/cytology , Blood Platelets/physiology , Platelet Adhesiveness/physiology , Fibrinogen/physiology , Fibrinogen/ultrastructure , Humans , Microscopy, Confocal , Platelet Glycoprotein GPIIb-IIIa Complex/physiology , Platelet Glycoprotein GPIIb-IIIa Complex/ultrastructure , Platelet Glycoprotein GPIb-IX Complex/physiology , Platelet Glycoprotein GPIb-IX Complex/ultrastructure , von Willebrand Factor/physiology , von Willebrand Factor/ultrastructure
20.
Ann Genet ; 41(1): 34-43, 1998.
Article in English | MEDLINE | ID: mdl-9599650

ABSTRACT

Von Willebrand disease (vWD), the most common congenital bleeding disorder in man, is related to quantitative and/or qualitative abnormalities of von Willebrand factor (vWF). This multimeric glycoprotein serves as carrier protein of factor VIII, an essential cofactor of coagulation in plasma, and promotes platelet adhesion to the damaged vessel and platelet aggregation. Distinct abnormalities of vWF are responsible for the three types of vWD. Types 1 and 3 are characterized by a quantitative defect of vWF whereas type 2, comprising subtypes 2A, 2B, 2M and 2N, refers to molecular variants with a qualitative defect of vWF. The knowledge of the structure of the vWF gene and the use of Polymerase Chain Reaction (PCR) have led to the identification of the molecular basis of vWD in a significant number of patients. Type 2A is characterized by a decreased platelet-dependent function of vWF associated with the absence of high molecular weight (HMW) multimers of vWF. Most of the type 2A mutations have been identified in the A2 domain of vWF which contains a proteolytic site, while a few others have been found within the propeptide and the C-terminal part of vWF which are involved in its multimerization and dimerization, respectively. In type 2B, defined by an increased affinity of vWF to platelet glycoprotein Ib (GPIb), various amino-acid (aa) substitutions or insertion have been localized within the A1 domain containing the GPIb binding site. In the latter domain have been also identified the few molecular abnormalities described in type 2M which is defined by a decreased platelet-dependent function not caused by the absence of HMW multimers. In type 2N, characterized by a defective binding of vWF to factor VIII, several aa substitutions have been identified within the factor VIII-binding domain in the N-terminal part of vWF. The identification of gene defects remains difficult in types 1 and 3. Whereas various abnormalities (total, partial or point deletions, point insertions, nonsense mutations) have already been identified in type 3, the molecular basis of type 1 is still unresolved in most cases. The characterization of the molecular basis of vWD is of fundamental interest in providing further insight into the structure-function relationship and the biosynthesis of vWF.


Subject(s)
Chromosomes, Human, Pair 12/genetics , von Willebrand Diseases/genetics , von Willebrand Factor/genetics , Binding Sites , Blood Platelets/metabolism , Chromosomes, Human, Pair 22/genetics , Dimerization , Endothelium, Vascular/metabolism , Factor VIII/metabolism , Female , Fetal Diseases/diagnosis , Fetal Diseases/genetics , Genes, Dominant , Genes, Recessive , Humans , Male , Models, Molecular , Molecular Weight , Mutation , Platelet Glycoprotein GPIb-IX Complex/metabolism , Polymerase Chain Reaction , Polymorphism, Genetic , Pregnancy , Prenatal Diagnosis , Protein Binding , Protein Conformation , Protein Precursors/chemistry , Protein Precursors/genetics , Pseudogenes , Structure-Activity Relationship , von Willebrand Diseases/classification , von Willebrand Diseases/diagnosis , von Willebrand Diseases/embryology , von Willebrand Factor/chemistry , von Willebrand Factor/physiology , von Willebrand Factor/ultrastructure
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