Characterisation of W1745C and S1783A, Two Novel Collagen Binding Defects in the A3 Domain of Von Willebrand Factor

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 424-424 ◽  
Author(s):  
Anne Riddell ◽  
Keith Gomez ◽  
Carolyn Millar ◽  
G. Mellars ◽  
Simon A Brown ◽  
...  
Keyword(s):  
Type I Collagen ◽  
Cysteine Residue ◽  
Binding Activity ◽  
Von Willebrand Disease ◽  
Clinical Samples ◽  
Type Iii Collagen ◽  
Type I ◽  
Collagen Binding ◽  
Type Iii ◽  
Von Willebrand

Abstract Investigation of three families with von Willebrand disease showed that haemorrhagic symptoms were associated with disproportionately reduced collagen binding activity whilst Ristocetin co-factor activity was commensurate with antigen and multimeric analysis was normal. Genetic analysis revealed heterozygosity for two novel mutations in two of the families: W1745C in exon 30 and S1783A in exon 31. In the third family the affected individuals were heterozygous for a previously-described mutation: S1731T in exon 30 but two unaffected individuals also carried this mutation. All three mutations lie in the A3 domain containing the main collagen binding site in VWF. In patients’ samples VWF:CB activity was measured using human type I and type III collagen. Patients heterozygous for W1745C and S1731T showed a reduction in binding to both collagens but more marked reduction in binding to type III collagen. Heterozygosity for S1731T resulted in mild impairment of type I collagen binding but normal binding to type III collagen. Site-directed mutagenesis was used to generate vectors containing the three mutations (S1731T, W1745C and S1783A) and also one containing a W1745A mutation. Mutated VWF was expressed in HEK293T cells both singly and in co-transfection with a wild-type VWF (wtVWF) vector. All VWF mutants were expressed at a similar rate to wtVWF. Multimeric analysis demonstrated that all the mutants had a similar multimeric structure compared to recombinant wtVWF. However recombinant-wtVWF (wtVWF) had a lower collagen binding to VWF antigen ratio (CB:Ag) compared to plasma VWF (0.39 type I collagen and 0.45 type III collagen vs >0.7 for plasma VWF). This is most likely due to the slight shift towards lower molecule weight multimers seen with recombinant VWF. CB:Ag ratios for the recombinant VWF showed the same pattern of binding to collagen type I and III as the clinical samples. The W1745A mutant demonstrated a similar CB:Ag ratio to W1745C. Kinetic analysis of binding to type I collagen demonstrated that W1745C, W1745A and S1783A did not bind and that S1731T bound with significantly less affinity compared to wtVWF (KD,app 27.1 ± 0.5nM and 7.3 ± 0.8nM respectively). Analysis of binding to type III collagen demonstrated that W1745C and W1745A both bound with ~ 8-fold reduced affinity (KD,app 16 ± 2.6nM and 21.3 ± 6.3nM) but wtVWF and S1731T bound with similar affinity, (KD,app 2.0 ± 0.1nM and 3.7 ± 0.85nM respectively). Analysis of the crystal structure of the VWF A3 domain showed that W1745 may interact with Y1780 and we noted the mutation Y1780A has also been shown to significantly reduce collagen binding. Measurement of free thiols present in VWF demonstrated that the new cysteine residue in W1745C is not involved in disulphide bond formation. These results indicate that it is the loss of W1745 rather than the creation of a new cysteine residue that is responsible for the loss of collagen binding activity. We therefore hypothesised that W1745 and Y1780 participate in an internal aromatic interaction that helps to maintain the structural configuration of A3. We sought confirmation by expressing another mutant; W1745F, replacing the tryptophan with another aromatic amino acid. As predicted this did not significantly affect collagen binding. In conclusion, our findings demonstrate that type 2 VWD may be arise from mutations in A3 causing abnormal collagen binding without other functional defects or abnormalities in multimer formation. This type of VWD may be under-recognised unless laboratories measure binding to both types I and III collagen. Mutations in A3 yield insights into the structural requirements for collagen binding may have differential effects on binding to collagen types I and III and can result in variable clinical phenotypes. Some mutations may not be consistently associated with bleeding symptoms.

Characterization of W1745C and S1783A: 2 novel mutations causing defective collagen binding in the A3 domain of von Willebrand factor

Blood ◽  
2009 ◽  
Vol 114 (16) ◽  
pp. 3489-3496 ◽  
Author(s):  
Anne F. Riddell ◽  
Keith Gomez ◽  
Carolyn M. Millar ◽  
Gillian Mellars ◽  
Saher Gill ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Type I Collagen ◽  
Binding Activity ◽  
Von Willebrand Disease ◽  
Site Directed Mutagenesis ◽  
Type Iii Collagen ◽  
Type I ◽  
Collagen Binding ◽  
Von Willebrand ◽  
Willebrand Factor

AbstractInvestigation of 3 families with bleeding symptoms demonstrated a defect in the collagen-binding activity of von Willebrand factor (VWF) in association with a normal VWF multimeric pattern. Genetic analysis showed affected persons to be heterozygous for mutations in the A3 domain of VWF: S1731T, W1745C, and S1783A. One person showed compound heterozygosity for W1745C and R760H. W1745C and S1783A have not been reported previously. The mutations were reproduced by site-directed mutagenesis and mutant VWF expressed in HEK293T cells. Collagen-binding activity measured by immunosorbent assay varied according to collagen type: W1745C and S1783A were associated with a pronounced binding defect to both type I and type III collagen, whereas the principal abnormality in S1731T patients was a reduction in binding to type I collagen only. The multimer pattern and distribution of mutant proteins were indistinguishable from wild-type recombinant VWF, confirming that the defect in collagen binding resulted from the loss of affinity at the binding site and not impairment of high-molecular-weight multimer formation. Our findings demonstrate that mutations causing an abnormality in the binding of VWF to collagen may contribute to clinically significant bleeding symptoms. We propose that isolated collagen-binding defects are classified as a distinct subtype of von Willebrand disease.

A Novel VWF A3 Domain Mutation with Absent Binding to Type I and Type III Collagen.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 541-541
Author(s):  
Veronica H. Flood ◽  
Carol A. Lederman ◽  
Jeffrey S. Wren ◽  
Pamela A. Christopherson ◽  
Kenneth D. Friedman ◽  
...  
Keyword(s):  
Index Case ◽  
Von Willebrand Disease ◽  
Type Iii Collagen ◽  
Type I ◽  
Healthy Controls ◽  
Wild Type ◽  
Collagen Binding ◽  
Type Iii ◽  
The Mean ◽  
Von Willebrand

Abstract Abstract 541 Von Willebrand factor (VWF) plays a key role in coagulation by tethering platelets to injured subendothelium through binding sites for collagen and platelet GPIb. Laboratory testing for von Willebrand disease (VWD) utilizes various assays of VWF protein and function, but collagen binding (VWF:CB) has not always been performed. Samples obtained from a large, multi-center US study of von Willebrand disease (VWD), the Zimmerman Program for the Molecular and Clinical Biology of VWD (ZPMCB-VWD), were evaluated for VWF:CB. Whole blood was collected for DNA sequencing and plasma samples were collected for VWF testing. VWF antigen (VWF:Ag), ristocetin cofactor activity (VWF:RCo), and VWF:CB were performed in the BloodCenter of Wisconsin hemostasis laboratory. A normal range for the VWF:CB was established using 233 healthy controls enrolled in the ZPMCB-VWD. The mean VWF:Ag was 105 IU/dL and the mean VWF:CB was 123 U/dL, with a mean VWF:CB/VWF:Ag ratio of 1.19. This yields a normal range of 0.86-1.51 (mean ± 2 SD). No sequence variations in the VWF A3 domain affecting VWF:CB were observed in the healthy controls. Collagen binding was then examined in subjects enrolled with a diagnosis of VWD. The index case in one family was noted to have a low VWF:CB/VWF:Ag ratio of 0.54. The VWF:Ag was 41 IU/dL, VWF:RCo was 44 IU/dL, VWF:CB was 22 U/dL, and multimer distribution was normal. Decreased VWF:CB/VWF:Ag was found in the father and sister of the index case, while the mother had normal VWF:CB/VWF:Ag. The index case initially presented with epistaxis and easy bruising. Her bleeding score was 8, as determined by the revised European Union questionnaire. DNA sequencing of exons 28-32 was performed and revealed a heterozygous mutation (5356C>G) predicting replacement of wild-type histidine 1786 by aspartic acid in all affected family members. This amino acid is located in a critical face of the A3 loop. The 1786D mutation was cloned into a full-length VWF expression vector and expressed, as was a previously reported collagen-binding mutation, 1731T. Collagen binding assays were performed using type I human placental collagen at 5 μg/mL or type III human placental collagen at 1 μg/mL to capture either plasma or recombinant VWF (rVWF). Both mutations had normal expression, at 97% and 87% of the expression seen with wild-type VWF, respectively. Multimer distribution was also normal, including presence of high molecular weight multimers. Plasma from family members heterozygous for the H1786D mutation showed a mean VWF:CB/VWF:Ag ratio of 0.23 ± 0.05 (mean ± SD) for type I collagen and 0.50 ± 0.11 for type III collagen. When the rVWF constructs were examined, the novel 1786D mutation showed essentially no binding to either type I or type III collagen, at <1% of wild-type rVWF. In contrast, the previously reported collagen binding mutation 1731T showed VWF:CB/VWF:Ag ratios of 45% for type I and 50% for type III collagen when compared to wild-type rVWF. As the proband had a minimally decreased VWF:Ag, we then examined collagen binding in ZPMCB-VWD subjects with VWF:Ag between 30 and 50 IU/dL. The mean VWF:CB/VWF:Ag ratio in this group was 1.02, with a range of 0.62 to 1.28. These results, taken together with the data in the healthy controls, suggest that decreased VWF:CB/VWF:Ag ratios may identify a specific defect in VWF function. In particular, the H1786D mutation appears to result in a complete loss of VWF's ability to bind either type I or type III collagen. The subjects with this mutation were all heterozygous, which may account for reduction, rather than absence, of in vitro VWF:CB. The VWF:CB may be useful in diagnosis of VWD, and a decreased VWF:CB/VWF:Ag ratio in the face of normal VWF multimer distribution may reflect specific loss of collagen binding ability leading to bleeding symptoms. Disclosures: Montgomery: GTI Diagnostics: Consultancy; Baxter: Consultancy; AstraZeneca: Consultancy; Bayer: Research Funding; CSL Behring: Membership on an entity's Board of Directors or advisory committees.

Collagen Binding Assay for von Willebrand Factor (VWF:CBA): Detection of von Willebrands Disease (VWD), and Discrimination of VWD Subtypes, Depends on Collagen Source

2000 ◽  
Vol 83 (01) ◽  
pp. 127-135 ◽  
Author(s):  
Emmanuel Favaloro
Keyword(s):  
Von Willebrand Factor ◽  
Binding Activity ◽  
Type Iii Collagen ◽  
Type I ◽  
Collagen Concentration ◽  
Collagen Binding ◽  
Type Iii ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Ristocetin Cofactor

SummaryA large number of different collagen preparations [n = 21] have been assessed for their ability to both detect von Willebrands Disease (VWD), and discriminate different VWD subtypes. Collagen preparations were tested at a range of concentrations and included: Type I, III and IV, and various mixtures of these, as aqueous supplied preparations and/or reconstituted from bulk lyophilised stock. Tissue sources for collagens ranged from human placenta to calf skin to equine tendon. Three of the collagen preparations tested did not support von Willebrand factor (VWF) binding in an ELISA process (therefore unable to detect VWD). The ability of the remaining preparations to detect VWF was variable, as was their ability to discriminate VWD subtypes. Detection of VWF and discrimination of VWD subtypes was not mutually inclusive. Thus, some collagen preparations provided excellent detection systems for VWF, but comparatively poorer discrimination of Type 2 VWD, while others provided good to acceptable detection and discrimination. Subtype discrimination was also dependent on the collagen concentration, and some batch to batch variation was evident with some preparations (particularly Type I collagens). Overall, best discrimination was typically achieved with Type I/III collagen mixtures, or Type III collagen preparations (where effectiveness was highly dependent on concentration). Good discrimination was also achieved with a commercial Type III collagen based VWF:CBA kit method. Results of the various ‘VWF:CBA assays’ are also compared with those using the Ristocetin Cofactor (VWF:RCof) assay (by platelet agglutination) and that using a commercial ‘VWF:RCof-alternative/ activity’ ELISA procedure. These latter methodologies tended to be less sensitive to VWF-discordance when compared to that detected by the majority of the VWF:CBA procedures. Abbreviations: FVIII:C Factor VIII: coagulant (assay); HMW High Molecular Weight [VWF]; PNP Pooled Normal Plasma; RIPA Ristocetin induced platelet aggregation procedure; VWD von Willebrands disease; VWF von Willebrand Factor; VWF:Ag von Willebrand Factor Antigen (assay); VWF: CBA Collagen Binding [Activity] Assay for VWF; VWF:RCof Ristocetin Cofactor Assay for VWF

scholarly journals Comparison of type I, type III and type VI collagen binding assays in diagnosis of von Willebrand disease

2012 ◽  
Vol 10 (7) ◽  
pp. 1425-1432 ◽  
Author(s):  
V. H. FLOOD ◽  
J. C. GILL ◽  
P. A. CHRISTOPHERSON ◽  
J. S. WREN ◽  
K. D. FRIEDMAN ◽  
...  
Keyword(s):  
Von Willebrand Disease ◽  
Type I ◽  
Binding Assays ◽  
Type Vi Collagen ◽  
Collagen Binding ◽  
Type Iii ◽  
Von Willebrand

Change in phenotype in long-term cultures of a clonal rat bone cell line: switch to the synthesis of α1 (I)-trimer collagen

1984 ◽  
Vol 62 (6) ◽  
pp. 462-469 ◽  
Author(s):  
Hardy Limeback ◽  
Kichibee Otsuka ◽  
Kam-Ling Yao ◽  
Jane E. Aubin ◽  
Jaro Sodek
Keyword(s):  
Collagen Synthesis ◽  
Type I Collagen ◽  
Bone Cell ◽  
Detectable Effect ◽  
Prostaglandin E ◽  
Intracellular Camp ◽  
Type Iii Collagen ◽  
Type I ◽  
Type Iii ◽  
Type V

A number of bone cell clones isolated from rat calvaria have been maintained in culture for more than 3 years. Several of these clones have undergone dramatic changes in phenotype. One of these clones, RGB 2.2, was observed originally to have a fibroblastic morphology in culture and to respond to parathyroid hormone (PTH), but not prostaglandin E2 (PGE2), with an increase in intracellular cAMP. Throughout several passages in early subcultures, these cells synthesized mostly type I collagen, with small amounts of type III and type V collagens. Whereas PTH had no detectable effect on collagen synthesis, PGE2 decreased the amount of total cell layer collagen, with the greatest effect on type III collagen, while increasing the proportion of type V collagen. Subsequent studies on these cells during 3 years in culture have indicated changes in their phenotype including a progressive change in morphology to a more cuboidal shape and a change in collagen synthesis, the cells producing large amounts of the "embryonic" collagen, α1(I) trimer. The reason(s) for the change in collagen expression is unknown, but may be the result of a change in which gene(s) is being expressed.

scholarly journals PERITONEAL ADHESIONS TYPE I, III AND TOTAL COLLAGEN ON POLYPROPYLENE AND COATED POLYPROPYLENE MESHES: EXPERIMENTAL STUDY IN RATS

2017 ◽  
Vol 30 (2) ◽  
pp. 77-82 ◽  
Author(s):  
Lucas Félix ROSSI ◽  
Manoel Roberto Maciel TRINDADE ◽  
Armando José D`ACAMPORA ◽  
Luise MEURER
Keyword(s):  
Type I Collagen ◽  
Cell Types ◽  
Type Iii Collagen ◽  
Type I ◽  
Abdominal Adhesions ◽  
Peritoneal Adhesions ◽  
Type Iii ◽  
Total Collagen

ABSTRACT Background: Hernia correction is a routinely performed treatment in surgical practice. The improvement of the operative technique and available materials certainly has been a great benefit to the quality of surgical results. The insertion of prostheses for hernia correction is well-founded in the literature, and has become the standard of treatment when this type of disease is discussed. Aim: To evaluate two available prostheses: the polypropylene and polypropylene coated ones in an experimental model. Methods: Seven prostheses of each kind were inserted into Wistar rats (Ratus norvegicus albinus) in the anterior abdominal wall of the animal in direct contact with the viscera. After 90 days follow-up were analyzed the intra-abdominal adhesions, and also performed immunohistochemical evaluation and videomorphometry of the total, type I and type III collagen. Histological analysis was also performed with hematoxylin-eosin to evaluate cell types present in each mesh. Results: At 90 days the adhesions were not different among the groups (p=0.335). Total collagen likewise was not statistically different (p=0.810). Statistically there was more type III collagen in the coated polypropylene group (p=0.039) while type I was not different among the prostheses (p=0.050). The lymphocytes were statistically more present in the polypropylene group (p=0.041). Conclusion: The coated prosthesis was not different from the polypropylene one regarding the adhesion. Total and type I collagen were not different among the groups, while type III collagen was more present on the coated mesh. There was a greater number of lymphocytes on the polypropylene mesh.

scholarly journals A matrix metalloproteinase-generated neoepitope of CRP can identify knee and multi-joint inflammation in osteoarthritis

2021 ◽  
Vol 23 (1) ◽  
Author(s):  
Louie C. Alexander ◽  
Grant McHorse ◽  
Janet L. Huebner ◽  
Anne-Christine Bay-Jensen ◽  
Morten A. Karsdal ◽  
...  
Keyword(s):  
Synovial Fluid ◽  
Matrix Metalloproteinase ◽  
Type I Collagen ◽  
Joint Inflammation ◽  
Cartilage Degradation ◽  
Womac Pain ◽  
Type Iii Collagen ◽  
Type I ◽  
Radiographic Imaging ◽  
Type Iii

Abstract Objective To compare C-reactive protein (CRP) and matrix metalloproteinase-generated neoepitope of CRP (CRPM) as biomarkers of inflammation and radiographic severity in patients with knee osteoarthritis. Methods Participants with symptomatic osteoarthritis (n=25) of at least one knee underwent knee radiographic imaging and radionuclide etarfolatide imaging to quantify inflammation of the knees and other appendicular joints. For purposes of statistical analysis, semi-quantitative etarfolatide and radiographic imaging scores were summed across the knees; etarfolatide scores were also summed across all joints to provide a multi-joint synovitis measure. Multiple inflammation and collagen-related biomarkers were measured by ELISA including CRP, CRPM, MMP-generated neoepitopes of type I collagen and type III collagen in serum (n=25), and CD163 in serum (n=25) and synovial fluid (n=18). Results BMI was associated with CRP (p=0.001), but not CRPM (p=0.753). Adjusting for BMI, CRP was associated with radiographic knee osteophyte score (p=0.002), while CRPM was associated with synovitis of the knee (p=0.017), synovitis of multiple joints (p=0.008), and macrophage marker CD163 in serum (p=0.009) and synovial fluid (p=0.03). CRP correlated with MMP-generated neoepitope of type I collagen in serum (p=0.045), and CRPM correlated with MMP-generated neoepitope of type III collagen in serum (p<0.0001). No biomarkers correlated with age, knee pain, or WOMAC pain. Conclusions To our knowledge, this is the first time that CRPM has been shown to be associated with knee and multi-joint inflammation based on objective imaging (etarfolatide) and biomarker (CD163) measures. These results demonstrate the capability of biomarker measurements to reflect complex biological processes and for neoepitope markers to more distinctly reflect acute processes than their precursor proteins. CRPM is a promising biomarker of local and systemic inflammation in knee OA that is associated with cartilage degradation and is independent of BMI. CRPM is a potential molecular biomarker alternative to etarfolatide imaging for quantitative assessment of joint inflammation.

scholarly journals Collagen Binding Provides a Sensitive Screen for Variant von Willebrand Disease

2013 ◽  
Vol 59 (4) ◽  
pp. 684-691 ◽  
Author(s):  
Veronica H Flood ◽  
Joan Cox Gill ◽  
Kenneth D Friedman ◽  
Pamela A Christopherson ◽  
Paula M Jacobi ◽  
...  
Keyword(s):  
Gel Electrophoresis ◽  
Reference Intervals ◽  
Von Willebrand Disease ◽  
Type Iii Collagen ◽  
Healthy Controls ◽  
Collagen Binding ◽  
Vessel Injury ◽  
Clinical Biology ◽  
Von Willebrand

BACKGROUND von Willebrand factor (VWF) is a multimeric protein that binds platelets and collagen, facilitating hemostasis at sites of vessel injury. Measurement of VWF multimer distribution is critical for diagnosis of variant von Willebrand disease (VWD), particularly types 2A and 2B, but the typical measurement by gel electrophoresis is technically difficult and time-consuming. A comparison of VWF collagen binding (VWF:CB) and VWF multimer distribution was performed to evaluate the utility of VWF:CB as a diagnostic test. METHODS Participants were enrolled in the Zimmerman Program for the Molecular and Clinical Biology of VWD. VWF:CB was analyzed with type III collagen and multimer distribution by agarose gel electrophoresis. The study population included 146 healthy controls, 351 individuals with type 1 VWD, and 77 with type 2 VWD. Differences between individuals with multimer group results within (controls) and outside the reference intervals were assessed with Mann–Whitney tests. RESULTS The mean VWF:CB/VWF antigen ratio was 1.10 for individuals with multimer distribution within the reference intervals and 0.51 for those with multimer distribution outside the reference intervals (P &lt; 0.001). Sensitivity of VWF:CB for multimer abnormalities was 100% for healthy controls, 99% for patients with type 1, and 100% for patients with type 2A and type 2B VWD using a VWF:CB/VWF antigen cutoff ratio of 0.6, and decreased to 99% for all patients with a ratio of 0.7. With the exception of individuals with novel or unclassified mutations, the VWF:CB was able to correctly categorize participants with variant VWD. CONCLUSIONS These findings suggest that VWF:CB may substitute for multimer distribution in initial VWD testing, although further studies are needed to validate the clinical utility of VWF:CB.

Type VI collagen induces cardiac myofibroblast differentiation: implications for postinfarction remodeling

2006 ◽  
Vol 290 (1) ◽  
pp. H323-H330 ◽  
Author(s):  
Jennifer E. Naugle ◽  
Erik R. Olson ◽  
Xiaojin Zhang ◽  
Sharon E. Mase ◽  
Charles F. Pilati ◽  
...  
Keyword(s):  
Cardiac Fibrosis ◽  
Type I Collagen ◽  
In Vivo Model ◽  
Type Iii Collagen ◽  
Type I ◽  
Myofibroblast Differentiation ◽  
Type Vi Collagen ◽  
Collagen Substrate ◽  
Type Iii

Cardiac fibroblast (CF) proliferation and differentiation into hypersecretory myofibroblasts can lead to excessive extracellular matrix (ECM) production and cardiac fibrosis. In turn, the ECM produced can potentially activate CFs via distinct feedback mechanisms. To assess how specific ECM components influence CF activation, isolated CFs were plated on specific collagen substrates (type I, III, and VI collagens) before functional assays were carried out. The type VI collagen substrate potently induced myofibroblast differentiation but had little effect on CF proliferation. Conversely, the type I and III collagen substrates did not affect differentiation but caused significant induction of proliferation (type I, 240.7 ± 10.3%, and type III, 271.7 ± 21.8% of basal). Type I collagen activated ERK1/2, whereas type III collagen did not. Treatment of CFs with angiotensin II, a potent mitogen of CFs, enhanced the growth observed on types I and III collagen but not on the type VI collagen substrate. Using an in vivo model of myocardial infarction (MI), we measured changes in type VI collagen expression and myofibroblast differentiation after post-MI remodeling. Concurrent elevations in type VI collagen and myofibroblast content were evident in the infarcted myocardium 20-wk post-MI. Overall, types I and III collagen stimulate CF proliferation, whereas type VI collagen plays a potentially novel role in cardiac remodeling through facilitation of myofibroblast differentiation.

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