Localization of binding sites within human von Willebrand factor for monomeric type III collagen

Biochemistry ◽  
1986 ◽  
Vol 25 (26) ◽  
pp. 8357-8361 ◽  
Author(s):  
Gerald J. Roth ◽  
Koiti Titani ◽  
Leon W. Hoyer ◽  
Mark J. Hickey
Keyword(s):  
Von Willebrand Factor ◽  
Binding Sites ◽  
Type Iii Collagen ◽  
Type Iii ◽  
Von Willebrand ◽  
Willebrand Factor

scholarly journals Localization of a collagen-interactive domain of human von Willebrand factor between amino acid residues Gly 911 and Glu 1,365

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1577-1583 ◽  
Author(s):  
M Kalafatis ◽  
Y Takahashi ◽  
JP Girma ◽  
D Meyer
Keyword(s):  
Amino Acid ◽  
Von Willebrand Factor ◽  
Binding Sites ◽  
Type Iii Collagen ◽  
Amino Acid Residues ◽  
Terminal Part ◽  
Human Type ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Scatchard Plots

A collagen-binding domain of von Willebrand factor (vWF) has been identified in the central part of the molecule by comparing the binding properties of vWF and Staphylococcus aureus V-8 protease-generated vWF fragments with collagen. The binding of purified human vWF to human type III collagen was found to be specific. At saturation, 38 to 50.2 micrograms of vWF bound per milligram of collagen. Scatchard plots derived from binding isotherms demonstrated the presence of at least two classes of binding sites. Purified vWF was digested with S aureus V- 8 protease into two complementary fragments (SpIII and SpII). SpII, the C-terminal end of vWF (amino acid residues 1,366 to 2,050), was totally devoid of affinity for collagen. Contrarily, purified SpIII, the N- terminal part of vWF (residues 1 to 1,365), totally displaced vWF binding and specifically bound to collagen. At saturation, 25 to 45 micrograms of SpIII bound per milligram of collagen. Scatchard plots demonstrated the presence of a single class of binding sites. SpIII was further digested with the same enzyme to generate SpI, a 52-kilodalton fragment from the C-terminal part of SpIII (residues 911 to 1,365). Spl induced a dose-dependent inhibition of both vWF and SpIII binding to collagen. A series of six monoclonal antibodies against SpIII that completely abolished vWF and SpIII interaction with collagen also bound to SpI. In conclusion, SpI extending between amino acid residues 911 and 1,365 of vWF contains a specific site that interacts with human type III collagen.

A3 Domain Is Essential for Interaction of von Willebrand Factor with Collagen Type III

1996 ◽  
Vol 75 (06) ◽  
pp. 950-958 ◽  
Author(s):  
Hanneke L Lankhof ◽  
Maggy van Hoeij ◽  
Marion E Schiphorst ◽  
Madelon Bracke ◽  
Ya-Ping Wu ◽  
...  
Keyword(s):  
Binding Site ◽  
Von Willebrand Factor ◽  
Binding Sites ◽  
Platelet Adhesion ◽  
Collagen Type ◽  
Collagen Type Iii ◽  
Collagen Binding ◽  
Type Iii ◽  
Von Willebrand ◽  
Willebrand Factor

Summaryvon Willebrand factor (vWF) mediates platelet adhesion at sites of vascular damage. It acts as a bridge between receptors on platelets and collagens present in the connective tissue. Two collagen binding sites have been identified on the A1 and A3 domain of the vWF subunit. To study the functional importance of these binding sites, we have made two deletion mutants that lack the A1 domain (residues 478-716; ΔA1-vWF; Sixma et al. Eur. J. Biochem. 196,369,1991 [1]) or the A3 domain (residues 910-1113; ΔA3-vWF). After transfection in baby hamster kidney cells overexpressing furin, the mutants were processed and secreted efficiently. Ristocetin or botrocetin induced platelet binding was normal for ΔA3-vWF as was binding to heparin and factor VIII. As reported by Sixma et al. (1) ΔAl-vWF still binds to collagen type III, indicating that the A3 domain is sufficient for the interaction. In the current study, we investigated the binding of ΔA3-vWF to collagen type III. When preincubated on collagen type III it did not support platelet adhesion under flow conditions, whereas it was able to support platelet adhesion when coated directly to a glass surface. The binding of 125I-ΔA3-vWF to collagen was specific but maximal binding was about 40 times less compared to 125I-vWF. When added at 25 times excess, ΔA3-vWF did not compete with 125I-vWF for binding to collagen type III, whereas ΔAl-vWF did. The binding of 125I-ΔA3-vWF could be blocked by excess unlabeled vWF but not by ΔA1-vWF. In conclusion, we demonstrate that the A3 domain in vWF contains the major collagen binding site. The major binding site present on the A3 domain and the minor site present on A1 bind to different sites on collagen.

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 Localization of a collagen-interactive domain of human von Willebrand factor between amino acid residues Gly 911 and Glu 1,365

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1577-1583 ◽  
Author(s):  
M Kalafatis ◽  
Y Takahashi ◽  
JP Girma ◽  
D Meyer
Keyword(s):  
Amino Acid ◽  
Von Willebrand Factor ◽  
Binding Sites ◽  
Type Iii Collagen ◽  
Amino Acid Residues ◽  
Terminal Part ◽  
Human Type ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Scatchard Plots

Abstract A collagen-binding domain of von Willebrand factor (vWF) has been identified in the central part of the molecule by comparing the binding properties of vWF and Staphylococcus aureus V-8 protease-generated vWF fragments with collagen. The binding of purified human vWF to human type III collagen was found to be specific. At saturation, 38 to 50.2 micrograms of vWF bound per milligram of collagen. Scatchard plots derived from binding isotherms demonstrated the presence of at least two classes of binding sites. Purified vWF was digested with S aureus V- 8 protease into two complementary fragments (SpIII and SpII). SpII, the C-terminal end of vWF (amino acid residues 1,366 to 2,050), was totally devoid of affinity for collagen. Contrarily, purified SpIII, the N- terminal part of vWF (residues 1 to 1,365), totally displaced vWF binding and specifically bound to collagen. At saturation, 25 to 45 micrograms of SpIII bound per milligram of collagen. Scatchard plots demonstrated the presence of a single class of binding sites. SpIII was further digested with the same enzyme to generate SpI, a 52-kilodalton fragment from the C-terminal part of SpIII (residues 911 to 1,365). Spl induced a dose-dependent inhibition of both vWF and SpIII binding to collagen. A series of six monoclonal antibodies against SpIII that completely abolished vWF and SpIII interaction with collagen also bound to SpI. In conclusion, SpI extending between amino acid residues 911 and 1,365 of vWF contains a specific site that interacts with human type III collagen.

Characterization of Partial Gene Deletions in Type III von Willebrand Disease with Alloantibody Inhibitors

1994 ◽  
Vol 72 (02) ◽  
pp. 180-185 ◽  
Author(s):  
David J Mancuso ◽  
Elodee A Tuley ◽  
Ricardo Castillo ◽  
Norma de Bosch ◽  
Pler M Mannucci ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Von Willebrand Disease ◽  
Pcr Analysis ◽  
Gene Deletions ◽  
Factor Gene ◽  
Type Iii ◽  
Large Deletions ◽  
Von Willebrand ◽  
Willebrand Factor

Summaryvon Willebrand factor gene deletions were characterized in four patients with severe type III von Willebrand disease and alloantibodies to von Willebrand factor. A PCR-based strategy was used to characterize the boundaries of the deletions. Identical 30 kb von Willebrand factor gene deletions which include exons 33 through 38 were identified in two siblings of one family by this method. A small 5 base pair insertion (CCTGG) was sequenced at the deletion breakpoint. PCR analysis was used to detect the deletion in three generations of the family, including two family members who are heterozygous for the deletion. In a second family, two type III vWD patients, who are distant cousins, share an -56 kb deletion of exons 22 through 43. The identification and characterization of large vWF gene deletions in these type III vWD patients provides further support for the association between large deletions in both von Willebrand factor alleles and the development of inhibitory alloantibodies.

PRIMARY BINDING SITE OF VON WILLEBRAND FACTOR IN THE SUBENDOTHELIUM WHICH MEDIATES PLATELET ADHESION IS NOT COLLAGEN

1987 ◽  
Author(s):  
Philip G de Groot ◽  
Jan A van Mourik ◽  
Jan J Sixma
Keyword(s):  
Monoclonal Antibody ◽  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Collagen Type ◽  
Collagen Type I ◽  
Extracellular Matrices ◽  
Type I ◽  
Type Iii ◽  
Von Willebrand ◽  
Willebrand Factor

We have studies the binding of von Willebrand factor (vWF) to extracellular matrices of endothelial cells and smooth muscle cells and to the vessel wall of human umbilical arteries in relation to its function in supporting platelet adhesion at high shear rates. CLB-RAg 38, a monoclonal antibody directed against vWF inhibits the binding of 125I-vWF extracellular matrices completely. The binding of 125I-vWF to subendothelium is not inhibited, because there are many different binding sites. CLB-RAg 38 inhibits platelet adhesion to extracellular matrices and subendothelium, in sofar as it is dependent on plasma vWF. CLB-RAg 38 has no effect on adhesion depending on vWF already bound to the matrix or subendothelium. CLB-RAg 38 does not inhibit binding of vWF to collagen type I and type III. Another monoclonal antibody against vWF, CLB-RAg 201, completely inhibits binding of vWF to collagen type I and type III. CLB-RAg 201 does not inhibit binding of 125I-vWF ot the extracellular matrices. CLB-RAg 201 partly inhibits platelet adhesion but this inhibition is also present when the adhesion depends on vWF already present in matrix or subendothelium, indicating that CLB-RAg 201 also inhibits the adhesion of platelets directly, this in contrast to CLB-RAg 38. The epitopes for CLB-RAg 201 and 38 were found on different tryptic fragments of vWF. These data indicate that vWF binds to subendothelium and to matrices of cultured cells by mechanism that is different from binding to collagen.

The Importance of the Carbohydrate Moiety of the Factor VIII/Von Willebrand Factor Protein in Binding to and Causing Agglutination of Human Platelets

1979 ◽  
Author(s):  
H.R. Gralnick ◽  
D.K. Morisato
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Binding Sites ◽  
Human Platelets ◽  
Galactose Oxidase ◽  
Scatchard Analysis ◽  
Human Fibrinogen ◽  
Number Of Binding Sites ◽  
Von Willebrand ◽  
Willebrand Factor

We have investigated the binding of radiolabelled factor VIII/von Willebrand factor (f. VIII/vWf) protein to human platelets (P) in the presence of ristocetin (R). In these atudies we have delineated the importance of the carbohydrate (CHO) moiety(s) in both the binding to the P and in cauaing agglutination of P. Binding of the f.VIII/vWf protein to human P was time and temperature dependent and dependent on the concentration of R. Binding was specific in that it could not be blocked by human fibrinogen but was inhibited by unlabelled f.VIII/vWf protein. In studies utilizing varying amounts of the f.VIII/vWf protein or by varying the number of P in the assay, the number of binding sites for the f. VIII/vWf protein were estimated at 9,500-9,800 per platelet. Scatchard analysis revealed 11,000 binding sites with 3,600 of high affinity and 7,400 of low affinity. Removal of the sialic acid of the f.VIII/vWf protein resulted in no significa nt change in its ability to bind to the P surface or cause agglutination in the presence, IR. Removal of the galactose by 6-galactosijase resulted in a 75% reduction of binding of the f.VIII/vWf protein and a 91% decrease in the agglutination of human P. Similar studies with galactose oxidase showed that oxidation of the penultimate galactose residue s results in a decrease in agglutination comparable to that seen with 6-galactosidase treatment. These studies indicate that the CHO moiety of the f.VIII/vWf protein is important in both binding to the P surface as well as causing agglutination of human P.

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