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.

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.

A Factor VIII Neutralizing Monoclonal Antibody and a Human Inhibitor Alloantibody Recognizing Epitopes in the C2 Domain Inhibit Factor VIII Binding to von Willebrand Factor and to Phosphatidylserine

1993 ◽  
Vol 69 (03) ◽  
pp. 240-246 ◽  
Author(s):  
Midori Shima ◽  
Dorothea Scandella ◽  
Akira Yoshioka ◽  
Hiroaki Nakai ◽  
Ichiro Tanaka ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Amino Acid ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Light Chain ◽  
Amino Acid Residues ◽  
Amino Terminal ◽  
Neutralizing Monoclonal Antibody ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryA neutralizing monoclonal antibody, NMC-VIII/5, recognizing the 72 kDa thrombin-proteolytic fragment of factor VIII light chain was obtained. Binding of the antibody to immobilized factor VIII (FVIII) was completely blocked by a light chain-specific human alloantibody, TK, which inhibits FVIII activity. Immunoblotting analysis with a panel of recombinant protein fragments of the C2 domain deleted from the amino-terminal or the carboxy-terminal ends demonstrated binding of NMC-VIII/5 to an epitope located between amino acid residues 2170 and 2327. On the other hand, the epitope of the inhibitor alloantibody, TK, was localized to 64 amino acid residues from 2248 to 2312 using the same recombinant fragments. NMC-VIII/5 and TK inhibited FVIII binding to immobilized von Willebrand factor (vWF). The IC50 of NMC-VIII/5 for the inhibition of binding to vWF was 0.23 μg/ml for IgG and 0.2 μg/ml for F(ab)'2. This concentration was 100-fold lower than that of a monoclonal antibody NMC-VIII/10 which recognizes the amino acid residues 1675 to 1684 within the amino-terminal portion of the light chain. The IC50 of TK was 11 μg/ml by IgG and 6.3 μg/ml by F(ab)'2. Furthermore, NMC-VIII/5 and TK also inhibited FVIII binding to immobilized phosphatidylserine. The IC50 for inhibition of phospholipid binding of NMC-VIII/5 and TK (anti-FVIII inhibitor titer of 300 Bethesda units/mg of IgG) was 10 μg/ml.

scholarly journals A monoclonal antibody to factor VIII inhibits von Willebrand factor binding and thrombin cleavage

Blood ◽  
1991 ◽  
Vol 77 (9) ◽  
pp. 1929-1936 ◽  
Author(s):  
JW Precup ◽  
BC Kline ◽  
DN Fass
Keyword(s):  
Monoclonal Antibody ◽  
Amino Acid ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Light Chain ◽  
Amino Acid Residues ◽  
Protein Fragments ◽  
Thrombin Cleavage ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract To study the interaction of human factor VIII (FVIII) with its various ligands, select regions of cDNA encoding FVIII light chain were cloned into the plasmid expression vector pET3B to overproduce FVIII protein fragments in the bacterium Escherichia coli. Partially purified FVIII protein fragments were used to produce monoclonal antibodies. One monoclonal antibody, 60-B, bound both an FVIII protein fragment (amino acid residues 1563 through 1909) and recombinant human FVIII, but not porcine FVIII. This antibody prevented FVIII-vWF binding and acted as an inhibitor in both the activated partial thromboplastin time (APTT) assay and a chromogenic substrate assay that measured factor Xa generation. The ability of the antibody to inhibit FVIII activity was diminished in a dose-dependent fashion by von Willebrand factor. This anti-FVIII monoclonal antibody bound to a synthetic peptide, K E D F D I Y D E D E, equivalent to FVIII amino acid residues 1674 through 1684. The 60-B antibody did not react with a peptide in which the aspartic acid residue at 1681 (underlined) was changed to a glycine, which is the amino acid present at this position in porcine FVIII. Gel electrophoretic analysis of thrombin cleavage patterns of human FVIII showed that the 60-B antibody prevented thrombin cleavage at light chain residue 1689. The coagulant inhibitory activity of the 60-B antibody may be due, in part, to the prevention of thrombin activation of FVIII light chain.

The N-Terminal Amino Acid Residues Ser764 and Lys773 of the D' Domain of Von Willebrand Factor Contribute to Factor VIII Binding

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2248-2248
Author(s):  
Lydia Castro-Núñez ◽  
Esther Bloem ◽  
Carmen van der Zwaan ◽  
Koen Mertens ◽  
Alexander B Meijer
Keyword(s):  
Amino Acid ◽  
Chemical Modification ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Terminal Region ◽  
Lysine Residue ◽  
Amino Acid Residues ◽  
N Terminus ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract 2248 Factor VIII (FVIII) circulates in a tight complex with its carrier protein von Willebrand factor (VWF). Activation of FVIII results in the dissociation of the FVIII-VWF complex after which FVIII can perform its role in the coagulation cascade. In the complex with VWF, FVIII is protected from rapid clearance from the circulation. Individuals with a mutation in VWF that impairs the ability of VWF to bind FVIII can therefore have a bleeding disorder caused by a low plasma level of FVIII. Mature VWF contains multiple domains of which the N-terminal D'-D3 domains have been shown to comprise the FVIII binding site. Detailed information about amino acid regions in VWF that contribute to the direct interaction with FVIII is, however, lacking. In the present study we have employed a chemical footprinting approach to identify amino acid regions of VWF that are involved in binding FVIII. To this end, the lysine amino acid residues of VWF were chemically modified in the presence of FVIII or activated FVIII. VWF was subsequently cleaved into peptides employing chymotrypsin. The identity of the peptides and whether or not they contained a modified lysine residue was assessed by nanoLC mass spectrometry. The results showed that the lysine residues of almost all identified peptides were modified to the same extent upon incubation of VWF with FVIII or activated FVIII. However, lysine residue 773 in the N-terminal peptide comprising the residues 766-SCRPPMVKL-774 was protected from chemical modification in the presence of FVIII. In addition, a peptide was identified in which the free amine group of serine 764 at the start of the D' domain was also differentially modified in the presence of FVIII or activated FVIII. We next studied the structure of a molecular model of the D' domain that was obtained by comparative homology modeling. Structure analysis revealed that the N-terminal region 764–773 is situated at the tip of the D' domain and that the amino acid residues Ser764 and Lys773 are in close proximity. This observation combined with the results obtained with the chemical footprinting approach implies that the residues Ser764 and Lys773 at the N-terminus of VWF are directly involved in the FVIII-VWF complex formation. Alternatively, upon binding of FVIII, there is a conformational change in this N-terminal region resulting into a differential accessibility of these residues for chemical modification. To further investigate on this issue, we constructed recombinant VWF variants in which the lysine residue 773 and the serine residue at position 764 were replaced by alanines. The variants Ser764Ala, Lys773Ala and WT-VWF were expressed in 293 cells and purified. The binding of Ser764Ala and Lys773Ala to FVIII was evaluated employing surface plasmon resonance (SPR) analysis. The data revealed that the N-terminal region of the VWF D' domain modulates the interaction with FVIII. The contribution of Ser764 and Lys733 was mainly reflected in the dissociation kinetics of the complex. We also assessed the association of the VWF variants to FVIII in a solid phase binding assay. In addition, we evaluated to what extent the VWF variants can compete with WT-VWF for binding FVIII. The results were in agreement with the findings obtained with SPR analysis, and demonstrated a modulatory role of the residues 764 and 773. Taken together, our data reveal that the residues Ser764 and Lys773 at the N-terminus of mature VWF contribute to the affinity of the FVIII-VWF complex. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Specific electrostatic interactions between charged amino acid residues regulate binding of von Willebrand factor to blood platelets

2017 ◽  
Vol 292 (45) ◽  
pp. 18608-18617 ◽  
Author(s):  
Gianluca Interlandi ◽  
Olga Yakovenko ◽  
An-Yue Tu ◽  
Jeff Harris ◽  
Jennie Le ◽  
...  
Keyword(s):  
Amino Acid ◽  
Von Willebrand Factor ◽  
Electrostatic Interactions ◽  
Blood Platelets ◽  
Amino Acid Residues ◽  
Von Willebrand ◽  
Willebrand Factor

scholarly journals A monoclonal antibody to factor VIII inhibits von Willebrand factor binding and thrombin cleavage

Blood ◽  
1991 ◽  
Vol 77 (9) ◽  
pp. 1929-1936
Author(s):  
JW Precup ◽  
BC Kline ◽  
DN Fass
Keyword(s):  
Monoclonal Antibody ◽  
Amino Acid ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Light Chain ◽  
Amino Acid Residues ◽  
Protein Fragments ◽  
Thrombin Cleavage ◽  
Von Willebrand ◽  
Willebrand Factor

To study the interaction of human factor VIII (FVIII) with its various ligands, select regions of cDNA encoding FVIII light chain were cloned into the plasmid expression vector pET3B to overproduce FVIII protein fragments in the bacterium Escherichia coli. Partially purified FVIII protein fragments were used to produce monoclonal antibodies. One monoclonal antibody, 60-B, bound both an FVIII protein fragment (amino acid residues 1563 through 1909) and recombinant human FVIII, but not porcine FVIII. This antibody prevented FVIII-vWF binding and acted as an inhibitor in both the activated partial thromboplastin time (APTT) assay and a chromogenic substrate assay that measured factor Xa generation. The ability of the antibody to inhibit FVIII activity was diminished in a dose-dependent fashion by von Willebrand factor. This anti-FVIII monoclonal antibody bound to a synthetic peptide, K E D F D I Y D E D E, equivalent to FVIII amino acid residues 1674 through 1684. The 60-B antibody did not react with a peptide in which the aspartic acid residue at 1681 (underlined) was changed to a glycine, which is the amino acid present at this position in porcine FVIII. Gel electrophoretic analysis of thrombin cleavage patterns of human FVIII showed that the 60-B antibody prevented thrombin cleavage at light chain residue 1689. The coagulant inhibitory activity of the 60-B antibody may be due, in part, to the prevention of thrombin activation of FVIII light chain.

scholarly journals Identification of a unique filamin A binding region within the cytoplasmic domain of glycoprotein Ibα

2005 ◽  
Vol 387 (3) ◽  
pp. 849-858 ◽  
Author(s):  
Susan L. CRANMER ◽  
Inna PIKOVSKI ◽  
Pierre MANGIN ◽  
Philip E. THOMPSON ◽  
Teresa DOMAGALA ◽  
...  
Keyword(s):  
Amino Acid ◽  
Von Willebrand Factor ◽  
Cytoplasmic Tail ◽  
Membrane Skeleton ◽  
Single Amino Acid ◽  
Amino Acid Residues ◽  
Filamin A ◽  
Binding Studies ◽  
Von Willebrand ◽  
Willebrand Factor

Binding of the platelet GPIb/V/IX (glycoprotein Ib/V/IX) receptor to von Willebrand factor is critical for platelet adhesion and aggregation under conditions of rapid blood flow. The adhesive function of GPIbα is regulated by its anchorage to the membrane skeleton through a specific interaction with filamin A. In the present study, we examined the amino acid residues within the cytoplasmic tail of GPIbα, which are critical for association with filamin A, using a series of 25-mer synthetic peptides that mimic the cytoplasmic tail sequences of wild-type and mutant forms of GPIbα. Peptide binding studies of purified human filamin A have demonstrated a major role for the conserved hydrophobic stretch L567FLWV571 in mediating this interaction. Progressive alanine substitutions of triple, double and single amino acid residues within the Pro561–Arg572 region suggested an important role for Trp570 and Phe568 in promoting GPIbα binding to filamin A. The importance of these two residues in promoting filamin A binding to GPIbα in vivo was confirmed from the study of Chinese-hamster ovary cells expressing GPIbα Trp570→Ala and Phe568→Ala substitutions. Phenotypic analysis of these cell lines in flow-based adhesion studies revealed a critical role for these residues in maintaining receptor anchorage to the membrane skeleton and in maintaining cell adhesion to a von Willebrand factor matrix under high-shear conditions. These studies demonstrate a novel filamin A binding motif in the cytoplasmic tail of GPIbα, which is critically dependent on both Trp570 and Phe568.

A Synthetic Factor VIII Peptide of Eight Amino Acid Residues (1677-1684) Contains the Binding Region of an Anti-Factor VIII Antibody which Inhibits the Binding of Factor VIII to von Willebrand Factor

1990 ◽  
Vol 63 (03) ◽  
pp. 403-406 ◽  
Author(s):  
Paul A Foster ◽  
Carol A Fulcher ◽  
Richard A Houghten ◽  
Theodore S Zimmerman
Keyword(s):  
Amino Acid ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Light Chain ◽  
Amino Acid Residues ◽  
Binding Region ◽  
Dependent Inhibition ◽  
Dose Dependent Inhibition ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryThe monoclonal anti-factor VIII (FVIII) antibody C4 has previously been reported to inhibit the binding of purified FVIII to immobilized von Willebrand factor (vWF). The binding area of C4 was identified to be within fifteen amino acid residues (1670-1684) based on the ability of a synthetic FVIII peptide consisting of amino acid residues 1670-1684 to completely inhibit the binding of C4 to FVIII. We now report the further localization of the binding region of C4 to within eight amino acid residues (1677-1684) of FVIII light chain. Nine new overlapping FVIII peptides were synthesized based on the amino acid sequence of the acidic region of FVIII light chain and tested, along with seven previously tested peptides, for the ability to inhibit C4 binding to FVIII in an ELISA assay. Three synthetic FVIII peptides 1670-1684, 1675-1690, and 1677-1684 demonstrated dose dependent inhibition of C4 binding to FVIII. The three reactive peptides contain residues 1677-1684 in common. Since C4 can completely inhibit the binding of FVIII to vWF, this report further localizes an eight amino acid residue region of FVIII which may be important in the mediation of vWF binding.

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