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 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.

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.

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.

A FACTOR VIII BINDING DOMAIN RESIDES WITHIN THE AMINO-TERMINAL 272 AMINO ACID RESIDUES OF VON WILLEBRAND FACTOR

1987 ◽  
Author(s):  
P A Foster ◽  
C A Fulcher ◽  
T Marti ◽  
K Titani ◽  
T S Zimmerman
Keyword(s):  
Monoclonal Antibody ◽  
Amino Acid ◽  
Factor Viii ◽  
Von Willebrand Factor ◽  
Dependent Manner ◽  
Amino Acid Residues ◽  
Amino Terminal ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Dose Dependent

We have identified a Factor VIII (FVIII) binding domain residing within the amino-terminal 272 amino acid residues of the mature von Willebrand Factor (vWF) subunit. Two dimensional crossed immunoelectrophoresis showed direct binding of purified human FVIII to purified human vWF. After proteolytic digestion of vWF with Staphylococcus aureus V8 protease, FVIII binding was seen only with the amino-terminal SP fragment III and not with the carboxy-terminal SP fragment II. A monoclonal anti-vWF antibody (C3) partially blocked FVIII binding to vWF and SP fragment III. FVIII also bound to vWF which had been adsorbed to polystyrene beads. This binding was inhibited in a dose dependent manner by whole vWF, SP fragment III, and by monoclonal antibody C3. Binding could not be inhibited by SP fragment I, which contains the middle portion of the vWF molecule, or by reduced and alkylated whole vWF. SP fragment II caused only minor inhibition. Trypsin cleavage of SP fragment III produced a 35-kDa fragment containing the amino-terminal 272 amino acid residues of vWF. This fragment reacted with monoclonal antibody C3 and inhibited the binding of FVIII to vWF in a dose dependent manner. The other major fragment obtained from this digestion was a two chain hetero-dimer composed of amino acid residues 273-511 and 674-728. This fragment did not inhibit FVIII binding. These studies demonstrate that a major FVIII binding site resides within the first 272 amino acid residues of vWF.

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 Studies on anti-von Willebrand factor (vWF) monoclonal antibody NMC-4, which inhibits both ristocetin- and botrocetin-induced vWF binding to platelet glycoprotein Ib

Blood ◽  
1991 ◽  
Vol 77 (1) ◽  
pp. 113-120 ◽  
Author(s):  
Y Fujimura ◽  
Y Usami ◽  
K Titani ◽  
K Niinomi ◽  
K Nishio ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Amino Acid ◽  
Von Willebrand Factor ◽  
Amino Acid Analysis ◽  
Disulfide Bonds ◽  
Platelet Glycoprotein ◽  
Amino Acid Residues ◽  
Tryptic Digest ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Anti-von Willebrand factor (vWF) monoclonal antibody NMC-4 completely inhibited vWF binding to platelet glycoprotein (GP) lb induced by either ristocetin or botrocetin at an IgG concentration of approximately 10 micrograms/mL, and also blocked binding of asialo-vWF to GP lb. NMC-4 coupled beads isolated a 97-Kd fragment (Fr) from a whole tryptic digest of vWF. The N-terminal sequencing of the nonreduced 97-Kd Fr, in combination with amino acid analysis, showed it to be a homodimer of residues 449 through 728 of the constituent subunit. Present data, together with the results obtained from previous studies, confirm the existence of one or three possible inter-subunit disulfide bonds between cysteine residues 459, 462, and 464. NMC-4 bound to reduced vWF Fr(s) more weakly than to nonreduced Fr(s), but it did not react with Fr III-T2 of vWF, a disulfide-linked twin heterodimer of residues 273 through 511 and 674 through 728 (Marti et al, Biochemistry 26:8099, 1987). Fr III-T2 completely inhibited ristocetin-induced vWF binding at a concentration of 100 mumol/L but had no effect on botrocetin-induced binding. In addition, both the N- and C-terminal polypeptides, residues 449 through 549 and 674 through 728, generated by subdigestion of the 52/48-Kd Fr (Fujimura et al, J Biol Chem 261:381, 1986), inhibited preferentially ristocetin-induced vWF binding without affecting to botrocetin-induced vWF binding. These findings suggest that amino acid residues 512 through 673 of the vWF subunit are involved in botrocetin-induced vWF binding.

scholarly journals Studies on anti-von Willebrand factor (vWF) monoclonal antibody NMC-4, which inhibits both ristocetin- and botrocetin-induced vWF binding to platelet glycoprotein Ib

Blood ◽  
1991 ◽  
Vol 77 (1) ◽  
pp. 113-120
Author(s):  
Y Fujimura ◽  
Y Usami ◽  
K Titani ◽  
K Niinomi ◽  
K Nishio ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Amino Acid ◽  
Von Willebrand Factor ◽  
Amino Acid Analysis ◽  
Disulfide Bonds ◽  
Platelet Glycoprotein ◽  
Amino Acid Residues ◽  
Tryptic Digest ◽  
Von Willebrand ◽  
Willebrand Factor

Anti-von Willebrand factor (vWF) monoclonal antibody NMC-4 completely inhibited vWF binding to platelet glycoprotein (GP) lb induced by either ristocetin or botrocetin at an IgG concentration of approximately 10 micrograms/mL, and also blocked binding of asialo-vWF to GP lb. NMC-4 coupled beads isolated a 97-Kd fragment (Fr) from a whole tryptic digest of vWF. The N-terminal sequencing of the nonreduced 97-Kd Fr, in combination with amino acid analysis, showed it to be a homodimer of residues 449 through 728 of the constituent subunit. Present data, together with the results obtained from previous studies, confirm the existence of one or three possible inter-subunit disulfide bonds between cysteine residues 459, 462, and 464. NMC-4 bound to reduced vWF Fr(s) more weakly than to nonreduced Fr(s), but it did not react with Fr III-T2 of vWF, a disulfide-linked twin heterodimer of residues 273 through 511 and 674 through 728 (Marti et al, Biochemistry 26:8099, 1987). Fr III-T2 completely inhibited ristocetin-induced vWF binding at a concentration of 100 mumol/L but had no effect on botrocetin-induced binding. In addition, both the N- and C-terminal polypeptides, residues 449 through 549 and 674 through 728, generated by subdigestion of the 52/48-Kd Fr (Fujimura et al, J Biol Chem 261:381, 1986), inhibited preferentially ristocetin-induced vWF binding without affecting to botrocetin-induced vWF binding. These findings suggest that amino acid residues 512 through 673 of the vWF subunit are involved in botrocetin-induced vWF binding.

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