von Willebrand Factor Protects Ca2+-Dependent Structure of the Factor VIII Light Chain.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1008-1008 ◽  
Author(s):  
Masahiro Takeyama ◽  
Midori Shima ◽  
Keiji Nogami ◽  
Masahiro Okuda ◽  
Yoshihiko Sakurai ◽  
...  
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Divalent Cation ◽  
Light Chain ◽  
Metal Ion ◽  
Exchange Resin ◽  
Cation Exchange Resin ◽  
The Other ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Factor VIII (FVIII) cofactor function is dependent on the presence of divalent cation, since its active form is metal-linked heterotrimer as well as factor V (FV). The activity of FVIII (FVIII:C) and FV in normal plasma were completely and selectively inactivated by the treatment with divalent cation exchange resin, imidinoacetate. However, FVIII antigen (FVIII:Ag) was preserved, suggesting that deprivation of FVIII:C by the resin was not due to absorption of FVIII but due to inactivation. Both FVIII:Ag and FVIII:C of recombinant FVIII were decreased by treatment with the resin. However, FVIII:Ag but not FVIII:C, was preserved in FVIII and von Willebrand factor complex (FVIII/VWF). Sandwich ELISA using anti-A3 and anti-A2 monoclonal antibodies revealed that association with heavy and the light chains were impaired in the resin-treated FVIII. These results suggested that FVIII was inactivated by deprivation of the metal ion by the resin and that VWF protected cation-dependent FVIII structure. Therefore, we tested if the resin deprives the metal ion such as Ca2+ from FVIII or FVIII/VWF. [Ca2+] in FVIII preparation was decreased from 1.30 to 0.07 mM by the resin. Furthermore, [Ca2+] was decreased from 0.39 to 0.01 mM in FVIII/VWF preparation. [Ca2+] was recovered completely by elution with 1 N HCl from the resins used in both preparations. FVIII:C of the resin-treated FVIII/VWF was partially recovered by addition of Ca2+, whilst FVIII:C of the resin-treated FVIII was not recovered. When FVIII was treated with the resin after addition of [Ca2+], the inactivation of FVIII was dose-dependently inhibited by ~20 % (at [Ca2+]: ~75 mM). On the other hand, the inactivation of FVIII was inhibited by ~60 % (at [Ca2+]: ~25 mM) in FVIII/VWF preparation. Present results demonstrated that FVIII was selectively inactivated by the cation exchange resin due to deprivation of Ca2+. Kinetic experiments by surface plasmon resonance using BIAcore demonstrated that the resin-treated FVIII as well as treated C2 didn’t interact with phospholipid and VWF. Furthermore, immunoblot analysis using the resin-treated FVIII revealed that anti-A2 monoclonal antibody reacted with the heavy chain, whilst anti-A3 and anti-C2 antibodies failed to react with the light chain. On the other hand, these antibodies reacted with the light chain in the experiment using FVIII/VWF, indicating that VWF protects antigenic conformation of the FVIII light chain. Present findings suggest another protection mechanism of VWF on FVIII through stabilization of Ca2+-dependent structure of the 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.

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.

scholarly journals Light Chain of Factor VIII Is Sufficient for Accelerating Cleavage of von Willebrand Factor by ADAMTS13 Metalloprotease

2012 ◽  
Vol 287 (39) ◽  
pp. 32459-32466 ◽  
Author(s):  
Wenjing Cao ◽  
Denise E. Sabatino ◽  
Ekaterina Altynova ◽  
Amy M. Lange ◽  
Veronica C. Casina ◽  
...  
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Light Chain ◽  
Von Willebrand ◽  
Willebrand Factor

SYNTHESIS. PROCESSING AND SECRETION OF HUMAN FACTOR VIII IN MAMMALIAN CELLS: REQUIREMENT FOR VON WILLEBRAND FACTOR

1987 ◽  
Author(s):  
Louise C Wasley ◽  
Andrew J Dorner ◽  
Randal C Kaufman
Keyword(s):  
Factor Viii ◽  
Cell Lines ◽  
Von Willebrand Factor ◽  
Light Chain ◽  
Human Factor ◽  
Conditioned Media ◽  
Mammalian Cell Lines ◽  
Human Factor Viii ◽  
Von Willebrand ◽  
Willebrand Factor

In the plasma factor VIII exists as a complex with von Willebrand factor (vWF). The cloning of the cDNA for factor VIII has provided the ability to develop mammalian cell lines which express high levels of factor VIII by using appropriatate expression plasmids and DNA cotransformation with selectable markers. We have studied the synthesis, processing, and secretion of factor VIII expressed in baby hamster kidney cells and in Chinese hamster ovary cells by 35S-methionine pulse and chase labeling and analysis by immunoprecipitation with specific antibodies which recognize the light and heavy chains of factor VIII. In both mammalian cell lines, factor VIII is synthesized as a primary translation product of 230 kDa. A significant amount remains within the endoplasmic reticulum in a stable complex with a glucose regulated protein of 78 kDa. The remainder traverses into the Golgi compartment where it is cleaved to the heavy and light chain forms. Very shortly thereafter the mature factor VIII appears in the conditioned media as the mature heavy and light chain species. Very little single chain factor VIII is secreted into the conditioned media. The accumulation of factor VIII in the conditioned media requires the presence of vWF factor. In the absence of vWF, the factor VIII appears as unassociated heavy and light chains which are rapidly degraded. Bovine, porcine, or human 3WF all effectively stabilize human factor VIII expressed in these rodent cell lines. These results suggest the presence of vWF promotes factor VIII chain association which stabilizes the factor VIII to proteolysis.

scholarly journals Characterization of des-(741–1668)-factor VIII, a single-chain factor VIII variant with a fusion site susceptible to proteolysis by thrombin and factor Xa

1995 ◽  
Vol 312 (1) ◽  
pp. 49-55 ◽  
Author(s):  
M J S H Donath ◽  
R T M de Laaf ◽  
P T M Biessels ◽  
P J Lenting ◽  
J W van de Loo ◽  
...  
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Heavy Chain ◽  
Light Chain ◽  
Factor Xa ◽  
Single Chain ◽  
Factor Ixa ◽  
Fusion Site ◽  
Von Willebrand ◽  
Willebrand Factor

A factor VIII variant has been characterized in which the heavy chain is directly fused to the light chain. Des-(741-1668)-factor VIII lacks the processing site at Arg1648, as Arg740 of the heavy chain is fused to Ser1669 of the light chain. The sequence of the fusion site is similar to that of other cleavage sites in factor VIII. The fusion site of des-(741-1668)-factor VIII was readily cleaved by both thrombin and factor Xa, and the same result was obtained for heavy chain cleavage. In contrast, des-(741-1668)-factor VIII cleavage by thrombin at position Arg1689 proceeded at a lower rate than the analogous cleavage by factor Xa, which presumably takes place at position Arg1721. The rate of cleavage at position Arg1689 by thrombin was also lower than that at the other processing sites. When des-(741-1668)-factor VIII was activated by thrombin, initial rates of factor Xa formation were similar to the rates obtained when plasma-derived factor VIII was activated by thrombin or factor Xa. Remarkably, activation of des-(741-1668)-factor VIII proceeded at a higher rate by factor Xa than by thrombin. These results indicate that factor VIII activation is strongly associated with cleavage at position Arg1689 or Arg1721. For the interaction between des-(741-1668)-factor VIII and von Willebrand factor, a Kd value of (0.8 +/- 0.3) x 10(-10) M was determined, which is similar to that of heterodimeric factor VIII. The affinity of single-chain des-(741-1668)-factor VIII for factor IXa was found to be 27 +/- 6 nM. The in vivo recovery and half-life of des-(741-1668)-factor VIII were assessed in guinea pigs. Upon infusion of des-(741-1668)-factor VIII at a dosage of 50 units/kg body weight, a rise of 1.0 +/- 0.3 unit/ml in factor VIII activity was obtained. The same recovery was determined for wild-type factor VIII. The half-life of des-(741-1668)-factor VIII was found to be 3 +/- 1 h, compared with 4 +/- 2 h for heterodimeric recombinant factor VIII. In conclusion, des-(741-1668)-factor VIII displays normal activity, is readily cleaved by thrombin and factor Xa at its fusion site, binds with high affinity to von Willebrand factor and factor IXa, and behaves like heterodimeric recombinant factor VIII in guinea pigs. By virtue of these properties, des-(741-1668)-factor VIII may prove useful for the treatment of bleeding episodes in patients with haemophilia A.

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.

Epitope Specific Discrepancies Between Antibody Mediated Inhibition Of Recombinant Factor VIII and Plasma Derived Factor VIII Containing Von Willebrand Factor Affecting All Domains Of Factor VIII

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1089-1089
Author(s):  
Tami Livnat ◽  
Amy L. Dunn ◽  
Shirley Azar-Avivi ◽  
Wallace Hunter Baldwin ◽  
Gili Kenet ◽  
...  
Keyword(s):  
Factor Viii ◽  
Von Willebrand Factor ◽  
Conflicts Of Interest ◽  
The Other ◽  
Factor Vii ◽  
Specific Response ◽  
Fviii Activity ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Kinetics Of

Abstract Background Previous results from our laboratory demonstrate an epitope specific response to high doses of FVIII within anti-C2 antibodies that was independent of antibody titer. In addition, for a panel of monoclonal antibodies (MAbs) directed against all FVIII domains the kinetics of inhibition influenced response to combinations of FVIII and recombinant factor VII (rFVIIa). The influence of inhibitor kinetics on response to treatment has also been demonstrated in inhibitor patient plasmas. The Bethesda assay detects the inhibitory capacity of anti-factor VIII (FVIII) antibodies to neutralize FVIII after 2 hours of incubation with pooled normal plasma (PNP). In this assay patient antibody is added to PNP as compared to the clinical scenario where recombinant FVIII (rFVIII) or plasma derived FVIII containing von Willebrand Factor (pdFVIII/VWF) is infused into the patient where antibody is already present. In this case the infused product is immediately available to interact with both VWF and anti-FVIII antibodies as compared to the Bethesda assay where VWF is already bound to FVIII when the antibody is added. Methods In this study we investigated the inhibitory kinetics of a panel of 20 anti-FVIII MAbs (Table) with known epitope specificity and inhibitory activity in a standard Bethesda assay. Inhibitor plasma consisted of a single MAb added to FVIII deficient plasma at 5 µg/ml. rFVIII and pd-FVIII/VWF were added to each inhibitor plasma and samples were sequentially removed at intervals between 5 and 90 minutes. FVIII activity was measured by a one-stage aPTT based assay. Results Of the MAb panel, 2 anti-A2 MAbs, 1D4 and 4A4, and the anti-A3 MAb F147 had full neutralization of both rFVIII and pd-FVIII/VWF. All 3 of these MAbs exhibit high inhibitory titers in the Bethesda assay. The majority of the other MAbs had improved neutralization kinetics and thus higher residual FVIII activity with pd-FVIII/VWF when compared to rFVIII. The figure below shows the residual FVIII activity at 15 minutes following the addition of rFVIII or pdFVIII/VWF into the inhibitor plasmas. Similar patterns were seen at the other time points. Three MAbs from the panel, two anti-A2 (4C7 and B157) and one anti-C2 (2-117), had significant inhibition of FVIII activity when rFVIII was added to the inhibitor plasma. This was not demonstrated in the standard Bethesda assay or when pd-FVIII/VWF was added to the inhibitor plasma. This demonstrates that the order of binding of VWF and anti-FVIII antibodies may be clinically relevant for a subset of FVIII epitopes. Conclusion The Bethesda assay in isolation neither predicts inhibitory kinetics nor defines response to various FVIII sources. FVIII source dependent neutralization kinetics and epitope mapping may be applied as additional tools for tailoring therapy in patients with inhibitors. Disclosures: No relevant conflicts of interest to declare.

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