scholarly journals Localization of a factor VIII-inhibiting antibody epitope to a region between residues 338 and 362 of factor VIII heavy chain.

1988 ◽  
Vol 85 (9) ◽  
pp. 3165-3169 ◽  
Author(s):  
J. Ware ◽  
J. R. Toomey ◽  
D. W. Stafford
Keyword(s):  
Factor Viii ◽  
Heavy Chain ◽  
Antibody Epitope

Acidic Region Residues 1680–1684 in the A3 Domain of Factor VIII Contain a Thrombin-Interactive Site Responsible for Proteolytic Cleavage at Arg1689

2021 ◽  
Author(s):  
Yuto Nakajima ◽  
Hiroaki Minami ◽  
Keiji Nogami
Keyword(s):  
Surface Plasmon Resonance ◽  
Factor Viii ◽  
Heavy Chain ◽  
Synthetic Peptides ◽  
C2 Domain ◽  
Wild Type ◽  
Cross Link ◽  
Domain Specific ◽  
Cofactor Activity ◽  
Acidic Region

AbstractFactor VIII (FVIII) is activated by thrombin-catalyzed cleavage at Arg372, Arg740, and Arg1689. Our previous studies suggested that thrombin interacted with the FVIII C2 domain specific for cleavage at Arg1689. An alternative report demonstrated, however, that a recombinant (r)FVIII mutant lacking the C2 domain retained >50% cofactor activity, indicating the presence of other thrombin-interactive site(s) associated with cleavage at Arg1689. We have focused, therefore, on the A3 acidic region of FVIII, similar to the hirugen sequence specific for thrombin interaction (54–65 residues). Two synthetic peptides, spanning residues 1659–1669 with sulfated Tyr1664 and residues 1675–1685 with sulfated Try1680, inhibited thrombin-catalyzed FVIII activation and cleavage at Arg1689. Treatment with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide to cross-link thrombin with either peptide showed possible contributions of both 1664–1666 and 1683–1684 residues for thrombin interaction. Thrombin-catalyzed activation and cleavage at Arg1689 in the alanine-substituted rFVIII mutants within 1663–1666 residues were similar to those of wild type (WT). Similar studies of 1680–1684 residues, however, demonstrated that activation and cleavage by thrombin of the FVIII mutant with Y1680A or D1683A/E1684A, in particular, were severely or moderately reduced to 20 to 30% or 60 to 70% of WT, respectively. Surface plasmon resonance-based analysis revealed that thrombin interacted with both Y1680A and D1683A/E1684A mutants with approximately sixfold weaker affinities of WT. Cleavage at Arg1689 in the isolated light-chain fragments from both mutants was similarly depressed, independently of the heavy-chain subunit. In conclusion, the 1680–1684 residues containing sulfated Tyr1680 in the A3 acidic region also contribute to a thrombin-interactive site responsible for FVIII activation through cleavage at Arg1689.

scholarly journals Heterogeneity of factor VIII antibodies: further immunochemical and biologic studies

Blood ◽  
1977 ◽  
Vol 49 (5) ◽  
pp. 807-817 ◽  
Author(s):  
MB Hultin ◽  
FS London ◽  
SS Shapiro ◽  
WJ Yount
Keyword(s):  
Factor Viii ◽  
Isoelectric Focusing ◽  
Heavy Chain ◽  
Light Chain ◽  
Major Peak ◽  
Igg Antibodies ◽  
Multiple Peaks ◽  
Light Chain Type ◽  
Apparent Homogeneity ◽  
Chain Type

Abstract Previous studies using immunoneutralization techniques have shown that many factor VIII inhibitors are IgG antibodies of a single light chain type. We have investigated this apparent homogeneity by immunoneutralization assay and liquid isoelectric focusing of inhibitor fractions from five hemophiliacs and two nonhemophiliacs. By immunoneutralization assay, inhibitors from four hemophiliacs and one nonhemophiliac were exclusively k light chain type: the fifth hemophilic inhibitor was predominantly k1 and the second nonhemophilic inhibitor was a mixture of k and gamma. However, heavy chain subtyping of the six predominantly or exclusively k inhibitors showed all to be mixtures of IgG4 and IgG1. By isoelectric focusing, two inhibitors showed multiple peaks of activity between pH 5 and 9. The remaining five showed predominant peaks of activity, which were solely IgGk1 between pH 5.8 and 7, with smaller peaks between pH 7 and 9. The most acidic major peak, focusing at pH 6, was IgG4 in the three cases tested. Two of these acidic peaks neutralized factor VIII more efficiently than other peaks in the same focusing profiles, suggesting greater affinity for factor VIII. These studies demonstrate that factor VIII inhibitors are composed of heterogenous subpopulations of immunoglobulins which can be separated by isoelectric focusing.

scholarly journals The Enhancing Effects of the Light Chain on Heavy Chain Secretion in Split Delivery of Factor VIII Gene

2007 ◽  
Vol 15 (10) ◽  
pp. 1856-1862 ◽  
Author(s):  
Lingxia Chen ◽  
Fuxiang Zhu ◽  
Juan Li ◽  
Hui Lu ◽  
Haiyan Jiang ◽  
...  
Keyword(s):  
Factor Viii ◽  
Heavy Chain ◽  
Light Chain ◽  
Factor Viii Gene ◽  
Split Delivery

Radioimmunoassay for quantitative measurement of factor VIII-heavy chain

1988 ◽  
Vol 68 (3) ◽  
pp. 307-312
Author(s):  
O. Nordfang ◽  
M. Ezban ◽  
P. Nilsson ◽  
J. B. Knudsen
Keyword(s):  
Factor Viii ◽  
Heavy Chain ◽  
Quantitative Measurement

Mechanism of Interaction between the Heavy Chain of Factor VIII and Thrombin.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1711-1711 ◽  
Author(s):  
Keiji Nogami ◽  
Qian Zhou ◽  
Hironao Wakabayashi ◽  
Timothy Myles ◽  
Lawrence L. Leung ◽  
...  
Keyword(s):  
Factor Viii ◽  
Heavy Chain ◽  
Active Site ◽  
Specific Activity ◽  
Solid Phase ◽  
Factor Xa ◽  
Site Directed Mutagenesis ◽  
Marginal Effect ◽  
Thrombin Cleavage ◽  
A2 Domain

Abstract Factor VIII is activated by proteolytic cleavages catalyzed by thrombin or factor Xa. An earlier study indicated that thrombin binding within the C2 domain facilitated cleavage at Arg1689 of factor VIII light chain (Nogami et al. (2000) J. Biol. Chem. 275, 25774–25780). However, thrombin-interactive region(s) within the heavy chain involved with cleaving the A1-A2 and A2-B domainal junctions remain to be determined. Several approaches were employed to examine the interactions between factor VIII heavy chain and thrombin. Fluorescence energy transfer using acrylodan-labeled A1 or A2 subunits (fluorescence donors) and a fluorescein-labeled, Phe-Pro-Arg-chloromethyl ketone active site-modified thrombin (Fl-FPR-thrombin; fluorescence acceptor) showed that FPR-thrombin bound to the A2 subunit with an ~6-fold higher affinity (Kd =36.6 nM) compared with the A1 subunit (Kd=234 nM). Solid phase binding assays using immobilized thrombin S205A, where the active-site Ser205 was converted to Ala by site directed mutagenesis, showed that the binding affinity of A2 subunit was ~3-fold greater than that of A1 subunit. Similar solid phase assays indicated that hirudin, a ligand for anion-binding exosite I of thrombin (ABE-I), effectively blocked thrombin interaction with A1 subunit while having little if any effect on its interaction with A2 subunit. Conversely, heparin, which binds ABE-II, blocked thrombin interaction with A2 subunit while showing only a marginal effect on A1 subunit binding. To identify an interactive site for thrombin in the A2 domain, we focused on two regions containing clustered acidic residues (389GluGluGluAspTrpAsp394 and 720GluAspSerTyrGluAsp725), which are localized near the N- and C-termini of the A2 domain, respectively. SDS-PAGE analyses using isolated factor VIII heavy chain as substrate showed peptides with the sequences 373–395 and 719–740 encompassing these acidic regions, blocked thrombin cleavage at both Arg372 (A1–A2 junction) and Arg740 (A2–B junction) while a 373–385 peptide did not block either cleavage. The 373–395 and 719–740 peptides competitively inhibited A2 binding to S205A thrombin in a solid phase assay (Ki=11.5 and 12.4 μM, respectively), and quenched the fluorescence of Fl-FPR-thrombin. These data demonstrate that both A2 terminal regions support interaction with thrombin. Furthermore, a B-domainless, factor VIII double mutant D392A/D394A was constructed and possessed specific activity equivalent to a severe hemophilia phenotype (<1% compared with wild type). This mutant was resistant to cleavage at Arg740 whereas cleavage at Arg372 was not appreciably affected. Thus the low specific activity of this mutant was attributed to small C-terminal extensions on the A2 subunit that were not removed following cleavage at Arg740. However, factor Xa cleavage of the mutant at Arg740 was not affected. These data suggest the acidic region comprising residues 389–394 in factor VIII A2 domain interacts with thrombin via ABE-II of the proteinase facilitating cleavage at Arg740.

Mechanisms of Plasmin-Catalyzed Inactivation of the Factor VIII.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1017-1017
Author(s):  
Keiji Nogami ◽  
Midori Shima ◽  
Tomoko Matsumoto ◽  
Katsumi Nishiya ◽  
Masahiro Takeyama ◽  
...  
Keyword(s):  
Factor Viii ◽  
Heavy Chain ◽  
Activated Protein C ◽  
Factor Xa ◽  
Protein S ◽  
Fold Increase ◽  
Clotting Factors ◽  
Fviii Activity ◽  
A1 Domain ◽  
Von Willebrand

Abstract Factor VIII (FVIII) functions as a cofactor for factor IXa in the intrinsic tenase complex. This tenase activity is down-regulated by activated protein C (APC) or factor Xa (FXa). Plasmin, the most potent fibrinolytic protease, inactivates FVIII as well as other clotting factors. However, the mechanism of FVIII inactivation by plasmin is poorly understood. FVIII activity reached to the peak value of ~2-fold increase at 3 min after the addition of plasmin in a one-stage clotting assay. Then, the activity was decreased rapidly and was undetectable within 30 min. This time-dependent reaction was not affected in the presence of von Willebrand factor and phospholipid. The activation of FVIII by plasmin was an ~50% level of that by FXa. The rate constant (min-1) of inactivation of FVIIIa by plasmin possessed ~11.3- and ~2.5-folds greater than those by FXa and APC in the presence of protein S, respectively. SDS-PAGE analysis showed that plasmin cleaved the 90~210-kDa heavy chain of FVIII to 50, 48,45, 40, and 38-kDa fragments via 90-kDa fragment. Using western blot and N-terminal sequence analyses, these fragments derived from the heavy chain were identified as A11-372, A1337-372-A2, A11-336, A2, and A137-336, respectively, by cleavages at Arg372, Arg740, Lys36 and Arg336 in the A1 domain. On the other hand, the 80-kDa light chain was cleaved to 67-kDa fragment via 70-kDa fragment by cleavages at Arg1721 and Arg1689, respectively, consistent with the pattern of cleavage by FXa. However, the cleavage at Arg336 by plasmin was much quicker than that at Arg372, contrast with that by FXa. Furthermore, this cleavage was faster than that by APC, consistent with rapid inactivation of FVIII. In addition, the cleavage at Arg336 of FVIIIa by plasmin was faster than that of isolated A1 or A1/A3-C1–C2 dimer, different with that by FXa. These results demonstrate the importance of cleavage at Arg336 for the mechanism of plasmin-catalyzed FVIII inactivation. Furthermore, this cleavage appears to be selectively modulated by the A2 domain that may interact with plasmin.

An Essential Role of the Factor VIII Light Chain in Facilitating Heavy Chain Secretion.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4034-4034
Author(s):  
Lingxia Chen ◽  
Juan Li ◽  
Hui Lu ◽  
Haiyan Jiang ◽  
Rita Sarkar ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Factor Viii ◽  
Heavy Chain ◽  
Light Chain ◽  
Hek293 Cells ◽  
Light Chains ◽  
Trans Splicing ◽  
In Cis

Abstract Blood coagulation Factor VIII (FVIII) is secreted as a heterodimer consisting of a heavy and light chain. Both in vitro and in vivo studies have demonstrated that these chains can be expressed independently. The expressed heavy and light chains can reassociate with recovery of biological activities. These observations have been particularly useful in a gene therapy setting since vector packaging capacity for adeno-associated virus (AAV) is a limiting factor. However, it has been demonstrated that the FVIII heavy chain is expressed ~10–100-fold less efficiently compared to the light chain when expressed independently. Previously the FVIII F309S mutation in the context of B-domainless FVIII (FVIII-BDD) and enhanced glycosylations within the B-domain have been shown to improve factor VIII expression and secretion. However, our in vitro studies indicate that these improvements in secretion were not retained when expressing the heavy chain alone with the same modifications. Other sequences, possibly in the light chain, may facilitate secretion. To investigate this further, we designed an intein trans-splicing strategy to control the addition of light chain to the heavy chain before secretion. Using HEK293 cells, we cotransfected seperate intein light chain and intein heavy chain plasmids and compared results to single plasmid transfected cells. 48 hours post-transfection, FVIII-specific ELISA results demonstrated that cotransfection of intein heavy chain and intein light chain had a significant influence on total heavy chain secretion compared to intein heavy chain expression alone. The co-transfected intein heavy chain and intein light chain were efficiently ligated together yielding a biologically active single chain FVIII derivative as demonstrated by clotting assays and Western blot analysis. Therefore, heavy chain secretion was directly enhanced by the attachment of the light chain to the C-terminus of the heavy chain. A similar phenomenon was not found when heavy and light chains were simply co-expressed in the same cell. It suggested that light chain functioned in cis. Hydrodynamic injection of plasmids with intein heavy chain and intein light chain into hemophilia A mice led to a much higher level of FVIII secretion. The amount of functional FVIII expression reached 3–6 units/ml at peak level. In the absence of intein light chain, FVIII heavy chain secretion was approximately 100 fold less efficient in vivo. To map the key elements of FVIII light in helping FVIII secretion, we made deletion variants in the light chain. These mutants had a dominant negative effect in reducing FVIII and FVIII heavy chain secretion while increasing the level of intracellular FVIII accumulation. Collectively our results are consistent with the conclusion that the FVIII light chain plays a critical role in facilitating heavy chain secretion in cis; probably through helping FVIII heavy chain maintain correct configuration and folding. The strategy to manipulate FVIII light chain addition through intein mediated trans-splicing reaction may also be explored for human gene therapy.

Tissue Factor-Dependent Activation of Factor VIII by Factor VIIa in the Early Phase of Blood Coagulation

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1036-1036
Author(s):  
Tetsuhiro Soeda ◽  
Keiji Nogami ◽  
Tomoko Matsumoto ◽  
Kenichi Ogiwara ◽  
Katsumi Nishiya ◽  
...  
Keyword(s):  
Factor Viii ◽  
Tissue Factor ◽  
Blood Coagulation ◽  
Heavy Chain ◽  
Light Chain ◽  
Early Phase ◽  
Factor Viia ◽  
Clotting Factors ◽  
Initiation Phase ◽  
A1 Domain

Abstract Factor VIIa (FVIIa), complexed with tissue factor (TF), is a trigger of blood coagulation through activation of factor X in the initiation phase. FVIIa can catalyze intrinsic clotting factors such as not only factor IX, but also factor VIII (FVIII). However the role and the mechanisms of the FVIIa-catalyzed FVIII are poorly understood. We first examined FVIIa-catalyzed FVIII activation in the presence of phospholipid (PL) using a one-stage clotting assay. The levels of FVIII activity elevated rapidly by ~4-fold within 30 sec after the addition of FVIIa (1 nM)-TF (1 nM)complex, and subsequently decreased to the initial level within 20 min. This time-dependent reaction was enhanced by the presence of TF and PL in dose-dependent manners, but was moderately inhibited (~50%) in the presence of von Willebrand factor at physiological concentration of 10 μg/mL. FVIII cleavage was evaluated using western blotting immediately after the addition of FVIIa-TF complex. The heavy chain of FVIII was proteolyzed more rapidly (at 15 sec) by cleavages at Arg740 (A2-B junction) and Arg372 (A1-A2 junction) by FVIIa-TF complex, whilst the cleavage at Arg336 in the A1 domain was appeared at ~2.5 min. However little cleavage of the light chain of FVIII was observed, supporting that cleavages at Arg740/Arg372 and Arg336 by FVIIa-TF complex contribute to the up- and down-regulation of FVIII(a) activity, respectively. Of interest, no proteolysis of isolated intact heavy chain was observed, indicating that the proteolysis of the heavy chain was governed by the presence of the light chain. Compared to FVIII activation by thrombin (0.1–1 nM), the activation by FVIIa (0.1–1 nM)-TF (1 nM) complex was observed more rapidly. The activation rate observed by the addition of FVIIa-TF complex was ~50-fold greater than that by thrombin. Kinetics by the chromogenic Xa generation assay showed the catalytic efficiency (kcat/Km; 8.9 min−1/32.8 nM) on FVIIa-TF complex-catalyzed FVIII activation showed ~4-fold greater than that on thrombin-catalyzed activation (kcat/Km; 7.5 min−1/86.4 nM). Furthermore, the catalytic efficiencies on cleavages at Arg740 and Arg372 of FVIII by FVIIa-TF complex were ~3- and ~20-fold greater compared to those by thrombin, respectively. These findings suggested that FVIIa-TF complex was a greater FVIII activator than thrombin in very early phase. In order to localize the binding region for FVIIa, we evaluated the interactions between FVIII subunit and Glu-Gly-Arg-active site modified FVIIa, lacking enzymatic activity, in a surface plasmon resonance-based assay. The heavy chain of FVIII bound to EGR-FVIIa with higher affinity than the light chain (Kd; 2.1 and 45 nM, respectively). Binding was particularly evident with the A2, A3, and C2 domains (Kd; 34, 37, and 44 nM, respectively), whilst the A1 domain failed to bind. In conclusion, we demonstrated that FVIIa-TF complex rapidly activated FVIII by proteolysis of the heavy chain and the activation was governed by the presence of the light chain. Furthermore, present results suggested the role of TF-dependent FVIII activation by FVIIa which is responsible for the initiation phase of blood coagulation.

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