scholarly journals The Role of Factor IXa and Factor VIII in the Activation of Factor X.

1977 ◽  
Author(s):  
Earl W. Davie ◽  
Gordon Vehar ◽  
Kazuo Fujikawa ◽  
Richard Di Scipio
Keyword(s):  
Factor Viii ◽  
Heavy Chain ◽  
Serine Proteases ◽  
Factor Xa ◽  
Basic Mechanism ◽  
Factor X ◽  
Amino Terminal ◽  
Factor Ixa ◽  
A Charge

Factor IXa and factor VIII participate in the middle phase of blood coagulation. These two proteins convert factor X to factor Xa in the presence of calcium ions and phospholipid. The coagulant activity of factor VIII is increased 50-100 fold by the addition of thrombin, and this activity is stabilized in the presence of CaCl2. The activated product (tentatively identified as activated factor VIII) was readily inhibited by diisopropyl phosphorofluoridate or antithrombin III, suggesting that it is a serine enzyme. The exact role of this enzyme in the conversion of factor X to factor Xa, however, is not known. When factor X (bovine or human) is converted to factor Xa, an activation peptide is cleaved from the amino-terminal end of the heavy chain. This gives rise to a new amino-terminal sequence of Ile-Val-Gly-Gly-in the heavy chain. No change occurs in the light chain during the activation reaction. These data indicate that the basic mechanism involved in the conversion of human and bovine factor X to factor Xa appears to be essentially identical and probably involves the formation of a charge relay system characteristic of the pancreatic serine proteases.

scholarly journals Cleavage of Factor VIII Heavy Chain Is Required for the Functional Interaction of A2 Subunit with Factor IXa

2001 ◽  
Vol 276 (15) ◽  
pp. 12434-12439 ◽  
Author(s):  
Philip J. Fay ◽  
Maria Mastri ◽  
Mary E. Koszelak ◽  
Hironao Wakabayashi
Keyword(s):  
Factor Viii ◽  
Heavy Chain ◽  
Fluorescence Anisotropy ◽  
Factor Xa ◽  
Factor X ◽  
Functional Interaction ◽  
Factor Ixa ◽  
Factor Viiia ◽  
A2 Domain ◽  
Stimulation Of

Factor VIII circulates as a noncovalent heterodimer consisting of a heavy chain (HC, contiguous A1-A2-B domains) and light chain (LC). Cleavage of HC at the A1-A2 and A2-B junctions generates the A1 and A2 subunits of factor VIIIa. Although the isolated A2 subunit stimulates factor IXa-catalyzed generation of factor Xa by ∼100-fold, the isolated HC, free from the LC, showed no effect in this assay. However, extended reaction of HC with factors IXa and X resulted in an increase in factor IXa activity because of conversion of the HC to A1 and A2 subunits by factor Xa. HC cleavage by thrombin or factor Xa yielded similar products, although factor Xa cleaved at a rate of ∼1% observed for thrombin. HC showed little inhibition of the A2 subunit-dependent stimulation of factor IXa activity, suggesting that factor IXa-interactive sites are masked in the A2 domain of HC. Furthermore, HC showed no effect on the fluorescence anisotropy of fluorescein-Phe-Phe-Arg-factor IXa in the presence of factor X, whereas thrombin-cleaved HC yielded a marked increase in this parameter. These results indicate that HC cleavage by either thrombin or factor Xa is essential to expose the factor IXa-interactive site(s) in the A2 subunit required to modulate protease activity.

FVIII Heavy Chain Enhances Tenase Activity Induced By FVIIIa Mimicking Bispesific Antibody, ACE910

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1481-1481
Author(s):  
Hiroaki Minami ◽  
Keiji Nogami ◽  
Takehisa Kitazawa ◽  
Kunihiro Hattori ◽  
Midori Shima
Keyword(s):  
Factor Viii ◽  
Heavy Chain ◽  
Light Chain ◽  
Research Funding ◽  
Factor Xa ◽  
Factor X ◽  
Advisory Committees ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Physiological Structure

Abstract Background: ACE910, asymmetric bispecific monoclonal antibodies to activated factor IX (IXa) and factor X, mimics the cofactor function of activated factor VIII (VIIIa) by modulating an optimal position on the tenase assembly. The estimated therapeutic range of ACE910 shows ~30% of thrombin generation in native tenase assembly, supporting that the structure on ACE910-mimicking tenase assembly is different from that on native tenase. Being close to physiological structure consisting from factor IXa, factor X, and factor VIIIa is important for potentiating the clotting function. We examined the effects of factor VIII subunits (light chain, heavy chain, A1 and A2, C2) on ACE910-tenase. Materials/Methods: The factor VIII light chain and heavy chain were isolated from EDTA-treated recombinant factor VIII following chromatography on SP- and Q- Sepharose columns. The A2 and A1 subunits were purified from thrombin-cleaved factor VIII heavy chain by Heparin-, SP- Sepharose columns. Purified factor Xa generation assays was examined with (i) factor VIII subunit (0-40 nM), ACE910 (10 µg/ml), phospholipid (PL) (40 µM), factor IXa (1 nM) and factor X (200 nM), (ii, iii) the A2 or heavy chain (40 nM), ACE910 (10 µg/ml), PL (40 µM), factor IXa and factor X (1 or 0-80 nM, and 0-300 or 200 nM, respectively). These mixtures were reacted for five minutes (i, ii) or one minute (iii). These assays were conducted at 37 °C. Results: (i) The factor Xa generation in ACE910-tenase complex in the absence of factor VIIIa was 10.1±2.2 nM. With the intact heavy chain and A2, amounts of factor Xa were increased dose-dependently, resulting in 1.3- and 1.2-fold increases, respectively. While, the light chain and A1 subunit failed to increase at all. (ii) Vmax for factor X in ACE910-tenase was 173.0±7.0 nM and Km was 31.2±3.9 nM. Vmax obtained with the heavy chain or A2 was 175.9±6.1 or 159.0±6.1 nM, whilst Km was 17.0±2.2 or 31.9±3.5 nM, respectively, indicating that the heavy chain enhanced the binding affinity for factor X in ACE910-tenase. (iii) Vmax for factor IXa in ACE910-tenase was 43.8±2.7 nM and Km was 36.9±4.8 nM. With the heavy chain or A2, Vmax was 46.8±3.0 or 45.0±3.1 nM, and Km was 36.4±3.0 or 32.1±4.9 nM, respectively, indicating that either the heavy chain or A2 did not enhance the catalytic activity and the binding affinity for factor IXa in ACE910-tenase. Conclusion: ACE910-tenase assembly seems to be close to physiological structure by the presence of intact heavy chain interacting with factor X. In addition, ACE910 may substitute the position such as the factor VIII(a) light chain associated with FIXa and FX on ACE910-tenase assembly defecting factor VIII. Disclosures Minami: Chugai Pharmaceutical Co., Ltd.: Research Funding. Nogami:Chugai Pharmaceutical Co., Ltd.: Membership on an entity's Board of Directors or advisory committees, Research Funding. Kitazawa:Chugai Pharmaceutical Co., Ltd.: Employment, Equity Ownership, Patents & Royalties. Hattori:Chugai Pharmaceutical Co., Ltd.: Employment, Equity Ownership, Patents & Royalties. Shima:Chugai Pharmaceutical Co., Ltd.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

The Role of Factor VIII in the Activation of Human Blood Coagulation Factor X by Activated Factor IX

1985 ◽  
Vol 54 (03) ◽  
pp. 654-660 ◽  
Author(s):  
K Mertens ◽  
A van Wijngaarden ◽  
R M Bertina
Keyword(s):  
Factor Viii ◽  
Substrate Inhibition ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Catalytic Efficiency ◽  
Enzyme Concentration ◽  
Factor Ix ◽  
Factor X ◽  
Factor Ixa

SummaryThe role of factor VIII in the activation of human factor X by factor IXa, Ca2+ and phospholipid has been investigated. Factor VIII stimulated the factor Xa formation after activation by factor Xa or thrombin; the activity of thrombin-activated factor VIII was about 4-fold that of factor Xa-activated factor VIII. The isolated procoagulant moiety of the factor VIII complex behaved identically to the complete complex, whereas the von Willebrand factor moiety did not participate in the factor Xa formation. Thrombin-activated factor VIII complex (factor Villa) was used to study the effect of factor Villa in kinetic experiments. The results revealed a complex kinetic behaviour, including substrate inhibition and non-linearity of the reaction rate with the enzyme concentration. Using previously obtained insight into the kinetics of factor X activation in the absence of factor VIII, the results were found to support the hypothesis that factor Villa participates in the factor Xa formation in a complex with phospholipid-bound factor IXa; the formation of the factor VUIa-factor IXa complex then increases the catalytic efficiency of the factor IXa by 500-fold.

scholarly journals The role of amino-terminal residues of the heavy chain of factor IXa in the binding of its cofactor, factor VIIIa

Blood ◽  
1994 ◽  
Vol 84 (6) ◽  
pp. 1837-1842 ◽  
Author(s):  
N Hamaguchi ◽  
SP Bajaj ◽  
KJ Smith ◽  
DW Stafford
Keyword(s):  
Heavy Chain ◽  
Specific Interaction ◽  
Factor Ix ◽  
Factor X ◽  
Mutant Proteins ◽  
Amino Terminal ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Critical Residues

Abstract The purpose of this study is to determine which residues of the factor IXa heavy chain are important for interaction with the cofactor of factor IXa, factor VIIIa. Because the monoclonal antibody (MoAb) FXC008 inhibits interaction between factors IXa and VIIIa, and because it also reacts with residues 181–310 of the factor IXa heavy chain, we used the computer-modelled structure of the factor IXa heavy chain to select charged surface residues likely to interact with FXC008 and/or factor VIIIa. We made mutations in the region of residues 181–310 of the heavy chain of factor IX, and replaced these amino acids individually with those located at the same position in factor X. The mutated factor IX retained complete clotting activity and thus interacted normally with factor VIIIa. Five mutant proteins (factor IXK214F, factor IXK228R, factor IXE240Q, factor IXK247V, and factor IXN260K) reacted with heavy chain-specific MoAbs FXC008 and A-5. Neither factor IXD276K nor factor IXR248H bound to FXC008. Factor IXR252V had reduced affinity to FXC008. Our results suggest the following: (1) factor IXa residues 214, 228, 240, 247, 248, 252, 260, and 276 are not involved in specific interaction with factor VIIIa; and (2) the FXC008 and factor VIIIa binding sites may not share critical residues.

scholarly journals The role of amino-terminal residues of the heavy chain of factor IXa in the binding of its cofactor, factor VIIIa

Blood ◽  
1994 ◽  
Vol 84 (6) ◽  
pp. 1837-1842
Author(s):  
N Hamaguchi ◽  
SP Bajaj ◽  
KJ Smith ◽  
DW Stafford
Keyword(s):  
Heavy Chain ◽  
Specific Interaction ◽  
Factor Ix ◽  
Factor X ◽  
Mutant Proteins ◽  
Amino Terminal ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Critical Residues

The purpose of this study is to determine which residues of the factor IXa heavy chain are important for interaction with the cofactor of factor IXa, factor VIIIa. Because the monoclonal antibody (MoAb) FXC008 inhibits interaction between factors IXa and VIIIa, and because it also reacts with residues 181–310 of the factor IXa heavy chain, we used the computer-modelled structure of the factor IXa heavy chain to select charged surface residues likely to interact with FXC008 and/or factor VIIIa. We made mutations in the region of residues 181–310 of the heavy chain of factor IX, and replaced these amino acids individually with those located at the same position in factor X. The mutated factor IX retained complete clotting activity and thus interacted normally with factor VIIIa. Five mutant proteins (factor IXK214F, factor IXK228R, factor IXE240Q, factor IXK247V, and factor IXN260K) reacted with heavy chain-specific MoAbs FXC008 and A-5. Neither factor IXD276K nor factor IXR248H bound to FXC008. Factor IXR252V had reduced affinity to FXC008. Our results suggest the following: (1) factor IXa residues 214, 228, 240, 247, 248, 252, 260, and 276 are not involved in specific interaction with factor VIIIa; and (2) the FXC008 and factor VIIIa binding sites may not share critical residues.

Activation of Human Coagulation Factor VIII by Activated Factor X, the Common Product of the Intrinsic and the Extrinsic Pathway of Blood Coagulation

1982 ◽  
Vol 47 (02) ◽  
pp. 096-100 ◽  
Author(s):  
K Mertens ◽  
R M Bertina
Keyword(s):  
Factor Viii ◽  
Human Factor ◽  
Intrinsic Factor ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Factor X ◽  
Extrinsic Factor ◽  
Extrinsic Pathway ◽  
Factor Ixa ◽  
The Common

SummaryThe intrinsic activation of human factor X has been studied in a system consisting of purified factors and in plasma. In both these systems factor Xa stimulated the activation of factor X by factor IXa plus factor VIII This is due to the activation of factor VIII by factor Xa. When this factor Xa is formed via the extrinsic pathway, the extrinsic factor X activator functions as a stimulator of the intrinsic factor X activator.

RAT HEPAT0CYTES IN CULTURE INACTIVATE FACTOR Xa.

1987 ◽  
Author(s):  
G D Qureshi ◽  
M Sun ◽  
C Gervin ◽  
H Evans
Keyword(s):  
Serine Proteases ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Rat Hepatocytes ◽  
Factor X ◽  
Clotting Factors ◽  
Hepatocyte Cultures ◽  
The Stability ◽  
Stabilization Period

Plasma contains zymogens of clotting factors, which under various stimuli are activated to serine proteases. Whereas much knowledge has been gained about the activation of clotting factors, relatively little is known about inactivation of these proteases. Antithrombin III has been shown to inactivate some activated clotting factors in plasma. Studies in intact animals have suggested that activated clotting factors are mainly inactivated in the liver. To investigate more fully the role of liver in inactivating the activated factors, we studied the stability of activated factor X(Xa) in hepatocyte cultures. Monolayer cultures on non-proliferating rat hepatocytes were prepared according to the method of Bissell et al. The culture medium was chemically defined and was free from serum or serum products. After the 24 h stabilization period, 0.5 units/ml of 100% activated bovine factor Xa was co-cultured with hepatocytes for 8 h. Samples were collected at 0, ½, 1 2, 4 and 8 h and tested for Xa activity using chromogenic substrate S-2222. At the end of 8 h only 41.07% of the initial Xa activity remained. Xa inactivation was not affected by a commercially prepared unfractionated heparin (1 unit/ml) and estradiol at 12.5, 25, 125 nM, a potentiator and inhibitor of antithrombin III, respectively. Inactivation of Xa in hepatocyte cultures was inhibited by the addition of cycloheximide (10-4M). Our data suggests that factor Xa is inactivated in hepatocyte cultures by one or more hepatic derived factors which do not meet the functional characteristics of antithrombin III.

Role of the Direct Interaction between Factor VIII C2 and Factor IXa Gla Domain in the Factor Xase Complex.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2687-2687
Author(s):  
Tetsuhiro Soeda ◽  
Keiji Nogami ◽  
Masahiro Takeyama ◽  
Kenichi Ogiwara ◽  
Kazuhiko Tomokiyo ◽  
...  
Keyword(s):  
Factor Viii ◽  
Light Chain ◽  
Dependent Manner ◽  
C2 Domain ◽  
Factor X ◽  
Calcium Dependent ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Gla Domain

Abstract Factor VIII functions as a cofactor for factor IXa in the anionic phospholipid surface-dependent conversion of factor X to Xa. It is well-known that the A2 and A3 domains of factor VIII interact with the catalytic domain and EGF2 domain of factor IXa, respectively. Recently, Furie et al. have reported that the Gla domain of factor IXa (factor IXa-GD) interacts with the light chain of factor VIII. However, the factor IXa-GD-interactive site on the light chain remained to be investigated. In the current study, the recombinant C2 (rC2) domain of factor VIII was prepared using a yeast secretion system. ELISA-based assay in the absence of phospholipid showed the Glu-Gly-Arg-active site modified factor IXa (EGR-factor IXa) bound to the immobilized rC2 domain dose-dependently, and the binding ability was maximum under the condition of 150 mM NaCl/1 mM CaCl2. This binding was competitively inhibited by the addition of excess of factor VIII or rC2 domain, supporting the specificity of this interaction. Furthermore, the presence of high ionic strength and the metal-ion chelator EDTA blocked this binding by ∼95 and ∼75%, respectively. Surface plasmon resonance-based assay showed that the binding affinity (Kd) of rC2 domain for EGR-factor IXa was 108 ± 15.5 nM. GD less-factor IXa, deleting the GD completely, failed to bind to rC2 domain. A monoclonal antibody against factor IXa-GD specific for calcium-dependent conformation (mAbIXa-GD) also inhibited (∼ 95%) the rC2 domain binding to EGR-factor IXa in a dose-dependent manner (IC50; 758 nM), suggesting the authentic of the C2 domain and factor IXa-GD interaction. The addition of rC2 domain or mAbIXa-GD inhibited the factor IXa-catalyzed factor X activation with factor VIIIa in the absence of phospholipid (IC50; 15.7 μM or 43.2 nM, respectively), whilst both any little affected in the absence of factor VIIIa. In addition, the ∼8-kDa C2 fragment obtained by V8 protease digestion (residues 2182–2259) bound directly to EGR-factor IXa. Taken together, these results indicate that factor VIII C2 domain directly interacts with factor IXa-GD via both the electrostatic- and calcium-dependent interactions. Furthermore, our results provide the first evidence for an essential role of the C2 domain in the association between factor VIII and factor IXa in the factor Xase complex.

Regulation of Factor VIIIa in the Intrinsic Factor Xase

1999 ◽  
Vol 82 (08) ◽  
pp. 193-200 ◽  
Author(s):  
Philip Fay
Keyword(s):  
Factor Viii ◽  
Intrinsic Factor ◽  
Factor Xa ◽  
Clinical Importance ◽  
Factor X ◽  
Subunit Dissociation ◽  
Protein Factor ◽  
Catalytic Rate Constant ◽  
Factor Ixa ◽  
Factor Viiia

IntroductionHemophilia A, the most common of the severe, inherited bleeding disorders, results from a deficiency or defect in the plasma protein factor VIII. The activated form of the protein serves as an essential cofactor for factor IXa in the conversion of factor X to factor Xa. This surface-bound complex of enzyme and cofactor is referred to as the intrinsic factor Xase. Factor VIIIa dramatically increases the catalytic rate constant for substrate conversion by an unclear mechanism. The activity and stability of the factor Xase appears to be regulated by the integrity of the cofactor. Factor VIIIa possesses a labile structure, and subunit dissociation results in the decay of Xase activity. Furthermore, factor VIIIa is a substrate for proteolytic inactivation by several enzymes, including factor IXa, the enzyme for which it serves as a cofactor. Although interest in the structure, function, and metabolism of factor VIII is commensurate with its biochemical and clinical importance, the molecular basis for its role in coagulation and the regulation of function through complex intramolecular and intermolecular interactions remain poorly understood.

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