The role of phospholipid and factor VIIIa in the activation of bovine factor X.

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.

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

Blood ◽

10.1182/blood.v84.6.1837.1837 ◽

1994 ◽

Vol 84 (6) ◽

pp. 1837-1842 ◽

Cited By ~ 12

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.

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

Blood ◽

10.1182/blood.v84.6.1837.bloodjournal8461837 ◽

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.

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Plasma Levels of Protein S, Protein C, and Factor X: Effects of Sex, Hormonal State and Age

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649925 ◽

1995 ◽

Vol 74 (05) ◽

pp. 1271-1275 ◽

Cited By ~ 36

Author(s):

C M A Henkens ◽

V J J Bom ◽

W van der Schaaf ◽

P M Pelsma ◽

C Th Smit Sibinga ◽

...

Keyword(s):

Regression Analysis ◽

Postmenopausal Women ◽

Protein C ◽

Blood Donors ◽

Premenopausal Women ◽

Protein S ◽

The Other ◽

Factor X ◽

Normal Ranges

SummaryWe measured total and free protein S (PS), protein C (PC) and factor X (FX) in 393 healthy blood donors to assess differences in relation to sex, hormonal state and age. All measured proteins were lower in women as compared to men, as were levels in premenopausal women as compared to postmenopausal women. Multiple regression analysis showed that both age and subgroup (men, pre- and postmenopausal women) were of significance for the levels of total and free PS and PC, the subgroup effect being caused by the differences between the premenopausal women and the other groups. This indicates a role of sex-hormones, most likely estrogens, in the regulation of levels of pro- and anticoagulant factors under physiologic conditions. These differences should be taken into account in daily clinical practice and may necessitate different normal ranges for men, pre- and postmenopausal women.

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The Activation of Human Factor X in Sodium Citrate: the Role of Factor VII

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648014 ◽

1976 ◽

Vol 36 (01) ◽

pp. 104-114 ◽

Cited By ~ 3

Author(s):

D. L Aronson ◽

A. J Mustafa

Keyword(s):

Sodium Citrate ◽

Human Factor ◽

Deae Cellulose ◽

Factor Ix ◽

Factor Vii ◽

Factor X

SummaryHuman factor X was purified by several different procedures yielding products which had varying amounts of factor VII and factor IX. Treatment with CHC13 during the fractionation of the factor X removed 95% of the factor VII and factor IX activity and the resulting factor X activated more slowly when incubated in 25% sodium citrate. Removal of residual factor VII by DEAE cellulose chromatography yielded a factor X which activated still more slowly and less completely. When the factor VII, removed by chromatography, was added to the chromatographed factor X, the ability to be activated in 25% sodium citrate was restored. Confirmatory evidence for the role of factor VII in this reaction was the inhibition of the conversion of the factor X by both DFP and SBTI.

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The Binding of Calcium Ions to Bovine Factor X by Rate Dialysis

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648668 ◽

1978 ◽

Vol 40 (02) ◽

pp. 350-357

Author(s):

Robert H Yue ◽

Menard M Gertler

Keyword(s):

Molecular Weight ◽

Dissociation Constant ◽

Calcium Ions ◽

Activation Process ◽

Factor X ◽

The Real ◽

High Affinity ◽

Binding Data ◽

Sequential Mechanism ◽

Bovine Factor

SummaryThe binding of Ca+2 to bovine factor X (molecular weight of 74,000) (Yue und Gertler 1977) was studied by the technique of rate dialysis and with the use of 45Ca+2. The binding data are consistent with a model of sequential mechanism. One mole of Ca+2 binds to the glycoprotein with a dissociation constant of 5.2 × 10-5 M and an additional 39 ± 4 moles of Ca+2 bind to this zymogen with a dissociation constant of 3.7 × 10-3M. The binding of the high affinity Ca+2 causes a functionally significant change in the zymogen, and (calcium) (factor X) complex is the real substrate in the activation process by the protease in Russell’s viper venom.

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The Formation of Intermediate Product I in a Purified System The Role of Factor IX or of its Precursor and of a Hageman Factor-PTA Fraction

Thrombosis and Haemostasis ◽

10.1055/s-0038-1654555 ◽

1961 ◽

Vol 6 (02) ◽

pp. 224-234 ◽

Cited By ~ 4

Author(s):

E. T Yin ◽

F Duckert

Keyword(s):

Intermediate Product ◽

Factor Ix ◽

Assay Method ◽

Lag Phase ◽

Factor X ◽

Haemophilia B ◽

Hageman Factor ◽

One Stage ◽

The One

Summary1. The role of two clot promoting fractions isolated from either plasma or serum is studied in a purified system for the generation of intermediate product I in which the serum is replaced by factor X and the investigated fractions.2. Optimal generation of intermediate product I is possible in the purified system utilizing fractions devoid of factor IX one-stage activity. Prothrombin and thrombin are not necessary in this system.3. The fraction containing factor IX or its precursor, no measurable activity by the one-stage assay method, controls the yield of intermediate product I. No similar fraction can be isolated from haemophilia B plasma or serum.4. The Hageman factor — PTA fraction shortens the lag phase of intermediate product I formation and has no influence on the yield. This fraction can also be prepared from haemophilia B plasma or serum.

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Interaction of manganese with bovine factor X.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)40119-0 ◽

1977 ◽

Vol 252 (14) ◽

pp. 4758-4761 ◽

Cited By ~ 11

Author(s):

S P Bajaj ◽

R Byrne ◽

T Nowak ◽

F J Castellino

Keyword(s):

Factor X ◽

Bovine Factor

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Interaction of the A1 Subunit of Factor VIIIa and the Serine Protease Domain of Factor X Identified by Zero-length Cross-linking

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615223 ◽

1998 ◽

Vol 80 (09) ◽

pp. 418-422 ◽

Cited By ~ 31

Author(s):

Kirsty Lapan ◽

Philip Fay

Keyword(s):

Serine Protease ◽

Heavy Chain ◽

Solid Phase ◽

Activated Protein C ◽

Cross Linking ◽

Factor X ◽

Serine Protease Domain ◽

C Terminus ◽

Factor Viiia ◽

Solid Phase Binding

SummaryWe have previously used a solid phase binding assay to localize a Factor X (FX) interactive site to the acidic C-terminus of the A1 subunit of FVIIIa (Lapan KA, Fay PJ. J Biol Chem 1997; 272: 2082-2088). The complex of FVIII-FX was made covalent following reaction with the zero-length cross-linking reagent 1-ethyl-3-(3-dimethylaminopropyl-)carbodiimide hydrochloride (EDC). Western blotting of the thrombin-cleaved complex showed that the A1 subunit of FVIIIa associated with FX heavy chain. The FX-A1 product was also detected following cross-linking to the A1/A3-C1-C2 dimer, but not the activated protein C-cleaved A1336/A3-C1-C2 form, indicating that a residue(s) in the region spanning Met337-Arg372 contributed to the intermolecular ion pair(s). A synthetic peptide to this acidic region (FVIII337-372) cross-linked to FX and the product was alkaline resistant indicating that amide linkage(s) were formed. Sequence analysis of the FX-FVIII337-372 adduct suggested that the first 12 NH2-terminal residues of the FX and peptide do not participate in cross-link formation. Conversion of the cross-linked product to FXa by RVV-X showed that the peptide was associated with the serine protease-forming domain of the heavy chain. These results indicate that the association of FVIIIa and FX occurs from a salt linkage(s) formed between residues of the A1 acidic C-terminus of the cofactor (within residues 349-372) and the serine protease-forming domain of the substrate.

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