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.

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.

The Functional Defect of Factor VIII Leiden, a Genetic Variant of Coagulation Factor VIII

1985 ◽  
Vol 54 (03) ◽  
pp. 650-653 ◽  
Author(s):  
K Mertens ◽  
A van Wijngaarden ◽  
R M Bertina ◽  
J J Veltkamp
Keyword(s):  
Factor Viii ◽  
Genetic Variant ◽  
Intrinsic Factor ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Coagulation Factor Viii ◽  
Factor X ◽  
Plasma Factor ◽  
Functional Defect ◽  
Factor X Activating Complex

SummaryFactor VIII Leiden is a genetic variant of coagulation factor VIII which has been detected in the plasma of a patient with mild haemophilia A. In this patient’s plasma factor VIII procoagulant antigen was in 5-fold excess over factor VIII procoagulant activity, indicating the presence of an abnormal factor VIII molecule. The variant factor VIII was isolated from the patient’s plasma, and its functional properties were studied in a factor X-activating system consisting of purified components. The isolated factor VIII Leiden was normally activated by factor Xa and by thrombin, but the activity of the factor Villa was about 3% of normal. The defect of factor Villa Leiden was studied by comparison with normal factor Villa in kinetic experiments of factor Xa formation. The results support the hypothesis that factor Villa Leiden has a reduced affinity for phospholipid-bound factor IXa in the intrinsic factor X-activating complex.

scholarly journals Functional assembly of intrinsic coagulation proteases on monocytes and platelets. Comparison between cofactor activities induced by thrombin and factor Xa.

1992 ◽  
Vol 176 (1) ◽  
pp. 27-35 ◽  
Author(s):  
M P McGee ◽  
L C Li ◽  
M Hensler
Keyword(s):  
Factor Viii ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Coagulation Cascade ◽  
Factor X ◽  
Regulatory Pathways ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Human Factor Viii

Generation of coagulation factor Xa by the intrinsic pathway protease complex is essential for normal activation of the coagulation cascade in vivo. Monocytes and platelets provide membrane sites for assembly of components of this protease complex, factors IXa and VIII. Under biologically relevant conditions, expression of functional activity by this complex is associated with activation of factor VIII to VIIIa. In the present studies, autocatalytic regulatory pathways operating on monocyte and platelet membranes were investigated by comparing the cofactor function of thrombin-activated factor VIII to that of factor Xa-activated factor VIII. Reciprocal functional titrations with purified human factor VIII and factor IXa were performed at fixed concentrations of human monocytes, CaCl2, factor X, and either factor IXa or factor VIII. Factor VIII was preactivated with either thrombin or factor Xa, and reactions were initiated by addition of factor X. Rates of factor X activation were measured using chromogenic substrate specific for factor Xa. The K1/2 values, i.e., concentration of factor VIIIa at which rates were half maximal, were 0.96 nM with thrombin-activated factor VIII and 1.1 nM with factor Xa-activated factor VIII. These values are close to factor VIII concentration in plasma. The Vsat, i.e., rates at saturating concentrations of factor VIII, were 33.3 and 13.6 nM factor Xa/min, respectively. The K1/2 and Vsat values obtained in titrations with factor IXa were not significantly different from those obtained with factor VIII. In titrations with factor X, the values of Michaelis-Menten coefficients (Km) were 31.7 nM with thrombin-activated factor VIII, and 14.2 nM with factor Xa-activated factor VIII. Maximal rates were 23.4 and 4.9 nM factor Xa/min, respectively. The apparent catalytic efficiency was similar with either form of factor VIIIa. Kinetic profiles obtained with platelets as a source of membrane were comparable to those obtained with monocytes. These kinetic profiles are consistent with a 1:1 stoichiometry for the functional interaction between cofactor and enzyme on the surface of monocytes and platelets. Taken together, these results indicate that autocatalytic pathways connecting the extrinsic, intrinsic, and common coagulation pathways can operate efficiently on the monocyte membrane.

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 contribution of Ca2+ and phospholipids to the activation of human blood-coagulation Factor X by activated Factor IX

1984 ◽  
Vol 223 (3) ◽  
pp. 607-615 ◽  
Author(s):  
K Mertens ◽  
R M Bertina
Keyword(s):  
Human Factor ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Activate Factor ◽  
Factor X ◽  
Formation Kinetic ◽  
Factor Ixa ◽  
Fold Stimulation

The role of the cofactors Ca2+ and phospholipid in the activation of human Factor X by Factor IXa was investigated. By use of a sensitive spectrophotometric Factor Xa assay, it was demonstrated that human Factor IXa can activate Factor X in the absence of cofactors. The presence of Ca2+ as the only cofactor resulted in a 7-fold stimulation of the Factor Xa formation. Kinetic analysis of the Ca2+-stimulated reaction showed that the apparent Km of Factor X was 4.6 microM, whereas the apparent Vmax. for Factor Xa formation was 0.0088 mol of Xa/min per mol of IXa. The presence of phospholipid as the only cofactor had no effect on the rate of Factor Xa formation. However, a several-hundred-fold stimulation was observed when Ca2+ and phospholipid were present in combination. The activation of Factor X in the presence of Ca2+ and phospholipid was found to be kinetically heterogeneous, involving both phospholipid-bound and free reactants. Quantitative data concerning the phospholipid binding of Factors IXa and X were used to study the relation between the rate of Factor Xa formation and the binding of enzyme and substrate to the phospholipid membrane. The results support the hypothesis that phospholipid-bound Factor X is the substrate in the phospholipid-stimulated reaction; however, phospholipid-bound and free Factor IXa seem to be equally efficient in catalysing the activation of phospholipid-bound Factor X.

The Mechanism of Activation of Human Coagulation Factor X

1979 ◽  
Author(s):  
K. Mertens ◽  
R.M. Bertina
Keyword(s):  
Active Site ◽  
Human Factor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Active Form ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Factor X ◽  
Process Factor ◽  
Molecular Events ◽  
Product Factor

During the coagulation process factor X is converted to a serine protease, factor Xa. The present study concerns the molecular events which occur during the activation of human factor X by Russell’s viper venom and by the purified proteins of the extrinsic and intrinsic activator. Conversion of factor X was detected by amidolytic assays and SDS/polyacrylamide-gel electrophoresis.The results show that all activators convert factor X (MW 72,000) to an active form. In the presence of phospholipid the initially formed factor Xa (MW 54,000) complicates the further sequence of reactions by catalysing a) the conversion of factor Xa to a second active form (MW 50,000), b) the conversion of factor X to an inactive product (MW 59,000) by splitting off a peptide containing the active site serine, and c) the further degradation of the 50,000 and 59,000 components to a smaller component (MW 40,000).Comparison of these data with those reported for bovine factor X suggests that the mechanism of activation of human factor X is more complicated. The inactivation of both factor Xa and factor X by product factor Xa might be considered as important regulatory principles.

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.

scholarly journals Identification of a binding site for blood coagulation factor IXa on the light chain of human factor VIII.

1994 ◽  
Vol 269 (10) ◽  
pp. 7150-7155
Author(s):  
P.J. Lenting ◽  
M.J. Donath ◽  
J.A. van Mourik ◽  
K. Mertens
Keyword(s):  
Factor Viii ◽  
Binding Site ◽  
Blood Coagulation ◽  
Light Chain ◽  
Human Factor ◽  
Coagulation Factor ◽  
Factor Ixa ◽  
Human Factor Viii

Cleavage at Arg740 Appears Essential for Thrombin-Catalyzed Cleavage at Arg372 during the Activation of Factor VIII.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1692-1692
Author(s):  
Jennifer Newell ◽  
Philip J. Fay
Keyword(s):  
Factor Viii ◽  
Time Course ◽  
Specific Activity ◽  
Factor Xa ◽  
Factor X ◽  
Wild Type ◽  
Thrombin Cleavage ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Initial Cleavage

Abstract Factor VIIIa serves as an essential cofactor for the factor IXa-catalyzed activation of factor X during the propagation phase of coagulation. The factor VIII procofactor is converted to factor VIIIa by thrombin-catalyzed proteolysis of three P1 positions at Arg372 (A1–A2 junction), Arg740 (A2–B junction), and Arg1689 (a3–A3 junction). Cleavage at Arg372 exposes a cryptic functional factor IXa-interactive site, while cleavage at Arg1689 liberates factor VIII from von Willebrand factor and contributes to factor VIIIa specific activity, thus making both sites essential for procofactor activation. However, cleavage at Arg740, separating the A2–B domainal junction, has not been rigorously studied. To evaluate thrombin cleavage at Arg740, we prepared and stably expressed two recombinant factor VIII mutants, Arg740His and Arg740Gln. Results from a previous study examining proteolysis at Arg372 revealed substantially reduced cleavage rates following substitution of that P1 Arg with His, whereas replacing Arg with Gln at residue 372 yielded an uncleavable bond at that site (Nogami et al., Blood, 2005). Specific activity values for the factor VIII Arg740His and Arg740Gln variants as measured using a one-stage clotting assay were approximately 50% and 18%, respectively, that of the wild type protein. SDS-PAGE and western blotting following a reaction of factor VIII Arg740His with thrombin showed reduced rates of cleavage at His740 as well as at Arg372 relative to the wild type. Alternatively, factor VIII Arg740Gln was resistant to thrombin cleavage at Gln740 and showed little, if any, cleavage at Arg372 over an extended time course. The mutant proteins assayed in a purified system by factor Xa generation showed a slight increase in activity for the Arg740His variant compared with the Arg740Gln variant in both the absence and presence of thrombin, and the activities for both variants were reduced compared with wild type factor VIII. These results suggest that cleavage at residue 740 affects subsequent cleavage at Arg372 and generation of the active cofactor factor VIIIa. Preliminary results obtained evaluating proteolysis of these mutants by factor Xa, which cleaves the same sites in factor VIII as thrombin, also revealed slow proteolysis at the P1 His and no cleavage at the P1 Gln. However, subsequent cleavage at Arg372 exhibited less dependence on initial cleavage at residue 740. These observations may explain the higher than predicted specific activity values obtained for the two variants and suggest a different mechanism of action for the two activating proteinases. Overall, these results support a model whereby cleavage of factor VIII heavy chain by thrombin is an ordered pathway with initial cleavage at Arg740 required to facilitate cleavage at the critical Arg372 site to yield the active cofactor.

Protein S Down-Regulates Factor VIIIa by Competing with Factor IXa.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1714-1714
Author(s):  
Masahiro Takeyama ◽  
Keiji Nogami ◽  
Kohei Tatsumi ◽  
Yuri Fujita ◽  
Ichiro Tanaka ◽  
...  
Keyword(s):  
Factor Viii ◽  
Factor Xa ◽  
Protein S ◽  
Factor X ◽  
Cleavage Rate ◽  
Physiological Concentration ◽  
C2 Domains ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Km Value

Abstract Factor VIII functions as a cofactor in the factor Xase complex responsible for phospholipid surface-dependent conversion of factor X to factor Xa by factor IXa. Factor VIIIa, activated form by thrombin and factor Xa, is down regulated by activated protein C (APC), and the reaction is enhanced by the presence of protein S, a cofactor for APC. It was previously reported that protein S inactivated directly factor Xa or factor Va, however, the direct regulation of factor VIII by protein S remains to be investigated. In the present study, surface plasmon resonance (SPR)-based assay showed that factor VIII bound directly to immobilized protein S (Kd; 70 nM). The isolated A2 and A3 domains also bound to protein S with similar modest affinity (Kd; 15 and 17 nM, respectively), whilst the isolated A1 and C2 domains failed to bind, suggesting the presence of protein S-binding sites within the A2 and A3 domain. Since it is known that factor IXa also interacts with the A2 and A3 domains in factor VIII, we examined the inhibitory effect of factor IXa on the factor VIII and protein S interaction in a SPR-based assay. Active-site modified (EGR−) factor IXa competitively inhibited the binding of protein S to both the A2 and A3-C1-C2 domains dose-dependently. Furthermore, Western blotting analysis using an anti-A1 monoclonal antibody revealed that Arg336 cleavage in factor VIII by factor IXa in the presence of protein S was slower with an ~1.8-fold lower cleavage rate than that in its absence, supporting that protein S competed the factor IXa interaction with factor VIII. Of interest, the reaction with protein S to factor VIII inhibited the generation of factor Xa dose-dependently in a factor Xa generation assay (IC50; 150 nM). The Km value for factor X obtained with factor Xase complex in the presence of physiological concentration of protein S was 19 nM, which was ~2-fold lower than that in its absence (45 nM). Whilst, the Km value for factor IXa in the presence of protein S was greater than 100 nM, which was ~5000-fold higher than that in its absence (21 pM). We demonstrate that protein S not only contributes to down-regulate factor VIIIa activity as a cofactor for APC, but also impairs the factor Xase complex by competing the binding of factor IXa to factor VIII.

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