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.

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.

scholarly journals Inhibition of human coagulation factor VIII by monoclonal antibodies. Mapping of functional epitopes with the use of recombinant factor VIII fragments

1989 ◽  
Vol 263 (1) ◽  
pp. 187-194 ◽  
Author(s):  
A Leyte ◽  
K Mertens ◽  
B Distel ◽  
R F Evers ◽  
M J M De Keyzer-Nellen ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Factor Viii ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Procoagulant Activity ◽  
Coagulation Factor Viii ◽  
Coagulation Cascade ◽  
Restriction Enzyme Digestion ◽  
Proteolytic Activation

The epitopes of four monoclonal antibodies against coagulation Factor VIII were mapped with the use of recombinant DNA techniques. Full-length Factor VIII cDNA and parts thereof were inserted into the vector pSP64, permitting transcription in vitro with the use of a promoter specific for SP6 RNA polymerase. Factor VIII DNA inserts were truncated from their 3′-ends by selective restriction-enzyme digestion and used as templates for ‘run-off’ mRNA synthesis. Translation in vitro with rabbit reticulocyte lysate provided defined radiolabelled Factor VIII fragments for immunoprecipitation studies. Two antibodies are shown to be directed against epitopes on the 90 kDa chain of Factor VIII, between residues 712 and 741. The 80 kDa chain appeared to contain the epitopes of the other two antibodies, within the sequences 1649-1778 and 1779-1840 respectively. The effect of antibody binding to these sequences was evaluated at two distinct levels within the coagulation cascade. Both Factor VIII procoagulant activity and Factor VIII cofactor function in Factor Xa generation were neutralized upon binding to the region 1779-1840. The antibodies recognizing the region 713-740 or 1649-1778, though interfering with Factor VIII procoagulant activity, did not inhibit in Factor Xa generation. These findings demonstrate that antibodies that virtually inhibit Factor VIII in coagulation in vitro are not necessarily directed against epitopes involved in Factor VIII cofactor function. Inhibition of procoagulant activity rather than of cofactor function itself may be explained by interference in proteolytic activation of Factor VIII. This hypothesis is in agreement with the localization of the epitopes in the proximity of thrombin-cleavage or Factor Xa-cleavage sites.

scholarly journals An Antibody Specific for Coagulation Factor IX Enhances the Activity of the Intrinsic Factor X-activating Complex

2004 ◽  
Vol 279 (39) ◽  
pp. 40445-40450 ◽  
Author(s):  
Randolf J. Kerschbaumer ◽  
Klaudia Riedrich ◽  
Martina Kral ◽  
Katalin Varadi ◽  
Friedrich Dorner ◽  
...  
Keyword(s):  
Intrinsic Factor ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Factor X ◽  
Coagulation Factor Ix ◽  
Factor X Activating Complex

scholarly journals Inhibition of the intrinsic factor X activating complex by protein S: evidence for a specific binding of protein S to factor VIII

Blood ◽  
1995 ◽  
Vol 86 (3) ◽  
pp. 1062-1071 ◽  
Author(s):  
SJ Koppelman ◽  
TM Hackeng ◽  
JJ Sixma ◽  
BN Bouma
Keyword(s):  
Factor Viii ◽  
Protein C ◽  
Intrinsic Factor ◽  
Activated Protein C ◽  
Protein S ◽  
Inhibitory Effect ◽  
Factor X ◽  
Ic50 Values ◽  
Factor X Activation ◽  
Factor X Activating Complex

Protein S is a vitamin K-dependent nonenzymatic anticoagulant protein that acts as a cofactor to activated protein C. Recently it was shown that protein S inhibits the prothrombinase reaction independent of activated protein C. In this study, we show that protein S can also inhibit the intrinsic factor X activation via a specific interaction with factor VIII. In the presence of endothelial cells, the intrinsic activation of factor X was inhibited by protein S with an IC50 value of 0.28 +/- 0.04 mumol/L corresponding to the plasma concentration of protein S. This inhibitory effect was even more pronounced when the intrinsic factor X activation was studied in the presence of activated platelets (IC50 = 0.15 +/- 0.02 mumol/L). When a nonlimiting concentration of phospholipid vesicles was used, the plasma concentration of protein S (300 nmol/L) inhibited the intrinsic factor X activation by 40%. Thrombin-cleaved protein S inhibited the endothelial cell-mediated factor X activation with an IC50 similar to that of native protein S (0.26 +/- 0.02 mumol/L). Protein S in complex with C4b-binding protein inhibited the endothelial cell-mediated factor X activation more potently than protein S alone (IC50 = 0.19 +/- 0.03 mumol/L). Using thrombin activated factor VIII, IC50 values of 0.53 +/- 0.09 mumol/L and 0.46 +/- 0.10 mumol/L were found for native protein S and thrombin-cleaved protein S, respectively. The possible interactions of protein S with factor IXa, phospholipids, and factor VIII were investigated. The enzymatic activity of factor IXa was not affected by protein S, and interaction of protein S with the phospholipid surface could not fully explain the inhibitory effect of protein S on the factor X activation. Using a solid-phase binding assay, we showed a specific, saturable, and reversible binding of protein S to factor VIII with a high affinity. The concentration of protein S where half-maximal binding was reached (B1/2max) was 0.41 +/- 0.06 mumol/L. A similar affinity was found for the interaction of thrombin-cleaved protein S with factor VIII (B1/2max = 0.40 +/- 0.04 mumol/L). The affinity of the complex protein S with C4B-binding protein appeared to be five times higher (B1/2max = 0.07 +/- 0.03 mumol/L). Because the affinities of the interaction of the different forms of protein S with factor VIII correspond to the IC50 values observed for the intrinsic factor X activating complex, the interaction of protein S with factor VIII may explain the inhibitory effect of protein S on the intrinsic factor X activating complex.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

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 Inhibition of the intrinsic factor X activating complex by protein S: evidence for a specific binding of protein S to factor VIII

Blood ◽  
1995 ◽  
Vol 86 (3) ◽  
pp. 1062-1071 ◽  
Author(s):  
SJ Koppelman ◽  
TM Hackeng ◽  
JJ Sixma ◽  
BN Bouma
Keyword(s):  
Factor Viii ◽  
Protein C ◽  
Intrinsic Factor ◽  
Activated Protein C ◽  
Protein S ◽  
Inhibitory Effect ◽  
Factor X ◽  
Ic50 Values ◽  
Factor X Activation ◽  
Factor X Activating Complex

Abstract Protein S is a vitamin K-dependent nonenzymatic anticoagulant protein that acts as a cofactor to activated protein C. Recently it was shown that protein S inhibits the prothrombinase reaction independent of activated protein C. In this study, we show that protein S can also inhibit the intrinsic factor X activation via a specific interaction with factor VIII. In the presence of endothelial cells, the intrinsic activation of factor X was inhibited by protein S with an IC50 value of 0.28 +/- 0.04 mumol/L corresponding to the plasma concentration of protein S. This inhibitory effect was even more pronounced when the intrinsic factor X activation was studied in the presence of activated platelets (IC50 = 0.15 +/- 0.02 mumol/L). When a nonlimiting concentration of phospholipid vesicles was used, the plasma concentration of protein S (300 nmol/L) inhibited the intrinsic factor X activation by 40%. Thrombin-cleaved protein S inhibited the endothelial cell-mediated factor X activation with an IC50 similar to that of native protein S (0.26 +/- 0.02 mumol/L). Protein S in complex with C4b-binding protein inhibited the endothelial cell-mediated factor X activation more potently than protein S alone (IC50 = 0.19 +/- 0.03 mumol/L). Using thrombin activated factor VIII, IC50 values of 0.53 +/- 0.09 mumol/L and 0.46 +/- 0.10 mumol/L were found for native protein S and thrombin-cleaved protein S, respectively. The possible interactions of protein S with factor IXa, phospholipids, and factor VIII were investigated. The enzymatic activity of factor IXa was not affected by protein S, and interaction of protein S with the phospholipid surface could not fully explain the inhibitory effect of protein S on the factor X activation. Using a solid-phase binding assay, we showed a specific, saturable, and reversible binding of protein S to factor VIII with a high affinity. The concentration of protein S where half-maximal binding was reached (B1/2max) was 0.41 +/- 0.06 mumol/L. A similar affinity was found for the interaction of thrombin-cleaved protein S with factor VIII (B1/2max = 0.40 +/- 0.04 mumol/L). The affinity of the complex protein S with C4B-binding protein appeared to be five times higher (B1/2max = 0.07 +/- 0.03 mumol/L). Because the affinities of the interaction of the different forms of protein S with factor VIII correspond to the IC50 values observed for the intrinsic factor X activating complex, the interaction of protein S with factor VIII may explain the inhibitory effect of protein S on the intrinsic factor X activating complex.(ABSTRACT TRUNCATED AT 400 WORDS)

Cloning and Characterization of the Murine Coagulation Factor X Gene

2000 ◽  
Vol 83 (05) ◽  
pp. 732-735 ◽  
Author(s):  
Adrian Cooper ◽  
Zhong Liang ◽  
Francis Castellino ◽  
Elliot Rosen
Keyword(s):  
Factor Viii ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Coagulation Factor Viii ◽  
Start Codon ◽  
Factor Vii ◽  
Factor V ◽  
Factor X ◽  
Coagulation Factor Vii ◽  
Coagulation Factor V

SummaryThe gene encoding murine coagulation factor X (fX) was isolated and characterized from a λFIX II library generated from murine genomic DNA. The 20130 bp sequence contains 18049 nucleotides that extend from the initiating methionine to the polyadenylation site. 1056 nucleotides 5’ of the start codon were determined and contain putative start sites for the FX mRNA as well as sites for binding of putative transcription factors. The sequence extends 1024 3’ of the polyadenylattion site.The gene contains 8 exons and 7 introns which were determined by comparing the mouse FX cDNA and gene sequences. The exonic structure of the gene is similar to that of the other mammalian vitamin K-dependent serine proteases of the coagulation system. These include an exon encoding the prepropepetide, the gladomain, a short helical stack, two exons for the two EGF domains, the activation pepetide, and two exons encoding the serine protease domain. The 5’ sequence of the mouse FX gene overlaps with the 3’ region of the FVII gene indicating that the murine FVII and FX gene are arranged in a head to tail arrangement as they are in humans. Abbreviations: fVII, coagulation factor VII; fIX, coagulation factor IX; fX, coagulation factor X; PC, Protein C; fV, coagulation factor V; fVa, activated coagulation factor V; fVIII, coagulation factor VIII; fVIIIa, activated coagulation factor VIII.

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