Coordinate Binding Studies of the Substrate (Factor X) with the Cofactor (Factor VIII) in the Assembly of the Factor X Activating Complex on the Activated Platelet Surface†

Biochemistry ◽  
2002 ◽  
Vol 41 (37) ◽  
pp. 11269-11276 ◽  
Author(s):  
Syed S. Ahmad ◽  
Peter N. Walsh
Keyword(s):  
Factor Viii ◽  
Factor X ◽  
Binding Studies ◽  
Platelet Surface ◽  
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)

FACTOR VIII MEDIATES BINDING OF FACTOR IX TO STIMULATED PLATELETS

1987 ◽  
Author(s):  
W Muntean ◽  
B Leschnik
Keyword(s):  
Factor Viii ◽  
Phospholipase C ◽  
Human Platelets ◽  
Final Concentration ◽  
Platelet Lysate ◽  
Factor Ix ◽  
Factor Vii ◽  
Factor X ◽  
Platelet Surface ◽  
Human Thrombin

In previous work we have shown that factor VII! binds to phospholipids of the membrane of stimulated platelets and that von Willebrand factor is not required for binding of factor VIII to platelets. Since factor VIII is a cofactor in the activation of factor X by factor IX, we investigated whether factor VIII enhances binding of factor IX to the platelet surface.Factor VIII and factor IX were purified by immunoadsorbent chromatography using specific rabbit antibodies. Washed human platelets (250/nl final concentration) stimulated by human thrombin and collagen were incubated with barbiturate buffer, or with purified factor IX (1 U/ml final concentration), or with factor IX (1 U/ml) in the presence of factor VIII (1 U/ml). Washed and stimulated platelets were also incubated with factor VIII and IX as above in the presence of different amounts of CaCl2. Platelets were then washed again and lysed by sonication. Factor VIII:Ag (immunoradiometric assay) and factor IX:Ag (ELISA) were measured in the platelet lysate prior to and after incubation of the lysate with phospholipase C.Platelet bound IX:Ag was significantly higher after incubation of stimulated platelets with factor IX in the presence of factor VIII than after incubation of platelets with buffer or with factor IX alone. CaCl2 proved to be essential for binding of factor IX to platelets even in the presence of factor VIII, but CaCl2 was not required for binding of factor VIII to platelets. Measurable VIII :Ag and IX:Ag increased significantly after incubation of the platelet lysate with phospholipase C.Our data suggest that factor VIII mediates binding of factor IX to phospholipids or receptors containing phospholipids on the membrane of stimulated platelets and thereby contributes to the assembly of the factor X activating complex on the platelet surface.

scholarly journals Formation and functioning of the factor IXa-VIII complex on the surface of endothelial cells

Blood ◽  
1987 ◽  
Vol 69 (2) ◽  
pp. 442-445
Author(s):  
K Varadi ◽  
S Elodi
Keyword(s):  
Endothelial Cells ◽  
Factor Viii ◽  
Maximum Activity ◽  
Lag Phase ◽  
Factor X ◽  
Slow Development ◽  
Binding Surface ◽  
Factor X Activation ◽  
Factor X Activating Complex ◽  
Optimal Assembly

The formation and functioning of the factor X activating complex on the surface of cultured human venous endothelial cells (HVEC) were investigated. To the HVEC monolayer human factors IXa, VIII, X, CaCl2, and S-2222 were added, and a gradually increasing activation of factor X was observed. The maximum activity of 88 nmol/L Xa/min was reached after a 12-minute lag phase. In the presence of thrombin-activated factor VIII (VIIIt) the same maximum activity developed in eight minutes, which suggests that VIIIt accelerates the formation of the IXa- VIII complex but does not influence its factor X-activating potential. Anti-VIII IgG did not affect the activity of the full-fledged complex. When anti-VIII IgG was added to the reaction mixture before factor VIII or during the lag phase of the reaction, it induced a concentration- dependent decrease of factor X activation. These results indicate that endothelial cells provide a binding surface for the IXa-VIII complex and that in the HVEC-bound complex factor VIII is protected from the effect of a specific antibody. However, the relatively slow development of the maximum activity indicates that HVEC only partially satisfy the surface criteria for the optimal assembly of the IXa-VIII complex.

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.

Characterization of Clotting Factors Associated with Blood Platelets after Gel Filtration

1973 ◽  
Vol 30 (02) ◽  
pp. 289-298
Author(s):  
Oddvar Tangen ◽  
Eva B. Lestrup ◽  
Herbert J. Berman
Keyword(s):  
Factor Viii ◽  
Blood Platelets ◽  
Gel Filtration ◽  
Factor Xa ◽  
Factor V ◽  
Factor X ◽  
Factor Viii Activity ◽  
Clotting Factors ◽  
Platelet Surface ◽  
Filtration Factor

SummaryAggregation of human and rabbit gel filtered platelets (GFP) has been studied in presence of Ca2+, activated factor X (Xa) and different plasma preparations. It was found that factor Xa by itself is not a platelet aggregating agent. However, the platelets aggregated immediately when platelet poor plasma (PPP) was added to a mixture of GFP, Ca2+ and factor Xa. Aggregation also occurred immediately when factor V-deficient plasma was substituted for PPP, but not when factor II-deficient plasma was used. In the absence of factor Xa, aggregation occurred on addition of factor V- or VIII-deficient plasma, but only after some delay. The platelet aggregation experiments and experiments with centrifugations and resuspensions of the platelets, clotting experiments, and gel filtration of platelet free plasma (PEP) led to the following conclusions : Factors II and X are totally removed from the platelets by gel filtration, factor V is closely associated with the platelet surface, and part of the factor VIII-activity in the plasma is eluted together with the GFP without being associated with the platelets. This factor VIII-activity belonged to an extremely large molecule or molecular complex with a Mw in the order of 2 - 5 · 107.

scholarly journals Formation and functioning of the factor IXa-VIII complex on the surface of endothelial cells

Blood ◽  
1987 ◽  
Vol 69 (2) ◽  
pp. 442-445 ◽  
Author(s):  
K Varadi ◽  
S Elodi
Keyword(s):  
Endothelial Cells ◽  
Factor Viii ◽  
Maximum Activity ◽  
Lag Phase ◽  
Factor X ◽  
Slow Development ◽  
Binding Surface ◽  
Factor X Activation ◽  
Factor X Activating Complex ◽  
Optimal Assembly

Abstract The formation and functioning of the factor X activating complex on the surface of cultured human venous endothelial cells (HVEC) were investigated. To the HVEC monolayer human factors IXa, VIII, X, CaCl2, and S-2222 were added, and a gradually increasing activation of factor X was observed. The maximum activity of 88 nmol/L Xa/min was reached after a 12-minute lag phase. In the presence of thrombin-activated factor VIII (VIIIt) the same maximum activity developed in eight minutes, which suggests that VIIIt accelerates the formation of the IXa- VIII complex but does not influence its factor X-activating potential. Anti-VIII IgG did not affect the activity of the full-fledged complex. When anti-VIII IgG was added to the reaction mixture before factor VIII or during the lag phase of the reaction, it induced a concentration- dependent decrease of factor X activation. These results indicate that endothelial cells provide a binding surface for the IXa-VIII complex and that in the HVEC-bound complex factor VIII is protected from the effect of a specific antibody. However, the relatively slow development of the maximum activity indicates that HVEC only partially satisfy the surface criteria for the optimal assembly of the IXa-VIII 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

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)

Studies of the Mechanism for Enhanced Cell Surface Factor VIIa/Tissue Factor Activation of Factor X on Fibroblast Monolayers after Their Exposure to N-Ethylmaleimide

1994 ◽  
Vol 72 (06) ◽  
pp. 848-855 ◽  
Author(s):  
Dzung The Le ◽  
Samuel I Rapaport ◽  
L Vijaya Mohan Rao
Keyword(s):  
Cell Surface ◽  
Tissue Factor ◽  
Factor Viia ◽  
Cell Damage ◽  
Specific Binding ◽  
Surface Membrane ◽  
Annexin V ◽  
Factor X ◽  
Binding Studies ◽  
Anionic Phospholipid

SummaryFibroblast monolayers constitutively expressing surface membrane tissue factor (TF) were treated with 0.1 mM N-ethylmaleimide (NEM) for 1 min to inhibit aminophospholipid translocase activity without inducing general cell damage. This resulted in increased anionic phospholipid in the outer leaflet of the cell surface membrane as measured by the binding of 125I-annexin V and by the ability of the monolayers to support the generation of prothrombinase. Specific binding of 125I-rVIIa to TF on NEM-treated monolayers was increased 3- to 4-fold over control monolayers after only brief exposure to 125I-rVIIa, but this difference progressively diminished with longer exposure times. A brief exposure of NEM-treated monolayers to rVIIa led to a maximum 3- to 4-fold enhancement of VIIa/TF catalytic activity towards factor X over control monolayers, but, in contrast to the binding studies, this 3- to 4-fold difference persisted despite increasing time of exposure to rVIIa. Adding prothrombin fragment 1 failed to diminish the enhanced VIIa/TF activation of factor X of NEM-treated monolayers. Moreover, adding annexin V, which was shown to abolish the ability of NEM to enhance factor X binding to the fibroblast monolayers, also failed to diminish the enhanced VIIa/TF activation of factor X. These data provide new evidence for a possible mechanism by which availability of anionic phospholipid in the outer layer of the cell membrane limits formation of functional VIIa/TF complexes on cell surfaces.

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.

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