scholarly journals Role of gamma-carboxyglutamic acid residues in the binding of factor IXa to platelets and in factor-X activation

Blood ◽  
1992 ◽  
Vol 79 (2) ◽  
pp. 398-405 ◽  
Author(s):  
R Rawala-Sheikh ◽  
SS Ahmad ◽  
DM Monroe ◽  
HR Roberts ◽  
PN Walsh
Keyword(s):  
Binding Sites ◽  
Factor Xa ◽  
Catalytic Efficiency ◽  
Factor Ix ◽  
Factor X ◽  
Apparent Dissociation Constant ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Carboxyglutamic Acid ◽  
Factor X Activation

Abstract To study the requirements for factor-IXa binding to platelets and factor-X activation, we examined the consequences of chemical modification (factor IXMOD) or enzymatic removal (factor IXDES) of gamma-carboxyglutamic acid (Gla) residues. In the presence of factor VIIIa and factor X, there were 344 (+/- 52) binding sites/platelet for factor IXaMOD (apparent dissociation constant [kdapp] = 4.5 +/- 0.9 nmol/L) and 275 (+/- 35) sites/platelet for factor IXaDES (kdapp = 5.0 +/- 0.8 nmol/L) compared with 580 (+/-65) sites/platelet for normal factor IXa (factor IXaN) (kdapp = 0.61 +/- 0.1 nmol/L) and 300 (+/-62) sites/platelet for factor IX (kdapp = 2.9 +/- 0.29 nmol/L). The concentrations of factor IXaN, factor IXaMOD and factor IXaDES required for half-maximal rates of factor-Xa formation were 0.67 nmol/L, 3.5 nmol/L, and 6.7 nmol/L. Whereas maximal velocities (Vmax) of factor Xa formation by factor IXaMOD (approximately 0.8 nmol/L.min-1) and factor IXaN (approximately 10.5 nmol/L.min-1), turnover numbers (kcat expressed as moles of factor Xa formed per minute per mole of factor IXa bound), and values of catalytic efficiency (kcat/Km) were normal, indicating that the decreased rates of factor X activation observed with factor IXaMOD and factor IXaDES are solely a consequence of the abnormal binding of these proteins to thrombin-activated platelets in the presence of factor VIIIa and factor X. Thus, factor IXa binding to platelets is mediated in part, but not exclusively, by high-affinity Ca2+ binding sites in the Gla domain of factor IX.

scholarly journals Role of gamma-carboxyglutamic acid residues in the binding of factor IXa to platelets and in factor-X activation

Blood ◽  
1992 ◽  
Vol 79 (2) ◽  
pp. 398-405 ◽  
Author(s):  
R Rawala-Sheikh ◽  
SS Ahmad ◽  
DM Monroe ◽  
HR Roberts ◽  
PN Walsh
Keyword(s):  
Binding Sites ◽  
Factor Xa ◽  
Catalytic Efficiency ◽  
Factor Ix ◽  
Factor X ◽  
Apparent Dissociation Constant ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Carboxyglutamic Acid ◽  
Factor X Activation

To study the requirements for factor-IXa binding to platelets and factor-X activation, we examined the consequences of chemical modification (factor IXMOD) or enzymatic removal (factor IXDES) of gamma-carboxyglutamic acid (Gla) residues. In the presence of factor VIIIa and factor X, there were 344 (+/- 52) binding sites/platelet for factor IXaMOD (apparent dissociation constant [kdapp] = 4.5 +/- 0.9 nmol/L) and 275 (+/- 35) sites/platelet for factor IXaDES (kdapp = 5.0 +/- 0.8 nmol/L) compared with 580 (+/-65) sites/platelet for normal factor IXa (factor IXaN) (kdapp = 0.61 +/- 0.1 nmol/L) and 300 (+/-62) sites/platelet for factor IX (kdapp = 2.9 +/- 0.29 nmol/L). The concentrations of factor IXaN, factor IXaMOD and factor IXaDES required for half-maximal rates of factor-Xa formation were 0.67 nmol/L, 3.5 nmol/L, and 6.7 nmol/L. Whereas maximal velocities (Vmax) of factor Xa formation by factor IXaMOD (approximately 0.8 nmol/L.min-1) and factor IXaN (approximately 10.5 nmol/L.min-1), turnover numbers (kcat expressed as moles of factor Xa formed per minute per mole of factor IXa bound), and values of catalytic efficiency (kcat/Km) were normal, indicating that the decreased rates of factor X activation observed with factor IXaMOD and factor IXaDES are solely a consequence of the abnormal binding of these proteins to thrombin-activated platelets in the presence of factor VIIIa and factor X. Thus, factor IXa binding to platelets is mediated in part, but not exclusively, by high-affinity Ca2+ binding sites in the Gla domain of factor IX.

scholarly journals A comparison of phospholipid and platelets in the activation of human factor VIII by thrombin and factor Xa, and in the activation of factor X

Blood ◽  
1988 ◽  
Vol 72 (5) ◽  
pp. 1761-1770
Author(s):  
P Neuenschwander ◽  
J Jesty
Keyword(s):  
Factor Viii ◽  
Factor Xa ◽  
Ionophore A23187 ◽  
Factor X ◽  
Apparent Dissociation Constant ◽  
Factor Ixa ◽  
Activated Platelets ◽  
Factor Viiia ◽  
Human Factor Viii ◽  
Factor X Activation

Two aspects of the activation of factor X by the intrinsic clotting pathway have been studied in purified human systems, in the presence of either purified phosphatidylserine:phosphatidylcholine vesicles (PS:PC) or platelets activated with ionophore A23187: (1) the activation of factor VIII by factor Xa and by thrombin, and (2) the activation of factor X by the factor IXa/VIIIa complex. Factor VIII activation by thrombin was unaffected in either rate or extent by the presence of PS:PC or activated platelets. In contrast, factor VIII activation by factor Xa required either PS:PC or platelets. The products of optimal factor VIII activation by the two enzymes, designated factor VIIIa(T) and factor VIIIa(Xa), are kinetically different in the activation of factor X by factor IXa, factor VIIIa(T) being approximately twice as active (in factor X activation) as factor VIIIa(Xa) in the presence of PS:PC or platelets. Factor VIIIa(Xa) can be converted to the more active VIIIa(T) by thrombin treatment, but the activity of factor VIIIa(T) is unchanged by factor Xa treatment. Factor X activation was also studied with optimally activated factor VIIIa(T), in the presence of PS:PC or activated platelets, as a function of factor IXa concentration in order to determine the apparent dissociation constant for the factor IXa-VIIIa interaction in the two cases. Activated platelets increased the apparent affinity more than fivefold.

scholarly journals A comparison of phospholipid and platelets in the activation of human factor VIII by thrombin and factor Xa, and in the activation of factor X

Blood ◽  
1988 ◽  
Vol 72 (5) ◽  
pp. 1761-1770 ◽  
Author(s):  
P Neuenschwander ◽  
J Jesty
Keyword(s):  
Factor Viii ◽  
Factor Xa ◽  
Ionophore A23187 ◽  
Factor X ◽  
Apparent Dissociation Constant ◽  
Factor Ixa ◽  
Activated Platelets ◽  
Factor Viiia ◽  
Human Factor Viii ◽  
Factor X Activation

Abstract Two aspects of the activation of factor X by the intrinsic clotting pathway have been studied in purified human systems, in the presence of either purified phosphatidylserine:phosphatidylcholine vesicles (PS:PC) or platelets activated with ionophore A23187: (1) the activation of factor VIII by factor Xa and by thrombin, and (2) the activation of factor X by the factor IXa/VIIIa complex. Factor VIII activation by thrombin was unaffected in either rate or extent by the presence of PS:PC or activated platelets. In contrast, factor VIII activation by factor Xa required either PS:PC or platelets. The products of optimal factor VIII activation by the two enzymes, designated factor VIIIa(T) and factor VIIIa(Xa), are kinetically different in the activation of factor X by factor IXa, factor VIIIa(T) being approximately twice as active (in factor X activation) as factor VIIIa(Xa) in the presence of PS:PC or platelets. Factor VIIIa(Xa) can be converted to the more active VIIIa(T) by thrombin treatment, but the activity of factor VIIIa(T) is unchanged by factor Xa treatment. Factor X activation was also studied with optimally activated factor VIIIa(T), in the presence of PS:PC or activated platelets, as a function of factor IXa concentration in order to determine the apparent dissociation constant for the factor IXa-VIIIa interaction in the two cases. Activated platelets increased the apparent affinity more than fivefold.

scholarly journals The anticoagulant mechanism of action of heparin in contact-activated plasma: inhibition of factor X activation

Blood ◽  
1985 ◽  
Vol 65 (5) ◽  
pp. 1226-1231 ◽  
Author(s):  
TB McNeely ◽  
MJ Griffith
Keyword(s):  
Antithrombin Iii ◽  
Factor Xa ◽  
Coagulation Factors ◽  
Factor Ix ◽  
Factor X ◽  
Clotting Time ◽  
Intrinsic Pathway ◽  
Blood Coagulation Factors ◽  
Factor Ixa ◽  
Factor X Activation

Abstract The effects of heparin on the activation of blood coagulation factors IX and X in contact-activated plasma were determined in the present study. In the presence and absence of 0.5 U/mL heparin, the amounts of factor IX that were cleaved 30 minutes after the addition of calcium and phospholipid to plasma exposed to glass (ie, contact activated) were essentially identical. In the absence of heparin, however, the plasma clotting time was between three and four minutes, while in the presence of heparin, the clotting time was approximately 40 minutes. More factor IXa was inhibited by antithrombin III in the presence of heparin than in its absence, but factor IXa levels sufficient for factor X activation appeared to be present in the heparinized plasma. Neither an increase in factor Xa nor a decrease in factor X was detected, however, in heparinized plasma. We conclude that the step in the intrinsic pathway of coagulation that is inhibited in the presence of heparin is at the level of factor X activation.

scholarly journals Activated clotting factors in factor IX concentrates

Blood ◽  
1979 ◽  
Vol 54 (5) ◽  
pp. 1028-1038 ◽  
Author(s):  
MB Hultin
Keyword(s):  
Antithrombin Iii ◽  
Initial Rate ◽  
Factor Xa ◽  
Factor Ix ◽  
Clinical Settings ◽  
Factor X ◽  
Clotting Factors ◽  
Factor Ixa ◽  
Human Factor Ix ◽  
Factor X Activation

Abstract The precise quantitation of activated factors in human factor IX concentrates has been accomplished with the use of recently developed, specific assays for factors IXa, Xa, and thrombin. The assay for factor IXa, which measures the initial rate of 3H-factor-X activation, was shown to be specific for factor IXa in the concentrates. Activated factor IX concentrates contained 1.0–2.3 microgram/ml of factor IXa; whereas the assays of unactivated concentrates were negative (less than 0.2 microgram/ml). The assays of factor Xa and thrombin, which measure the initial rate of p-nitroaniline release from S-2222 and S-2238, respectively, showed similar small amounts of factor Xa (4–34 ng/ml) and thrombin (12–76 ng/ml) in the activated and unactivated concentrates. The nonactivated partial thromboplastin time of the concentrates correlated significantly with the factor IXa content, but not with factor Xa or thrombin. Antithrombin III antigen in 3 of 4 concentrates was several-fold higher than antithrombin III activity, suggesting the presence of antithrombin III complexed with activated factors. These results support the hypothesis that the degree of activation of factor IX concentrates is related primarily to the concentration of factor IXa, which may be responsible for the thrombogenicity of these concentrates in some clinical settings.

scholarly journals The role of the second growth-factor domain of human factor IXa in binding to platelets and in factor-X activation

1995 ◽  
Vol 310 (2) ◽  
pp. 427-431 ◽  
Author(s):  
S S Ahmad ◽  
R Rawala ◽  
W F Cheung ◽  
D W Stafford ◽  
P N Walsh
Keyword(s):  
Growth Factor ◽  
Human Factor ◽  
Factor Ix ◽  
Factor X ◽  
Binding Studies ◽  
Factor Ixa ◽  
Equilibrium Binding ◽  
Activated Platelets ◽  
Factor Viiia ◽  
Factor X Activation

To study the structural requirements for factor IXa binding to platelets, we have carried out equilibrium binding studies with human factor IXa after replacing the second epidermal growth factor (EGF) domain by the corresponding polypeptide region of factor X. The chimeric protein, factor IX(Xegf2), and the wild-type, factor IXwt, produced in embryonic kidney cells 293 were radiolabelled with 125I and activated with factor XIa. Direct binding studies with thrombin-activated platelets showed normal stoichiometry and affinity of binding of factor IXawt in the presence of factor VIIIa (2 units/ml) and factor X (1.5 microM). However, under similar experimental conditions, factor IXa(Xegf2) was bound to a smaller number of sites (396 sites/platelet) with decreased affinity, i.e. a dissociation constant (Kd) of 1.4 nM, compared with normal factor IXa, factor IXaN (558 sites/platelet; Kd 0.67 nM), or factor IXawt (590 sites/platelet; Kd 0.61 nM). The concentrations of factor IXaN and factor IXawt required for half-maximal rates of factor-X activation were 0.63 nM and 0.7 nM, indicating a close correspondence of the Kd, app. for binding of factor IXawt to the factor-X activating complex on activated platelets to the Kd obtained in equilibrium binding studies. In contrast, kinetic parameters for factor-X activation by factor IXa(Xegf2) showed a decreased affinity (Kd 1.5 nM), in agreement with results of binding studies. These studies with factor IX(Xegf2) suggest that the EGF-2 domain may be important for specific high-affinity factor IXa binding to platelets in the presence of factor VIIIa and factor X.

scholarly journals Regions 301–303 and 333–339 in the catalytic domain of blood coagulation Factor IX are Factor VIII-interactive sites involved in stimulation of enzyme activity

1999 ◽  
Vol 339 (2) ◽  
pp. 217-221 ◽  
Author(s):  
Joost A. KOLKMAN ◽  
Peter J. LENTING ◽  
Koen MERTENS
Keyword(s):  
Catalytic Domain ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Factor X ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Α Helix ◽  
Factor X Activation ◽  
Affinity Interaction ◽  
Stimulation Of

The contribution of the Factor IX catalytic domain to Factor VIIIa binding has been evaluated by functional analysis of Factor IX variants with substitutions in α-helix region 333–339 and region 301–303. These regions were found to play a prominent role in Factor VIIIa-dependent stimulation of Factor X activation, but do not contribute to the high-affinity interaction with Factor VIIIa light chain. We propose that complex assembly between Factor IXa and Factor VIIIa involves multiple interactive sites that are located on different domains of these proteins.

Factor VIII-Factor IX Interactions: Molecular Sites Involved in Enzyme-Cofactor Complex Assembly

1999 ◽  
Vol 82 (08) ◽  
pp. 209-217 ◽  
Author(s):  
Patrick Celie ◽  
Joost Kolkman ◽  
Peter Lenting ◽  
Koen Mertens
Keyword(s):  
Factor Viii ◽  
Tissue Factor ◽  
Factor Viia ◽  
Three Dimensional ◽  
Factor Ix ◽  
Coagulation Cascade ◽  
Factor X ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Factor X Activation

IntroductionThe activation of factor X is one of the steps in the coagulation cascade that is driven by the assembly of an activated serine protease with a membrane-bound cofactor. In the initial phase of coagulation, factor X is activated by the complex of activated factor VII (factor VIIa) and tissue factor. Subsequently, during the so-called propagation phase, factor X activation is catalyzed by the complex of activated factor IX (factor IXa) and activated factor VIII (factor VIIIa). In these complexes, factor VIIa and factor IXa are the factor X-activating enzymes, whereas tissue factor and factor VIIIa serve as non-enzymatic cofactors.1 Factors VIIa and IXa are highly homologous to other cofactor-dependent enzymes, such as activated factor X (factor Xa) and activated protein C, both in amino acid sequence, domain organization, and three-dimensional structure.2 Factor VIIa and IXa further share low or negligible activity towards their natural substrate factor X, unless in complex with their physiological cofactors.Although tissue factor and factor VIIIa serve similar roles as biological amplifiers, they are structurally different. Tissue factor is a small, transmembrane protein with an extracellular part comprising 219 amino acids. Factor VIII is much larger (2,332 amino acids), circulates in plasma, and requires proteolytic processing to exert its biological activity.3 When cofactors are assembled with their respective enzymes, a dramatic increase in enzymatic activity occurs. The underlying molecular mechanism, however, remains poorly understood.During the past few years, remarkable progress has been made in understanding the molecular details of enzyme-cofactor assembly within the coagulation cascade. Crystallography has provided high-resolution structures of tissue factor4 and the various cofactor-dependent coagulation enzymes.2 Moreover, the crystal structure of the factor VIIa—tissue factor complex has been resolved and has allowed the identification of the molecular sites involved in enzyme-cofactor interaction.5,6 Such details are still lacking, however, for the factor IXa—factor VIIIa complex. Current views are derived from three-dimensional models generated by homology modeling based on structurally-related proteins, such as nitrite reductase,7 ceruloplasmin,8 and galactose oxidase.9 Despite their inherent limitations, these models greatly facilitate the interpretation of previous functional studies on factor X activation. As such, the availability of molecular models may be considered an important step toward resolving the structure of the factor IXa—factor VIIIa complex and understanding the role of complex assembly and defects thereof. This chapter provides an overview of the current developments in this field.

Kinetics of Factor X Activation by Factor IXa the Effects of Phospholipid and Factor VIIIa

1979 ◽  
Author(s):  
G.V. Dieyen ◽  
J. Rosing ◽  
G. Tans ◽  
H.C. Hemker
Keyword(s):  
Factor Xa ◽  
Clotting Factor ◽  
Factor X ◽  
Clotting Factors ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Contrast Factor ◽  
Ca Ions ◽  
Factor X Activation ◽  
Kinetics Of

The kinetics of factor X activation by activated clotting factor IXa was measured either in the presence or absence of phospholipid, Ca-ions and factor VIIIa. This study was carried out with purified bovine clotting factors. The rate of factor Xa formation was measured using the chromogenic substrate S 2222. Both the Km for factor X and the Vmax of factor Xa formation were determined for different compositions of the factor X activating complex in order to get more insight in the role of phospholipid, Ca2+ and factor VIII in factor X activation. With factor IXa alone, the activation of factor X is a very inefficient process. The presence of phospholipids predominantly lowers the Km for factor X while in contrast factor VIIIa mainly increases the Vmax of factor Xa formation. The implications of these findings for the mechanism of, factor X activation in the intrinsic pathway will be discussed.

scholarly journals Stabilization of thrombin-activated porcine factor VIII:C by factor IXa phospholipid

Blood ◽  
1984 ◽  
Vol 63 (6) ◽  
pp. 1303-1308 ◽  
Author(s):  
P Lollar ◽  
GJ Knutson ◽  
DN Fass
Keyword(s):  
Sodium Dodecyl ◽  
Factor Xa ◽  
Factor Ix ◽  
Phospholipid Vesicles ◽  
Factor X ◽  
Irreversible Inhibitor ◽  
Factor Ixa ◽  
Major Alteration ◽  
Factor X Activation ◽  
Enzymatic Complex

Abstract The activation of porcine factor X by an enzymatic complex consisting of activated factor IX (factor IXa), thrombin-activated factor VIII:C (factor VIII:Ca), phospholipid vesicles, and calcium was studied in the presence of an irreversible inhibitor of factor Xa, 5-dimethylamino- naphthalene-1-sulfonyl-glutamyl-glycyl-arginyl- chloro met hyl ketone ( DEGR -CK). The formation of factor Xa was measured continuously by monitoring the increase in solution fluorescence intensity that occurs upon formation of DEGR -factor Xa. Omission of any component from the enzymatic complex reduced the reaction rate to a negligible level. In the presence of fixed excess factor IXa, the velocity of factor X activation was linearly dependent on the concentration of factor VIII:C, and thus, provided a plasma-free assay of factor VIII:C. Activation of factor VIII:C by 0.1 NIH U/ml thrombin in the presence of factor IXa, phospholipid vesicles, and calcium, followed at variable time intervals by the addition of factor X and DEGR -CK, was complete within 5 min, as judged by the fluorometric assay, and resulted in little or no loss of factor VIII:C activity over a period of 20 min; whereas, activation in the absence of either IXa or phospholipid vesicles decreased the half-life of factor VIII:C to approximately 5 min. Analysis of 125I-factor VIII:C-derived activation peptides by sodium dodecyl sulfate polyacrylamide gel radioelectrophoresis revealed identical results, regardless of whether factor IXa and/or phospholipid vesicles were included in the activation, suggesting that the lability of factor VIII:Ca is not due to a major alteration of its primary structure. We conclude that the activated porcine factor VIII:C molecule is stabilized markedly because of its interaction with factor IXa and phospholipid.

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