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PubMed1995

Functional consequences of the Ser334-->Pro mutation in a human factor X variant (factor XMarseille).

A factor X molecular variant was identified in a 55-year-old woman at a routine preoperative coagulation screening. Plasma factor X antigen was normal, whereas factor X activity was decreased when factor X was activated by either the extrinsic pathway (21%), the intrinsic pathway (21%) or the factor X activator from Russell viper venom, RVV-X (26%). Factor XMarseille was isolated from plasma by immunoaffinity chromatography and compared with normal factor X purified by the same method. Activation of factor XMarseille by factor IXa or by RVV-X in a purified system showed that the rate of cleavage was decreased, whereas once produced, factor XaMarseille had a normal catalytic efficiency for either the peptide substrate S-2765 (D-Arg-Gly-Arg-NH-Np) or prothrombin. The rate of inhibition of factor XaMarseille by antithrombin III was also normal. Defective proteolysis of factor XMarseille by factor IXa or by RVV-X was the consequence of a threefold decrease in the kcat for the activation of factor XMarseille while the Km of RVV-X or factor IXa for factor X was normal. We have determined the molecular basis of the defect in the factor XMarseille gene by amplification of all eight exons, single-strand conformational polymorphism analysis of the amplified exons and subsequent sequence analysis. The patient was homozygous for a T-->C mutation in exon VIII, resulting in the substitution of Ser334 by proline. From comparison of three-dimensional models of various serine proteases, it appears that Ser334 is located within a surface-exposed variable region of factor X. This observation suggests that the Ser334-->Pro mutation either is responsible for a misalignment of the active sites of specific factor X activators in close proximity to the cleavage site, or that the Ser-->Pro mutation alters the spatial orientation of the cleavage site by nonlocal modifications of factor X structure.

Animals↗
PubMed1992

Diffusion control in blood coagulation. Activation of factor X by factors IXa/VIIIa assembled on human monocyte membranes.

This study examines mechanisms that regulate the activation of blood coagulation proteases on intact cell membranes. The activation of factor X by factors IXa and VIIIa assembled on viable monocytes is presented as a biologically relevant model for membrane-dependent proteolysis of coagulation zymogens. The hypothesis that this reaction is limited by diffusion was tested by comparing predicted with observed concentration dependence, temperature dependence, and effective rate coefficient. Rates of factor X catalysis were measured using a chromogenic substrate specific for the product, factor Xa. The value of KR and of K1/2, i.e. concentrations giving half-maximal rates in reciprocal functional titrations with substrate and enzyme, respectively, were directly correlated with the concentration of the titrated component. Arrhenius plots constructed over temperatures encompassing 10-35 degrees C were biphasic with downward concavity. Apparent activation energies were 6.01 +/- 0.93 and 35.84 +/- 8.9 kcal/mol for the interval above and below the inflection point, respectively. The effective rate coefficient calculated from apparent kinetic parameters was 3.58 +/- 0.1 x 10(12) M-1 s-1. This rate is similar to the maximal rate of collision between factor X molecules and the monocyte, i.e. 2.9 x 10(12) M-1 s-1 estimated from the steady-state von Smoluchowski equation for uniformly reacting spherical particles. The observed agreement between predicted and experimental results indicates that under biologically relevant conditions, the rate of factor X activation by the intrinsic protease is controlled by diffusion of factor X toward the catalytic site.

Blood Coagulation↗
PubMed1986

Preparation and properties of derivatives of bovine factor X and factor Xa from which the gamma-carboxyglutamic acid containing domain has been removed.

Limited proteolysis of bovine blood coagulation Factor X by chymotrypsin produces a derivative in which the light chain is cleaved between Tyr 44 and Lys 45. Two peptide products, residues 1-44 of the Factor X light chain and a modified zymogen, Factor X(-GD) have been isolated and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, elution behavior on anion-exchange chromatography, amino acid composition, and by partial amino acid sequence determination. Factor X(-GD) no longer contains the 12 gamma-carboxyglutamic acid residues of the native zymogen and thus serves as a model for investigation of the properties conferred on Factor X by the presence of gamma-carboxyglutamic acid. Cleavage of Factor X at Tyr 44 by chymotrypsin is inhibited by Ca2+ and Mg2+ ions. Factor X(-GD) is activated by the coagulation factor activator of Vipera russellii venom, but at less than 1% of the rate of activation of native Factor X. The susceptibility of Tyr 44 to chymotryptic cleavage implies that this residue is on the surface of the light chain of Factor X. Factor Xa(-GD) is indistinguishable from native Factor Xa in its activity on Benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide, on prothrombin alone, and on prothrombin plus Factor Va. In the presence of phospholipid the rate of prothrombin activation catalyzed by Factor Xa(-GD) is the same as in the absence of phospholipid.

1-Carboxyglutamic Acid↗
PubMed1988

Inhibition of prothrombin activation by factor X and factor IX Gla-peptides.

In the present study, human factor X and factor IX were each digested with chymotrypsin, and the Gla-peptide from each protein was purified by QAE-Sephadex chromatography. The effect of each Gla-peptide on the activation of human prothrombin by a complex of factor Xa, phospholipid, and calcium was studied using an amidolytic assay for generated thrombin. Prothrombin activation was half-maximally inhibited by factor X Gla-peptide at a concentration of 0.7 microM. Factor IX Gla-peptide was markedly less inhibitory and inhibited this reaction half-maximally at a concentration of 3.7 microM. Kinetic analyses revealed that the factor X Gla-peptide inhibited this reaction in an apparent competitive manner, whereas the factor IX Gla-peptide yielded an exponential Dixon plot. Heat decarboxylation experiments revealed that 3-4 gamma-carboxyglutamic acid residues are critical for the expression of inhibitory activity in each peptide. These studies indicate that, in spite of their structural homology, the ability of each of these Gla-peptides to act as a prothrombinase inhibitor is markedly different.

Factor IX↗
PubMed1980

Mechanized amidolytic technique for determination of factor X and factor-X antigen, and its application to patients being treated with oral anticoagulants.

We describe a mechanized chromogenic assay for factor X, the results of which correlate well with those for the one-stage clotting assays for factor X in which it is activated either via the extrinsic pathway by thromboplastin or directly by Russell's viper venom. We purified human factor X and raised monospecific antibodies to it in rabbits. We used our chromogenic assay for factor X to develop a factor-X-inhibitor neutralization assay for determination of factor-X antigen. Patients receiving oral anticoagulant treatment had significantly different factor-X activities after activation via thromboplastin or with Russell's viper venom. The concentration of factor-X antigen, although decreased, significantly exceeded factor-X clotting activity or chromogenic activity in this group of patients. Results of the chromogenic assay for factor X correlated well with results of "Thrombotest," a clotting test introduced by Owren (Lancet ii: 754, 1959) to control anticoagulant therapy. For patients taking oral anticoagulant drugs, the therapeutic range by our assay is 180 to 300 units/L.

Anticoagulants↗
PubMed1977

A comparison of human prothrombin, factor IX (Christmas factor), factor X (Stuart factor), and protein S.

Human prothrombin, factor IX, and factor X have been idolated in high yield and characterized as the their amino-terminal sequence, molecular weight, amino acid composition, and migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An additional human plasma protein, called protein S, has also been purified and its properties have been compared with those of prothrombin, factor IX, and factor X. Prothrombin (mol wt 72 000), factor IX (mol wt 57 000), and protein S (mol wt 69 000) are single-chain glycoproteins, while factor X (mol wt 59 000) is a glycoprotein composed of two polypeptide chains held together by a disulfide bond(s). The amino-terminal sequence of the light chain of human factor X is homologous with prothrombin, factor IX, and protein S. The heavy chain of human factor X is slightly larger than the heavy chain of bovine factor X and differs from bovine factor X in its amino-terminal sequence.

Amino Acid Sequence↗
PubMed1990

Inhibition of factor X and factor V activation by dermatan sulfate and a pentasaccharide with high affinity for antithrombin III in human plasma.

There is evidence that by catalyzing thrombin inhibition, several glycosaminoglycans can inhibit the thrombin-mediated amplification reactions of coagulation and thereby delay prothrombin activation. The two amplification reactions can apparently be catalysed by endogenously generated factor Xa and thrombin. This study provides evidence which suggests that on a molar basis, an agent which can only catalyse thrombin inhibition is approximately 10 times more effective than an agent which can only catalyse factor Xa inhibition in their ability to inhibit intrinsic prothrombin activation. We determined the concentrations of each of heparin, dermatan sulfate and a pentasaccharide with high affinity for antithrombin III, to delay intrinsic prothrombin activation for at least 15s. Heparin catalyses both thrombin and factor Xa inhibition; dermatan sulfate catalyses only thrombin inhibition, while the pentasaccharide only catalyses factor Xa inhibition. Efficient prothrombin activation, which coincided with both factor X activation and factor V proteolysis, was first observed 45s after CaC12 was added to contact-activated plasma. Heparin (approximately 0.1 microM) prolonged by at least 30 s the time required for the activation of the three clotting factors to begin. The minimum concentrations of the pentasaccharide and dermatan sulfate to delay the activation of prothrombin, factors X and V were approximately 50 microM and approximately 5 microM, respectively. Thus, each anticoagulant could inhibit intrinsic prothrombin activation only when it inhibited activation of both factors X and V. A combination of approximately 5 microM pentasaccharide and approximately 0.05 microM dermatan sulfate similarly delayed the activation of all three clotting factors. Thus, while catalysis of thrombin inhibition is a more effective pathway than catalysis of factor Xa inhibition for delaying prothrombin activation, the simultaneous catalysis of thrombin and factor Xa inhibition can synergistically improve the ability of a sulfated polysaccharide to delay prothrombin activation.

Animals↗
PubMedDate not supplied

Coagulation protein function. IV. Effect of acetaldehyde upon factor X and factor Xa, the proteins at the gateway to the common coagulation pathway.

Acetaldehyde (AcH) (447 mM) exerts an inhibition on Factor Xa, as followed by a clotting assay, but does not inhibit the hydrolysis of the synthetic fluorogenic substrate, N-tBOC-Ile-Glu-Gly-Arg-7-amido-4-methylcoumarin. These data suggest that AcH, although not reacting at the catalytic site of Factor Xa nor at the binding site for the synthetic substrate, does interact with the functional groups on the enzyme that bind to its natural substrate, prothrombin. As a consequence of such interaction, the charge and conformation of Factor Xa is altered, thereby limiting effective activation of prothrombin. Additionally, alkylation of factor Xa may also affect its capacity to associate with Factor Va for the activation of prothrombin. AcH also reacts with Factor X, prolonging clotting times when the zymogen is activated with Russell's viper venom (RVV). It also reduces the rate of hydrolysis of the fluorogenic substrate after activation of the alkylated zymogen by RVV. These data lead to the considerations that AcH-modified Factor X is no longer as effectively activated by RVV due to an alteration of its charge/conformation. Additional possibilities include a likely alkylation of the Factor Xa moiety of Factor X by AcH such that the activation product has an altered charge/conformation compared to native Factor Xa, including possible alkylation of its binding site(s) for prothrombin. The reduced rate of hydrolysis of the synthetic fluorogenic substrate for Factor Xa by the alkylated, activated Factor X lends further support to the generation of a modified Factor Xa by RVV, which may have a lower binding or catalytic rate for the fluorogenic substrate. These results support the suggestion that chronic consumption of alcohol may prolong the reported coagulation times as a result of reaction of alcohol's primary metabolite, AcH, with clotting factors, thereby reducing their physiological potential.

Acetaldehyde↗
PubMed2002

Phospholipid-binding domain of factor VIII is involved in endothelial cell-mediated activation of factor X by factor IXa.

Apparently quiescent, nonapoptotic endothelial cells mediate the activation of factor X by activated factor IX in the presence of its cofactor, activated factor VIII. In a previous study, we reported that during the activation of factor X, the interaction of the cofactor with the endothelial cell membrane clearly differs from the interaction of the cofactor with artificial lipid membranes. In the present study, we identified the peptide domain of factor VIII involved in the assembly of the enzyme-cofactor complex on the endothelial cell surface. With the use of monoclonal antibodies against different peptide sequences on the factor VIII light chain, it was observed that the lipid-binding region of the C2 domain on the factor VIII light chain mediates the assembly of the factor X-activating complex on the endothelial cell surface. In addition, a synthetic peptide that constitutes region Ala2318-Tyr2332 of the C2 domain and that is known for its ability to inhibit the binding of factor VIII to artificial lipid membranes also showed inhibition of the cofactor activity of factor VIII on endothelial cells. Thus, the carboxy-terminal part of the factor VIII light chain not only contains sites involved in lipid binding but also contains sites involved in complex assembly on the endothelial cell membrane.

Annexin A5↗
PubMed1979

Acquired, transient factor X (Stuart factor) deficiency in patient with mycoplasma pneumonial infection.

A case of severe haemorrhagic diathesis due to acquired deficiency of factor X (both immunologically and in procoagulant activity) is presented. The clinical and serological features of this case indicated mycoplasma pneumonial infection. Factor X in the peripheral blood did not appear to be influenced by administration of vitamin K, prothrombin-complex concentrate, fresh plasma or fresh whole blood. Circulating inhibitors of blood coagulation were absent and systemic amyloidosis could not be demonstrated. After 20 d, factor X spontaneously returned to normal. In view of the absence of other known causes of factor X deficiency, a possible relationship with mycoplasma pneumonial infection is suggested.

Blood Transfusion↗
PubMed1994

The role of human factor X activation peptide in activation of factor X by factor IXa.

We studied the interaction of factor X activation peptide (XAP) with factor IXa and factor Xa and the effect of XAP on factor IXa-catalyzed activation of factor X. XAP associated with factor Xa in the presence of 5 mM Ca2+ was dissociated from factor Xa by gel chromatography using Ultrogel AcA54 in 5 mM EDTA, or in 8 M urea-0.1% SDS. An exogenous isolated XAP inhibited the factor IXa-catalyzed factor X activation both in the presence and absence of factor VIIIa. 4-Amidinophenylmethylsulfonyl (aPMS)-factor Xa independent of XAP also inhibited the factor X activation more effectively than XAP alone in the presence of factor VIIIa. However, aPMS-factor Xa independent of XAP hardly inhibited the factor X activation in the absence of factor VIIIa. The binding of 125I-labeled factor X to the aPMS-factor IXa fixed to a microwell plate was inhibited by unlabeled factor X or XAP, but not by aPMS-factor Xa with or without XAP. Factor IXa directly bound to XAP and aPMS-factor Xa with XAP, but did not bind to aPMS-factor Xa without XAP. These findings suggest that the region of XAP in factor X directly interacts with factor IXa, and factor Xa region other than XAP interacts with factor VIIIa. Desialation or deletion of N-linked carbohydrates of XAP reduced the inhibitory activity of XAP for the factor X activation by factor IXa to approximately 50% of that of the intact XAP. This suggests that the sialic acids in the carbohydrate chains of the XAP region partly contribute to the interaction with factor IXa during its activation.

Amino Acid Sequence↗
PubMed1978

Activation of bovine factor X (Stuart factor)--analogy with pancreatic zymogen-enzyme systems.

The activation of bovine coagulation factor X has been studied by kinetic and spectrophotometric measurements. The pH dependence of the hydrolysis of specific ester substrates by activated factor Xa can be ascribed to two independently ionizing groups with pKa values of 6.9 and 8.8, respectively. The rates of reaction of factor X, before and after activation, with the active-site titrant methanesulfonyl fluoride, suggest that the reactivity of the active-site serine residue in factor X is similar to that in trypsinogen and in factor Xa similar to that in trypsin. Analogous comparisons using diisopropyl phosphofluoridate as the titrant suggest that a hydrophobic binding site is absent in both the enzyme and zymogen. This conclusion is consistent with the lack of change in circular dichroism when acyl derivatives of factor V are converted to their acyl enzyme counterparts.

Animals↗
PubMed1992

Tissue factor residues 157-167 are required for efficient proteolytic activation of factor X and factor VII.

The cell surface receptor tissue factor (TF) initiates coagulation by supporting the proteolytic activation of factors X and IX as well as VII to active serine proteases. Architectural similarity of TF to the cytokine receptor family suggests a strand-loop-strand structure for TF residues 151-174. Site-directed Ala exchanges in the predicted surface loop demonstrated that residues Tyr157, Lys159, Ser163, Gly164, Lys165, and Lys166 are important for function. Addition of side chain atoms at the Ser162 position decreased function, whereas the Ala exchange was tolerated. The dysfunctional mutants bound VII with high affinity and fully supported the catalysis of small peptidyl substrates by the mutant TF.VIIa complex. Lys159-->Ala substitution was compatible with efficient activation of factor X, whereas the Try157-->Ala exchange and mutations in the carboxyl aspect of the predicted loop resulted in diminished activation of factor X. The specific plasma procoagulant activity of all functionally deficient mutants increased 7- to 200-fold upon the supplementation of VIIa suggesting that TF residues 157-167 also provide important interactions that accelerate the activation of VII to VIIa. These data are consistent with assignment of the TF 157-167 region as contributing to protein substrate recognition and cleavage by the TF.VIIa complex.

Amino Acid Sequence↗
PubMed1975

Activation of bovine factor X (Stuart factor): conversion of factor Xaalpha to factor Xabeta.

Bovine factor X (molecular weight 55,100) is a blood coagulation factor present in plasma in a precursor or zymogen form. It is a glycoprotein which has been isolated as a two-chain structure held together by one or more disulfide bonds. During the coagulation process, factor X is converted to a serine protease by the hydrolysis of a specific peptide bond in the amino-terminal region of the heavy chain. This cleavage occurs between Arg-51 and Ile-52, giving rise to factor Xaalpha (molecular weight 45,300) and an activation peptide (molecular weight 9500). Factor Xaalpha is then converted to factor Xabeta (molecular weight 42,600) by hydrolysis of a second specific peptide bond in the carboxyl-terminal region of the heavy chain. This cleavage occurs between Arg-290 and Gly-291, giving rise to a second glycopeptide (molecular weight 2700). Factor Xaalpha and factor Xabeta have equivalent coagulant activity, indicating that the cleavage of the second peptide bond is unrelated to the activation process.

Amino Acid Sequence↗
PubMed1984

Kinetic studies of the activation of factor X by factors IXa and VIII:C in the absence of thrombin.

The activation of factor X by the components of the intrinsic coagulation pathway has been studied using a preparation of bovine factor VIII which did not require 'activation' with thrombin prior to its participation in the reaction. Measurements of the kinetic parameters indicated that factor VIII:C stimulated the formation of factor Xa via a 170-fold increase in the Vmax; factor VIII:C had little effect on the Km for factor X.

Animals↗
PubMed2002

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.

The assembly of the factor X activating complex on the platelet surface requires the occupancy of three receptors: (1) enzyme factor IXa, (2) cofactor factor VIII(a), and (3) substrate factor X. To further evaluate this three-receptor model, simultaneous binding isotherms of (125)I-factor X and (131)I-factor VIII(a) to activated platelets were determined as a function of time and also as a function of the concentrations of both ligands in the presence of active site-inhibited factor IXa (45 nM) and 5 mM CaCl(2). In the presence of active site-inhibited factor IXa and factor VIIIa there are two independent factor X binding sites: (1) low affinity, high capacity (approximately 9000 sites/platelet; K(d) approximately 380 nM) and (2) low capacity, high affinity (1700 sites/platelet; K(d) approximately 30 nM). A single specific and selective factor X binding site was expressed (1200 sites/platelet; K(d) approximately 9 nM) when the shared factor X/factor II site was blocked by excess factor II (4 microM). In the presence of active site-inhibited factor IXa (4 nM) and factor II (4 microM), factor X binds to 3-fold more platelet sites than procofactor VIII with relatively low affinity (K(d) approximately 250 nM). The activation of procofactor VIII to factor VIIIa increases the affinity of binding to platelets of both factor VIIIa ( approximately 4-fold to K(d) approximately 0.8-1.5 nM) and factor X ( approximately 25-50-fold to K(d) approximately 5-9 nM). In the presence of excess zymogen factor IX, which blocks the shared factor IX/factor IXa binding site, the substrate, factor X, and the active cofactor, factor VIIIa, form a 1:1 stoichiometric complex. These coordinate binding studies support the conclusion that factor X initially binds to a high-capacity, low-affinity platelet binding site shared with prothrombin, which then presents factor X to a specific high-affinity site consisting of factor VIIIa bound to a high-affinity, low-capacity receptor on activated platelets.

Binding Sites↗
PubMed1991

Interaction of feedback control and product inhibition in the activation of factor X by factors IXa and VIII.

A simple numerical model of the activation of factor X by factors IXa and VIII has been constructed in order to identify and examine the major controls that operate in a nonflowing system in the presence of (1) inhibitors of factor Xa and (2) feedback activation of factor VIII by factor Xa. The model confirms, and allows parameter estimation for, (1) the control of factor Xa yield by factor VIIIa decay; (2) the control of generation-curve area by the rate of factor Xa inhibition; and (3) the reduction in the factor VIIIa decay rate in the presence of factor IXa. Beyond confirmation of existing data, the model also predicts that below a definite, but very low, threshold level of factor IXa (less than or equal to 10 pM), minimal feedback activation of factor VIII will occur. The concentration of factor IXa at which the threshold is observed in simulations is dependent on the rate of inhibition of factor Xa.

Enzyme Activation↗
PubMed2001

Binding of plasminogen and tissue plasminogen activator to plasmin-modulated factor X and factor Xa.

Previous work in our laboratory has suggested that the fibrinolytic enzyme plasmin (Pn) inactivates coagulation factors X (FX) and Xa (FXa) in the presence of Ca(2+) and anionic phospholipid (aPL), producing fragments which bind plasminogen (Pg) and accelerate tissue plasminogen activator (t-PA). Our goals here were to determine if the Pn-mediated fragments of FX or FXa remain associated, whether they directly bind t-PA, and to quantify their interaction with Pg. Binding to aPL, benzamidine-Sepharose, or the active-site inhibitor dansyl-Glu-Gly-Arg-chloromethyl ketone demonstrated that Pn cleavage yielded noncovalent heterodimers of a fragment containing the aPL-binding domain (FXgamma(47) or FXagamma(33)) and a 13-kDa fragment (FXgamma(13) or FXagamma(13)). Both ligand blotting and surface plasmon resonance (SPR) showed that Pn-cleaved FX and FXa bound t-PA directly when Pn-treatment was effected in the presence of aPL and Ca(2+). Using SPR, apparent K(d) values of 1-3 microM and 0.3-0.4 microM were measured directly and by competition for the FXgamma(47/13)-Pg and FXagamma(33/13)-Pg interactions, respectively. For the first time, Pg-binding to a receptor was shown to be Ca(2+) enhanced, although primarily mediated by C-terminal lysine residues. Mathematical modeling of kinetic data suggesting two Pg per FXgamma(47/13) or FXagamma(33/13) was consistent with our conclusion that each subunit of FXgamma(47/13) or FXagamma(33/13) contains a C-terminal lysine. Earlier X-ray structures show that these Lys residues are distal from each other and the membrane, supporting the model where each interacts with a separate Pg. t-PA acceleration by FXgamma(47/13) or FXagamma(33/13) may therefore involve simultaneous presentation of two substrate molecules.

Anions↗