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PubMed1995

Inhibition of leukocyte-endothelial cell interactions and inflammation by peptides from a bacterial adhesin which mimic coagulation factor X.

Factor X (factor ten) of the coagulation cascade binds to the integrin CD11b/CD18 during inflammation, initiating procoagulant activity on the surface of leukocytes (Altieri, D.C., O.R. Etingin, D.S. Fair, T.K. Brunk, J.E. Geltosky, D.P. Hajjar, and T. S. Edgington. 1991. Science [Wash.DC]. 254:1200-1202). Filamentous hemagglutinin (FHA), an adhesin of Bordetella pertussis also binds to the CD11b/CD18 integrin (Relman D., E. Tuomanen, S. Falkow, D.T. Golenbock, K. Saukkonen, and S.D. Wright. 1990. Cell. 61:1375-1382). FHA and the CD11b/CD18 binding loops of Factor X share amino acid sequence similarity. FHA peptides similar to Factor X binding loops inhibited 125I-Factor X binding to human neutrophils and prolonged clotting time. In addition, ETKEVDG and its Factor X analogue prevented transendothelial migration of leukocytes in vitro and reduced leukocytosis and blood brain barrier disruption in vivo. Interference with leukocyte migration by a coagulation-based peptide suggests a novel strategy for antiinflammatory therapy.

Adhesins, Bacterial↗
PubMed1976

Platelet aggregation and adhesiveness in classical factor X deficiency and in the abnormal factor X (factor X Friuli) coagulation disorder.

Platelet aggregation to common inductors and to Ristocetin, Thrombofax and Ionophore is normal in congenital factor X deficiency and in factor X Friuli coagulation disorders. Washed normal platelets resuspended in the patient's plasma and in adsorbed normal plasma showed a normal aggregation. On the contrary, normal platelets resuspended in normal serum failed to aggregate. These studies indicate that factor X plays no role in normal platelet aggregation.

Adenosine Diphosphate↗
PubMed2003

A novel type I factor X variant (factor X Cys350Phe) due to loss of a disulfide bond in the catalytic domain.

We report a novel mutation within the coagulation factor X (FX) that we have designated FX Padua 4. The phenotype and genotype of the proband and family members were studied. The proband was a child affected by a complex neurological syndrome who, after birth, experienced severe bleeding. The proband showed a laboratory pattern characterized by a severe reduction of FX activity and FX antigen, suggesting a true deficiency. Molecular analysis disclosed a new FX mutation localized in the catalytic domain responsible for a Cys350Phe substitution. The proband was homozygous for this mutation. The proband's mother and father showed a heterozygous pattern and had approximately one-half the normal FX activity and FX antigen. Residual purified FX Cys350Phe had an identical behavior to normal FX as showed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Molecular modeling confirms that the mutation leads to the disruption of a disulfide bridge in the catalytic region of FX. Comparison with other topologically equivalent mutations in other vitamin K-dependent proteins suggests that this disruption could adversely affect protein folding/stability, accounting for the cross-reactive material negative phenotype.

Adult↗
PubMed2001

A new factor X defect (factor X Padua 3): a compound heterozygous between true deficiency (Gly(380)-->Arg) and an abnormality (Ser(334)-->Pro).

We report a novel mutation in Factor X (FX) gene which results in a phenotype without any bleeding tendency. The proband has been found to be a compound heterozygote between a novel FX true deficiency (Gly(380)-->Arg) and a previously reported dysfunctional mutation Ser(334)-->Pro (FX Marsiglia). Prothrombin time (PT) and partial thromboplastin time (PTT) were moderately prolonged and were fully corrected by the addition of normal serum. Her FX activity level varied between 8% and 19% of normal according to the method used whereas the FX antigen level was 40% of the normal control value. All the exons and intron/exon junctions of the FX gene were studied using a combined approach of polymerase chain reaction and conformation sensitive gel electrophoresis. A transversion G to A in exon 8 resulting in the replacement of Gly380 by Arg was found in the proband, in the father and in a proband's brother, whereas heterozygous FX Marsiglia was present in the proband's mother and her sister. Gly380 is strictly linked to Ser379, a component of the catalytic triad. The substitution of Gly for Arg causes the introduction of a large charged amino acid which could affect the catalytic function of FX leading to secretion problem, accounting for the cross-reactive material (CRM) negative phenotype.

Adult↗
PubMed1994

Baculovirus-mediated expression of the epidermal growth factor-like modules of human factor IX fused to the factor XIIIa transamidation site in fibronectin. Evidence for a direct interaction between the NH2-terminal epidermal growth factor-like module of factor IXa beta and factor X.

Factor IX is a vitamin K-dependent procoagulant zymogen of a serine protease. In the presence of Ca2+ the active form of factor IX (factor IXa beta) forms a complex with factor VIIIa on suitable phospholipid surfaces such as aggregated platelets. This macromolecular complex rapidly activates factor X. We have previously provided data that suggest an interaction between the NH2-terminal epidermal growth factor (EGF)-like module of factor IXa beta and the substrate factor X. In an alternative approach to study this protein-protein interaction, we have expressed three recombinant baculovirus constructs encoding the EGF-like modules of human factor IX and a truncated form of fibronectin in a system based on the infection of insect cells (Spodoptera frugiperda 21). This strategy allows a simple one-step purification of the recombinant proteins on a gelatin-Sepharose column, followed by removal of the gelatin-binding part derived from fibronectin by proteolytic cleavage. The fusion proteins were isolated at yields of 20-50 micrograms/ml culture medium. The recombinant EGF-like modules contained 0.2-0.4 mol of erythro-beta-hydroxyaspartic acid/mol of protein, i.e. similar to the amount found in factor IX from human plasma, and appeared to be glycosylated at Ser-53. The NH2-terminal EGF-like module, which contained a transamidation acceptor site derived from fibronectin, was cross-linked by factor XIIIa in solution to intact and Gla-domainless factor X. There was no evidence of cross-linking to activated factor X or to factor X fragments containing only the gamma-carboxyglutamic acid module and the two EGF-like modules. The cross-linking results suggest a specific interaction between the NH2-terminal EGF-like module of factor IXa beta and the heavy chain of unactivated factor X. This interaction, albeit weak as judged by competition experiments, may be important for the targeting of factor X to the factor IXa beta-factor VIIIa complex on biological membranes and for the subsequent dissociation of factor Xa from the complex after activation.

Amino Acid Sequence↗
PubMed1982

The acceleration by polylysine of the activation of factor X by factor IXa.

The present study reports that polylysine can function as a cofactor in the conversion of factor X to factor Xa by factor IXa. In the presence of polylysine, factor X is converted to factor Xa by factor IXa as demonstrated by both clotting and amidolytic assays. The activation of factor X by factor IXa requires the prior activation of factor IX to IXa by factor XIa. Conversion of factor X to factor Xa by factor IXa is not observed in the absence of polylysine. The activation reaction proceeds optimally at pH 8.0 with an equal weight ratio of polylysine to factor X. The effect of polylysine is readily reversed by low concentrations of NaCl or elevated temperature suggesting that electrostatic interactions are of primary importance in the polylysine facilitation of the activation of factor X by factor IXa.

Animals↗
PubMed1993

Carbohydrate residues modulate the activation of coagulation factor X.

Factor X is a plasma protein involved in both the intrinsic and extrinsic pathways of blood coagulation. Post-translational modifications of the protein involve gamma-carboxylation of specific glutamic acid residues, beta-hydroxylation of one aspartic acid residue, and N- and O-linked glycosylation. Even though it is known that gamma-carboxylation is instrumental in regulating biological activity, the role of glycosylation in the function and properties of factor X has not been previously investigated. We utilized lectin binding and glycosidase treatment to investigate the functional role of carbohydrates on the activation peptide of factor X. Sambucus nigra agglutinin, a lectin that binds to sialic acid terminally linked alpha(2-6) to galactose or N-acetyl-galactosamine inhibits activation of human factor X in a dose-dependent manner. Inhibition of activation was observed for both intrinsic (factor IXa/VIIIa) and extrinsic (factor VIIa/tissue factor) pathway complexes. In accordance with this, selective removal of sialic acid residues on the activation peptide of factor X by neuraminidase also results in a drastic reduction of activation of the zymogen by these complexes. Corresponding reduction of activity in classical clotting assays (activated partial thromboplastin time and prothrombin time) also agrees with this observation. These results suggest a possible role of N-linked carbohydrates in the activation of factor X.

Animals↗
PubMed1989

Molecular recognition in the activation of human blood coagulation factor X.

Factor X can be activated by the extrinsic activation complex (factor VIIa:tissue factor), the intrinsic activation complex (factor IXa:factor VIIIa) and by an enzyme from Russell's viper venom (RVV-X). To identify the regions on the surface of factor X that mediate its association with these three activators, we have prepared 21 synthetic peptides representing 65% of the primary structure of factor X. Only 3 of the 21 peptides inhibited the rate of factor X activation, indicating the regions represented by these three peptides are involved in factor X association. Using purified components, the rate of factor Xa formation was inhibited in a dose-dependent manner by these three peptides with the same relative potency of inhibition in each of the activation systems. The observed relative potencies were: peptide 267-283 greater than or equal to peptide 284-303 greater than peptide 417-431. Kinetic analyses indicated that the three peptides inhibited factor X activation in a non-competitive manner, and in mixed inhibitor assays the peptides were shown to be mutually exclusive of one another. In coagulation-based assays, the potency of inhibition by each peptide was decreased. However, in Russell's viper venom-X-initiated assays peptide 417-431 was the best inhibitor. Fab fragments of antibodies raised to these peptides and affinity purified on factor X-agarose columns inhibited both the purified and coagulation-based assays in a dose-dependent manner. Using the x-ray crystal structure of chymotrypsinogen as a model, these three peptides were found to be located spatially close to one another on the surface of factor X and opposite to the region where factor X is cleaved for activation. These data are consistent with a model in which the three activators combine with factor X through a recognition site composed of multiple loci that is distal to the potential cleavage site. This interaction aligns the active sites of these three enzymes in the correct orientation to cleave factor X at the same arginyl-isoleucyl bond.

Amino Acid Sequence↗
PubMed1994

Biochemistry of factor X.

Factor X circulates as a serine protease which is converted to the active form at the point of convergence of the intrinsic and extrinsic coagulation pathways. Subsequently, the enzymatic species, factor Xa, is involved in macromolecular complex formation with its cofactor factor Va, a phospholipid surface and calcium to convert prothrombin into thrombin. The gene encoding factor X shares a number of structural and organisational features in common with the other vitamin K-dependent coagulation proteins, suggesting that they have evolved from a common ancestral gene. Each of the exons encoding these proteins can be considered as a module coding for a homologous domain in each protein. These structural domains in factor X are responsible for specific functional properties including gamma-carboxylase recognition, calcium binding, phospholipid surface interaction, as well as cofactor and substrate binding. Studies of recombinant proteins and proteolytic fragments continue to provide significant insight into structure-function relationships of the protein modules within factor X.

Animals↗
PubMed1988

Separation of human factor X from factor Xa by reversed-phase high-performance liquid chromatography.

Human factor X is the vitamin K-dependent proenzyme of a plasma serine protease that participates in the cascade of events leading to blood coagulation. It is converted to its active form, factor Xa, after specific cleavage by other plasma proteases or the protease from Russel's Viper venom. We have separated Factor X from factor Xa by reversed-phase high-performance liquid chromatography using an increasing gradient of acetonitrile in 0.1% trifluoroacetic acid. The factor X and factor Xa activities were well separated from each other on a wide-pore diphenyl column (Whatman Protesil 300) in less than 30 min. Both factor X and factor Xa activities were found to be essentially unaffected by the solvent system. This system was used to evaluate the purity of several factor X and factor Xa preparations. The kinetics of the Russel's Viper venom catalyzed conversion of factor X to factor Xa was also studied by using this chromatography system. A time-dependent decrease in the protein peak corresponding to factor X and a corresponding increase in the factor Xa protein peak was observed upon incubation with Russel's Viper venom.

Chromatography, DEAE-Cellulose↗
PubMed2004

Mechanisms of interactions of factor X and factor Xa with the acidic region in the factor VIII A1 domain.

The 337-372 sequence of the factor VIIIa A1 subunit contains interactive sites for both zymogen factor X and the active enzyme, factor Xa. Solid phase binding studies indicated that factor Xa possessed a >20-fold higher affinity for the isolated A1 subunit of factor VIIIa compared with factor X. Heparin completely inhibited zero-length cross-linking of the 337-372 peptide to factor Xa but not to factor X. In the presence of calcium, factor Xa showed greater affinity for heparin than factor X. Studies using factor Xa mutants in which heparin-binding exosite residues were individually replaced by Ala showed that the R240A mutant was defective in recognition of the Lys36 cleavage site, generating the A137-372 intermediate with approximately 20% the catalytic efficiency of wild type. This defect likely resulted from an approximately 4-fold increase in Km for the A1 substrate because kcat values for the wild type and mutant were equivalent. Cleavage of the A1-A2 domain junction by factor Xa R240A was not blocked by the 337-372 peptide. Studies using mutant factor VIII where clustered acidic residues in the 337-372 segment were replaced by Ala showed that a factor VIIIa D361A/D362A/D363A mutant possessed a approximately 1.6-fold increase in Km for factor X compared with wild type. However, similar Km values were observed for recombinant factor X and R240A substrates. These results indicate that the binding regions of factor X and factor Xa for A1 domain overlap and that both utilize acidic residues 361-363. Furthermore, factor Xa but not factor X interacts with high affinity at this site via residues contained within the heparin-binding exosite of the proteinase.

Alanine↗
PubMed1974

A comparison of bovine prothrombin, factor IX (Christmas factor), and factor X (Stuart factor).

A comparison has been made of the electrophoretic behavior, chemical composition, amino-terminal sequence, and immunological properties of bovine prothrombin, factor IX (Christmas factor), and factor X (Stuart factor). Some immunological crossreactivity was found between the antibody to prothrombin and factor X although prothrombin and factor X differ substantially in amino-acid and carbohydrate composition. Considerable amino-acid sequence homology was found in the amino-terminal portion of prothrombin, factor IX, and the light chain of factor X. These data provide further evidence to support the hypothesis that at least three of the vitamin K-dependent clotting factors have evolved from a common ancestral gene.

Amino Acid Sequence↗
PubMed1978

Activation of factor X by factors IXa and VIII; a specific assay for factor IXa in the presence of thrombin-activated factor VIII.

We studied the activation of factor X by the intrinsic pathway of blood coagulation using a new assay of factor X activation. When factor X tritiated in its sialic acid residues is activated, activation can be measured by the release of tritiated activation peptide, and the initial rate of activation can be determined under varying conditions. In the presence of phospholipid and calcium ions, factor IXa activated factor X slowly without factor VIII, and this activation was blocked by a specific factor IX inhibitor. These data provide strong evidence that factor IXa is the enzyme responsible for factor X activation by the intrinsic pathway. The role of factor VIII was also investigated. Factor VIII could be reproducibly thrombin activated and then stabilized by the addition of 2 mM benzamidine hydrochloride; this suggests that inactivation is due to proteolysis. Neither unactivated nor thrombin-activated factor VIII produced factor X activation without factor IXa. With a constant level of factor IXa, factor X activation was directly proportional to the level of activated factor VIII. With a constant level of activated factor VIII, factor X activation was proportional to the factor IXa concentration. This observation was exploited to develop a specific, sensitive assay for factor IXa.

Factor IX↗