Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Factor IXa”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 667 records · Page 37Linked to original sources
PubMed1996

Purification, characterization and cDNA cloning of a novel anticoagulant of the intrinsic pathway, (prolixin-S) from salivary glands of the blood sucking bug, Rhodnius prolixus.

The salivary glands of the blood sucking insect, Rhodnius prolixus, have an anticoagulant, prolixin-S, which was reported as a specific inhibitor of intrinsic coagulant pathway. Prolixin-S was purified from the salivary glands extract of Rhodnius prolixus by gel filtration and anion exchange HPLC by assaying prolongation of activated partial thromboplastin time (APTT). The isolated protein specifically inhibited factor IXa-catalyzed activation of factor X in the presence of Ca2+ and phospholipids irrespective of the presence or absence of factor VIIIa. The anticoagulant factor had red color and a specific absorbance peak at 402 nm and thus it was identified as a heme protein. A Rhodnius prolixus salivary gland cDNA library was prepared, screened with an antibody against prolixin-S and its complete cDNA sequence was determined. cDNA and deduced amino acid sequences showed that prolixin-S is a novel anticoagulant of 19,922 Da, which has no sequence homology with any other anticoagulant reported so far.

Amino Acid Sequence↗
PubMed1998

Cloning, expression, and characterization of mouse tissue factor pathway inhibitor (TFPI).

Tissue factor pathway inhibitor (TFPI) acts to regulate the initiation of coagulation by first inhibiting factor Xa. The complex of factor Xa/TFPI then inhibits the factor VIIa/tissue factor complex. The cDNA sequences of TFPI from several different species have been previously reported. A high level of similarity is present among TFPIs at the molecular level (DNA and protein sequences) as well as in biochemical function (inhibition of factor Xa, VIIa/tissue factor). In this report, we used a PCR-based screening method to clone cDNA for full length TFPI from a mouse macrophage cDNA library. Both cDNA and predicted protein sequences show significant homology to the other reported TFPI sequences, especially to that of rat. Mouse TFPI has a signal peptide of 28 amino acid residues followed by the mature protein (in which the signal peptide is removed) which has 278 amino acid residues. Mouse TFPI, like that of other species, consists of three tandem Kunitz type domains. Recombinant mouse TFPI was expressed in the human kidney cell line 293 and purified for functional assays. When using human clotting factors to investigate the inhibition spectrum of mouse TFPI, it was shown that, in addition to human factor Xa, mouse TFPI inhibits human factors VIIa, IXa, as well as factor XIa. Cloning and expression of the mouse TFPI gene will offer useful information and material for coagulation studies performed in a mouse model system.

Amino Acid Sequence↗
PubMed1984

A coagulation pathway on bovine aortic segments leading to generation of Factor Xa and thrombin.

Previous studies have demonstrated the binding of Factors IX and IXa to cultured bovine aortic endothelial cells. The present study examines the interaction of Factors IX, IXa, and Xa with the luminal surface of calf aortas, shown by microscopic examination to have a continuous layer of endothelium. Radioimmunoassay of Factor IX showed that 74 fmol/10(6) cells of Factor IX could be eluted from freshly prepared aortic segments. Binding of 3H-Factors IX and IXa to aortic segments was saturable, and comparable to binding in previous studies using cultured endothelial cells. Preincubation of aortic segments with 3H-Factor IXa and von Willebrand factor (VWF)/Factor VIII, followed by washing and addition of Factor X, resulted in formation of Factor Xa. The addition of prothrombin to these activation mixtures resulted in formation of thrombin. Exogenous phospholipid and Factor V were not required for Factor X and prothrombin activation on the intact native endothelium. Incubation of 125I-Factor Xa with the vessel segments resulted in most of the tracer being complexed with antithrombin III originally present on the aortic segment (3.8 pmol antithrombin III/10(6) cells). The Factor Xa-antithrombin III complex was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis exclusively in the supernatants. 125I-Factor Xa not complexed with antithrombin III bound specifically to the vessel segment. The time course of binding was biphasic, consisting of an initial more rapid reversible phase followed by a slower irreversible phase. The latter phase correlated with the formation of a covalent complex (Mr, 76,000) between 125I-Factor Xa and a vessel-localized protein presumably distinct from antithrombin III. The activation of prothrombin by vessel-bound Factor Xa was inhibited by anti-bovine Factor V IgG, suggesting that there is interaction of Factor Xa with a Factor V-like molecule provided by the endothelial cell surface. Addition of antibody to antithrombin III prevented formation of Factor Xa-antithrombin III and thrombin-antithrombin III complexes in the supernatant and increased apparent thrombin activity 30-50-fold. These studies demonstrate that freshly obtained vessels with a continuous layer of native endothelium can support activation of Factor X and prothrombin: vessel-bound Factor IXa can activate Factor X in the presence of VWF/Factor VIII. Factor Xa can also bind to the vessel and participate in the activation of prothrombin. The apparent efficiency of prothrombin activation, however, is dampened by the presence of functional antithrombin III on the vessel wall.

Animals↗
PubMed1994

Crystal structure of the extracellular region of human tissue factor.

Tissue factor is a cell-surface glycoprotein receptor which initiates the blood coagulation cascade after vessel injury by interacting with blood clotting factor VII/VIIa and which is implicated in various pathological processes. When bound to tissue factor, factor VII is readily converted to the active protease factor VIIa by trace amounts of factors Xa, IXa or VIIa. Human tissue factor consists of 263 residues, the first 219 of which comprise the extracellular region. We have determined the crystal structure of the extracellular region at a resolution of 2.2 A. Tissue factor consists of two immunoglobulin-like domains associated through an extensive, novel, interdomain interface region. The binding site for factor VII lies at the interface region and involves residues from domain 1 and an extended loop (binding 'finger') of domain 2. This is the first reported structure of a representative of the class 2 cytokine receptor family, which also includes interferon-alpha, interferon-gamma (refs 2, 3) and interleukin-10 (ref. 4) receptors.

Amino Acid Sequence↗
PubMed1995

Blood coagulation factor IX-binding protein from the venom of Trimeresurus flavoviridis: purification and characterization.

The coagulation factor IX/factor X-binding protein (IX/X-bp) from the venom of Trimeresurus flavoviridis is a heterogeneous two-chain protein, and the structure of each chain is similar to that of the carbohydrate-recognition domain of C-type lectins, such as asialoglycoprotein receptors, pancreatic stone protein, and the Fc epsilon receptor for immunoglobulin E. Analysis of the binding properties of IX/X-bp revealed that it binds to the gamma-carboxyglutamic acid (Gla)-containing domains of factors IX and X [Atoda, H. et al. (1994) Eur. J. Biochem. 224, 703-708]. In the present study, we isolated another anticoagulant protein that binds to factor IX but is not to factor X. This protein, designated IX-bp, inhibited factor IXa-induced clotting but not factor Xa-induced clotting, whereas IX/X-bp inhibits both. The concentration of IX-bp for half-maximal binding to solid-phase bovine factor IX was 0.4 nM whereas IX-bp did not bind to factor X even at 40 nM. The binding of IX-bp to solid-phase factor IX was inhibited by the addition of Gla-domain peptide of factor IX, indicating that IX-bp binds to the Gla-domain region of factor IX. IX-bp had two Ca(2+)-binding sites with different affinities for Ca2+ ions. At pH 7.5, the apparent Kd values for these sites were 14 and 130 microM, respectively. IX-bp was a two-chain protein (27.5-kDa band before reduction and 16.8- and 15.7-kDa bands after reduction on SDS-PAGE) and it reacted with immunoglobulin G against IX/X-bp. The complete amino acid sequence of IX-bp was determined. The 16.8-kDa chain (A chain) of IX-bp consisted of 129 residues, of which 19 were different from those in the A chain of IX/X-bp (129 residues). The sequence of the 15.7-kDa chain (B chain) was identical to that of the B chain of IX/X-bp (123 residues). We conclude that IX-bp is a protein that is structurally similar to but functionally different from IX/X-bp. The difference of binding specificity between IX-bp and IX/X-bp presumably arises from the sequence differences in the A chains.

Amino Acid Sequence↗
PubMed1997

Search for mutations in the genes for coagulation factors V and VIII with a possible predisposition to activated protein C resistance.

A total of 74 non-pregnant women with a previous episode of thrombosis were investigated for activated protein C (APC) resistance in the aPTT-based and factor IXa-X-based assays and for the presence of mutations in all APC-cleavage sites in the heavy chains of factor V and factor VIII. DNA fragments were amplified with the polymerase chain reaction (PCR) and those encoding for the Arg-306 and Arg-506 (factor V) and for Arg-740 (factor VIII) cleavage sites were subjected to restriction enzyme analysis. DNA fragments of 29 selected patients corresponding to the Arg-306 and Arg-679 cleavage sites in factor V, and to the Arg-336 and Arg-562 cleavage sites in factor VIII were sequenced. APC resistance was found in 40 cases, using the aPTT-based assay and in 35, using the factor IXa-X-based assay (23 patients were APC resistant in both assays), whereas 22 individuals had a normal response to APC. Forty-three patients carried Arg-506 to Gln mutation in factor V. No other polymorphism or mutation was found in the genes for factors V or VIII in the vicinity of the APC cleavage sites. It was concluded that the difference in response to APC in the two assays may not be explained by the presence of mutations in the APC cleavage sites of factor V and factor VIII in this group of patients. The data do not exclude the presence of mutations elsewhere in the factor V or factor VIII genes.

Adult↗
PubMed2000

Properties of anti-factor VIII inhibitor antibodies in hemophilia A patients.

Blood coagulation factor VIII functions in the intrinsic pathway of blood coagulation as a cofactor by enhancing the assembly of its complex with factors IX and X on the surface of activated platelets. This requires factor VIII interaction with these two proteins, von Willebrand factor (vWF), and phospholipids on the platelet surface. Once factor VIII and factor IX are activated by proteolytic cleavage, the complex is able to activate factor X to factor Xa by proteolysis. In hemophilia A patients with severe factor VIII deficiency, about 30% respond to factor VIII infusion therapy immunologically to produce antibodies that inactivate the infused factor VIII and others that are noninhibitory. An assay that measures only the inhibitor antibodies demonstrated that the factor VIII A2, A3, and C2 domains are the most immunogenic, and domains A1 and B are poorly immunogenic or not immunogenic. The specific antibody responses to A2, A3, and C2 vary considerably among individuals, and epitopes for inhibitor antibodies have been determined for all three. The anti-C2 inhibitors prevent factor VIII binding to phospholipids and vWF, and anti-A3 inhibitors prevent binding to factor IX (IXa). An inhibitor binding site for factor X has been localized to the A1 domain acidic region, leading to inhibition of factor VIII/factor X binding by antibodies. This inhibitor mechanism is rare. Because a second binding site for factor IX was localized to the factor VIII A2 domain, it is likely but not proven that prevention of factor IX binding to factor VIII is the inhibitor mechanism for this epitope.

Animals↗
PubMed2001

Modeling human zymogen factor IX.

Modern theoretical techniques are employed to provide complete three dimensional structure for the zymogen and activated forms of human coagulation factors IX and IXa. These structures are fully calcium bound and equilibrated in an electrically neutral aqueous environment. The relationship of structure to mutational data is examined. We find that a substantial relative orientational change of the catalytic domain occurs on activation. Also, we find that the electrostatistically dipolar nature of the catalytic domain is substantially modified upon activation, with cleavage of the negatively charged activation peptide leaving behind a largely hydrophobic face in factor IXa. While the backbone atoms of the catalytic residues have little relative movement, nearby loops are found that do move. The presence or absence of these changes likely defines specificity.

Amino Acid Substitution↗
PubMed2002

Unusual benefits of macromolecular shielding by polyethylene glycol for reactions at the diffusional limit: the case of factor VIIai and tissue factor.

Protein modification with poly(ethylene glycol) (PEG) can prolong circulatory lifetime and lower protein antigenicity in an animal. These benefits may arise from the proposed mechanism of PEG action, molecular shielding of the protein surface, and lowered interaction with other macromolecules. Proteins that depend on macromolecule association for their function would not seem good targets for PEG modification as the benefits may be mitigated by loss of function. Indeed, high loss of function applied to PEG-modified factor VIIa and to active site-blocked blood clotting factors Xa or IXa was studied. A surprising finding was that PEG-modified, active site-blocked factor VIIa (PEG-VIIai, PEG-40 000) retained 40% of its function despite an 18-fold increase in circulatory lifetime. The discrepancy between functional loss and increased circulatory lifetime was consistent with a process that was limited by the diffusion step of assembly rather than the chemical binding step. The impact of PEG-40 000 on diffusion of VIIai is small (about 3-fold) relative to its potential impact on molecular shielding during the chemical binding step of association. These properties extended to a mutant of VIIai (P10Q/K32E, QE-VIIai) that has 25-fold higher function than wild-type factor VIIai. Overall, properties of PEG-modified proteins can suggest features of the kinetic mechanism and may provide enhanced proteins for anticoagulation therapy.

Amino Acid Chloromethyl Ketones↗
PubMed1999

New insights into how blood clots: implications for the use of APTT and PT as coagulation screening tests and in monitoring of anticoagulant therapy.

Blood coagulation occurs efficiently on cell surfaces such as activated platelets and monocytes, and fibroblasts. It is initiated by limited amounts of tissue factor (TF) exposed at the sites of vascular injury that complexes with trace amounts of circulating factor VIIa (FVIIa). Additional FVIIa-TF complexes are formed from FVII-TF involving positive feedback loops, including FVIIa-TF as well as factors Xa and IXa as they are formed in subsequent steps. For sustained normal coagulation to proceed, effective in vivo activation of factor X requires the participation of factor IXa generated via the FVIIa-TF complex. This may, in part, be due to effective inhibition of factor Xa and FVIIa-TF complex by tissue factor pathway inhibitor that results in blockage of direct activation of factor X by the FVIIa-TF complex. Additional generation of factor Xa at injury sites may then proceed via the FIXa-VIIIa pathway. Thrombin generated from prothrombin via complex formation of prothrombin with FXa and FVa on phospholipid surfaces (prothrombinase complex) powerfully accelerates coagulation by activation of FVIII and FV, and sustains coagulation through activation of FXI. Thus, in light of our current understanding of how blood clots in vivo, it is clear that both prothrombin time (PT) and activated partial thromboplastin time (APTT) are highly artificial in vitro systems with major limitations. Nevertheless, these tests are quite useful as global screening tests for abnormalities in the intrinsic or extrinsic, as well as common, pathways of coagulation and for monitoring of anticoagulant therapy.

Anticoagulants↗
PubMed2003

The assembly of the factor X-activating complex on activated human platelets.

Platelet membranes provide procoagulant surfaces for the assembly and expression of the factor X-activating complex and promote the proteolytic activation and assembly of the prothrombinase complex resulting in normal hemostasis. Recent studies from our laboratory and others indicate that platelets possess specific, high-affinity, saturable, receptors for factors XI, XIa, IX, IXa, X, VIII, VIIIa, V, Va and Xa, prothrombin, and thrombin. Studies described in this review support the hypothesis that the factor X-activating complex on the platelet surface consists of three receptors (for the enzyme, factor IXa; the substrate, factor X; and the cofactor, factor VIIIa), the colocalization of which results in a 24 million-fold acceleration of the rate of factor X activation. Whether the procoagulant surface of platelets is defined exclusively by procoagulant phospholipids, or whether specific protein receptors exist for the coagulant factors and proteases, is currently unresolved. The interaction between coagulation proteins and platelets is critical to the maintenance of normal hemostasis and is pathogenetically important in human disease.

Blood Coagulation Factors↗
PubMed2002

A human antibody that inhibits factor IX/IXa function potently inhibits arterial thrombosis without increasing bleeding.

10C12, a human antibody F(ab')2, which specifically binds to the gamma-carboxyglutamic acid domain of factor IX/factor IXa (F.IX/IXa), interferes with all known coagulation processes in which F.IX/IXa is involved. In a rabbit model of carotid artery injury, intravenous administration of 10C12 or heparin decreased thrombosis dose dependently. The dose that resulted in a 90% reduction of thrombus mass (ED90) was a 30-microg/kg bolus of 10C12 or a 100-U/kg bolus plus 1.0 U x kg(-1) x min(-1) infusion of heparin. Heparin, at and below the ED90, significantly prolonged coagulation times and cuticle bleeding times. In contrast, 10C12 had no effect on coagulation or bleeding times at doses up to 4 times the ED90. To further evaluate the effect of 10C12 on bleeding, it was compared with heparin in a novel model of blood loss. At the ED90 of heparin, blood loss induced by a standardized injury to the vasculature of the rabbit tibia increased to more than 2 times that of saline controls. In contrast, the dose of 10C12 required to produce a similar increase in blood loss was more than 30 times the ED90. The antithrombotic potency and relative safety of this fully human antibody suggests that it may have therapeutic value for treatment of thrombotic disorders.

Animals↗
PubMed1985

Development of procoagulant binding sites on the platelet surface.

Activation of coagulation factor X by a complex of factors IXa-VIIIa and prothrombin by a complex of factor Xa.Va is markedly enhanced in the presence of a negatively-charged phospholipid surface. A suitable phospholipid surface is provided by a platelet lysate but not by a suspension of intact platelets, due to the internal localization of phosphatidylserine in the platelet membrane. Upon stimulation of platelets with a combination of collagen and thrombin, or calcium ionophore A23187 or treatment with diamide, alterations in the distribution of membrane phospholipids take place resulting in the exposure of significant amounts of phosphatidylserine at the platelet surface. As a consequence, an increased number of intrinsic factor X and prothrombinase complexes can be assembled at the platelet surface thus leading to an acceleration of factor Xa and thrombin formation. Studies with pathological platelets have shown that neither release nor aggregation are essential to provoke prothrombinase activity. The relatively high prothrombinase activity of non-stimulated Bernard-Soulier platelets is in agreement with the slightly altered phospholipid distribution in these platelets, in which more phosphatidylserine is exposed at the outer surface. Disturbances in the membrane bilayer structure as well as changes in the plasma membrane-cytoskeleton interaction are considered as possible explanations for the increased transbilayer movement of phosphatidylserine.

Bernard-Soulier Syndrome↗
PubMed2006

A potent anti-coagulant RNA aptamer inhibits blood coagulation by specifically blocking the extrinsic clotting pathway.

A potent antidote-controlled aptamer, as an anticoagulant, has reportedly overcome the complications of acute bleeding by the administration of available anticoagulants. In the present study, we provide evidence that the aptamer binds specifically to factors IX and IXa and inhibits their functions. Furthermore, we demonstrated that the aptamer inhibits blood coagulation by interfering with the extrinsic pathway, blocking the complex of factor VIIa and tissue factor interactions with factor IX. The results from the previous and present studies suggest that the aptamer probably binds in the vicinity of the EGF1 and EGF2 domains of factor IX.

Anticoagulants↗
PubMed1995

Enhanced plasmin inhibition by a reactive center lysine mutant of the Kunitz-type protease inhibitor domain of the amyloid beta-protein precursor.

The Alzheimer's disease related protein, amyloid beta-protein precursor (A beta PP), contains a domain homologous to Kunitz-type serine protease inhibitors (KPI). The recombinant KPI domain of A beta PP is a potent inhibitor of coagulation factors XIa and IXa and functions as an anticoagulant in vitro. Here we report the expression, purification, and characterization of a reactive center lysine mutant of the KPI domain of A beta PP (KPI-Lys17). An expression plasmid for the KPI-Lys17 domain of A beta PP encoded amino acids 285-345 of the A beta PP cDNA containing a lysine substitution at arginine 17 in the KPI domain. The secreted 61-amino acid product was purified to homogeneity and functionally characterized. The protease inhibitory properties of the KPI-Lys17 domain were compared to those of the native KPI domain of A beta PP. Both KPI domains equally inhibited trypsin, chymotrypsin, and coagulation factors IXa and Xa. However, the KPI-Lys17 domain was an approximately 25-fold less effective inhibitor of coagulation factor XIa resulting in markedly less prolongation of the activated partial thromboplastin time compared to the native KPI domain of A beta PP. On the other hand, the KPI-Lys17 domain was an approximately 10- and 5-fold better inhibitor of plasmin in a chromogenic substrate assay and in a fibrinolytic assay, respectively, than the native KPI domain of A beta PP. Together, these studies suggest that the KPI-Lys17 domain has enhanced anti-fibrinolytic and diminished factor XIa inhibitory properties compared to the native KPI domain of A beta PP.

Amino Acid Sequence↗
PubMed1996

Cells and the activation of factor VII.

Binding of factor VII to tissue factor (TF) present on or released from cells initiates TF-dependent coagulation in vivo. Earlier data obtained with relipidated TF suspensions led to the hypothesis that all factor VII bound to limiting TF sites exposed after tissue injury could be rapidly activated to VIIa/TF complexes by low concentrations of factor Xa, IXa, and VIIa. However, newer data on the activation of factor VII bound to TF expressed on cultured cell monolayers support the hypothesis that not all VII/TF complexes formed at a site of tissue injury are readily activated to VIIa/TF complexes during hemostasis. This is possibly related to why recombinant factor VIIa can bypass impaired IXa/VIIIa/phospholipid complex function in hemostasis.

Blood Cells↗
PubMed1985

A new low molecular weight heparin fragment (PK 10169): in vitro and in vivo studies.

The depolymerized heparin fragment PK 10169 was compared with conventional mucosal sodium heparin. The inhibition of factors Xa and IXa by heparin and by PK 10169 was similar on a weight base whilst the inhibition of thrombin by PK 10169 was at least 5 times weaker than by heparin. Subcutaneous injection of PK 10169 was not followed by prolongation of the thrombin time. The APTT was considerably less prolonged after PK than after heparin. Platelet reaction was increased by heparin but was not influenced by PK 10169. In vitro the euglobulin lysis time (ELT) was shortened after addition of heparin to plasma but not after addition of PK 10169. After injection, however, there was an equal shortening of the ELT by both substances. Advantages of PK 10169 over heparin are therefore a weaker anticoagulant effect and the missing influence on platelet functions.

Anticoagulants↗