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PubMed2004

Novel missense mutation in the coagulation factor IX catalytic domain associated with severe haemophilia B--Factor IXDelhi.

Factor IX is a vitamin K-dependent serine protease, which exists as a zymogen in the blood. On activation to factor IXa, by factor XIa or tissue factor-factor VIIa complex, it forms tenase complex with factor VIIIa, in the presence of Ca2+. This tenase complex enzymatically converts factor X to factor Xa, thereby bringing about the coagulation cascade. Mutations in factor IX gene have been shown to cause haemophilia B, which is inherited as an X-linked recessive disorder. Herein we report a novel missense mutation at the nucleotide position 30829-T > A in the exon 8 of factor IX gene. This transversion leads to the substitution of histidine 236 to glutamine. This resulting abnormal protein has been named factor IXDelhi. Molecular modelling was performed to predict the molecular pathology of this mutation. We predict that this change in the catalytic domain may affect the surface loop that accommodates Ca2+, thereby leading to severe bleeding disorder.

Catalytic Domain↗

PubMed2002

Nonpeptide factor Xa inhibitors: DPC423, a highly potent and orally bioavailable pyrazole antithrombotic agent.

DPC423, 1-[3-(aminomethyl)phenyl]-N-[3-fluoro-2'-(methylsulfonyl)[1,1'-biphenyl]-4-yl]-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide, is a synthetic, orally bioavailable, competitive, and selective inhibitor of human coagulation factor Xa (K(i) [nM]: factor Xa, 0.15; trypsin, 60; thrombin, 6000; plasma kallikrein, 61; activated protein C, 1800; factor IXa, 2200; factor VIIa, >15,000; chymotrypsin, >17,000; urokinase, >19,000; plasmin, >35,000; tissue plasminogen activator, >45,000; complement factor I, 44,000 [IC(50)]). In vitro, DPC423 produced anticoagulant effects in human plasma in which it doubled prothrombin time, activated partial thromboplastin time, and Heptest clotting time at 3.1 +/- 0.4, 3.1 +/- 0.4, and 1.1 +/- 0.5 microM, respectively. In dogs, DPC423 had a good pharmacokinetic profile with an oral bioavailability of 57%, a plasma clearance of 0.24 L/kg/h, and a plasma half-life of 7.5 h. In rabbit and rat models of arteriovenous shunt thrombosis, DPC423 was an effective antithrombotic agent with an IC(50) of 150 and 470 nM, respectively. The antithrombotic effect of DPC423 is likely to be related to the inhibition of factor Xa but not to the inhibition of thrombin or due to direct inhibition of platelet aggregation. Therefore, based on potency, selectivity, efficacy, and oral bioavailability, DPC423 was selected for clinical development as an oral anticoagulant for the potential treatment of thrombotic disorders. Preliminary human data suggest that DPC423 is orally bioavailable in humans and has a long plasma half-life.

Administration, Oral↗

PubMed1996

Expression of Kunitz protease inhibitor--containing forms of amyloid beta-protein precursor within vascular thrombi.

BACKGROUND: The presence of patent neovessels within vascular occlusions in chronic thromboembolic pulmonary hypertension suggests that local mechanisms exist to regulate the coagulation system. This study investigated the expression of a potent inhibitor of Factor IXa and Factor XIa (ie, protease nexin-2/ amyloid beta-protein precursor, A beta PP) in the organized vascular occlusions harvested from patients with this disease. METHODS AND RESULTS: Immunohistochemical analysis revealed intense immunoreactivity for A beta PP in the single layer of cells that line the neovessels. A positive signal was also detected by in situ hybridization analysis with the use of a 35S-UTP-labeled antisense riboprobe that recognizes the various alternatively spliced mRNA forms of this molecule. To identify the forms of A beta PP produced within the thrombi, total RNA was extracted from the thrombi, reverse transcribed, and subjected to amplification with the use of the polymerase chain reaction (PCR) and primers that flank the region encoding the alternatively spliced 56-amino acid Kunitz-type protease inhibitor (KPI) domain. The major PCR products consisted of 255 bp and 312 bp and corresponded to transcripts encoding this domain (ie, A beta PP751 and A beta PP770). In situ hybridization analysis with the use of a 35S-UTP-labeled antisense riboprobe complementary to the region encoding the KPI domain confirmed the presence of these mRNA species in nucleated cells lining the neovessels. CONCLUSIONS: The expression of KPI-containing isoforms of A beta PP in thrombus endothelial cells may represent one mechanism utilized in this disease to shift the local hemostatic balance and preserve regional vessel patency.

Adult↗

PubMed1985

Interaction of antithrombin III with bovine aortic segments. Role of heparin in binding and enhanced anticoagulant activity.

Bovine antithrombin III (AT III) interaction with the luminal surface of bovine aortic segments with a continuous layer of endothelium was examined. Incubation of 125I-AT III with vessel segments, previously washed free of endogenous AT III, demonstrated specific, time-dependent binding to the protease inhibitor to the endothelium. Half-maximal binding was observed at an added AT III concentration of 14 nM. Binding of 125I-AT III to the vessel wall was reversible (50% dissociated in 4 min), and addition of either heparin or Factor Xa accelerated displacement of 125I-AT III from the vessel segment. Dissociation of 125I-AT III from the vessel segment in the presence of factor Xa coincided with the formation of a Factor Xa-125I-AT III complex. Inactivation of Factor IXa and Factor Xa by AT III was facilitated in the presence of vessel segments. Pretreatment of vessel segments with highly purified Flavobacterium heparinase precluded the vessel-dependent augmentation of AT III anticoagulant activity as well as specific binding of 125I-AT III to the vessel endothelium. In contrast, pretreatment of the vessel segments with chrondroitinases (ABC or AC) had no detectable effect on 125I-AT III binding or on AT III anticoagulant activity. AT III binding to vessel segments was competitively inhibited by increasing concentration of platelet factor 4. Binding of the protease inhibitor to vessel segments was inhibited by chemical modification of AT III lysyl or tryptophan residues. These AT III derivatives retained progressive inhibitory activity. These data suggest that heparin-like molecules are present on the aortic vessel wall and mediate binding of AT III to the vessel surface, as well as enhancing the anticoagulant activity of AT III at these sites.

Animals↗

PubMed2005

[Prophylaxis and treatment of venous thromboembolism: the role of new antithrombotic drugs].

Venous thromboembolic disease (VTE) is a major cause of morbidity and mortality. Unfractioned heparin, low-molecular weight heparins and vitamin K antagonists, currently used in the prophylaxis and treatment of VTE, are effective and relatively safe. Otherwise, they have some important limitations (narrow therapeutic window, highly variable dose-response relationship; limitation by the need of parenteral administration for heparins and the risk of heparin-induced thrombocytopenia) which provide opportunities for new antithrombotic drugs. These drugs include: inhibitors of factors IXa and factor VIIa/tissue factor complex, agents that enhance the protein C anticoagulant pathway, direct and antithrombin-dependent Xa inhibitors, direct and indirect thrombin inhibitors. Fondaparinux and idraparinux, two new indirect parenteral factor Xa inhibitors, have been studied in well conducted trials, showing interesting results both in the prevention and in the treatment of VTE. Ximelagatran, the first orally available thrombin inhibitor, represents a promising new anticoagulant drug for the prophylaxis of VTE after major orthopedic surgery and for the treatment of deep vein thrombosis.

Anticoagulants↗

PubMed1991

Pharmacokinetics of activated protein C in guinea pigs.

Protein C is a vitamin K-dependent zymogen of the serine protease, activated protein C (APC), an important regulatory enzyme in hemostasis. In view of the potential of human APC as an anticoagulant and profibrinolytic agent, the pharmacokinetics and tissue distribution of APC were studied in guinea pigs. The plasma elimination of a trace dose of 125I-APC was biphasic following an initial rapid elimination of approximately 15% of the injected dose within 1 to 2 minutes. This rapid removal of 125I-APC from the circulation was found to be a result of an association with the liver regardless of the route of injection. Essentially identical results were obtained with active site-blocked forms of APC generated with either diisopropylfluorophosphate or D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone, which indicates that the active site was not essential for the liver association. Accumulation of all three forms of APC in the liver peaked at 30 minutes and then declined as increasing amounts of degraded radiolabeled material appeared in the gastrointestinal tract and urine. Removal of the gamma-carboxyglutamic acid (gla) domain of diisopropylphosphoryl-APC resulted in a 50% reduction in the association with liver and an accumulation in the kidneys. Protein C and protein S were cleared from the circulation at rates approximately one-half and one-fourth, respectively, that of APC. Both in vitro and in vivo, APC was found to form complexes with protease inhibitors present in guinea pig plasma. Complex formation resulted in a more rapid disappearance of the enzymatic activity of APC than elimination of the protein moiety. These findings indicate two distinct mechanisms for the elimination of APC. One mechanism involves reaction with plasma protease inhibitors and subsequent elimination by specific hepatic receptors. The other mechanism involves the direct catabolism of APC by the liver via a pathway that is nonsaturable over a substantial dose range and independent of the active site. This pattern of elimination is distinctly different from that observed with the homologous coagulation enzymes thrombin, factor IXa, and factor Xa.

Animals↗

PubMed1985

The role of endothelium in the homeostatic balance of haemostasis.

As the cells forming the luminal vascular surface, endothelial cells are strategically positioned to play an important role in the regulation of coagulation. They cannot be regarded as an inert surface lining the vessel wall since they possess multiple activities. Anticoagulant properties include provision of a cell surface with heparin-like molecules (which can serve as binding sites for antithrombin III), synthesis of thrombomodulin (which alters the substrate specificity of thrombin), maintenance of a low level of tissue factor and generation of prostacyclin. In addition to these anticoagulant properties, endothelial cells can play a role in procoagulant reactions. Studies which have examined mechanisms underlying the localization of thrombotic processes have suggested the possible involvement of endothelial cells in procoagulant events. Endothelial cells have been found to propagate factor X and prothrombin activation once factor IXa and factor Xa have been formed. Factors regulating the balance of plasminogen activator and inhibitor synthesis are also under study. Perturbation of endothelial cells with induction of tissue factor and production of platelet-activating factor and thromboxane provides a model of the thrombotic state in which endothelium can promote coagulation. The multiple properties of endothelial cells indicate that a continuous blood flow in an unperturbed region of the vessel wall results from a complex interplay of anticoagulant and procoagulant activities. In a perturbed region, endothelial cells might initiate coagulation and the thrombin formed on the surface of endothelial cells might then lead to recruitment of platelets.

Antithrombin III↗

PubMed1985

The functional defect of factor VIII Leiden, a genetic variant of coagulation factor VIII.

Factor 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 VIIIa was about 3% of normal. The defect of factor VIIIa Leiden was studied by comparison with normal factor VIIIa in kinetic experiments of factor Xa formation. The results support the hypothesis that factor VIIIa Leiden has a reduced affinity for phospholipid-bound factor IXa in the intrinsic factor X-activating complex.

Calcium↗

PubMed1984

Phospholipids as dynamic participants in biological processes.

Phospholipids are described as active biological molecules. Three distinctly different roles are examined. The first centers on protein-lipid interactions and the lipid requirement expressed by certain enzymes. This category is illustrated by two soluble proteins of the blood coagulation scheme, Factor IXa and Factor Xa, and by an integral membrane protein, the (Ca2+ + Mg2+)-ATPase of human erythrocytes. The next two examples depict phospholipids as active participants in membrane-mediated events. In the first of these, termed the phosphoinositide effect, a phospholipid becomes a substrate during membrane signaling, and its products presumably act as second messengers. In the second example, a phospholipid is a signal that, among other reactions, induces the phosphoinositide effect. Here, the phospholipid (platelet activating factor) serves as a lipid chemical mediator. These examples show that phospholipids behave not only as structural molecules but also as dynamic, functionally important components of cells.

Blood Coagulation↗

PubMed1983

Binding to phospholipid protects factor VIII from inactivation by human antibodies.

The addition of purified factor IXa and phospholipid to factor VIII concentrate protected the VIII:C from inactivation by human antibodies. This protective effect was shown to be due largely to the phospholipid. Addition of phospholipid alone gave substantial protection against even high-titer antibodies, as shown by measurements of thrombin generation and VIII:C assays. Increasing concentrations of phospholipid led to significant reductions in the amount of VIII C:Ag detected by an IRMA method, up to 70% of the original VIII C:Ag being "lost" at the highest concentration of phospholipid. These results indicate that phospholipid binding plays an important part in the procoagulant activity of factor VIII and that human antibodies to VIII:C are directed largely at the phospholipid binding site. The addition of phospholipid to factor VIII concentrates could have important clinical applications in the treatment of hemophiliacs with antibodies to factor VIII.

Antibodies↗

PubMed1983

Amino acid sequence of human beta-factor XIIa.

Human factor XII was activated by limited proteolysis with trypsin, and the resulting beta-factor XIIa (Mr = 30,000) was isolated by DEAE-Sephacel column chromatography. The complete amino acid sequence of beta-factor XIIa was then determined on peptides produced by enzymatic digestion with either trypsin, chymotrypsin, or Staphylococcus aureus V8 protease and by chemical cleavage at methionyl and tryptophyl bonds. beta-Factor XIIa is a glycoprotein composed of a heavy chain (243 amino acid residues) and a light chain (9 amino acid residues), and these two chains are held together by a disulfide bond. The carbohydrate is attached to asparagine residue 61 in the heavy chain. The amino acid sequence of the heavy chain shows a high degree of homology to the corresponding regions of other plasma serine proteases, such as plasmin, thrombin, factor IXa and factor Xa, as well as the pancreatic digestive enzymes. These results demonstrate that factor XII is the precursor of a typical serine protease that participates in the coagulation cascade.

Amino Acid Sequence↗

PubMed1982

Activation of human coagulation factor VIII by activated factor X, the common product of the intrinsic and the extrinsic pathway of blood coagulation.

The 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.

Blood Coagulation↗

PubMed1978

Properties of the factor Xa binding site on human platelets.

The affinity (Ka) of human coagulation Factor Xa for thrombin-treated (to stimulate the release reaction) platelets has been determined to be 3 to 4 x 10(10) M-1 by equilibrium binding studies using 125I-labeled Xa. The binding of Factor Xa to platelets results in an increase of 300,000-fold in the apparent enzymatic activity of Xa in the conversion of prothrombin to thrombin. The activity of platelet surface Xa is approximately 15-fold greater than that observed with optimum concentrations of bovine Factor V and phospholipids in place of platelets. Ca2+ is required for the Xa-platelet interaction; the optimum concentration is 2.5 mM. Related coagulation factors, including Factor X, Factor IXa, diisopropylphosphoryl Factor Xa, and prothrombin do not complete with Factor Xa for the Xa binding sites. The rate of thrombim formation at saturating amounts of Xa is directly proportional to the number of platelets from 1 x 10(7) to 5 x 10(8) platelets/ml. Factor Xa bound to platelets is not inactivated by antithrombin III. An antibody that inhibits both human and bovine coagulation Factor V activity blocks both Xa binding to released platelets and the rapid thrombin formation associated with this binding, suggesting that Factor V from platelets is involved in the Xa-platelet interaction.

Binding Sites↗

PubMed1993

[Role of phospholipids in hemostasis].

This article reviews the significance of phospholipids in the haemostatic process. The plasma membranes of activated human blood platelets provide a catalytic phospholipid surface on which the "tenase" complex (factor IXa-factor VIIIa) and the "prothrombinase" complex (factor Xa-factor Va) can be assembled. The formation of a procoagulant platelet surface involves the exposure of anionic phospholipids e.g. phosphatidylserine, and is associated with shedding of microvesicles from the membranes of activated platelets. Moreover, tissue-factor, which plays a key role in blood coagulation by initiating the extrinsic coagulation pathway, requires the presence of phospholipids for optimal biological activity. The phospholipid dependency of the coagulation system explains the prolongation of phospholipid dependent clotting tests in patients with phospholipid directed antibodies such as lupus anticoagulants.

Blood Coagulation↗

PubMed1997

Antithrombin III-independent effect of depolymerized holothurian glycosaminoglycan (DHG) on acute thromboembolism in mice.

A previous study in this laboratory showed that depolymerized holothurian glycosaminoglycan (DHG) has two different antithrombin III (ATIII)-independent inhibitory effects on the in vitro blood coagulation system: heparin cofactor II (HCII)-dependent inhibition of thrombin, and ATIII- and HCII-independent inhibition of factor X activation by factor IXa-factor VIIIa complex (Nagase et al. Blood 85, 1527-1534, 1995). In the present study, we compared the antithrombotic effects of DHG in normal and in ATIII-deficient mice with those of unfractionated heparin (UFH) and low molecular weight heparin (LMWH). DHG, unlike UFH and LMWH, exerted an in vivo antithrombotic effect even in mice with decreased plasma ATIII activity (about 30% of normal). We then compared the anticoagulant and antithrombotic effects of DHG in mice with those of high molecular weight (HMW)-DHG, low molecular weight (LMW)-DHG, and dermatan sulfate (DS). In terms of in vitro anticoagulant activity assessed by use of purified human components, DHGs (DHG, HMW-DHG, and LMW-DHG) had different anti-thrombin activity in the presence of HCII and anti-factor Xase activities, which differences were dependent on the molecular weight. With respect to in vivo antithrombotic activity, DHG, HMW-DHG, and LMW-DHG showed almost the same inhibitory effect on acute thromboembolism in mice (minimum effective dose [MED]: > 0.3 mg/kg). Since the antithrombotic activities of DHGs were not correlated with the anticoagulant-specific activities, the contribution of the two anticoagulant activities to the in vivo antithrombotic effect of DHGs remains unknown. However, DHG was more effective against acute thromboembolism in mice than DS (MED > 1 or > 3 mg/kg), which showed no inhibitory activity toward factor Xase. Therefore, it seems that factor Xase inhibition contributes greatly to the antithrombotic effect of DHG and that DHG exerts this effect in mice mainly by inhibiting factor Xase.

Animals↗

PubMed1976

Mechanism of activation of bovine factor VII. Products of cleavage by factor Xa.

Coagulation Factor VII from bovine plasma is a glycoprotein containing a single peptide chain. The NH2-terminal sequence of Ala-Asx-Gly-Phe-Leu- is homologous with the NH2 termini of prothrombin, Factor IX, and the light chain of Factor X. Factor Xa in the presence of calcium ions and phospholipid cleaves Factor VII at an Arg-Ile bond in the sequence Arg-Ile-Val-Gly-Gly-, producing a two-chain molecule with at least 85 times the coagulant activity of single-chain Factor VII and a new NH2-terminal sequence homologous with the corresponding chains of thrombin, Factor IXa and Factor Xa. A second slower cleavage at an Arg-Gly bond destroys Factor VII activity. Bovine Factor VII, unlike prothrombin, Factor IX, and Factor X, is rapidly inhibited by diisopropylphosphorofluoridate (iPr2PF). [3H]iPr2PF is readily incorporated into one-chain, two-chain, and three-chain forms of Factor VII up to ratios of approximately 0.9 moles of [3H]diisopropylphosphate per mole of protein. The radioactive peptides generated from each form of [32P]iPr2PF-inhibited Factor VII by tryptic and thermolytic digestion were found to migrate together on paper electrophoresis. This indicates that the iPr2PF is incorporated stoichiometrically into the same specific site in each form.

Amino Acid Sequence↗

PubMed2001

Factor VIIIa cofactor activity shows enhanced ionic strength sensitivity in the absence of phospholipid.

Factor VIIIa, a cofactor for the protease factor IXa, is a trimer of A1, A2 and A3-C1-C2 subunits. In the absence of phospholipid (PL), the k(cat) for factor VIIIa-dependent, factor IXa-catalyzed conversion of factor X was markedly less than that observed in the presence of PL (approx. 150 min(-1)) and decreased as the ionic strength of the reaction increased. At low salt concentration, the k(cat) (5.5 min(-1)) was approx. 8-fold greater than observed at near physiologic ionic strength (0.7 min(-1)). However, this level of salt showed minimal effects on the intermolecular affinities of factor VIIIa (or isolated A2 subunit) for factor IXa or on the K(m) for factor X. Alternatively, the association of A2 subunit with A1 subunit was sensitive to increases in salt and paralleled the reduction in k(cat) observed with factor VIIIa. This instability was not observed in PL-containing reactions. Fluorescence energy transfer between acrylodan-A2 and fluorescein-A1/A3-C1-C2 dimer showed a requirement for both PL and factor IXa for maximal association of A2 with dimer. These results indicate that in the presence of factor IXa, the salt-dependent dissociation of factor VIIIa subunits is significantly enhanced in the absence of PL, promoting a reduced k(cat) for the cofactor-dependent generation of factor Xa.

Factor IXa↗

PubMed1982

Role of human factor VIII in factor X activation.

The cofactor function of human Factor VIII in Factor X activation was investigated by an initial-rate assay of 3H-Factor X activation in the presence of human factor IXa, Ca2+, and either phospholipid or fresh washed human platelets. Purified Factor VIII that has not been activated by thrombin or Factor Xa supports Factor X activation after a lag of several minutes. A specific inhibitor of Factor Xa, which had no inhibitory activity against Factor IXa, markedly prolonged this lag, whereas specific thrombin inhibitors did not prolong the lag. These data support the conclusion that unactivated Factor VIII has no ability to support Factor X activation in a purified system until it is activated by Factor Xa feedback during the lag period. When Factor VIII was optimally preactivated by thrombin, the lag was completely abolished, regardless of the order of addition of the other reactants or the phospholipid source. These data indicate that there is no slow, time-dependent ordering of the reactants at the phospholipid or activated platelet surface if Factor VIII has been preactivated. Unactivated platelets did not support Factor X activation by Factors IXa and VIII. The effect of activated Factor VIII on the kinetics of bovine Factor X activation was primarily to increase the Vmax (54-fold), whereas with human Factor X, Factor VIII both increased the Vmax 56-fold and decreased the Km sixfold to 0.14 microM, similar to the plasma concentration of Factor X. Therefore, a change in the plasma factor X concentration would be expected to have a major effect on the rate of Factor X activation in vivo.

Blood Coagulation↗