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PubMed1997

The importance of the binding of factor Xa to phospholipids in the inhibitory mechanism of tissue factor pathway inhibitor: the transmembrane and cytoplasmic domains of tissue factor are not essential for the inhibitory action of tissue factor pathway inhibitor.

To investigate the inhibitory mechanism of tissue factor pathway inhibitor (TFPI), an attempt was made to examine the inhibitory activity of TFPI toward the factor VIIa-truncated tissue factor (TF1-219) complex, which lacks its transmembrane and cytoplasmic domains. Factor VIIa-TF1-219 activity was significantly inhibited by TFPI-factor Xa complex in the presence of phospholipids, but was not in the absence of phospholipids. In addition, TFPI did not inhibit factor VIIa-TF1-219 activity in the presence of gamma-carboxyglutamic acid-domainless factor Xa. The ability of TFPI-factor Xa complex to inhibit factor VIIa-TF1-219 activity was totally dependent on the presence of phospholipids and was neutralized by prothrombin fragment 1 in a dose-dependent manner. These results indicate that the transmembrane and cytoplasmic domains of tissue factor are not essential for the inhibitory mechanism of TFPI and confirm that the binding of factor Xa to phospholipids through its gamma-carboxyglutamic acid domain is essential for this reaction.

Anticoagulants↗
PubMed1991

Low molecular weight heparin-catalyzed inactivation of factor Xa and thrombin by antithrombin III--effect of platelet factor 4.

Low molecular weight (LMW) heparin preparations have unknown distributions of ATIII-binding material, so mean molecular weights as such might bear little information on their anti-factor Xa and anti-thrombin activities, and on the neutralization of these activities by platelet factor 4 (PF4). These properties were investigated in pure systems with proteins of human origin. Pseudo-first order rate constants of inactivation of factor Xa and thrombin by antithrombin III were determined as function of heparin concentration, in the presence of 4.0 mM CaCl2. Despite a large variation in the mean molecular weights, the ratios of the anti-factor Xa over the anti-thrombin activities were essentially the same for the 4th International Standard for heparin (0.46), the 1st International Standard for LMW heparin (0.32), CY216 (0.42) and enoxaparin (0.50). The ultra LMW heparin CY222 had only a 2-times higher ratio (0.98). Analysis of CY216 subfractions, obtained by gel filtration, showed that the heparin molecules of the upper region of the molecular weight distribution are responsible for the anti-thrombin, but also to a large extent for the anti-factor Xa activities. The results indicate that depolymerization of unfractionated heparin does not result in an increased anti-factor Xa/anti-thrombin ratio, because in the presence of Ca(2+)-ions the rate constants of inactivation of factor Xa are lowered as compared to those of native heparin. PF4-dependent neutralization of anti-factor Xa and anti-thrombin activities of fixed concentrations of the LMW heparins was studied by measuring rate constants as function of PF4 concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Antithrombin III↗
PubMed2001

Discovery of transition state factor Xa inhibitors as potential anticoagulant agents.

Factor Xa is an attractive biological target in the discovery and development of either parenteral or orally active anticoagulant agents. Several strategies have been utilized at COR Therapeutics in the pursuit of tri-peptide based transition state mimetic factor Xa inhibitors with high aqueous solubility. Some of these inhibitors have displayed excellent in vitro potency in inhibiting factor Xa in the prothrombinase complex. More importantly, these compounds showed strong in vivo antithrombotic efficacy without significant bleeding complications in several animal thrombosis models. These results demonstrated that small molecule factor Xa inhibitors could be advantageous over Warfarin and LMWH. For the discovery and development of orally active anticoagulant agents, small organic molecules as reversible factor Xa inhibitors were explored. From a medicinal chemistry perspective, significant insight has been gained regarding the in vivo antithrombotic efficacy and pharmacokinetic behaviors of each class of factor Xa inhibitors. This review will focus on the design and discovery of transition state factor Xa inhibitors as potential parenteral anticoagulant agents. Several excellent comprehensive review articles on factor Xa inhibitors have appeared recently [1-4].

Animals↗
PubMed1999

Crystallographic analysis of potent and selective factor Xa inhibitors complexed to bovine trypsin.

Factor Xa is a serine protease which activates thrombin (factor IIa) and plays a key regulatory role in the blood-coagulation cascade. Factor Xa is, therefore, an important target for the design of anti-thrombotics. Both factor Xa and thrombin share sequence and structural homology with trypsin. As part of a factor Xa inhibitor-design program, a number of factor Xa inhibitors were crystallographically studied complexed to bovine trypsin. The structures of one diaryl benzimidazole, one diaryl carbazole and three diaryloxypyridines are described. All five compounds bind to trypsin in an extended conformation, with an amidinoaryl group in the S1 pocket and a second basic/hydrophobic moiety bound in the S4 pocket. These binding modes all bear a resemblance to the reported binding mode of DX-9065a in bovine trypsin and human factor Xa.

Animals↗
PubMed2000

Identification of basic residues in the heparin-binding exosite of factor Xa critical for heparin and factor Va binding.

We recently demonstrated that a template mechanism makes a significant contribution to the heparin-accelerated inactivation of factor Xa (FXa) by antithrombin at physiologic Ca(2+), suggesting that FXa has a potential heparin-binding site. Structural data indicate that 7 of the 11 basic residues of the heparin-binding exosite of thrombin are conserved at similar three-dimensional locations in FXa. These residues, Arg(93), Lys(96), Arg(125), Arg(165), Lys(169), Lys(236), and Arg(240) were substituted with Ala in separate constructs in Gla domainless forms. It was found that all derivatives cleave Spectrozyme FXa with similar catalytic efficiencies. Antithrombin inactivated FXa derivatives with a similar second-order association rate constant (k(2)) in both the absence and presence of pentasaccharide. In the presence of heparin, however, k(2) with certain mutants were impaired up to 25-fold. Moreover, these mutants bound to heparin-Sepharose with lower affinities. Heparin concentration dependence of the inactivation revealed that only the template portion of the cofactor effect of heparin was affected by the mutagenesis. The order of importance of these residues for binding heparin was as follows: Arg(240) > Lys(236) > Lys(169) > Arg(165) > Lys(96) > Arg(93) >/= Arg(125). Interestingly, further study suggested that certain basic residues of this site, particularly Arg(165) and Lys(169), play key roles in factor Va and/or prothrombin recognition by FXa in prothrombinase.

Binding Sites↗
PubMed2007

Computational modeling of factor Xa inhibition by immobilized tissue factor pathway inhibitor.

Coating surfaces of implanted devices with anticoagulants can reduce thrombosis and studies using a recombinant form of endogenous tissue factor pathway inhibitor (rTFPI) are promising. The anticoagulant function of immobilized rTFPI is thought to occur primarily by its inhibition of plasma clotting factor Xa (FXa); however the kinetics of this reaction at a surface are as yet unknown. To better understand the surface inhibition reaction under flow conditions, a theoretical model was developed delineating the roles of mass transport and reaction kinetics for an in vitro parallel plate device used in prior experimental studies [Hall et al., J. Biomech. Eng. 120:484-490, 1998]. As a first approximation, the kinetics of inhibition of FXa by rTFPI reported for static, homogeneous systems was considered. The unsteady convection-diffusion equation was solved for different wall-shear rates and inlet concentrations of FXa using the computational fluid dynamics software CFD-ACE (ESI Software Group). The results show that the heterogeneous inhibition reaction is diffusion controlled prior to saturation of the rTFPI. The experimental results compare favorably with the model at the lower shear rates (100-400 s(-1)). At higher shear rates (>400 s(-1)) the theoretical results follow the same trend as the experimental results but show a greater inhibition of FXa, implying an effect of flow or shear on the inhibition reaction.

Animals↗
PubMed1991

Regulation of activated protein C by factor Xa.

In this study, the ability of factor Xa to protect factor Va from proteolysis by activated protein C (APC) was verified. Interestingly, factor X was found to exert a similar effect with a dose-dependence identical to that of factor Xa. The effects of factor X and Xa were abrogated in the presence of protein S. To further assess the interactions of factor Va with factors Xa, X and APC, direct binding studies were performed. Factor X and Xa bound to factor Va with equal efficacy. Both proteins displaced APC from its factor Va binding site. These interactions were calcium-dependent. Although the binding of factor Xa devoid of its principal calcium binding site (the Gla-domain) to factor Va was identical to that observed using the native protein, its APC inhibitory effects were significantly reduced. These findings suggest that the Gla-domain of factor X (Xa) is pivotal in the protection of factor Va from APC.

Binding Sites↗
PubMed2005

Functional mapping of charged residues of the 82-116 sequence in factor Xa: evidence that lysine 96 is a factor Va independent recognition site for prothrombin in the prothrombinase complex.

It has been hypothesized that two antiparallel structures comprised of residues 82-91 and 102-116 in factor Xa (fXa) may harbor a factor Va- (fVa-) dependent prothrombin recognition site in the prothrombinase complex. There are 11 charged residues in the 82-116 loop of human fXa (Glu-84, Glu-86, Lys-90, Arg-93, Lys-96, Glu-97, Asp-100, Asp-102, Arg-107, Lys-109, and Arg-115). With the exception of Glu-84, which did not express, and Asp-102, which is a catalytic residue, we expressed the Ala substitution mutants of all other residues and evaluated their proteolytic and amidolytic activities in both the absence and presence of fVa. K96A and K109A activated prothrombin with 5-10-fold impaired catalytic efficiency in the absence of fVa. All mutants, however, exhibited normal activity toward the substrate in the presence of fVa. K109A also exhibited impaired amidolytic activity and affinity for Na(+); however, both fVa and higher Na(+) restored the catalytic defect caused by the mutation. Analysis of the X-ray crystal structure of fXa indicated that Glu-84 may interact by a salt bridge with Lys-109, explaining the lack of expression of E84A and the lower activity of K109A in the absence of fVa. These results suggest that none of the residues under study is a fVa-dependent recognition site for prothrombin in the prothrombinase complex; however, Lys-96 is a recognition site for the substrate independent of the cofactor. Moreover, the 82-116 loop is energetically linked to fVa and Na(+) binding sites of the protease.

Alanine↗
PubMed1988

Kinetics of inactivation of membrane-bound factor Va by activated protein C. Protein S modulates factor Xa protection.

Kinetic analyses were done to determine what effect factor Xa and protein S had on the activated protein C (APC)-catalyzed inactivation of factor Va bound to phospholipid vesicles or human platelets. In the presence of optimal concentrations of phospholipid vesicles and Ca2+, a Km of 19.7 +/- 0.6 nM factor Va and a kcat of 23.7 +/- 10 mol of factor Va inactivated/mol of APC/min were obtained. Added purified plasma protein S increased the maximal rate of factor Va inactivation only 2-fold without effect on the Km. Protein S effect was unaltered when the phospholipid concentration was varied by 2 orders of magnitude. The reaction on unactivated human platelets yielded a Km = 12.5 +/- 2.6 nM and kcat = 6.2 +/- 0.6 mol of factor Va inactivated/mol of APC/min. Added purified plasma protein S or release of platelet protein S by platelet activation doubled the kcat value without affecting the Km. Addition of a neutralizing anti-protein S antibody abrogated the effect of plasma protein S or platelet-released protein S, but was without effect in the absence of plasma protein S or platelet activation. Studies with factor Xa indicated that factor Xa protects factor Va from APC-catalyzed inactivation by lowering the effective concentration of factor Va available to interact with APC. From these data a dissociation constant of less than 0.5 nM was calculated for the interaction of factor Xa with membrane-bound factor Va. Protein S abrogated the ability of factor Xa to protect factor Va from inactivation by APC without affecting the interaction of factor Xa with factor Va. These combined data suggest that one physiological function of protein S is to allow the APC-catalyzed inactivation of factor Va in the presence of factor Xa.

Blood Platelets↗
PubMed1992

Expression, purification, and characterization of inactive human coagulation factor Xa (Asn322Ala419).

We have expressed in Chinese hamster ovary cells a catalytically inactive form of human factor Xa (factor rXai). A recombinant precursor of human factor Xa was inactivated by two point mutations in the serine protease catalytic triad, Asp322Asn and Ser419Ala. A two-step purification to homogeneity of the secreted material involved immunoaffinity followed by heparin-agarose chromatography. Two forms were identified; a fully processed dimer (70%) and a partially processed monomer (30%). Limited N-terminal amino acid sequencing of factor rXai detected the predicted residues and gamma-carboxyglutamic acid content was 90% of human plasma control. Although devoid of measurable proteolytic activity, factor rXai competitively inhibited plasma factor Xa assembly into functional prothrombinase complexes (Ki = 3 x 10(-10) M). Factor rXai also inhibited plasma clotting in a dose-dependent manner. The possible use of recombinant catalytically inactive proteins as a general approach for pharmacological regulation of human diseases is discussed.

Amino Acid Sequence↗
PubMed1985

Activation of porcine factor VIII:C by thrombin and factor Xa.

The activation of porcine factor VIII:C by thrombin and by factor Xa was studied by a chromogenic substrate assay and by sodium dodecyl sulfate-polyacrylamide gel radioelectrophoresis of 125I-labeled factor VIII:C activation products. In the chromogenic assay, the kinetics of factor VIII:C dependent activation of factor X by factor IXa in the presence of calcium and phosphatidylserine/phosphatidylcholine vesicles were measured with N-benzoyl-L-isoleucyl-L-glutamylglycyl-L-arginine p-nitroanilide (S2222) as substrate. Substrate dependence of initial rates of the reaction at fixed factor IXa, factor VIII:C, lipid, and calcium obeyed Michaelis-Menten kinetics. At fixed factor IXa, factor X, lipid, and calcium the initial rates of the reaction varied linearly with lower factor VIII:C concentrations and plateaued at higher concentrations. The linear initial rate dependence formed the basis of a rapid, plasma-free assay of activated factor VIII:C. The activation of factor VIII:C by thrombin or factor Xa and the enzyme-independent rate of spontaneous inactivation were studied under conditions of excess enzyme. A model of the activation kinetics was developed and fit to the data by a nonlinear least-squares technique. From the model, the catalytic efficiencies (kcat/Km) of factor VIII:C activation by thrombin and factor Xa were 5.0 X 10(6) M-1 s-1 and 1.1 X 10(6) M-1 s-1, respectively. By comparison with published values of the catalytic efficiencies of several other coagulation enzymes for various substrates, both thrombin and factor Xa are efficient enzymes toward factor VIII:C.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1997

Selective inhibition of the prothrombinase complex: factor Va alters macromolecular recognition of a tick anticoagulant peptide mutant by factor Xa.

The prothrombinase complex assembles through reversible interactions between the protease, factor Xa, the cofactor, factor Va, and acidic phospholipid membranes in the presence of calcium ions. Changes in macromolecular recognition by factor Xa which may result from its interaction with factor Va in the prothrombinase complex have been probed using a recombinant derivative of tick anticoagulant peptide where Arg3 has been replaced with Ala (R3A-TAP). In contrast to the wild type inhibitor, R3A-TAP was a weak competitive inhibitor of factor Xa (Ki = 794 nM). The inhibition of the prothrombinase complex by R3A-TAP was characterized by slow, tight-binding kinetics with an increased affinity of approximately 4000-fold (Ki* = 0.195 nM) relative to that of solution-phase factor Xa. Stopped-flow measurements using p-aminobenzamidine (PAB) demonstrated that the reaction between solution-phase factor Xa and R3A-TAP could be adequately described by a single reversible step with rate constants that were consistent with equilibrium binding measurements. The rate-limiting bimolecular combination of R3A-TAP and factor Xa was competitive with PAB binding of the protease. In contrast, the reaction of R3A-TAP with prothrombinase measured using PAB yielded biphasic stopped-flow traces, indicating a multistep pathway for the reaction of the inhibitor with the enzyme complex. The kinetic measurements were consistent with the initial formation of a ternary complex between R3A-TAP, prothrombinase, and PAB followed by two unimolecular steps which lead to PAB dissociation from the enzyme. In this case, prior occupation of the active site by PAB had no effect on the bimolecular reaction between R3A-TAP and prothrombinase. Thus, the interaction of factor Xa with factor Va on the membrane surface alters recognition of R3A-TAP by the protease, leading to changes in the thermodynamics as well as in the observed kinetic mechanism for the reaction. Therefore, a single amino acid substitution in TAP reveals large changes in macromolecular recognition by factor Xa as a consequence of its interaction with the cofactor within the prothrombinase complex.

Animals↗
PubMed2005

Pharmacological characterization of the active synthetic factor Xa inhibitors M55551 and M55165.

Factor Xa plays an important role in blood coagulation and is widely regarded as an attractive target for antithrombotic drug development. M55551 and M55165 (1-arylsulfonyl-3-piperazinone derivatives) are novel synthetic factor Xa inhibitors. In vitro, M55551 and M55165 competitively inhibited factor Xa with K(i) values of 3.2 nM and 2.3 nM, respectively, and prolonged clotting time in human and rat plasma. Pharmacokinetic analysis of these compounds revealed that M55551 was intravenously active with a short half-life (0.2 h) and that M55165 exhibited good bioavailability (31%) with a long half-life (3.9 h). Therefore, the antithrombotic effects of M55551 and M55165 were compared with those of the intravenous anticoagulant argatroban and the oral anticoagulant warfarin. Intravenous administration of M55551 and oral administration of M55165 inhibited thrombus formation at 0.3 mg/kg and 10 mg/kg, respectively, without significant prolongation of bleeding time. In contrast, although argatroban (0.3 mg/kg) and warfarin (1 mg/kg) also inhibited thrombus formation, significant prolongation of bleeding time was observed at dosages of 3 mg/kg and 1 mg/kg, respectively. These results suggest that M55551 and M55165 are potent factor Xa inhibitors that are active upon intravenous and oral administration, respectively, and that may prove clinically useful for the treatment of thrombosis while minimizing bleeding risks.

Administration, Oral↗
PubMed2003

Effect of vascular injury on inhibition of venous thrombosis with ZK-807834, a direct inhibitor of factor Xa.

Inhibition of factor Xa with the small molecule inhibitor ZK-807834 (Mr 527 Da, Ki 0.11 nM) attenuates progression of thrombosis, but the ED50 is substantially lower for venous compared with arterial thrombosis in experimental animals. To determine whether this reflects differences in the extent of vascular injury, we compared the dose-response of ZK-807834 for inhibition of venous thrombosis induced with a cotton thread and copper wire device in the presence and absence of balloon catheter-induced injury to the vena cava in rabbits. ZK-807834 administration over 2 h (total dosages of 0.0023-2.3 micro mol kg-1, n = 6/group) resulted in dose-dependent reductions in clot weight compared with vehicle controls, but the ED50 was 0.03 micro mol kg-1 for non-injured veins and 0.42 micro mol kg-1 for injured veins. We conclude that vascular injury invokes a tissue factor-mediated response that increases the dose requirements for inhibition of venous thrombosis with ZK-807834.

Amidines↗
PubMed2002

The discovery of YM-60828: a potent, selective and orally-bioavailable factor Xa inhibitor.

Since Factor Xa (FXa) is well known to play a central role in thrombosis and hemostasis, inhibition of FXa is an attractive target for antithrombotic strategies. As a part of our investigation of a non-peptide, orally available FXa inhibitor, we found that a series of N-[(7-amidino-2-naphthyl)methyl]aniline derivatives possessed potent and selective inhibitory activities. Structure--activity relationship (SAR) of the substituent (R(1)) on the central aniline moiety suggested that increasing lipophilicity caused a detrimental effect on anticoagulant activity (prothrombin time assay) in plasma. Several compounds bearing a hydrophilic substituent in R(1) showed not only potent FXa inhibitory activities but also high anticoagulant activities. The best compound in this series was sulfamoylacetic acid derivative (YM-60828) which was a potent, selective and orally bioavailable FXa inhibitor and was chosen for clinical development.

Administration, Oral↗
PubMed1998

Transient high affinity binding of tissue factor pathway inhibitor-factor Xa complexes to negatively charged phospholipid membranes.

The interaction of tissue factor pathway inhibitor (TFPI), factor Xa, and TFPI-factor Xa complexes with negatively charged phospholipid membranes composed of 25 mol % phosphatidylserine and 75 mol % phosphatidylcholine was studied by ellipsometry. The binding of TFPI alone was negligible; factor Xa bound with moderate affinity, with a dissociation constant Kd = 42 nM. Formation of the TFPI-factor Xa complex drastically enhanced the affinity for phospholipid membranes, Kd = 5 nM, compared to that of either protein alone. TFPI1-161, a TFPI variant lacking the third Kunitz domain and the positively charged C-terminus did not enhance binding affinity of the factor Xa. Analysis of the kinetics of adsorption and desorption confirmed the equilibrium binding data, although upon longer residence at the lipid membrane the desorption rate of TFPI-factor Xa complexes became slower, indicating an increase in affinity with longer residence of the TFPI-factor Xa complexes at the membrane. In contrast, binding of TFPI-factor Xa complexes in the presence of an excess factor Xa was transient; maximal binding is followed by a slow desorption of the complex. Immunoblot analysis revealed that this desorption was accompanied with cleavage of TFPI by membrane-bound factor Xa. Collectively, our results show that phosphatidylserine containing membranes will accumulate tightly bound TFPI-factor Xa complexes, and that uncomplexed, phospholipid-bound, factor Xa, will cause limited proteolysis of TFPI accompanied by simultaneous release of these complexes from the phospholipid membrane.

Adsorption↗