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PubMed2001

Inhibition of factor Xa suppresses the expression of tissue factor in human monocytes and lipopolysaccharide-induced endotoxemia in rats.

BACKGROUND: Activated factor X (FXa) is involved in hemostasis, thrombogenesis, inflammation, and cellular immune response. Tissue factor (TF) is an initiator of blood coagulation. We investigated whether FXa induces TF expression in human peripheral monocytes and whether treatment with FXa inhibitor reduces TF expression in an experimental model of rat endotoxemia. METHODS: Human peripheral mononuclear cells were used to determine TF expression induced by FXa. Experimental rat endotoxemia was induced by intravenous bolus injection of lipopolysaccharide (LPS). A specific FXa inhibitor, DX-9065a, was administered subcutaneously immediately after LPS injection. RESULTS: FXa induced TF expression in monocytes without intervention of thrombin and the expression was suppressed by FXa inhibitor. In the experimental model of rat endotoxemia, TF and TF mRNA expression levels in the liver were reduced by DX-9065a. Moreover, administration of DX-9065a suppressed the rise in plasma concentrations of thrombin-antithrombin III complex (TAT) and monocyte chemoattractant protein-1 (MCP-1). CONCLUSIONS: Our results indicated that FXa can induce TF expression in human peripheral monocytes and that inhibition of FXa reduces TF expression in the liver of rat endotoxemia. These results suggest that FXa is an important factor for TF expression in sepsis.

Animals↗

PubMed1987

Inhibition of human blood coagulation factor Xa by alpha 2-macroglobulin.

The inactivation of activated factor X (factor Xa) by alpha 2-macroglobulin (alpha 2M) was studied. The second-order rate constant for the reaction was 1.4 X 10(3) M-1 s-1. The binding ratio was found to be 2 mol of factor Xa/mol of alpha 2M. Interaction of factor Xa with alpha 2M resulted in the appearance of four thiol groups per molecule of alpha 2M. The apparent second-order rate constants for the appearance of thiol groups were dependent on the factor Xa concentration. Sodium dodecyl sulfate gradient polyacrylamide gel electrophoresis was used to study complex formation between alpha 2M and factor Xa. Under nonreducing conditions, four factor Xa-alpha 2M complexes were observed. Reduction of these complexes showed the formation of two new bands. One complex (Mr 225,000) consisted of the heavy chain of the factor Xa molecule covalently bound to a subunit of alpha 2M, while the second complex (Mr 400,000) consisted of the heavy chain of factor Xa molecule and two subunits of alpha 2M. Factor Xa was able to form a bridge between two subunits of alpha 2M, either within one molecule of alpha 2M or by linking two molecules of alpha 2M. Complexes involving more than two molecules of alpha 2M were not formed.

Electrophoresis, Polyacrylamide Gel↗

PubMed2000

Factor Xa: simulation studies with an eye to inhibitor design.

Factor Xa is a serine protease which activates thrombin and plays a key regulatory role in the blood-coagulation cascade. Factor Xa is at the crossroads of the extrinsic and intrinsic pathways of coagulation and, hence, has become an important target for the design of anti-thrombotics (inhibitors). It is not known to be involved in other processes than hemostasis and its binding site is different to that of other serine proteases, thus facilitating selective inhibition. The design of high-affinity selective inhibitors of factor Xa requires knowledge of the structural and dynamical characteristics of its active site. The three-dimensional structure of factor Xa was resolved by X-ray crystallography and refined at 2.2 A resolution by Padmanabhan and collaborators. In this article we present results from molecular dynamics simulations of the catalytic domain of factor Xa in aqueous solution. The simulations were performed to characterise the mobility and flexibility of the residues delimiting the unoccupied binding site of the enzyme, and to determine hydrogen bonding propensities (with protein and with solvent atoms) of those residues in the active site that could interact with a substrate or a potential inhibitor. The simulation data is aimed at facilitating the design of high-affinity selective inhibitors of factor Xa.

Amino Acid Sequence↗

PubMed1986

Effect of limited modification of amino groups on the reactivity of human factor Xa.

The basis of the specificity of human coagulation factor Xa has been probed with a reagent that reacts with nucleophiles, N-succinimidylpropionate. At pH 8.0 and 0.25 mM N-succinimidylpropionate, 0.4 microM factor Xa lost approx. 90% of its activity toward prothrombin in 4 min. The decay was first-order, k = 0.64 min-1, which increased to 0.98 min-1 in 1 mM Ca2+, and the dependence of k upon pH was consistent with primary amines being the target. The rate of modification was unaffected by the presence of a tetrapeptide substrate during modification; likewise, activity toward a tripeptide p-nitroanilide was unaltered during exposure of factor Xa to N-succinimidylpropionate with or without Ca2+. In addition, inhibition by antithrombin III was retained with a somewhat enhanced rate after modification; however, the acceleration of this by heparin was significantly less. Kinetic determination of the number of residues modified gave a reaction order of 2.0, while reaction with N-succinimidyl[3H]propionate yielded labeled factor Xa containing 1.0 mol N-succinimidylpropionate/mol factor Xa and 50% normal clotting activity, or 2.0 mol N-succinimidylpropionate/mol and 1% activity, respectively. Thus, one nucleophilic group is required for the reaction of factor Xa with prothrombin but not for the hydrolysis of peptides or recognition of antithrombin III. The decay of clotting activity of the factor X zymogen in N-succinimidylpropionate was much slower though still Ca2+-dependent. Conversely, the reaction of a related compound--N-succinimidyl(4-hydroxyphenyl)propionate or Bolton-Hunter reagent--with factor Xa broadly resembled that of N-succinimidylpropionate but the decay curves indicated more complex kinetics. Therefore, the target groups vary in their accessibility to modification according to the structural characteristics of both the protein and the reagent.

Antithrombin III↗

PubMed1992

Mechanisms of binding of recombinant extrinsic pathway inhibitor (rEPI) to cultured cell surfaces. Evidence that rEPI can bind to and inhibit factor VIIa-tissue factor complexes in the absence of factor Xa.

Extrinsic pathway inhibitor plays a key role in modulating tissue factor-dependent blood coagulation. We have studied binding of radioiodinated recombinant extrinsic pathway inhibitor (rEPI) to cultured cell surfaces. rEPI in the absence of added reactants bound to a limited extent to three cell lines studied. Binding of rEPI to two cell lines possessing surface tissue factor, but not to a cell line lacking surface tissue factor, was markedly increased in the presence of both factor VIIa and factor Xa, and calcium ions. Moreover, some increased tissue factor-dependent binding was also demonstrated with factor VIIa alone. Binding isotherms of rEPI to factor VIIa-tissue factor obtained with an ovarian carcinoma cell line were hyperbolic. Scatchard plots indicated the following: a Kd value of 4.5 +/- 1.5 nM and 335,000 +/- 84,000 sites/cell when factor Xa was present; a Kd value of 11.9 +/- 3.5 nM and 236,000 +/- 68,000 sites/cell when factor Xa was absent. In functional studies, high concentrations of rEPI, e.g. 27-67.5 nM, were found to inhibit factor VIIa-tissue factor-catalyzed release of activation peptide from tritiated factor IX in the absence of factor Xa. Whereas factor Xa was thus shown not to be required for rEPI to inhibit factor VIIa-tissue factor catalytic activity, its presence markedly enhanced rEPI's inhibitory function. Since the local concentration of extrinsic pathway inhibitor achieved at a site of tissue injury is unknown, the physiologic significance of the observation of extrinsic pathway inhibitor-induced inhibition of factor VIIa-tissue factor activity in the absence of factor Xa is not clear. However, factor Xa-independent inhibition could play a significant role when large doses of rEPI are administered in experimental studies of thrombosis.

Antibodies↗

PubMed1981

Cofactor dependence of factor Xa incorporation into the prothrombinase complex.

The blood coagulation serine protease, factor Xa, was dansylated with the active site-directed inhibitor, dansyl-glutamyl-glycyl-arginyl chloromethyl ketone. The Ca2+-dependent interactions of the inactivated factor Xa with its cofactors, phospholipid and activated factor V (factor Va), were studied through alterations of fluorescence polarization values of dansyl moiety of the modified enyme. In the presence of phospholipid and Ca2+, factor Va and factor Xa interacted with 1:1 stoichiometry, an interaction characterized by markedly enhanced polarization. The factor Va-independent interaction of factor Xa with phospholipid was also observed, characterized by dissociation constant Kd=2.7 x 10-6 M and stoichiometry of 66 mol of phospholipid/mol of factor Xa. The interaction of factor Xa with vesicles in the absence of factor Va exhibited considerably lower polarization values than in the presence of factor Va. These data obtained by direct spectral measurements are in agreement with the inferences drawn previously from studies of kinetics that the prothrombinase complex consists of 1:1 stoichiometric complex of factor Xa and phospholipid-bound factor Va, and that the enzymatic complex assembles in the absence of the natural substrate, prothrombin.

Amino Acid Chloromethyl Ketones↗

PubMed2002

Crystal structures of two potent nonamidine inhibitors bound to factor Xa.

There has been intense interest in the development of factor Xa inhibitors for the treatment of thrombotic diseases. Our laboratory has developed a series of novel non-amidine inhibitors of factor Xa. This paper presents two crystal structures of compounds from this series bound to factor Xa. The first structure is derived from the complex formed between factor Xa and compound 1. Compound 1 was the first non-amidine factor Xa inhibitor from our lab that had measurable potency in an in vitro assay of anticoagulant activity. The second compound, 2, has a molar affinity for factor Xa (K(iapp)) of 7 pM and good bioavailability. The two inhibitors bind in an L-shaped conformation with a chloroaromatic ring buried deeply in the S1 pocket. The opposite end of these compounds contains a basic substituent that extends into the S4 binding site. A chlorinated phenyl ring bridges the substituents in the S1 and S4 pockets via amide linkers. The overall conformation is similar to the previously published structures for amidine-based inhibitors complexed with factor Xa. However, there are significant differences in the interactions between the inhibitor and the protein at the atomic level. Most notably, there is no group that forms a salt bridge with the carboxylic acid at the base of the S1 pocket (Asp189). Each inhibitor forms only one well-defined hydrogen bond to the protein. There are no direct charge-charge interactions. The results indicate that electrostatic interactions play a secondary role in the binding of these potent inhibitors.

Amidines↗

PubMed2002

Effects of a synthetic factor Xa inhibitor (JTV-803) on various laboratory tests.

Synthetic direct inhibitors of factor Xa are capable of prolonging the global anticoagulant assay times in a concentration-dependent fashion. The relative degree of thrombin generation inhibition at an equivalent prolongation is not similar to the results observed with heparins and oral anticoagulant drugs. In addition, the direct factor Xa inhibitors prolong the Russell's viper venom test (RWT) and Heptest clotting times. Ecarin clotting time (ECT) and thrombin time (TT) remain unaffected. The kinetics of factor Xa inhibition are markedly different than those observed with pentasaccharide and heparins. Therefore, the methods developed for heparins and pentasaccharides may not be applicable for the monitoring of factor Xa inhibitors. To test the feasibility of using the prothrombin time (PT), International Normalized Ratio (INR), activated partial thromboplastin time (aPTT), Heptest, thrombin time, RVVT, ECT, and a modified anti-Xa amidolytic assay, to monitor a synthetic factor Xa inhibitor, normal human pool plasma samples were spiked with a synthetic factor Xa inhibitor in the concentration range of 0 to 1 microg/mL and 0 to 25 microg/mL. Different laboratory tests were performed and INR and other ratios were calculated. The anticoagulant effects on whole blood were measured using the activated clotting time (ACT). Further studies on the effect of factor Xa inhibitor on platelet aggregation; factor II, VII, and X functional levels; and fibrinopeptide A (FPA) generation were carried out at equivalent INR levels in comparison to oral anticoagulant and antithrombin agents. FPA generation at equivalent anticoagulant level in comparison to heparin (twice the baseline) was also carried out. Factor Xa inhibitor produced a concentration-dependent prolongation of the ACT. ACT was doubled at a concentration of 4 to 5 microg/mL. There was a marked difference in the prolongation of the PT by a synthetic factor Xa inhibitor dependent on the ISI of the PT reagent used. When the results were calculated to determine INR, marked variations were noted between the recombinant thromboplastin and rabbit brain thromboplastin. The rabbit brain thromboplastin reagent gave markedly high INR values. Similar results were observed when different aPTT reagents were studied. In the anti-Xa assay, modification of the incubation time was employed to extend the proper sensitivity range. These studies warrant further investigation to understand the mechanism of action of factor Xa inhibitors.

Anticoagulants↗

PubMed1995

Prothrombin contributes to the assembly of the factor Va-factor Xa complex at phosphatidylserine-containing phospholipid membranes.

The activation of prothrombin is catalyzed by prothrombinase, a complex of factor Xa and factor Va assembled on a negatively charged phospholipid membrane. We used a tubular flow reactor to identify the relative contributions of factor Va, prothrombin, and the negatively charged phosphatidylserine to the assembly of prothrombinase. Perfusion of phospholipid-coated capillaries with a mixture of factor Xa, factor Va, and prothrombin resulted in a steady-state rate of thrombin production that increased with (i) the phosphatidylserine content of the phospholipid bilayer, (ii) the factor Va concentration, and, most interestingly, (iii) the prothrombin concentration of the perfusion solution. Incorporation of 20 mol % phosphoatidylethanolamine, a phospholipid with poor ability to promote prothrombinase activity, into a 5 mol % phosphatidylserine membrane also increased the steady-state rate of thrombin production. Direct measurements of the amount of prothrombinase in the flow reactor demonstrated that increased catalytic activities were the result of an increased steady-state amount of membrane-associated prothrombinase. Thus, similar turnover numbers of prothrombin activation (3100 min-1) were calculated, irrespective of the phosphatidylserine content of the membrane. We established for membranes with low phosphatidylserine content (< 10 mol%) a linear relationship between the prothrombinase activity and the arithematical product of the factor Va concentration in the perfusion solution and the prothrombin concentration near the catalytic surface. Our results indicate that, in addition to factor Va, prothrombin also is essential to the assembly of prothrombinase at macroscopic surfaces with low phosphatidylserine content. The data further suggest that the prothrombin concentration near the surface, controlled by the prothrombinase activity and mass transfer, is an important regulator of the prothrombinase surface density.

Factor Va↗

PubMed1989

Unfractionated heparin inhibits thrombin-catalysed amplification reactions of coagulation more efficiently than those catalysed by factor Xa.

We have proposed previously that the steps in coagulation most sensitive to inhibition by heparin are the thrombin-dependent amplification reactions, and that prothrombinase is formed in heparinized plasma only after Factor Xa activates Factor VIII and Factor V. These propositions were based on the demonstration that both heparin and Phe-Pro-Arg-CH2Cl completely inhibited 125I-prothrombin activation for up to 60 s when contact-activated plasma (CAP) was replenished with Ca2+. Furthermore, the addition of thrombin to CAP before heparin or Phe-Pro-Arg-CH2Cl completely reversed their inhibitory effects. Additional support for the above hypotheses is provided in this study by demonstrating that, when the activity of thrombin is suppressed by heparin (indirectly) or by Phe-Pro-Arg-CH2Cl (directly), exogenous Factor Xa reverses the ability of these two agents to inhibit prothrombin activation. Prothrombin activation was initiated by adding Factor Xa (1 nM) or thrombin (1 or 10 nM) simultaneously with CaCl2 to CAP. In the absence of heparin or Phe-Pro-Arg-CH2Cl, prothrombin activation was seen 15 s later in either case. Heparin failed to delay, and Phe-Pro-Arg-CH2Cl delayed for 15 s, prothrombin activation in CAP supplemented with Factor Xa. In contrast, heparin and Phe-Pro-Arg-CH2Cl completely inhibited prothrombin activation for at least 45 s in CAP supplemented with 1 nM-thrombin. Heparin failed to delay prothrombin activation in CAP supplemented with 10 nM-thrombin, whereas Phe-Pro-Arg-CH2Cl completely inhibited prothrombin activation in this plasma for 45 s. These results suggest that in CAP: (1) Factor Xa can effectively activate Factor VIII and Factor V when the proteolytic activity of thrombin is suppressed; (2) heparin-antithrombin III is less able to inhibit Factor Xa than thrombin; (3) suppression of the thrombin-dependent amplification reactions is the primary anticoagulant effect of heparin.

Antithrombin III↗

PubMed1992

Molecular recognition sites on factor Xa which participate in the prothrombinase complex.

Coagulation factor X, when activated to factor Xa by proteolytic cleavage, itself becomes an active serine protease which participates as a component of the macromolecular prothrombinase complex along with factor Va, phospholipid, and calcium ions. To identify specific structural regions on factor Xa responsible for mediating its function in activating prothrombin, we used 21 synthetic peptides corresponding to 65% of the primary structure of factor X as potential inhibitors of prothrombin activation. Using purified components, thrombin formation was inhibited by seven peptides in a dose-dependent noncompetitive manner. Antibodies to selected inhibitory peptides affinity purified on a factor Xa-agarose column inhibited thrombin formation in a dose-dependent manner, indicating that the corresponding regions on factor Xa are surface-exposed. Kinetic analyses varying the order of reagent addition suggested that peptides 211-222, 254-269, and 263-274 were highly effective in preventing the factor Xa-factor Va interaction. Peptides 275-287 and 415-425 were considered to derive from a distal region involved in substrate binding, based upon mixed inhibition kinetic analyses and assuming that inhibitory peptides not inhibitory in factor Va binding are related to a specific region of substrate interaction. Cross-linking studies confirmed that peptides 263-274 and 263-276 could bind specifically to the light chain of factor V/Va. These findings provide the basis for further pursuing the precise definition of interactive sites on factor Xa using site-directed mutagenesis and molecular modeling.

Amino Acid Sequence↗

PubMed1986

Factor Xa inactivation by a heparinized hydrogel.

Incubating Factor Xa and plasma in the presence of "beads" of a heparinized hydrogel (heparin-PVA) resulted in a loss of Factor Xa activity as measured by clotting time or chromogenic substrate assays. This loss in activity was attributed to the activity of the heparin immobilized to the polyvinyl alcohol (PVA) hydrogel. The immobilized heparin retained an apparent activity approximately 1% that of a similar mass of heparin in solution. The loss in apparent activity was in part attributed to diffusion effects which limited the accessibility of the enzyme and inhibitor to the heparin immobilized in the interior of the gel "beads". The chromogenic substrate activity of adsorbed Factor Xa was also measured and was found to decrease after exposure to antithrombin III to similar extents regardless of the sequence of exposure. This suggests that Factor Xa and antithrombin III have similar affinities for this immobilized heparin, unlike the situation for thrombin (Thromb-Res., 20, 543-554, 1980).

Factor X↗

PubMed1994

Mutational analysis of antistasin, an inhibitor of blood coagulation factor Xa derived from the Mexican leech Haementeria officinalis.

Antistasin is a Factor Xa inhibitor that is present in the salivary glands of the Mexican leech Haementeria officinalis. The antistasin protein consists of 119 amino acids, of which residues 1-55 (domain I) are 56% similar to residues 56-110 (domain II). Of the nine C-terminal amino acids (residues 111-119; domain III), four are positively charged. The reactive site for Factor Xa is located in domain I. In this study we assessed the role of separate domains and of individual amino acids in the reactive site for the inhibition of Factor Xa. A series of mutants was constructed and expressed in Chinese hamster ovary (CHO) cells. In vitro chromogenic assays for Factor Xa show that domain I is sufficient for inhibition of Factor Xa. Domains II and III neither contain any intrinsic Factor Xa inhibitory activity, nor contribute to the activity of domain I. Furthermore, domain II does not become a Factor Xa inhibitor by partially adaptating its sequence towards that of the reactive site in domain I. Mutation of the cysteine at position 33 is not crucial for Factor Xa inhibition, suggesting a relatively rigid reactive site loop structure.

Amino Acid Sequence↗

PubMed1998

Effector protease receptor 1 mediates the mitogenic activity of factor Xa for vascular smooth muscle cells in vitro and in vivo.

The binding of 125I-factor Xa to human aortic smooth muscle cell (SMC) monolayers was studied. At 4 degreesC, 125I-factor Xa bound to a single class of binding sites with a dissociation constant value of 3.6+/-0.7 nM and a binding site density of 11,720+/-1,240 sites/cell (n = 9). 125I-factor Xa binding was not affected by factor X, thrombin, or by DX9065, a direct inhibitor of factor Xa, but was inhibited by factor Xa (IC50 = 5.4+/-0.2 nM; n = 9) and by antibodies specific for the effector cell protease receptor 1 (EPR-1), a well-known receptor of factor Xa on various cell types. A factor X peptide duplicating the inter-EGF sequence Leu83-Leu88-(Gly) blocked the binding of 125I-factor Xa to these cells in a dose-dependent manner (IC50 = 110+/-21 nM). Factor Xa increased phosphoinositide turnover in SMCs and when added to SMCs in culture was a potent mitogen. These effects were inhibited by DX9065 and by antibodies directed against EPR-1 and PDGF. Increased expression of EPR-1 was identified immunohistochemically on SMCs growing in culture and in SMCs from the rabbit carotid artery after vascular injury. When applied locally to air-injured rabbit carotid arteries, antibodies directed against EPR-1 (100 mug/ artery) strongly reduced myointimal proliferation 14 d after vascular injury (65-71% inhibition, P < 0.01). DX9065 (10 mg/kg, subcutaneous) inhibited myointimal proliferation significantly (43% inhibition, P < 0.05). These findings indicate that SMCs express functional high affinity receptors for factor Xa related to EPR-1, which may be of importance in the regulation of homeostasis of the vascular wall and after vascular injury.

Animals↗

PubMed2003

Clinical trials with factor Xa inhibition in the prevention of postoperative venous thromboembolism.

Factor Xa inhibition is a selective mechanism used for anticoagulation therapy. Clinical trials of drugs designed to inhibit factor Xa for the prevention of venous thromboembolism (VTE) after major orthopedic surgery are discussed. Hemostasis is a balanced process of blood coagulation and clot dissolution. Factor Xa is located at a key point in the clotting cascade, since it bridges the extrinsic and intrinsic pathways of this process. Inhibition of factor Xa via antithrombin (AT) by fondaparinux has been studied extensively. Fondaparinux selectively binds to AT, producing rapid anticoagulation with an approximate 300-fold potentiation of the natural inhibitory activity of AT against factor Xa. The anticoagulant demonstrates a linear dose response and is eliminated renally. Comparative trials conducted in major hip and knee surgeries established the efficacy and safety of fondaparinux in this setting. Overall, fondaparinux significantly reduced the incidence of VTE by day 11 (6.8%) compared with enoxaparin (13.7%)--common odds reduction of 55.2%. No difference in the incidence of clinically relevant bleeding (e.g, major bleeding leading to death or reoperation, or occurring in a critical organ) was observed. An increase in the occurrence of major bleeding was observed when the first dose was administered prior to 6 hours after surgical closure, resulting in the recommendation that the first dose be given 6 to 8 hours postsurgically. With strict adherence to recommendations for its use, fondaparinux may be used safely in certain special populations. Results from these large controlled trials predict that this factor Xa inhibitor will receive the highest recommendation in subsequent evidence-based guidelines for the prevention of VTE following major hip and knee surgeries, including surgery for hip fracture repair.

Antithrombin III↗

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↗

PubMed1998

Factor Xa induces cytokine production and expression of adhesion molecules by human umbilical vein endothelial cells.

Proinflammatory effects induced by the serine protease factor Xa were investigated in HUVEC. Exposure of cells to factor Xa (5-80 nM) concentration dependently stimulated the production of IL-6, IL-8, and monocyte chemotactic protein-1 (MCP-1) and the expression of E-selectin, ICAM-1, and VCAM-1, which was accompanied by polymorphonuclear leukocyte adhesion. The effects of factor Xa were blocked by antithrombin III, but not by the thrombin-specific inhibitor hirudin, suggesting that factor Xa elicits these responses directly and not via thrombin. IL-1alpha and TNF-alpha were not implicated, since neither the IL-1 receptor antagonist nor a TNF-neutralizing Ab could suppress the factor Xa responses. Active site-inhibited factor Xa and factor Xa depleted from gamma-carboxyglutamic acid residues were completely inactive. The effector cell protease receptor-1 (EPR-1) seems not to be involved since anti-EPR-1 Abs failed to inhibit cytokine production. Moreover, neither the factor X peptide Leu83-Leu88, representing the inter-epidermal growth factor sequence in factor Xa that mediates ligand binding to EPR-1, nor the peptide AG1, corresponding to the EPR-1 sequence Ser123-Pro137 implicated in factor Xa binding, inhibited the factor Xa-induced cytokine production. In conclusion, these findings indicate that factor Xa evokes a proinflammatory response in endothelial cells, which requires both its catalytic and gamma-carboxyglutamic acid-containing domain. The receptor system involved in these responses induced by factor Xa remains to be established.

Cells, Cultured↗

PubMed1997

Coagulation factor Xa induces endothelium-dependent relaxations in rat aorta.

The relaxing effect of coagulation factor Xa on phenylephrine-contracted rat aortic rings was compared with the effect of thrombin and trypsin. All three proteases induced a dose-dependent relaxation in the presence of an intact endothelium. EC50 values were 3 +/- 1, 24 +/- 9, and 16 +/- 1 nmol/L for thrombin, trypsin, and factor Xa, respectively. Whereas thrombin induced rapid relaxations followed by partial recontraction, trypsin and factor Xa induced slower sustained effects. Factor Xa-induced relaxations were not affected by hirudin at high concentrations (1 mumol/L) but were abolished by DX9065A, a specific inhibitor of the catalytic activity of factor Xa. Furthermore, no relaxations to factor Xa could be elicited in the presence of the NO synthase inhibitor N omega-nitro-L-arginine methyl ester (100 mumol/L), whereas relaxations were not altered in the presence of the inactive enantiomer N omega-nitro-D-arginine methyl ester (100 mumol/L). Addition of factor Xa together with thrombin induced relaxations that were larger than those induced by thrombin alone, whereas factor Xa had no additional effects on trypsin-induced relaxations. Further-more, factor Xa relaxed thrombin-desensitized aortic rings but was ineffective in trypsin-desensitized tissues. These data suggest that factor Xa acts on a cleavable endothelial receptor that induces NO release, resulting in the relaxation of precontracted rat aortic rings. Factor Xa does not act through endothelial thrombin receptors but may activate another cleavable trypsin-sensitive receptor.

Animals↗