Xa Activity Research Articles

A widely applicable set of descriptors

Three sets of molecular descriptors computable from connection table information are defined. These descriptors are based on atomic contributions to van der Waals surface area, log P (octanol/water), molar refractivity, and partial charge. The descriptors are applied to the construction of QSAR/QSPR models for boiling point, vapor pressure, free energy of solvation in water, solubility in water, thrombin/trypsin/factor Xa activity, blood-brain barrier permeability, and compound classification. The wide applicability of these descriptors suggests uses in QSAR/QSPR, combinatorial library design, and molecular diversity work.

Pharmacologic modulation of thrombin generation associated with human clots by human purified antithrombin alone or in the presence of low molecular weight heparin or unfractionated heparin

Procoagulant activities associated with human clots may contribute to thrombus extension. We investigate the inhibition of clot-associated factor Xa and thrombin activities by purified human antithrombin either alone or as combination with a low molecular weight heparin (enoxaparin) as compared with unfractionated heparin (UFH). The standard clots were prepared by recalcification of frozen platelet-poor human plasma. Clot-associated thrombin was measured on the clot after clot incubation in recalcified buffer or recalcified prothrombin solution. The enzymatic reaction was measured using a specific substrate for thrombin (CBS 3447). The thrombin concentration was determined both on the clots and in the reaction mixtures. In parallel, prothrombin fragment 1.2 and thrombin-antithrombin complexes (TAT) were measured using enzyme-linked immunosorbent assay methods. We demonstrated that in the presence of purified human prothrombin and antithrombin (AT), a partial inhibition of clot associated thrombin activity correlated with an increase of TAT complexes. However, antithrombin was unable to inhibit thrombin generation induced by the clot-associated factor Xa. Enoxaparin (low molecular weight heparin) and UFH did not enhance clot-bound thrombin inhibition induced by AT. We conclude that clot-bound thrombin is accessible to human antithrombin alone. AT is also able to inhibit thrombin generated by factor Xa-associated clot. However, neither a low molecular weight heparin or UFH enhanced the effect of AT alone.

Platelet-dependent and procoagulant mechanisms in arterial thrombosis

The results of experiment and clinical studies support the hypothesis that initiation and progression of arterial thrombosis are dependent on a dynamic interaction between platelet- and coagulation-dependent mechanisms. Although treatment of patients with unstable coronary syndromes acutely with glycoprotein IIb/IIIa inhibitors has already been shown to be remarkably effective in improving short- and long-term prognosis, strategies that inhibit factor thrombin and/or Xa activity may also have a role either alone or in combination with platelet inhibitors in treating patients with coronary thrombosis.

EFFICACY AND PHARMACOKINETICS OF NEUTRALASE[trade mark sign] IN CABG

Introduction. Neutralase[trade mark sign] (heparinase I) is a 42.5 kD enzyme which neutralizes heparin by cleaving it into primarily di- and tetrasaccharide fragments. [1] The activated clotting time (ACT) of heparinized volunteers returned to baseline after Neutralase administration, [2] leading to this phase II dose ranging study in elective coronary artery bypass graft (CABG) patients. Methods. Six institutions enrolled 49 patients with IRB approval into 3 sequential dosing groups in an open-label design: 5 mcg/kg (n=12), 7 mcg/kg (n=25) and 10 mcg/kg (n=12). Porcine mucosal heparin anticoagulation was reversed with bolus Neutralase at cardiopulmonary bypass (CPB) conclusion. Measurements included ACT (3 min prior to bolus and 3, 6, and 9 min thereafter), Neutralase blood concentrations, anti-Factor IIa and Xa activity (multiple timepoints) and chest tube drainage (CTD) over 24 hrs. An analysis-of-variance model with treatment as a main effect was used in assessing mean ACT and median (mean rank) chest tube drainage. Results. Figure 1 demonstrates a dose response relationship for ACT return to baseline at 9 min. Neutralase rapidly and completely eliminated anti-IIa activity, and decreased anti-Xa activity to 20% at 9 min and 6% at 12 hrs. Mean (SD) plasma half-life was 12.1 (2.9) min compared to 6.6 (2.1) min in volunteers. Figure 2 displays 12 hr CTD data.Figure 1Figure 2Discussion. The data support 7 mcg/kg as an appropriate dose for investigational clinical use after CPB: 5 mcg/kg yields more bleeding, while 10 mcg/kg yields an equivalent blood loss; and both 7 and 10 mcg/kg are clinically adequate for heparin reversal.

Combined Use of Markers of Muscle Necrosis and Fibrinogen Conversion in the Early Differentiation of Myocardial Infarction and Unstable Angina

Intracoronary formation of blood clots on ruptured arteriosclerotic plaques is considered the main cause of acute myocardial infarction (AMI) (1). After such ruptures, exposed tissue factor binds to factor VIIa from plasma, and the resulting tissue factor-factor VIIa complex activates factor X toward factor Xa, the enzyme converting prothrombin to thrombin. By cleavage of fibrinopeptides A and B, thrombin produces desAABBfibrin monomers that polymerize into still-soluble complexes called “thrombus precursor proteins” (TpPs). New antigens formed on these complexes were used for a TpP assay (2). Because the acute thrombotic event precedes coronary occlusion and muscle necrosis, detection of activated coagulation potentially allows early detection of AMI. Until now, attempts in this field have focused on markers for factor Xa and thrombin activity, such as prothrombin fragment 1.2 and thrombin-antithrombin complexes. However, these markers are not necessarily closely related to the actual formation of fibrin clots (3), especially in chronically hypercoagulable patients, and TpP could perform better in this respect. We therefore studied plasma concentrations of TpP and fibrin monomers (FMs) in patients with suspected AMI. The results were compared with two small cytosolic cardiac marker proteins, myoglobin (Mb) and fatty acid-binding protein (FABP), that are early markers for necrosis (4 , 5) and with a highly …

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.

Identification and Characterization of Anticoagulant Activities in the Saliva of the Lone Star Tick, Amblyomma americanum (L.)

Anticoagulant activities against both the extrinsic and intrinsic coagulation pathways were identified in the saliva of partially fed female lone star ticks, Amblyomma americanum (L.). The activities of factor Xa and thrombin in the common pathway of the coagulation cascade were inhibited by tick saliva. The greatest anticoagulant activities were found in the saliva of ticks weighing more than 200 mg. The anticoagulant activities in tick saliva could be detected without preincubation of tick saliva with sheep plasma, but preincubation significantly increased the activities. Tick saliva anticoagulant activities were abolished by boiling for 15 min or being treated with trypsin for 1 hr. Phosphatidylcholine (3 mM) and phospholipase A2 inhibitor oleyloxyethyl phosphorylcholine (0.2 mM) did not affect the anticoagulant activities significantly, suggesting that the phospholipase A2 activity found in tick saliva does not contribute to the anticoagulant activities. Size exclusion high performance liquid chromatography revealed that the molecular weights of the anticoagulant activities were approximately 16,000 D. The anticoagulant activities in tick saliva are believed to play an important role in facilitating tick feeding by helping overcome the host hemostatic system.

Absolute and Comparative Subcutaneous Bioavailability of Ardeparin Sodium, a Low Molecular Weight Heparin

Ardeparin sodium (Normiflo, Wyeth-Ayerst) is a low molecular weight heparin undergoing clinical evaluation as an antithrombotic agent. The objective of this study was to evaluate the absolute and comparative bioavailability of ardeparin following subcutaneous administration of three different formulations [two formulations of ardeparin at 10,000 anti-factor Xa (aXa) U/ml, but with different preservatives, and a 20,000 aXa U/ml formulation]. The study was conducted using a randomized 4-period crossover design (three subcutaneous treatments and one intravenous treatment) in 24 healthy subjects, and the pharmacokinetics of ardeparin were characterized by plasma anti-factor IIa (aIIa) and anti-factor Xa (aXa) activities. The mean absolute bioavailability of ardeparin based on aIIa activity ranged from 62% to 64% and the mean absolute bioavailability based on aXa activity ranged from 88% to 97%. Based on bioequivalence testing criteria, the three ardeparin formulations were bioequivalent.

The role of thrombin in coronary artery thrombosis

The critical role of thrombin in mediating platelet-rich thrombosis has been defined extensively in experimental and, more recently, clinical studies. The results of these studies suggest that the extent of thrombin activity after atherosclerotic plaque rupture is dependent on tissue factor-mediated activation of the coagulation system and activation of factor Xa. Local factor Xa-Va complex activity, in turn, is dependent on platelet adhesion and activation, which are potentiated by thrombin. Thus, the initiation and progression of coronary artery thrombosis seems to be dependent on a dynamic interaction between procoagulant and platelet-related factors that result in the elaboration of thrombin and platelet-rich thrombosis.

Effects of low molecular weight heparin on a severely antithrombin III-decreased diseminated intravascular coagulation model in rabbits

The effect of dalteparin, a low molecular weight heparin, on severely antithrombin III (ATIII)-decreased disseminated intravascular coagulation (DIC) model was compared with that of unfactionated heparin (heparin). The DIC model in rabbits was produced by continuous infusion of thrombin in combination with bolus injection of latex. After a 3 hr infusion of thrombin, plasma ATIII activity was lowered to 30 % of normal plasma. Platelet number, fibrinogen content and α 2 plasmin inhibitor (α 2PI) activity were also decreased. Dalteparin (25–100 IU/kg/hr) and heparin (25–100 U/kg/hr) inhibited the decrease in ATIII activity, platelet number and fibrinogen content, and had no effect on α 2PI activity. Activated partial thromboplastin time (APTT) was prolonged by heparin (50 and 100 U/kg/hr), but not by dalteparin (25–100 IU/kg/hr). The ratio of anti-factor Xa (F.Xa) activity to anti-thrombin activity for dalteparin (50 IU/kg/hr) was higher than that for heparin (50 U/kg/hr). With the addition of exogenous ATIII, the ratio of anti-F.Xa to anti-thrombin for heparin increased, but that for dalteparin did not change. However, the increased ratio for heparin was still lower than the unchanged ratio for dalteparin. These results suggest that both dalteparin and heparin have the ability to rectify the abnormal parameters of severely ATIII-decreased DIC, and that the effects of dalteparin are mainly involved with anti-F.Xa activity whereas the effects of heparin are via anti-thrombin activity.

An Introduction to Low-Molecular-Weight Heparins and Their Use in the Treatment of Deep Venous Thrombosis.

Low-molecular-weight heparins (LMWHs) are fractions of standard heparin (SH) with an average molecular weight of 4000--5000 daltons. They have the ability to inhibit the activity of factor Xa in the coagulation cascade but are unable to significantly change the activity of thrombin (factor IIa). Therefore, LMWHs have antithrombotic properties, but they do not prolong the activated partial thromboplastin time (APTT). In contrast to SH, the pharmacokinetic profiles of LMWHs are different. LMWHs exhibit minimal binding with plasma proteins, endothelial cells and platelet factor IV, a longer half-life, and linear elimination. Due to these properties, the clinical response is more predictable than that seen with SH. In addition, monitoring of clotting factors and subsequent dosage adjustment are not necessary to ensure efficacy when using a LMWH. These agents may have a decreased risk of thrombocytopenia and bleeding. LMWHs have been used in the initial treatment of deep venous thrombosis (DVT) and have been found to be safe and efficacious. When compared to SH, the LMWHs studied have been more effective in improving venographic scores and short-term treatment, but further evaluation is needed to determine if a benefit exists in preventing recurrent DVT.

Chrono-Pharmacological Study of Once Daily Curative Dose of a Low Molecular Weight Heparin (200IU antiXa/kg of Dalteparin) in Ten Healthy Volunteers

Low molecular weight heparin (LMWH) is currently prescribed for the treatment of deep vein thrombosis at the dose of 100 IU antiXa/kg twice daily or at a dose of 175 IU antiXa/kg once daily with a similar efficacy. We decided to study the chrono-pharmacology of curative dose of LMWH once daily administrated according to the one previously described with unfractionated heparin (UFH). Ten healthy volunteers participated in an open three-period crossover study according to three 24 h cycles, separated by a wash-out interval lasting 7 days: one control cycle without injection, two cycles with subcutaneous injection of 200 IU antiXa/kg of Dalteparin (Fragmin) at 8 a.m. or at 8 p.m. Parameters of heparin activity were analysed as maximal values and area under the curve. Activated partial thromboplastin time (APTT), thrombin time (TT), prothrombin time (PT) and tissue factor pathway inhibitor (TFPI) were higher after 8 p.m. injection than after 8 a.m. injection (p < 0.05) while no chrono-pharmacological variation of anti factor Xa (AXa) activity was observed. Thus the biological anticoagulant effect of 200 IU antiXa/kg of Dalteparin seems to be higher after an evening injection than after a morning injection. A chrono-therapeutic approach with LMWH, as prescribed once daily, deserves further investigation since our results suggest that a preferential injection time may optimise the clinical efficacy of these LMWH.

Neutralization of the anticoagulant activity of low molecular weight heparin LU 47311 (Clivarin®) in man by protamine chloride

Bleeding induced by unfractionated heparin (UFH) can be antagonized by protamine as shown by normalization of thrombin time and aPTT. In order to learn about the neutralization capacity of protamine against the anticoagulant effects of LU 47311 a comparison study vs UFH was performed in 12 healthy male volunteers. Whereas the prolongation of aPTT and thrombin time induced by both heparins was reversed, inhibition of anti F Xa activity was not. A anti F Xa activity following injection of UFH was immediately antagonized by only 20 – 40 % with LU 47311. A rebound phenomenon of LU 47311 after protamine chloride was not detected. The platelet system remained unchanged.

Isolation and characterization of an intracellular serine proteinase inhibitor from a monkey kidney epithelial cell line.

A recent report described a thrombin inhibitory activity in the soluble fraction of human placenta and the cytosolic fraction of K562 cells. Isolation and characterization of the functionally inactive 35-38-kDa placental form of this protein revealed that it was a novel serine proteinase inhibitor (Coughlin, P. B., Tetaz, T., and Salem, H. H. (1993) J. Biol. Chem. 268, 9541-9547). In the present study, we observed a 67-kDa sodium dodecyl sulfate (SDS)-stable complex when 125I-thrombin was incubated with the cytosolic fraction of a monkey kidney epithelial cell line, BSC-1. This complex was not observed in either the particulate cell fraction extracted with 0.2% Triton X-100 or medium conditioned by cells, suggesting that the thrombin-complexing factor is confined to the cytoplasm. The cytoplasmic antithrombin activity was purified to apparent homogeneity from the cytosol of BSC-1 cells previously pulsed with [35S]methionine by a combination of heparin-agarose chromatography, Mono Q fast protein liquid chromatography, and anhydrotrypsin-Affi-Gel 10 affinity chromatography. Analysis of the affinity-purified preparation by SDS-polyacrylamide gel electrophoresis and fluorography revealed a single protein with an apparent molecular mass of 38 kDa. The purified 38-kDa protein inhibited the amidolytic activities of thrombin, trypsin, urokinase, and factor Xa but not that of elastase. Incubation of the 38-kDa protein with excess thrombin identified approximately 60% of the labeled 38-kDa protein in an SDS-stable 67-kDa complex. The purified 38-kDa inhibitor was cleaved with cyanogen bromide and the isolated peptides subjected to microsequencing. Amino acid sequence obtained for a region within this protein exhibited significant homology with human antithrombin III and plasminogen activator inhibitors 1 and 2. This homologous peptide contained the full complement of residues designated as highly conserved in helix F of the greater serine proteinase inhibitor superfamily. In addition, an internal sequence of GGGGDIHQGF was found in the monkey cytoplasmic inhibitor, which is identical to that reported for an internal sequence of the human placental inhibitor. These findings confirm the existence of a novel cytoplasmic serine proteinase inhibitor in mammalian cells and provide additional details of its molecular properties. The physiological function of this novel serine proteinase inhibitor in cytoplasm is unknown.

低分子量Ｈｅｐａｒｉｎ（ＦＲ‐８６０）の実験的播種性血管内凝固症候群に対する効果

Effects of low molecular weight heparin (FR-860) on experimental disseminated intravascular coagulation (DIC) models in rats were compared with those of conventional unfractionated heparin (UF-heparin) and other anticoagulants. In the endotoxin-induced DIC model, FR-860 (12.5-200 U/kg/hr) and UF-heparin (25-200 U/kg/hr) inhibited dose-dependently the decreases in platelet counts, fibrinogen, antithrombin III activity and alpha 2-plasmin inhibitor activity, and they also inhibited the increases in fibrin de-products and thrombus formation in the glomerular capillary bed. Neither gabexate mesilate (FOY, 10 mg/kg/hr) nor nafamostat mesilate (FUT, 0.1 mg/kg/hr) improved endotoxin-induced DIC. FR-860 showed comparable potency to UF-heparin in plasma anti-factor Xa (F.Xa) activity. However, FR-860 was weaker than UF-heparin in prolongation of activated partial thromboplastin time. In the thrombin-induced DIC model, both FR-860 and UF-heparin significantly improved, as seen in the endotoxin-induced DIC model, the changes in coagulation and fibrinolytic parameters and suppressed the production of pulmonary thrombus. On the other hand, both FOY and FUT showed significant but weak improvement in this model. In addition, FR-860 inhibited the enhancement of fibrinolysis and the production of pulmonary thrombus in the lactic acid-induced DIC model. These results suggest that the efficacy of FR-860 on DIC in rats is comparable to that of UF-heparin and that the efficacy can be attributed to its anti-F.Xa activity. FR-860 can be expected to be a useful therapeutic drug for DIC.

Interaction of Feedback Control and Product Inhibition in the Activation of Factor X by Factors IXa and VIII

A simple numerical model of the activation of factor X by factors IXa and VIII has been constructed in order to identify and examine the major controls that operate in a nonflowing system in the presence of (1) inhibitors of factor Xa and (2) feedback activation of factor VIII by factor Xa. The model confirms, and allows parameter estimation for, (1) the control of factor Xa yield by factor VIIIa decay; (2) the control of generation-curve area by the rate of factor Xa inhibition; and (3) the reduction in the factor VIIIa decay rate in the presence of factor IXa. Beyond confirmation of existing data, the model also predicts that below a definite, but very low, threshold level of factor IXa (less than or equal to 10 pM), minimal feedback activation of factor VIII will occur. The concentration of factor IXa at which the threshold is observed in simulations is dependent on the rate of inhibition of factor Xa.

Binding of human factors X and Xa to HepG2 and J82 human tumor cell lines. Evidence that factor Xa binds to tumor cells independent of factor Va.

Previous studies demonstrated that several cultured human tumor cell lines potentiate the conversion of prothrombin to thrombin by factor Xa and calcium in the absence of exogenous factor Va. In the present study, the specific binding of radioiodinated preparations of human factor X and factor Xa to a human hepatocellular carcinoma cell line (HepG2) that constitutively synthesizes a factor V/Va molecule, and a human bladder carcinoma cell line (J82) that does not synthesize factor V/Va, was examined. Radioiodinated factor Xa bound specifically to J82 and HepG2 cells, whereas no significant specific binding of 125I-factor X to either cell was observed. The binding isotherm of 125I-factor Xa to each tumor cell line exhibited a hyperbolic profile, and Scatchard analysis demonstrated a single class of binding site for factor Xa on each cell surface with Kd values of 1.66 +/- 0.39 and 1.64 +/- 0.52 nM and 566,000 +/- 71,000 and 28,000 +/- 6,000 binding sites/cell for HepG2 and J82 cells, respectively. Thrombin formation by cell-bound factor Xa was hyperbolic and saturable at 5 nM factor Xa on each cell line. Hanes-Woolf plots of the prothrombin activation data indicated that half-maximal rates of thrombin formation occurred at factor Xa concentrations of 1.50 +/- 0.43 nM and 1.42 +/- 0.48 nM on HepG2 and J82 cells, respectively. Pretreatment of J82 cells with polyclonal anti-human factor V IgG had no measurable effect on either the binding of 125I-factor Xa or prothrombin activation. However, pretreatment of HepG2 cells with anti-human factor V IgG inhibited prothrombin activation in a dose-dependent manner, but did not inhibit the binding of factor Xa to this cell. When both cell lines were preincubated with exogenous human factor Va, the binding of factor Xa to either HepG2 or J82 cells was marginally affected. These data indicate that HepG2 and J82 cells have cell surface factor Xa binding sites proximal to, but independent of, cell surface factor Va of either exogenous or endogenous origin.

Chemical Composition, Particle Size Range, and Biological Activity of some Low Molecular Weight Heparin Derivatives

Several low molecular weight (LMW) heparin sodium derivatives from different sources, as well as some related regular heparin sodium preparations, were examined for chemical composition by high field (300 MHz) 1H NMR spectroscopy, for particle size range by quasi‐elastic light scattering (QELS) methods, and for anti‐coagulation potency and anti‐factor Xa activity by the standard U.S. Pharmacopeial assays described for regular heparin. The NMR spectra provided insight into possible modes of depolymerization used to generate the LMW heparins, as well as into the presence of dermatan sulfate or other chemical contaminants. The QELS analysis permitted the heparin preparations to be characterized and compared by virtue of their distinctive particle size distributions.

Procoagulant activity of human tumours: existence of Xa and thrombin-like activities

We have analysed 15 infiltrating duct carcinomas of the breast, 10 gastrointestinal adenocarcinomas, one each of the thyroid and larynx, and four mesenchymal tumours for the presence and the nature of procoagulant activity (PCA). The metastatic tumours had a significantly higher PCA (P = 0.01-0.001) as compared to the non-metastatic tumours in the respective groups, and almost 20-25 times the activity as compared to normal tissue (P = 0.001). Although the majority of the tumours had FVII-dependent tissue thromboplastin-like activity, some of the tumour homogenates revealed the presence of an FVII-independent PCA. Unlike the known alternate PCA, which acts via factor X activation, this PCA was factor X independent. It caused clot formation in FX-deficient plasma (six cases) and purified fibrinogen solution (four cases), indicating the presence of a Xa-like enzyme or a thrombin-like activity respectively.

Beneficial effect of low molecular weight heparin on the hemodialysis model in dogs

The effect of FR-860, one of low molecular weight heparins, was investigated on the hemodialysis model in dogs for comparison with that of conventional unfractionated heparin (UF-heparin). In a bolus injection model, FR-860 at 12.5 ∼ 100 U/kg prolonged the dialysis time (the time until the arterial circuit pressure reaching 500 mmHg) in a dose-dependent manner and UF-heparin at 50 U/kg also prolonged it. Additionally, FR-860 (12.5 ∼ 50 U/kg/hr) and UF-heparin (25 and 50 U/kg/hr) continuously inhibited the rise of the arterial circuit pressure in an infusion model. In both models, the efficacy of FR-860 was more potent than that of UF-heparin. FR-860 and UF-heparin decreased the amount of blood loss remaining in the dialyzer in the infusion model. During hemodialysis, both FR-860 and UF-heparin showed activated plasma anti-F.Xa activity, prolongation of aPTT and such of thrombin time in a dose-dependent manner in those models. However, FR-860 was higher in anti-F.Xa activity and weaker in prolongation of aPTT and thrombin time than UF-heparin. These results suggest that FR-860 is more beneficial in in efficacy and higher in safety against the bleeding risk than UF-heparin hemodialysis.