Human pharmacological studies of a defined low molecular weight heparin fraction (Fragminr) evidence for a simultaneous inhibition of factor Xa and IIa (thrombin)

The effect of a low molecular weight heparin (FragminR) (20–80 U/kg) was studied in 10 healthy male volunteers after intravenous administration. Clotting ab-abnormalities are extensive shortly after injection, lasting for 1 – 2 hours depending on heparin dosage. Dilute thrombin time, showing a close correlation to anti-F-Xa activity, is as sensitive as anti-F-Xa. The parallel time course of dilute thrombin time and anti-F-Xa activity clearly demonstrates that LMWH has an effect on F-IIa lasting as long as that of F-Xa. After subcutaneous administration clotting abnormalities are only slight with 40–60 U/kg, in contrast to the intravenous study. The platelet system remains unchanged.     

Comparison of the anticoagulant and antithrombotic effects of synthetic thrombin and factor Xa inhibitors

The anticoagulant effect of selected synthetic inhibitors of thrombin and factor Xa was studied in vitro in commonly used clotting assays. The concentrations of the compounds doubling the clotting time in the various assays were mainly dependent on their thrombin inhibitory activity. Factor Xa inhibitors were somewhat more effective in prolonging the prothrombin time compared to the activated partial thromboplastin time, whereas the opposite was true of thrombin inhibitors. In vivo, in a venous stasis thrombosis model and a thrombo-plastin-induced microthrombosis model in rats the thrombin inhibitors were effective antithrombotically whereas factor Xa inhibitors of numerically similar Ki value for the respective enzyme were not effective at equimolar dosage. The results are discussed in the light of the different prerequisites and conditions for inhibition of thrombin and factor Xa in the course of blood clotting.     

Assay of unfractionated and LMW heparin with chromogenic substrates: Twin methods with factor Xa and thrombin

Utilizing two newly synthesized chromogenic substrates (CS), two different assay methods for heparin in plasma have been developed. The assay with bovine factor Xa and the highly reactive “Substrate FXa-1” (CH₃OCO-D-CHA-Gly-Arg-pNA-AcOH) measures both unfractionated (UF) heparin and low molecular weight (LMW) heparin within a single standard curve in the 0.05–1.5 U/ml plasma range. The very similar (and less expensive) assay with bovine thrombin and “Substrate Th-1” (2AcOH-H-D-CHG-Ala-Arg-pNA), measures OF heparin, but not LMW heparin. The standard curves are highly reproducible (CV 3.5–4.7%). For clinical work, a linear standard curve is obtained with three standards and lin-log plot. The “within run” SD was 0.007–0.026 U/ml. Mean recovery of 0.5 U/ml heparin added to 10 pathological plasma samples ranged 0.46–0.53 U/ml (SD 0.034–0.040). Activities of three of heparin and three LMW heparin preparations are reported.     

Inherited AT-III deficiency: fast and slow inactivation of thrombin and factor Xa

A family with inherited AT-III deficiency is presented. This variant is unlike those formerly described, as the plasma concentration is high, the thrombin inhibition with heparin is slow, and factor Xa inhibition is decreased.     

Inactivation of bovine and human thrombin and factor Xa by antithrombin III studied with amidolytic methods

Antithrombin III (At-III) purified from human plasma was incubated with thrombin (T), activated factor X (Xa) or mixtures of T and Xa. The amount of enzymes inactivated by the amount of At-III in one ml of normal plasma was determined. - At-III which had inactivated one enzyme, was incapable of inactivating the other. Inactivation of Xa and T by At-III was found to proceed like a second order reaction. The inactivation rate was highly dependent on the At-III concentration, the half-time values of the enzyme activities decreasing with increasing At-III concentration. With excess inhibitor, variations in the concentration of enzyme had little influence on the inactivation rate. Bovine Xa was more rapidly inactivated than bovine T. In contrast, human T was more rapidly inactivated than human Xa.     

Effect of the novel direct factor Xa inhibitor DX-9065a on thrombin generation and inhibition among patients with stable atherosclerotic coronary artery disease

INTRODUCTION: Thrombin, a pluripotential effector enzyme with prothrombotic, proinflammatory, and mitogenic properties, plays a pivotal role in the pathobiology and clinical expression of atherothrombotic coronary artery disease. Existing anticoagulant drugs have not been shown to attenuate thrombin generation or activity consistently. We sought to investigate the effect of DX-9065a on thrombin generation and inhibition in patients with stable CAD. DX-9065a is a small-molecule, synthetic, direct inhibitor of factor Xa. MATERIALS AND METHODS: Peripheral venous blood samples were collected serially during and after administration of either placebo or 1 of 4 weight-adjusted regimens of DX-9065a, in 73 patients with stable CAD participating in the XaNADU-1B study. RESULTS AND CONCLUSIONS: At baseline, the median (25th, 75th) prothrombin activation fragment 1.2 (F1.2) level was 2.56 (2.05, 3.20) nmol/L, and the median d-dimer level was 0.26 (0.19, 0.38) mug FEU/L. There were significant relationships between measured plasma DX-9065a concentrations and both F1.2 (4.9% decrease for each doubling of DX-9065a) (P<0.0001) and d-dimer (5.5% decrease for each doubling of DX-9065a) (P=0.001). F1.2 was suppressed (below baseline) at 96 h after administration of DX-9065a. Coronary thrombotic events did not occur during or after study drug administration. DX-9065a, the first in a class of small-molecule, direct, selective and reversible factor Xa inhibitors, reduces thrombin generation and fibrin formation among patients with stable CAD. The effect is concentration-dependent and persists for at least 96 h following drug cessation, without biochemical or clinical evidence of rebound.     

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)     

Role of heparin in the inactivation of thrombin, factor Xa, and plasmin by antithrombin III

To characterize the mode of action of heparin, the kinetic behaviour of the inhibition of thrombin, factor Xa, and plasmin by antithrombin III was studied in the presence and absence of heparin. Following the concentration dependence of inactivation, in both cases a linear dependence of the apparent first-order inactivation rate constant on the antithrombin III concentration was found. These results are explained by enzyme-heparin interactions. Thus, heparin is believed to act as an activator of the enzymes that makes them more susceptible to antithrombin III.

Values of kinetic constants for the inactivation reaction of the several enzymes were determined. From these values it is concluded that heparin influences primarily the thrombin-antithrombin III interaction.     

Interaction of thrombin and factor Xa with bovine vascular endothelial cells, smooth muscle cells and rat hepatoma cells

The aims of the present investigation were to characterize the binding and inhibition of thrombin and factor Xa to bovine vascular endothelial cells (EC), bovine smooth muscle cells (SMC), and rat hepatoma cells (RHC), and to evaluate the effects of plasma constituents on their inhibition. The enzymatic activities of bovine thrombin and factor Xa were assayed using chromogenic substrates. After 10 min incubation with the cells, thrombin activity in the solution had decreased by about 20% and was subsequently recovered on the cell surfaces. When the cells with the surface-bound thrombin were incubated with defibrinogenated plasma or antithrombin III (AT-III) for 30 sec only about 10% and 20-40%, respectively, of the initial activity could be recovered. In similar experiments with factor Xa, initial activity in the solution had decreased by 10% after 10 min incubation, and was subsequently recovered from the cell surfaces. After 30 sec incubation with AT-III, no cell surface-bound factor Xa activity was detected, whereas 10% of the bound factor Xa activity was recovered after incubation with defibrinogenated plasma. It is concluded that thrombin and factor Xa are taken up and inhibited by EC, SMC and RHC cell surfaces in similar ratios, suggesting that cell surface-mediated inhibition of clotting factors is not restricted to vascular wall cells. The inactivation of factor Xa was dependent on AT-III, however, the inactivation of thrombin was further promoted by an additional unidentified plasma constituent.     

Time courses of the antithrombotic effects, bleeding enhancing effects and interactions with factors Xa and thrombin after administration of low molecular weight heparinoid Org 10172 or heparin to rats

Time response curves of the anti-thrombotic effects, bleeding enhancing effects, effects on APTT, anti-Xa activities, anti-thrombin activities and thrombin generation inhibitory activities of the low molecular weight heparinoid Org 10172 and heparin have been compared in rats. The time courses of these effects were similar for heparin but quite different for Org 10172. Org 10172 induced anti-thrombotic and anti-Xa effects which lasted approximately 3 times longer than those at the same anti-Xa doses of heparin, whereas the bleeding enhancing effects and effects on APTT of Org 10172 were of shorter duration than those of heparin. The half-life of the anti-thrombin effect after Org 10172 seemed somewhat longer than after heparin administration. Thrombin generation inhibition by Org 10172 showed a slightly longer duration than by heparin. The similarities between the time courses of the anti-thrombotic effect and the anti-Xa activity after Org 10172 administration suggest that the most appropriate parameter to monitor Org 10172 treatment is the plasma anti-Xa level.     

Inhibition of the generation of thrombin and factor Xa by a fucoidan from the brown seaweed Ecklonia kurome.

The effects of a fucoidan (C-II), which was purified from the brown seaweed Ecklonia kurome, on the generation of thrombin and factor Xa have been investigated by measuring the amidolytic activities by using the respective specific chromogenic substrates in both plasma and purified systems. C-II inhibited significantly the generation of thrombin in both the intrinsic and the extrinsic pathways, although the intrinsic inhibitory effect by C-II was more remarkable than the extrinsic one. On the other hand, C-II was a good inhibitor of the factor Xa generation in the intrinsic pathway, while it was a poor one in the extrinsic pathway. In the purified systems C-II also inhibited the formation of prothrombin-activating complex (i.e., prothrombinase), but not its activity. The concentration of C-II required for 50% inhibition of thrombin generation was about one-tenth to one-seventh of that of the activity of the generated thrombin in plasma. These results indicate that C-II has an inhibitory effect on the generation of thrombin by blocking the formation of prothrombinase and by preventing the generation of intrinsic factor Xa in addition to its antithrombin activity, and also that the generation-inhibitory effect is more remarkable than C-II's enhancement effect on the antithrombin activity by heparin cofactor II in plasma.     

Linear diffusion of thrombin and factor Xa along the heparin molecule explains the effects of extended heparin chain lengths

QUESTION: How does the size of the heparin moiety in the anti-thrombin (AT)-heparin complex influence its anticoagulant properties? APPROACH: Of 52 heparin fractions of precise Mr between 2800 and 37,000 we determined the dissociation constant (Kd) of the binding of the enzyme to the AT-heparin complex and the decay constant (kdec) of thrombin and factor Xa at 1 microM of that complex. RESULTS: The Kd of thrombin or factor Xa is constant when expressed in terms of the concentration of sugar units, i.e. the enzymes bind the better the longer the heparin. Thrombin (Kd=1.86+/-0.13 microM) binds 11 times tighter than factor Xa (Kd=20.2 +/-1.5 microM). Factor Xa inactivation velocity is proportional to the concentration of pentasaccharide-bound AT if Mr<10,000 but decreases at higher Mr. Thrombin inactivation is constant per pentasaccharide with twelve adjacent monosaccharides (C-domain). CONCLUSION: The data fit a model in which thrombin and factor Xa bind at a random site on the heparin chain and, via one-dimensional diffusion, reach the AT that is bound to its specific binding site on the heparin. Factor Xa, but not thrombin, can dissociate from heparin before reaching bound AT.     

Inhibitory effects of TFPI on thrombin and factor Xa generation in vitro - modulatory action of glycosaminoglycans

The effect of tissue factor pathway inhibitor (TFPI) on thrombin and factor Xa generation was studied in an in vitro system using a prothrombin complex concentrate. It was found that TFPI, via the direct inhibition of factor Xa and the tissue factor/factor VIIa complex, inhibited both the further generation of factor Xa and the generation of thrombin in a concentration-dependent manner. The generation of thrombin (IC₅₀ 255 ng/ml) was more pronounced than that of factor Xa (IC₅₀ 684 ng/ ml). The inhibitory activity of TFPI was significantly enhanced when unfractionated heparin was present in the assay system at a concentration of 10 μg/ml which did not show any inhibitory effects on protease generation in the same system. Furthermore, the influence of TFPI at subthreshold concentrations (100 ng/ml and 200 ng/ ml, resp.) on the inhibitory action of unfractionated heparin (UFH), a low molecular weight heparin (LMWH), heparan sulfate (HS) and the synthetic heparin pentasaccharide (PS) was investigated. Whereas in the concentration range used (0.3–40 μg/ml) these glycosaminoglycans did not inhibit thrombin and factor Xa generation, after supplementation of the system with TFPI a concentration-dependent inhibition of the generation of the proteases up to 40–50 % was seen for UFH, LMWH and HS. TFPI did not increase the activity of PS.     

A new factor Xa inhibitor (lefaxin) from the Haementeria depressa leech

The salivary complex of the leech Haementeria depressa produces potent anticoagulant components. Among them, a protein named lefaxin inhibits factor Xa (FXa). Lefaxin was purified to homogeneity from dissected salivary complexes by gel filtration in Sephadex G-150 followed by two ion exchange chromatography steps in Mono-Q. Inhibition of FXa by lefaxin was demonstrated by the inhibition of its amidolytic activity, measured with chromogenic substrate S-2765 (apparent K(I) of 4 nM), and of its ability to inhibit thrombin generation in the prothrombinase complex (EC50 of 40 nM). Lefaxin has a molecular weight of 30 kDa and an isoelectric point of 5.7. It is made of a polypeptide chain whose N-terminal sequence shows no similarity with that of other FXa inhibitors (antistasin and ghilianten) isolated from leech saliva. On the other hand, the N-terminal sequence of lefaxin presents significant sequence similarity with nitric oxide carrier proteins myohemerythrin from the annelid Nereis diversicolor and prolixin S from the triatoma Rhodnius prolixus. Interestingly, prolixin S also proved to be an anticoagulant protein acting on FXa.     

In vitro pharmacological profile of 3-N-(2-fluoroethyl)spiperone

The binding affinities of spiperone and 3-N-(2-fluoroethyl)spiperone (FESP) have been compared for several rodent brain receptor sites and for inhibition of monoamine release and uptake sites. FESP and spiperone have almost identical profiles, namely a high affinity for dopamine-D2 and serotonin-S2 receptors, a low affinity for alpha 1-adrenergic receptors, and negligible binding to other sites. These results suggest that available data on spiperone binding may be applied to the interpretation of PET data obtained with FESP.     

Human in vitro pharmacodynamic profile of the selective Factor Xa inhibitor ZK-807834 (CI-1031)

ZK-807834 (also known as CI-1031) is a small molecule that potently and selectively inhibits Factor Xa. Studies in animals have shown that ZK-807834 attenuates thrombosis and thrombus progression after fibrinolysis and exhibits an increased antithrombotic-to-bleeding risk ratio compared with conventional agents. The present study describes the human in vitro anticoagulant and pharmacodynamic profile ZK-807834. Consistent with its selective inhibition of Factor Xa, ZK-807834 in the range of 0.3-0.5 microM prolonged prothrombin time (PT) and activated partial thromboplastin time (aPPT) twofold without affecting thrombin time (TT). Intersubject variability of in vitro anticoagulant activity was nominal and gender-independent. ZK-807834 inhibited Factor Xa in clot-bound prothrombinase with an average IC50 of 10+/-7 (S.D.) nM. ZK-807834 exhibited no direct effect on ADP- or collagen-induced platelet aggregation. Based on the potency and specificity, ZK-807834 may represent an important advance in the development of selective and safe antithrombotics.     

A meta-analysis of phase III randomized controlled trials with novel oral anticoagulants in atrial fibrillation: Comparisons between direct thrombin inhibitors vs. factor Xa inhibitors and different dosing regimens

Aims

Previous studies evaluating the ability of novel oral anticoagulants (NOAC) to prevent thromboembolism in patients with non-valvular atrial fibrillation (AF) have identified differences between the efficacy and safety of the drugs tested. Whether these differences reflect differences in direct thrombin or Xa inhibition, different dosing regimens or specific aspects of each agent or trial has not yet been explored.

Methods

A search was performed on MEDLINE, EMBASE and COCHRANE, and ongoing studies were tracked on clinicaltrials.gov. Phase III randomized controlled trials of direct thrombin inhibitors (DTI) and factor Xa inhibitors (FXaI) vs. warfarin in patients with AF were eligible. Data were pooled using random-effects, according to the Mantel-Haenszel model. Sensitivity analyses were performed on DTI, FXaI, once-daily and twice-daily regimens.

Results

Seven studies were pooled, including a total of 80,290 patients. Both DTI and FXaI outperformed warfarin regarding stroke or systemic embolism, intracranial bleeding, total and cardiovascular mortality. No significant differences were found between DTI and FXaI or between once-daily and twice-daily regimens. Some drugs performed worse than warfarin regarding some secondary endpoints, including: edoxaban 30 mg bid on ischaemic stroke, dabigatran on acute myocardial infarction, dabigatran 150 mg bid and rivaroxaban 20mgod on gastrointestinal bleeding.

Conclusion

Our pooled data do not support the hypothesis of a significant class-effect of DTI or FXaI, nor the benefit of once-daily vs. twice-daily dosing in the setting of AF, reinforcing that the choice of NOAC should be adapted to the specific patient and focused on the agent itself, rather than the pharmacological class or dosing regimen.