piperidines and napsagatran

In addition to its pivotal role in blood coagulation, thrombin is one of the most important agonists for platelet recruitment and aggregation. Thrombin inhibitors impede thrombin-induced platelet aggregation but have no effect on aggregation induced by other agonists. A review is presented of selective thrombin inhibitors now in clinical investigation, some of which are also orally active.

Topics: Administration, Oral; Adult; Animals; Antithrombins; Arginine; Blood Platelets; Boron Compounds; Clinical Trials as Topic; Dogs; Drug Evaluation, Preclinical; Glycine; Hirudin Therapy; Hirudins; Humans; Naphthalenes; Oligopeptides; Peptide Fragments; Pipecolic Acids; Piperidines; Recombinant Proteins; Sulfonamides; Thrombin

The effect of oral warfarin on the pharmacokinetics and pharmacodynamics of the synthetic direct thrombin inhibitor napsagatran was investigated.. In an open, randomised, two-way crossover study, 12 healthy male volunteers were infused napsagatran (80 micrograms/min) for 48 h. Each subject was administered 25 mg warfarin (Coumadin) at the start of the infusion in either the first or second treatment period. Sampling was performed regularly over the treatment period and 24 h thereafter for measurement of plasma levels of napsagatran, activated partial thromboplastin time (APTT) and prothrombin time (PT).. The pharmacokinetic parameters of napsagatran were not significantly influenced by co-administration of warfarin. Napsagatran administration was followed by increases in APTT and PT. Co-administration of warfarin increased the AUEC (area under the effect curve) calculated for the period 0-48 h (corrected for baseline) for APTT by 45% (95% CI: 28-65%) and for PT by 438% (95% CI: 272-678%) compared to the treatment with napsagatran alone.. Warfarin has no effect on the pharmacokinetics of napsagatran, but has a marked influence on the pharmacodynamic parameters (APTT, PT) of napsagatran. In clinical practice, this interaction between the two compounds should be taken into account. The PT cannot be used to monitor the effect of oral anticoagulants during the switch from this group of direct thrombin inhibitors to full oral anticoagulant therapy.

Topics: Adult; Anticoagulants; Antithrombins; Area Under Curve; Cross-Over Studies; Humans; Male; Models, Biological; Naphthalenes; Partial Thromboplastin Time; Piperidines; Prothrombin Time; Warfarin

Direct thrombin inhibitors belong to a new class of antithrombotic drugs whose effects on blood coagulation in vivo in patients suffering from acute thrombotic conditions have not yet been fully explored.. One hundred and five patients with acute proximal deep-vein thrombosis were randomized to receive a continuous intravenous infusion of napsagatran, a novel synthetic thrombin inhibitor, at a fixed dose of 5 mg/h (n = 36) or 9 mg/h (n = 25) for five days, or APTT-adjusted unfractionated heparin (UFH, n = 44) for the same time. In these patients, thrombin activity and thrombin generation could be assessed by measuring thrombin-antithrombin III complexes (TAT) and prothrombin fragment 1+2 (F1+2), respectively, on three occasions. At baseline, TAT and F1+2 did not differ among the three groups. On Day 2 (steady state), TAT significantly decreased in all groups, and the decrease was significantly more pronounced in the patients given higher-dose napsagatran. F1+2 decreased significantly only in UFH-treated patients. Two hours after cessation of the infusion, the TAT levels increased in the two napsagatran groups but not in the UFH group, whilst F1+2 went back to the baseline levels in the napsagatran-treated patients but remained low in the UFH-treated patients. There was no rebound effect.. The data presented suggest that direct thrombin inhibition with napsagatran at 9 mg/h is more potent than UFH in attenuating thrombin activity, but is less potent than UFH in inhibiting thrombin generation. The real significance of these findings will have to be substantiated in further trials with clinically relevant endpoints.

Topics: Antithrombins; Enzyme Inhibitors; Fibrinolytic Agents; Heparin; Humans; Naphthalenes; Piperidines; Single-Blind Method; Thrombin; Venous Thrombosis

One hundred and ten patients with acute proximal deep-vein thrombosis were randomized in a sequential dose-finding design, to receive continuous intravenous infusion of napsagatran, a novel synthetic thrombin-inhibitor, at a fixed dose of 5 mg/h (n = 37) or 9 mg/h (n = 26), or APTT-adjusted unfractionated heparin (n = 47). Oral anticoagulants were started on the 2nd day and the study drug was discontinued from the 5th treatment day, as soon as the International Normalized Ratio was above 2. Control venogram (97 venogram pairs evaluable) after 5-8 days of treatment showed improvement in 3 napsagatran-treated patients (versus none in heparin-treated patients) and worsening in 4 napsagatran-treated patients (versus 2 in heparin-treated patients). The venographic Marder's score did not change among the treatment groups. New lung scan perfusion defects (99 scintigram pairs evaluable) occurred in 4 (11%), 4 (21%), and 4 (10%) patients in the napsagatran (5 mg/h) group, in the napsagatran (9 mg/h) group, and in the heparin control group, respectively. There was no statistically significant difference in any of these endpoints between the 3 groups. No major bleeding was observed and the rare minor bleedings occurred at a similar rate in the three treatment groups. In conclusion, the ADVENT trial has shown data that suggest comparable efficacy and safety of a synthetic, direct thrombin inhibitor (napsagatran) and conventional heparin therapy for treatment of proximal DVT. These results suggest that synthetic direct thrombin inhibitors are a promising class of antithrombotic agents which deserves further development in this field.

Topics: Adult; Aged; Antithrombins; Blood Coagulation Tests; Female; Follow-Up Studies; Heparin; Humans; Infusions, Intravenous; Male; Middle Aged; Naphthalenes; Partial Thromboplastin Time; Piperidines; Thrombophlebitis

The use of in vitro data for quantitative predictions of transporter-mediated elimination in vivo requires an accurate estimation of the transporter Michaelis-Menten parameters, V(max) and K(m), as a first step. Therefore, the experimental conditions of in vitro studies used to assess hepatic uptake transport were optimized regarding active transport processes, nonspecific binding, and passive diffusion (P(dif)). A mechanistic model was developed to analyze and accurately describe these active and passive processes. This two-compartmental model was parameterized to account for nonspecific binding, bidirectional passive diffusion, and active uptake processes based on the physiology of the cells. The model was used to estimate kinetic parameters of in vitro transport data from organic anion-transporting peptide model substrates (e.g., cholecystokinin octapeptide deltorphin II, fexofenadine, and pitavastatin). Data analysis by this mechanistic model significantly improved the accuracy and precision in all derived parameters [mean coefficient of variations (CVs) for V(max) and K(m) were 19 and 23%, respectively] compared with the conventional kinetic method of transport data analysis (mean CVs were 58 and 115%, respectively, using this method). Furthermore, permeability was found to be highly temperature-dependent in Chinese hamster ovary (CHO) control cells and artificial membranes (parallel artificial membrane permeability assay). Whereas for some compounds (taurocholate, estrone-3-sulfate, and propranolol) the effect was moderate (1.5-6-fold higher permeability at 37 degrees C compared with that at 4 degrees C), for fexofenadine a 16-fold higher passive permeability was seen at 37 degrees C. Therefore, P(dif) was better predicted if it was evaluated under the same experimental conditions as V(max) and K(m), i.e., in a single incubation of CHO overexpressed cells or rat hepatocytes at 37 degrees C, instead of a parallel control evaluation at 4 degrees C.

Topics: Algorithms; Animals; Biological Transport, Active; CHO Cells; Computer Simulation; Cricetinae; Cricetulus; Diffusion; Estrone; Fatty Acids, Monounsaturated; Fluvastatin; Hepatocytes; Indoles; Kinetics; Male; Membranes, Artificial; Models, Biological; Naphthalenes; Oligopeptides; Organic Anion Transporters; Permeability; Pharmaceutical Preparations; Pharmacokinetics; Piperidines; Quinolines; Rats; Rats, Wistar; Sincalide; Temperature; Terfenadine

Antibody mediated inhibition of tissue factor (TF) function reduces thrombus size in ex vivo perfusion of human blood over a TF-free surface at venous shear rates suggesting that TF might be involved in the mechanism of deep vein thrombosis. Moreover, TF-bearing monocytes and polymorphonuclear (PMN) leukocytes were identified in human ex vivo formed thrombi and in circulating blood. To understand the role of TF in thrombus growth, we applied a rabbit venous thrombosis model in which a collagen-coated thread was installed within the jugular vein or within a silicon vein shunt. The effect of an inhibitory monoclonal antirabbit TF antibody (AP-1) or Napsagatran, a specific inhibitor of thrombin, was quantified by continuously monitoring 125I-fibrinogen incorporation into the growing thrombi. The antithrombotic effect obtained with the anti-TF antibody was comparable to the effect observed with the thrombin inhibitor napsagatran suggesting that in this animal model the thrombus propagation is highly TF dependent. Immunostaining revealed that TF was mostly associated with leukocytes within the thrombi formed in the jugular vein or in the silicon vein shunt. Ex vivo perfusion experiments over collagen-coated coverslips demonstrated the presence of TF-bearing PMN leukocytes in circulating blood. The results suggest that in rabbits venous thrombus growth is mediated by clot-bound TF and that blocking the TF activity can inhibit thrombus propagation.

Topics: Animals; Antibodies, Monoclonal; Blood Vessel Prosthesis; Fibrinogen; Immunohistochemistry; Jugular Veins; Leukocytes; Naphthalenes; Piperidines; Rabbits; Thromboplastin; Venous Thrombosis

During studies on warfarin, heparin and various anticoagulants with novel mechanisms of action, the activated partial thromboplastin time (aPTT) and the (apparent) international normalized ratio (INR) from a bedside monitor (Coagucheck Plus(R)) were compared with laboratory assay results. Data were compared using the Bland and Altman method of comparison where systematic differences result in significant slopes of the regression line. During heparin treatment, the bedside monitor largely underestimated the aPTT (slope = -0.80). During treatment with the direct thrombin inhibitor napsagatran (slope = 0.99), the pentasaccharides Org31540/SR90107A (slope = 0.77) and SanOrg34006 (slope = 0.35), and warfarin (slope = 0.60), the bedside monitor underestimated the aPTT at lower aPTT levels, while at higher aPTT levels it overestimated the laboratory values. The bedside monitor slightly overestimated the INR during treatment with warfarin (slope = 0.33). Apparent INR was largely overestimated during treatment with Org31540/SR90107A (slope = 1.38), SanOrg34006 (slope = 0.97), Napsagatran (slope = 1.23), and recombinant tissue factor pathway inhibitor (slope = 1.48, P < 0.001 for all regression lines). These results indicate that a substantial disagreement in aPTT or (apparent) INR exists between the bedside monitor and laboratory assay during treatment with the studied 'classic' and novel anticoagulants. The amount of disagreement depended on the anticoagulant given.

Topics: Anticoagulants; Antithrombins; Drug Monitoring; False Negative Reactions; False Positive Reactions; Fibrinolytic Agents; Heparin; Humans; Laboratories; Naphthalenes; Oligosaccharides; Partial Thromboplastin Time; Piperidines; Point-of-Care Systems; Reproducibility of Results; Warfarin

The objective of this work was to assess the pharmacokinetics of napsagatran, a low molecular weight thrombin inhibitor, after intravenous administration in a variety of laboratory animals, and prospectively to help design the first pharmacokinetic studies in man. Napsagatran is actively excreted into the bile and urine of various species and pronounced species-differences in its pharmacokinetics are observed. It is, therefore, an interesting compound to use in tests of the limitations of presently available inter-species scaling methods. The present data suggest that allometric exponent values which are consistent with the values expected for physiological processes and small organic molecules are not necessarily associated with successful predictions in man when active transport processes are involved in the disposition of the compounds. For example, compared with the values observed in man, the clearance (CL), non-renal clearance (CL(nr)) and the volume of distribution at steady state (Vd(ss)) were over-predicted by 3-, 7- and 2-fold, respectively, by use of allometry. Of the species tested, the cynomolgus monkey seemed to be the most useful for predicting kinetics in man when the approach based on concentration-time transformations was used. Thus, for half-life (t(1/2)), CL and Vd(ss), the observed mean values of 1.7 h, 459 mL min(-1) and 24 L kg(-1) in man were very close to the values predicted from the cynomolgus monkey (1.7 h, 652 mL min(-1) and 22 L kg(-1), respectively). The results show that there are large inter-species differences for kidney and liver excretion of napsagatran. This is probably because of the involvement of active transport processes, which compromised the kinetic extrapolation from animal to man, although a more thorough investigation of the transporters involved in the disposition of napsagatran is necessary to enable better understanding of the species differences observed.

Topics: Animals; Antithrombins; Biological Transport, Active; Dogs; Humans; Macaca fascicularis; Male; Naphthalenes; Piperidines; Rabbits; Rats; Species Specificity

We examined the effect of a specific thrombin inhibitor, Ro 46-6240, alone and combined with an antagonist of the platelet GP IIb/IIIa, Ro44-9883, on the response to tissue-type plasminogen activator in a canine model of thrombolysis. Platelet activity was determined by measuring the excretion of 2,3-dinorthromboxane (TX)B2, an enzymatic metabolite of TXA2. Ro 46-6240 administered before tissue-type plasminogen activator induced a dose-dependent prolongation of the activated partial thromboplastin time and prothrombin time. The time to reperfusion decreased dose-dependently (P < .01) to 10 +/- 6 min vs. 52 +/- 5 min in controls. Ro 46-6240 also prevented reocclusion, which occurred in every case in control experiments. Urinary excretion of 2,3-dinor-TXB2 increased from 3 +/- 1 to 37 +/- 9 ng/mg creatinine in controls after reperfusion. This increase was reduced in a dose-dependent fashion by Ro 46-6240, such that at the highest dose, urinary 2,3-dinor-TXB2 after reperfusion was 5.6 +/- 1 ng/mg creatinine. Similar functional and biochemical effects were seen when a subthreshold dose of Ro 46-6240 was combined with Ro 44-9883. At the dose used, Ro 44-9883 alone abolished platelet aggregation ex vivo but failed to modify the response to tissue-type plasminogen activator or the excretion of 2,3-dinor-TXB2 after reperfusion (51 +/- 6 ng/mg creatinine, n = 3). However, the combination of Ro 44-9883 and Ro 46-6240 reduced the time to reperfusion (40 +/- 8 vs. 68 +/- 15 min; n = 7, P < .05), prevented reocclusion and abolished the rise in urinary 2,3-dinor-TXB2 (5 +/- 1 ng/mg creatinine, n = 4). These findings suggest that thrombin mediates platelet activation during coronary thrombolysis. The increased platelet activity results in platelet aggregation and a subsequent increase in TXA2 formation.

Topics: Acetates; Animals; Antithrombins; Coronary Vessels; Dogs; Drug Interactions; Naphthalenes; Piperidines; Platelet Aggregation; Platelet Aggregation Inhibitors; Thrombin; Thromboxane A2; Tyrosine

Inhibition of the tissue factor/factor VIIa (TF/F.VIIa) complex attenuates thrombosis in different animal models of arterial thrombosis. However, it remains unclear to what extent the antithrombotic effects are associated with changes in hemostatic functions and how this compares with inhibition of thrombin, an enzyme acting at a later stage in the coagulation cascade. The antithrombotic and the antihemostatic effects of a monoclonal anti-TF antibody (AP-1) were compared in a model of arterial thrombosis to those of a direct thrombin inhibitor (napsagatran) and heparin. In anesthetized rabbits transient arterial thrombi were induced by mechanical damage to the subendothelium of a moderately stenosed carotid artery. Recurrent formation and dislodgement of thrombi resulted in cyclic flow variations (CFVs) which were monitored over 2 hours. Rabbits received intravenously either a placebo (control), a monoclonal anti-rabbit TF antibody (AP-1, 0.05 mg/kg as an i.v. bolus repeated every 15 min, a specific low molecular weight thrombin inhibitor (napsagatran, 3 microg/kg/min) or heparin (3 and 13 microg/kg/min). The effect of the inhibitors on the hemostatic system was studied in a separate set of rabbits by measuring template bleeding times (BT) in the ear arterioles, marginal ear vein and the nail cuticle of the foreleg. AP-1 and napsagatran showed a similar antithrombotic activity (78% and 80% abolition of the CFVs, respectively), whereas either low or high dose heparin was poorly effective (43% and 40% inhibition of CFVs, respectively). At these antithrombotic doses and even at 4-fold higher dosage, AP-1 did not significantly alter the BT, whereas napsagatran and heparin prolonged the ear vessels and cuticle BT in a dose-dependent manner. These results suggest that in contrast to direct thrombin inhibition, the blockade of the TF/F. VIIa function did not result in a concomitant prolongation of the bleeding time. Thus, dissociation of antithrombotic and antihemostatic effects indicates that inhibition of the coagulation system at its initial stage represents a promising approach for the development of new anticoagulants.

Topics: Animals; Antibodies, Monoclonal; Anticoagulants; Antithrombins; Bleeding Time; Blood Coagulation; Disease Models, Animal; Hemodynamics; Heparin; Naphthalenes; Piperidines; Rabbits; Thrombin; Thromboplastin; Thrombosis

A series of inhibitors of factor Xa (FXa) were investigated using the thrombin generation assay to evaluate the potency and specificity needed to efficiently block thrombin generation in activated human plasma. By inhibiting FXa the generation of thrombin in plasma is delayed and decreased. Inhibitor concentrations which cause 50 percent inhibition of thrombin generation (IC50) correlate in principle with the Ki values for inhibition of free FXa. Recombinant tick anticoagulant peptide (r-TAP) is able to inhibit thrombin generation with considerably low IC50 values of 49 nM and 37 nM for extrinsic and intrinsic activation, respectively. However, the potent synthetic, low molecular weight inhibitors of FXa (Ki values of about 20 nM) are less effective in inhibiting the generation of thrombin with IC50 values at micromolar concentrations. The overall effect of inhibitors of FXa in the thrombin generation assay was compared to that of thrombin inhibitors. On the basis of similar Ki values for the inhibition of the respective enzyme, synthetic FXa inhibitors are less effective than thrombin inhibitors. In contrast, the highly potent FXa inhibitor r-TAP causes a stronger reduction of the thrombin activity in plasma than the most potent thrombin inhibitor hirudin.

Topics: Animals; Anticoagulants; Arginine; Arthropod Proteins; Cattle; Dipeptides; Factor Xa; Factor Xa Inhibitors; Fibrinolytic Agents; Hirudins; Humans; Intercellular Signaling Peptides and Proteins; Kinetics; Molecular Structure; Naphthalenes; Partial Thromboplastin Time; Peptides; Pipecolic Acids; Piperidines; Propionates; Rabbits; Recombinant Proteins; Serine Proteinase Inhibitors; Sulfonamides; Sulfones; Thrombin; Thromboplastin

Napsagatran, a new synthetic direct thrombin inhibitor, was compared with heparin in a canine model of coronary thrombosis and concomitantly in an ex vivo perfusion chamber model. Occlusive thrombosis of the left circumflex coronary artery was induced by electrical injury. In parallel, arterial subendothelium was exposed to native blood using an annular perfusion chamber for 5, 10 and 20 min at a wall shear rate of 650/s. Dogs received saline, heparin (40 and 70 U/kg/h) or napsagatran (3 and 10 microgram/kg/min). Heparin (40 U/kg/h) and napsagatran (3 microgram/kg/min) delayed or prevented in vivo thrombotic occlusion, but only napsagatran (10 microgram/kg/min) significantly decreased the intracoronary thrombus when compared with saline. High-dose heparin (70 U/kg/h) or napsagatran (10 microgram/kg/min) decreased the platelet-rich thrombus after a 20-min chamber perfusion. Neither heparin nor napsagatran decreased the thrombus volume after a 5-min perfusion. Heparin (70 U/kg/h) and napsagatran (10 microgram/kg/min) prolonged the activated partial thromboplastin time differently (>x6 and x1.4, respectively, P<0.01), whereas the activated clotting time was prolonged equally (x2.5). Thus napsagatran in this model shows arterial antithrombotic effects similar to those of heparin. The chamber experiments suggest that neither compound affects the initiation of platelet thrombus formation. In arterial thrombosis, the activated clotting time has a higher predictive value than the activated partial thromboplastin time when a direct thrombin inhibitor is compared with heparin.

Topics: Animals; Antithrombins; Coronary Thrombosis; Disease Models, Animal; Dogs; Female; Fibrinolytic Agents; Heparin; Male; Naphthalenes; Partial Thromboplastin Time; Perfusion; Piperidines

To further define the anticoagulant activity of Ro 46-6240, a novel, synthetic, thrombin inhibitor, we compared its effect on extrinsic and intrinsic thrombin generation in human platelet-poor plasma with that of recombinant hirudin and standard heparin. The time course of thrombin generation was followed with a chromogenic substrate assay. The total amount of active thrombin formed was quantified by calculating the area under the thrombin generation curve. Ro 46-6240 and r-hirudin delayed thrombin formation in a concentration-dependent manner in both activation systems whereas heparin showed this effect only in the intrinsic system. Heparin was the most potent inhibitor of extrinsic and intrinsic thrombin generation with IC50 values of 20 and 27 nM, respectively. Ro 46-6240 was nearly as potent as r-hirudin for inhibiting extrinsic thrombin generation (IC50 418 vs 229 nM) and intrinsic thrombin generation (IC50 463 vs 343 nM) despite a much lower affinity of Ro 46-6240 for thrombin (Ki apparent: 0.3 nM) in a purified buffer system. The similar potency of the small active-site thrombin inhibitor compared to the larger hirudin may be explained by different kinetic mechanisms for inhibition of thrombin and by a higher accessibility to the phospholipid surface where thrombin generation takes place. In conclusion, our results show that a specific small thrombin inhibitor efficiently inhibits and delays thrombin generation in human coagulating plasma. This reduced thrombin generation might be caused by inhibition of thrombin-mediated feedback reactions during blood coagulation.

Topics: Antithrombins; Binding Sites; Chromogenic Compounds; Dipeptides; Heparin; Hirudins; Humans; Hydrolysis; Kinetics; Naphthalenes; Piperidines; Recombinant Proteins; Thrombin

Specific thrombin inhibitors are considered to be more potent antithrombotics than heparin. However, the relation between the systemic anticoagulation generated by thrombin inhibitors and their antithrombotic effect is not well defined. In a guinea pig carotid thrombosis model, the activated clotting time (ACT), the activated partial thromboplastin time (aPTT), and thrombin-generation tests were evaluated for their ability to predict the arterial antithrombotic effect of direct thrombin inhibitors such as hirudin and Ro 46-6240 compared with heparin.. Thrombosis of the carotid artery was induced by subendothelial damage in guinea pigs, and the subsequent cyclic flow variations were monitored. The effects of pretreatment with intravenous heparin, hirudin, and Ro 46-6240 were tested. After doubling the baseline aPTT, 1 IU.kg-1.min-1 heparin was inactive, whereas either hirudin or Ro 46-6240 (30 micrograms.kg-1.min-1) prevented thrombus formation by 80%. Heparin (10 IU.kg-1.min-1) induced the same antithrombotic effect but with indefinite aPTT prolongation. However, for similar prolongation of the ACT, the three compounds had equivalent antithrombotic effects. Thrombin generation was predictive of the antithrombotic effect of the thrombin inhibitors but not of heparin.. The arterial antithrombotic effect of direct thrombin inhibitors, when compared with those of heparin, should be evaluated by the ACT and not the aPTT or thrombin-generation assays. For a "therapeutic" aPTT prolongation, thrombin inhibitors induce higher systemic anticoagulation than does heparin and thus might unduly have higher bleeding liability.

Topics: Animals; Antithrombins; Carotid Artery Thrombosis; Guinea Pigs; Heparin; Hirudin Therapy; Humans; In Vitro Techniques; Male; Naphthalenes; Partial Thromboplastin Time; Piperidines; Predictive Value of Tests; Rabbits; Whole Blood Coagulation Time

Clot-bound thrombin remains active and is less accessible to heparin-antithrombin III than fluid-phase thrombin. To determine whether clot-bound human thrombin is more susceptible to inactivation by direct thrombin inhibitors, the activity of a novel synthetic competitive thrombin inhibitor Ro 46-6240, recombinant hirudin and unfractionated heparin were compared with fluid-phase thrombin and clot-bound thrombin. Fibrinopeptide A generated in human plasma was used as an index of thrombin activity. Hirudin was the most potent inhibitor of fluid-phase and clot-bound thrombin. However, Ro 46-6240 inhibited clot-bound thrombin three times more potently than fluid-phase thrombin (IC50 19 vs 56 ng/ml) while hirudin was two times (IC50 8 vs 3 ng/ml) and heparin six times (IC50 1,205 vs 200 ng/ml) less active against clot-bound thrombin compared with fluid-phase thrombin. The relative selectivity for clot-bound thrombin is not a unique property of Ro 46-6240 since two other synthetic thrombin inhibitors tested inhibited clot-bound thrombin more effectively than fluid-phase thrombin and a third was equally active against both forms of thrombin. In contrast, the affinities of two chromogenic substrates were similar for both forms of thrombin. This study shows that direct thrombin inhibitors inhibit clot-bound thrombin more potently than heparin and suggests that an apparent selectivity for clot-bound thrombin can be achieved with some synthetic thrombin inhibitors. Further studies have to show whether the high potency of these direct thrombin inhibitor translates into antithrombotic efficacy in clinical situations with pre-existing clots.

Topics: Binding, Competitive; Blood Coagulation; Chromogenic Compounds; Fibrinopeptide A; Heparin; Hirudins; Humans; Naphthalenes; Piperidines; Recombinant Proteins; Thrombin

Thrombin is involved in the pathogenesis of venous and arterial thrombosis. This study addressed the question of the relative importance of thrombin in the early and late phases of thrombogenesis. The effect of Ro 46-6240 (1.43 mg/kg bolus and 0.1 mg/kg per minute i.v.), a novel, selective thrombin inhibitor on thrombogenesis induced by rabbit aorta subendothelium, was measured ex vivo in a perfusion chamber model after a short (5-minute) and long (30-minute) exposure time to rabbit native blood. The role of the perfusion time was assessed at shear rates of 100/s, 650/s, and 2600/s. These shear rates mimic blood flow conditions found in veins, arteries, and small or stenosed arteries, respectively. Fibrin deposition and platelet thrombus formation on subendothelium were evaluated by microscopic morphometry. In the presence of Ro 46-6240, fibrin deposition was abolished at both perfusion times and at all shear rates. In the 5-minute experiments, thrombus height was reduced by Ro 46-6240 at shear rates of 100/s (85%) and 650/s (35%) but not at a shear rate of 2600/s, whereas thrombus area was not affected at any shear rate. In contrast, both thrombus height and thrombus area were reduced (60% to 90%) by Ro 46-6240 in the 30-minute perfusion groups at all wall shear rates. The antithrombotic effect of Ro 46-6240 after 30-minute perfusion was confirmed by the minimal increase in the pressure difference between the entrance and the exit of the perfusion chamber compared with the control groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Aorta; Blood Flow Velocity; Blood Platelets; Endothelium, Vascular; Female; Fibrin; Male; Naphthalenes; Piperidines; Rabbits; Thrombin; Thrombosis

TNF-alpha induces changes in endothelial cell functions, such as upregulation of tissue factor, resulting in endothelial procoagulant activity which may play a role in disseminated intravascular coagulation. The procoagulant activity of TNF-alpha-stimulated endothelial cell monolayers was studied in a human ex vivo native (nonanticoagulated) blood flow system using the three thrombin inhibitors recombinant hirudin, Ro 46-6240, and heparin. Under venous blood flow conditions (shear rate 65 s-1) recombinant hirudin, Ro 46-6240, and heparin inhibited fibrin deposition on the endothelial cells by 50% at concentrations of 14, 28, and 412 ng/ml, respectively. The highest tested concentrations of the thrombin inhibitors reduced the postchamber fibrinopeptide A levels from 713 +/- 69 to < 70 ng/ml. Surprisingly, even at relatively high inhibitor concentrations, some local fibrin deposits were found on TNF-alpha-stimulated cells, suggesting that some endothelial cells possess higher procoagulant activity than others. Therefore, the surface expression pattern of tissue factor, the primary initiator of coagulation in this system, was examined by immunogold-silver staining. The results showed that the tissue factor density on the cell surface varied strongly among TNF-alpha-stimulated endothelial cells. Using TNF receptor-selective agonistic mutants of TNF-alpha, it was demonstrated further that the heterogenous surface expression of tissue factor was mediated entirely by the 55-kD TNF receptor and did not involve the 75-kD TNF receptor. We conclude that in this system TNF-alpha induces heterogenous tissue factor expression which may lead to a high local thrombin concentration, such that even in the presence of thrombin inhibitors focal fibrin deposition occurs.

Topics: Blood Circulation; Blood Coagulation; Blood Platelets; Cell Membrane; Dose-Response Relationship, Drug; Endothelium, Vascular; Fibrin; Fibrinopeptide A; Heparin; Hirudins; Humans; Immunohistochemistry; Leukocytes; Naphthalenes; Piperidines; Thrombin; Thromboplastin; Tumor Necrosis Factor-alpha; Umbilical Veins