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The objectives of this study were to investigate whether the affinity of thrombin for small-molecule, active site-directed thrombin inhibitors and substrates is affected by the presence of thrombomodulin (TM), and to what extent thrombin inhibitors inhibit TM-bound thrombin. Inhibition of human alpha-thrombin was studied in the presence and absence of solubilised rabbit lung TM in a buffer containing CaCl(2). TM inhibited thrombin-induced proteolysis of human fibrinogen with a dissociation constant (K(D)) of 4 nmol/l. With at least 16-fold molar excess of TM over thrombin the affinity of thrombin both for the small thrombin substrates (S-2366 and S-2238) and the reversible, active site-directed thrombin inhibitors (inogatran and melagatran) increased twofold. In contrast, the ability of hirudin to inhibit thrombin was reduced by TM, since hirudin competes with TM in binding to thrombin. The effect of thrombin inhibitors on protein C activation by thrombin bound to human kidney cells transfected with cDNA for human TM was also studied. The mean binding capacity of the transfected cells was approximately 320,000 quantified by flow cytometry with antibodies against TM. Hirudin, inogatran and melagatran inhibited the activation of protein C by thrombin complexed with cell-bound TM in a dose-dependent manner, with mean IC(50) values+/-S.D. of 4.4+/-0.8, 20.0+/-1.1 and 6.4+/-0.2 nmol/l, respectively. Antithrombin inhibited protein C activation with an IC(50) value of 290+/-10 nmol/l, which was enhanced fourfold (IC(50) 60 nmol/l) by the addition of heparin 0.5 U/ml. Heparin alone, up to a concentration of 1 U/ml, had no effect on the activation of protein C. Small direct thrombin inhibitors thus inhibited both free and TM-bound thrombin and therefore also inhibited the activation of protein C. Whether this will influence their clinical efficacy or safety versus heparin and warfarin, which also inhibit protein activation, respectively, lowers the concentration of protein C, remains to be studied in clinical trials.

SummaryMelagatran, a new, competitive and rapid inhibitor of thrombin with a molecular mass of 429 Da is described. Melagatran is well tolerated when administered in very high doses, and the oral bioavailability in the dog is relatively high. The aim of the study was to determine, in the preclinical setting, the degree of selectivity against the fibrinolytic system required for entering the clinical development phase. Melagatran was compared with two structurally similar thrombin inhibitors, inogatran and H 317/86. The potent inhibition of thrombin by melagatran was demonstrated by a low inhibition constant (Ki) for thrombin (0.002 μmol/l) and prolongation of clotting time to twice the control value in coagulation assays at low concentrations (0.010, 0.59 and 2.2 μmol/l for thrombin time, activated partial thromboplastin time and prothrombin time, respectively). Furthermore, thrombin-induced platelet aggregation was inhibited at the same concentration (IC50-value 0.002 μmol/l) as the Ki-value for thrombin. In two assays of global fibrinolysis, inhibition was observed at a concentration of 1.1 μmol/l in a euglobulin plasma fraction model, while no inhibition was observed at a concentration of ≤10 μmol/l in a plasma model. In an in vivo model of endogenous fibrinolysis in the rat, inhibition of fibrinolysis was observed at ≥1.0 μmol/l. In all assays, except the Ki-ratio determinations, the compounds could be graded with regard to selectivity against the fibrinolytic system: inogatran > melagatran > H 317/86. For melagatran, inhibition of fibrinolysis was not observed at concentrations below the upper limit of the proposed therapeutic plasma concentration interval (

The thrombin inhibitor inogatran is a synthetic peptidomimetic with a molecular weight of 439 dalton. In vitro studies have shown that inogatran is a classical competitive inhibitor of the active site of thrombin with a K i of 15 × 10 −9 mol/l. Inogatran doubles the thrombin clotting time in human plasma at 20 × 10 −9 mol/l, APTT at 1.2 × 10 −6 mol/l, and prothrombin time at 4 × 10 −6 mol/l. The effects on rat and dog plasma are similar although slightly weaker. IC 50 for inhibition of thrombin-induced aggregation of human platelets is 17 × 10 −9 mol/l. Inogatran has no effect on platelet aggregation induced by ADP or collagen. Up to a concentration of 10 × 10 −6 mol/l inogatran does not inhibit t-PA-induced fibrinolysis as seen in an ECLT system. Inogatran has good selectivity for thrombin as compared to several other serine proteases occurring in the blood. It is concluded that the properties of inogatran in vitro make the compound suitable for further studies in animals and man. Copyright © 1997 Elsevier Science Ltd

The binding of a series of low-molecular-mass, active-site-directed thrombin inhibitors (399-575 Da) to human alpha-thrombin was investigated by surface plasmon resonance technology (BIACORE), stopped-flow spectrophotometry, and isothermal titration microcalorimetry (ITC). The equilibrium constants K(D) (nM to microM range) at 25 degrees C obtained from the BIACORE analysis correlated well with the inhibition constants K(i) in a chromogenic inhibition assay. The interactions between thrombin and three potent inhibitors, melagatran, inogatran, and CH-248, were further investigated at temperatures between 278 and 310K. A one-to-one binding stoichiometry found with ITC was supported by BIACORE data. K(i) and K(D) values increased with the temperature, mainly due to higher values for dissociation rate constants. The changes in enthalpy, DeltaH, and entropy, DeltaS, determined from the linear van't Hoff plots (R coefficient > 0.99), were linearly correlated by chemical compensation. Both techniques indicated clear differences in DeltaS for the three inhibitors, with a strong correlation to the number of rotational bonds. Immobilization of thrombin increased the binding stability at higher temperature and reduced the DeltaH by 20 kJ mol(-1). DeltaH values obtained from the inhibition kinetics and BIACORE were thus not identical, but correlated well with ITC data obtained at 37 degrees C. The two thermodynamic techniques allowed further differentiation between compounds of similar affinity; furthermore, kinetic analysis, hence analysis of the transition state, is complementary to ITC. A direct BIACORE binding assay might be a useful alternative to more elaborate inhibition studies.

Suboptimal gastrointestinal absorption is a problem for many direct thrombin inhibitors. The studies presented herein describe the new oral direct thrombin inhibitor H 376/95, a prodrug with two protecting residues added to the direct thrombin inhibitor melagatran. Absorption properties in vitro: H 376/95 is uncharged at intestinal pH while melagatran is charged. H 376/95 is 170 times more lipophilic (octanol water partition coefficient) than melagatran. As a result, the permeability coefficient across cultured epithelial Caco-2 cells is 80 times higher for H 376/95 than for melagtran. Pharmacokinetic studies in healthy volunteers: H 376/95 is converted to melagatran in man. Oral bioavailability, measured as melagatran in plasma, is about 20% after oral administration of H 376/95, which is 2.7–5.5 times higher than after oral administration of melagatran. The variability in the area under the drug plasma concentration vs. time curve (AUC) is much smaller with oral H 376/95 (coefficient of variation 20%) than with oral melagatran (coefficient of variation 38%). Pharmacodynamic properties: H 376/95 is inactive towards human α-thrombin compared with melagatran [inhibition constant (Ki) ratio, 185 times], a potential advantage for patients with silent gastrointestinal bleeding. In an experimental thrombosis model in the rat, oral H 376/95 was more effective than the subcutaneous low molecular weight heparin dalteparin in preventing thrombosis. Conclusion: By the use of the prodrug principle, H 376/95 endows the direct thrombin inhibitor melagatran with pharmacokinetic properties required for oral administration without compromising the promising pharmacodynamic properties of melagatran.

The sensitivity of BIACORE technology is sufficient for detection and characterization of binding events involving low-molecular-weight compounds and their immobilized protein targets. The technology requires no labeling and provides information on the stability of the compound/target complex with a single injection of the compound. This is useful for qualifying hits obtained in a primary screen and in lead optimization. Although immobilized targets can be reused, the surface may slowly deteriorate, solvent effects can distort binding levels during injection of compounds, and some compounds may exhibit broad protein selectivity rather than target specificity. A reliable direct binding assay for compounds binding to immobilized thrombin using a combination of two reference surfaces, a dextran surface for subtraction and calibration of solvent effects and a protein surface for identification of compounds that tend to bind proteins, has been developed. Eleven compounds with known binding specificity to thrombin and 159 additional compounds were investigated. All compounds with known binding specificity were identified at 1 and 10 microM concentration. One additional compound was scored as positive. The direct binding assay compared favorably with two competitive assay formats, a surface competitive assay and a inhibitor in solution assay, that were examined in parallel.

Twenty-nine patients were operated on with the Charnley hip prosthesis. All the patients were given dextran 70 as thrombosis prophylaxis. Deep vein thrombosis (DVT) was diagnosed in 10 patients with the radioactive fibrinogen uptake test and phlebography. Variables of coagulation and fibrinolysis were studied before and after surgery. Tissue plasminogen activator (t-PA) activity in the plasma without venous occlusion decreased postoperatively, but there was no correlation with DVT. The t-PA activity in venous occlusion plasma was not reduced after surgery. Plasminogen activator inhibitor (PAI-1) levels were raised immediately postoperatively. There was a significant correlation between preoperative PAI-1 activity and development of postoperative DVT (P less than 0.05). Patients developing DVT had higher levels of PAI-1 postoperatively than patients not developing DVT. A defective fibrinolytic system, as defined by high PAI-1 activity, thus predisposed to postoperative DVT.

Tissue plasminogen activator (t-PA) in plasma was separated from inhibitors by adsorption on lysine-Sepharose. It was then determined indirectly by measuring the plasmin generated from plasminogen with poly-lysine as stimulator, in a chromogenic, parabolic rate assay. The reaction proceeded with tissue plasminogen activator and plasmin(ogen) adsorbed on the gel, and followed the kinetics described for similar parabolic rate assays in soluble systems. The assay was standardized against melanoma plasminogen activator (m-PA) and had the sensitivity range of 0.001-0.020 IU (4-80 pg). Anti-m-PA IgG quenched the activity generated in plasma on venous occlusion and part of the activity in pre-occlusion plasma. The method was sensitive to purified urokinase. The basic plasma values in resting normal individuals were: mean 0.08, range 0.01-0.26 X 10(3) IU/l (n = 19), and after 20 min of venous occlusion: mean 2.48, range 0.24-4.34 X 10(3) IU/l (n = 10). The assay correlates well with a fibrin plate method, r = 0.96.

The physiologically important α2-antiplasmin has been measured by aid of a chromogenic tripeptide substrate. Low values in patients’ plasmas are found in situations with increased fibrinolysis such as streptokinase therapy and liver cirrhosis, whereas high values are found postoperatively, postpartum and after an acute thrombosis.

An α 2 -macroglobulin-plasmin complex was prepared by incubation of plasmin with an excess of purified α 2 -macroglobulin. The conditions for complex formation were analysed, and formation of a complex was confirmed by crossed immunoelectrophoresis and by activity measurements on fibrin and p-tosyl-L-arginine methyl ester. The experiments indicated binding of no more than one mole of plasmin per mole of α 2 -macroglobulin. The activity of the α 2 -macroglobulin-plasmin complex and free plasmin on the plasmin specific substrate H-D-Val-Leu-Lys-p-nitroanilid was investigated under various conditions. The activity of the complex was found to be about 50% of that of free plasmin. An increase in ionic strength increased the activity of free plasmin, while the activity of the complex was essentially unaffected. There were no differences in pH-optima or Michaelis constant for free and α 2 -macroglobulin-bound plasmin.

Reduced fibrinolytic activity increases the risk of recurrent thromboembolism and it has been suggested thatit also plays a role in postoperative thrombosis.Material and methods. Fibrinolytic parameters were analysed in 29 patients submitted to total hip replacement. Dextran 70 was given as thrombosis prophylaxis. Blood samples weretaken pre operatively, one day and oneweek postoperatively. Venous occlusion test was done in all patients.125 I-fibrinogen test was used for deep vein thrombosis (DVT) screening.Positive test was confirmed with phlebography. Fibrinolytic activity was measured on fibrin plates. Tissueplasminogen activator (t-PA) and itsspecific inhibitor (PAI) were analysed with photometric and immunologicalmethods.Results. The first postoperative dayt-PA activity decreased and PAI increased significantly. One week after operation only PAI showed significatdifference from preoperative values.10 of the patients developed DVT.The PAI level significantly higher in DVT patients preoperatively. Thisdifference in PAI level was significant compared with non-DVT patients also one day and one week postoperatively.Conclusion. The recently discovered t-PA inhibitor (PAI) seems to be correlated to postoperative thrombosis in total hip surgery.

Fifteen patients with at least two bone fractures and damages of soft tissues, mainly after traffic accidents, were investigated for changes in the fibrinolytic system. Seventeen healthy students were controls. The patients were conventionally treated in an intensive care unit. Blood samples, in all 140, were taken on the day of admission and then daily for about one week. The blood was analysed for plasminogen with electroimmunoassay and with chromogenic substrate after streptokinase activation, for α 2-antiplasmin with electroimmunoassay and two types of activity measurements (chromogenic substrate), for α 2-antiplasmin - plasmin complexes with RIA technique, and for α 2-macroglobulin with electroimmunoassay and activity measurements. In addition fibrinogen, fibrinogen-fibrin degradation products and fibrinolytic activity on fibrin plates were estimated.Plasminogen was somewhat low the first days after trauma lowest 69 % ± 22 % (M ± SD), day 3]. It then slowly increased towards the end of the investigation (highest 123% ± 21%). The last days of investigation the immunological method also gave higher values than the activity method, α 2 Antiplasmin was low directly after the trauma (lowest 67% ± 18%, day 1 ) and increased gradually (highest 115% ± 19%, day 6 ). The first days after trauma immunological method gave higher values than the activity methods, α 2 Antiplasmin - plasmin complexes varied, but showed occasionally high values in particular the first day. α 2 -Macroglobulin decreased only slightly on day 5-6, when it also showed a difference between the immunological and activity methods.It is concluded that the fibrinolytic system is activated immediately after a trauma. Later there is an increase of plasminogen, α 2 antiplasmin and fibrinogen, probably due to an acute phase reaction. Some reasons for the methodological discrepancies are discussed. Serial analysis of α 2-antiplasmin activity seems to be a relevant method for evaluation of fibrinolysis in a complex clinical situation.