The effects of human thrombomodulin on the inactivation of thrombin by its serum inhibitors

Thrombomodulin is an endothelial cell protein which accelerates thrombin-dependent protein C activation by over 1000 fold. In this study, the effect of thrombomodulin on the inactivation of thrombin by its serum inhibitors was evaluated. 125I-thrombin was incubated at 37 degrees C with serum and the resulting complexes separated by SDS-PAGE. Antithrombin III was the major complex formed with some 125I-thrombin bound to heparin cofactor II and higher molecular weight fractions. Inclusion of thrombomodulin at increasing concentrations inhibited 125I-thrombin binding to antithrombin III and the higher molecular weight fractions but had little effect on thrombin-heparin cofactor II complex formation. Similar results were obtained using a purified antithrombin III/heparin cofactor II system. Kinetic studies, using purified antithrombin III, revealed that thrombomodulin acts as a weak competitive inhibitor towards antithrombin III (Ki = 39 nM). We postulate that in the microcirculation, where the ratio of thrombomodulin to antithrombin III is relatively high, thrombin bound to thrombomodulin may be protected from inactivation by antithrombin III and can thus promote efficient activation of protein C.     

The influence of direct and antithrombin-dependent thrombin inhibitors on the procoagulant and anticoagulant effects of thrombin

INTRODUCTION: Clinical trials evaluating direct thrombin inhibitors in unstable coronary artery disease (CAD) have been disappointing. The hypothesis tested in the present study was that these agents may inhibit the anticoagulant effect of thrombin to a further extent than the procoagulant effect of thrombin. MATERIALS AND METHODS: We studied both reversible and irreversible thrombin inhibitors and compared the effects of each inhibitor on activated protein C (APC) generation vs. the effect on fibrinopeptide A (FPA) generation. A mixture of protein C, thrombin inhibitor, fibrinogen, fibrin polymerisation blocker and thrombin was incubated with thrombomodulin (TM)-expressing human saphenous vein endothelial cells (HSVECs). The inhibitors investigated were melagatran, inogatran, hirudin, hirugen, D-Phe-D-Pro-D-arginyl chloromethyl ketone (PPACK), and antithrombin (AT) alone or in combination with unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH). RESULTS: All agents, except hirugen, inhibited APC and FPA generation in a dose-dependent manner. FPA inhibition/APC inhibition ratios, based on IC50 for inogatran, melagatran, hirudin, PPACK, AT, AT-UFH and AT-LMWH were 1.73, 0.85, 0.55, 2.1, 0.5, 0.65 and 3.1 respectively. CONCLUSIONS: All agents, except hirugen, inhibited APC and FPA generation approximately to a similar extent. Thus, it can be inferred that the poor efficacy of thrombin inhibitors in recent clinical trials in patients with unstable CAD is unlikely to be a consequence of their effects on the protein C system.     

Further studies on the interaction between thrombin and GP Ib using crossed immunoelectrophoresis. Effect of thrombin inhibitors

The platelet surface protein GP Ib (glycocalicin-related protein) has been shown to be retarded by thrombin-Sepharose 4B in a crossed immunoelectrophoresis system. The interaction between GP Ib and thrombin was abolished when thrombin was blocked either at the active serine site with tosyl-lysine-chloromethyl-ketone (TLCK) or phenylmethylsulfonylfluoride (PMSF) or at the fibrinogen binding site (macromolecular binding site) with N-bromosuccinimide (NBS) or heparin, indicating that both sites have to be freely accessible for the retention of the glycocalicin-related protein by thrombin.     

Interaction of calmodulin inhibitors and protein kinase C inhibitors with voltage-dependent calcium channels

We compared the relative abilities of a series of calmodulin inhibitors and protein kinase C inhibitors to influence 45Ca2+ influx through voltage-dependent Ca2+ channels in PC12, a clonal neural cell line. K+-depolarization-dependent 45Ca2+ uptake was reduced by the calmodulin inhibitors calmidazolium, trifluoperazine, W-7, W-13, and W-5 at concentrations comparable to those that affect calmodulin, while the protein kinase C inhibitors polymyxin B and H-7 were weak or ineffective. The Ca2+ channel antagonist properties of calmodulin inhibitors should be considered in interpreting their effects on Ca2+-dependent cellular events.     

The influence of synthetic thrombin inhibitors on the thrombin-antithrombin reaction

The reaction of the antithrombin III of blood plasma with thrombin is inhibited in the presence of the reversible inhibitor, 4-amidinophenylpyruvic acid (APPA) and prevented when the active site of thrombin is chemically blocked by reaction with the irreversible inhibitor, 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF). These results support the assumption that the thrombin-antithrombin reaction proceeds like an enzyme-substrate reaction.     

Antithrombotic and haemorrhagic effects of synthetic and naturally occurring thrombin inhibitors

The effects of the synthetic thrombin inhibitor N-(2-naphthylsulfonylglycyl)-4-amidinophenylalanine piperidide (βNas-Gly-(pAm)Phe-Pip) and the naturally occurring inhibitors hirudin and heparin on the bleeding time were studied in mice and rats by the method of transection of the tail tip and of standardized incision of the tail. With both methods the thrombin inhibitors prolonged the bleeding time in dependence on the dose and the plasma concentration obtained. The transection bleeding time was influenced by the inhibitors in a similar manner, whereas in the case of incision of the tail heparin caused a more pronounced effect than hirudin and βNas-Gly-(pAm)Phe-Pip. Comparison of the antithrombotic actions of the inhibitors with their effects on the bleeding time showed that, in contrast to the selective thrombin inhibitors hirudin and βNas-Gly-(pAm) Phe-Pip, antithrombotically effective doses of heparin induced a clear prolongation of bleeding time.     

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.     

Anticoagulant and antithrombotic properties of synthetic sulfated bis-lactobionic acid amides

Sulfated bis-lactobionic acid amides, a new class of polyanions with heparin-like properties, were synthesized and their antithrombotic and anticoagulant activities were determined. Compared to heparin and Fragmin^R, a low molecular weight heparin, the substances exhibited moderate to low anticoagulant activities in aPTT, thrombin clotting time and Heptest assays. In amidolytic assays no anti-Ila activity and only exceedingly low anti-Xa activity was observed. The antithrombotic activity of the bis-lactobionic acid amides was determined using two thrombosis models. In rabbit and rat models thrombi were induced by a combination of endothelial damage and reduction of blood flow or only by endothelial damage. At least one of the bis-lactobionic acid amides (LW 10082) exhibited a considerable antithrombotic activity which was similar to low molecular weight heparin.     

Antithrombotic effects and bleeding time of thrombin inhibitors and warfarin in the rat.

Warfarin limits the synthesis of y-glutamyl carboxylated forms of coagulation factors, factor II, factor VII, factor IX, and factor X, protein C, and protein S and as a result impairs the function of these proteins. In contrast, direct inhibitors of thrombin only affect one enzyme in the coagulation cascade. The aim of this study was to investigate the antithrombotic effect and the slope of the dose-response curves of the multifactorial coagulation inhibitor warfarin in comparison with the single factor low-molecular-weight thrombin inhibitors melagatran and inogatran. An arterial thrombosis model in rats was used, and vessel damage was induced by topical application of ferric chloride to the carotid artery. The slopes of the dose-response curves were 3.6, 1.8, 1.1, and 1.2, for warfarin, heparin, inogatran, and melagatran, respectively. For warfarin the antithrombotic effect increased from 23% to 81% when the dose was doubled. In contrast, 10-fold increases in the doses of inogatran and melagatran were necessary to obtain a similar increase in antithrombotic effect. The doses needed to obtain 80% antithrombotic effect for heparin, warfarin, and melagatran were investigated in a tail transection bleeding model. For heparin, this dose significantly prolonged the bleeding time and the blood loss; for warfarin, only the total bleeding time was increased while for melagatran there was no increase in bleeding. We conclude that, thrombin inhibitors affecting only one enzyme in the coagulation cascade seem preferable to inhibitors affecting multiple enzymes, such as warfarin, due to shallower dose-response curves and a wider therapeutic interval.     

Dual effects of thrombin and a 14-amino acid peptide agonist of the thrombin receptor on septal cholinergic neurons

We have compared the effects of thrombin and of the 14-amino acid peptide agonist (TRAP-14) of the thrombin protease activated receptor (PAR) on cholinergic neurons in pure cultures of rat septal neurons and in co-cultures of septal neurons and glial cells. In pure septal cultures, low concentrations of thrombin (up to 10 nM) did not affect choline acetyltransferase (ChAT) activity, a marker of cholinergic neurons, or 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction, an index of cell viability. However, 100 nM thrombin decreased ChAT activity and MTT reduction by 44 and 17%, respectively. In co-cultures, a low concentration of thrombin (1 nM) increased ChAT activity (+75%), whereas a high concentration (100 nM) decreased it (−83%). At this high concentration, thrombin was neurotoxic, as indicated by a large decrease in MTT reduction (−80%). Thrombin effects on ChAT activity were mimicked by TRAP-14 both in pure septal cultures (no effect at 0.1 μM and −63% at 100 μM) and in co-cultures (+25% at 0.1 μM and −28% at 100 μ M). In contrast, this peptide did not affect MTT reduction. These dual effects of thrombin and TRAP-14 on ChAT activity in co-cultures, were also observed on pure cultures of septal cells supplied with NGF. The activation and inhibition by TRAP-14 of the expression of ChAT activity in septal neuron/glial cell cultures were inhibited by a 9-amino acid peptide antagonist of thrombin PAR. Thus, the effects of thrombin on cholinergic neurons seem to be mainly mediated by thrombin PAR and glial cells seem to play a major role in these thrombin actions.     

Effects of COX-1 and COX-2 inhibitors on the firing of rat midbrain dopaminergic neurons--possible involvement of endogenous kynurenic acid

Kynurenic acid (KYNA) is an endogenous glutamate-receptor antagonist with a preferential action at the glycine-site of the NMDA-receptor. In the present in vivo study, the importance of brain KYNA to modulate the activity of dopamine (DA) neurons in the ventral tegmental area (VTA) was analyzed by utilizing the decrease in brain KYNA formation induced by the cyclooxygenase (COX)-2 inhibitor parecoxib. A reduction in brain KYNA concentration (39-44%) by parecoxib (25 mg/kg, i.v., 1 h or, i.p., 3.5 h) was associated with a decreased firing rate and burst firing activity. In concordance, an increase in brain KYNA concentration (150-300%), induced by the COX-1 inhibitor indomethacin (50 mg/kg, i.v., 1 h or, i.p., 3.5 h), produced opposite effects, that is, increased firing rate and burst firing activity. The decrease and increase in neuronal firing of VTA DA neurons by the COX-inhibitors was reversed by L-701,324 (antagonist at the NMDA-glycine site; 0.06-2 mg/kg, i.v.) and by D-cycloserine (partial agonist at the NMDA-glycine site; 2-32 mg/kg, i.v.), respectively. In addition, the parecoxib-induced decrease in firing rate and burst firing activity was effectively blocked by pretreatment with kynurenine (5 mg/kg, i.p., 30 min), the immediate precursor of KYNA. Present results suggest that the action of COX-inhibitors on the firing of VTA DA neurons are linked to their effects on KYNA formation and that endogenous KYNA is tonically modulating the neuronal activity of VTA DA neurons. Such a modulatory action of KYNA should be of importance for the functioning of mesocorticolimbic DA pathway.     

Interaction of human blood platelet aggregation inhibitors with phospholipid films

A series of carbamoylpiperidines and related entities, a class of compounds only recently identified by the authors as potent human blood platelet aggregation inhibitors, showed correspondingly strong levels of “specific” interaction, in monomolecular film systems, with some phospholipids viewed as prototypes of actual platelet plasma membrane constituents. The data strongly corroborate our previously articulated view that our compounds possess appropriate hydrophobic character to penetrate the lipid bilayer of the platelet plasma membrane, subsequently are capable of generating sufficient quantities of their cationic species to counteract massively stimulus-induced mobilization of Ca⁺⁺ ions, and thereby restrain or void Ca⁺⁺-dependent phospholipase activity.     

Direct thrombin inhibitors built on the azaphenylalanine scaffold provoke degranulation of mast cells

The main structural feature of direct thrombin inhibitor LK-732 responsible for the appropriate interaction at the thrombin active site is a strong basic group. A possibility that a strong basic group of LK-732 might contribute to the mast cell degranulation effect and consequent reduction of tracheal air flow (TAF) and fall of mean arterial blood pressure (MAP) in rats was investigated in the present study. At doses up to 5 mg/kg (i.v.), LK-732 did not cause significant changes of TAF and MAP. At 7 mg/kg (i.v.), a sudden reduction of TAF and a fall of MAP was observed within 5 min after LK-732 administration (75% mortality, p = 0.007). A less basic direct thrombin inhibitor LK-658 (21 mg/kg, i.v.) did not significantly disturb TAF and MAP. A reduction of TAF and a fall of MAP caused by LK-732 (7 mg/kg, i.v.) was almost completely abolished in rats with degranulated mast cells (0% mortality, p = 0.008). LK-732 concentration-dependently degranulated rat peritoneal mast cells in vitro (pEC(50) = 1.92 +/- 0.05 muM). A structure-activity relationship (SAR) study revealed that the terminal basic groups attached to the aromatic ring are responsible for the mast cell degranulation effect. A good correlation was observed between mast cell degranulation and pK(b) of analogues of LK-732 (R(2) = 0.49), but not between mast cell degranulation and thrombin K(i) (R(2) = 0.23). LK-732-induced reduction of TAF, the fall of MAP and high mortality originate from LK-732-induced mast cell degranulation. As judged by the SAR study, this effect could be overcome by reducing the basicity of LK-732.     

Heparin-like effect of polymethacrylic acid on the reaction between thrombin and antithrombin-III

The effect of polymethacrylic acid on the reaction between thrombin and antithrombin-III was investigated. It was found that polymethacrylic acid accelerated the inactivation rate of thrombin by antithrombin-III, whereas this polyanion did not affect thrombin activity. It is suggested that polymethacrylic acid may simulate the action of heparin on thrombin and antithrombin-III.     

Thrombin generation in severe haemophilia A and B: the endogenous thrombin potential in platelet-rich plasma

Thrombin generation was investigated in platelet-rich plasma (PRP) from 11 healthy controls, 17 patients with severe haemophilia A and 7 patients with severe haemophilia B. Mean endogenous thrombin potential (ETP) in arbitrary fluorescence units (FU) was 226.9 +/- 44.6, 186.4 +/- 22.5, 154.2 +/- 41.3 in controls, haemophilia A and B, respectively, all at a platelet count of 200 x 10(9)/l (p = 0.004 for controls vs. haemophilia A, p = 0.003 for controls vs. haemophilia B, no significant difference between haemophilia A and B). The contribution of FVIII to thrombin generation in haemophilia A was 1.31 +/- 0.16 FU/% of FVIII:C activity, while for FIX in haemophilia B this was 0.80 +/- 0.21 FU/% of FIX activity. There was an almost linear relationship between increasing platelet count and thrombin generation up to a mean platelet count of 100 x 10(9)/l. Further increase in platelet count has only a marginal influence on thrombin generation. Platelets increase ETP in haemophilia A by 0.184 +/- 0.022 FU/10(9) platelets/l and in haemophilia B by 0.319 +/- 0.085 FU/10(9) platelets/l, and this was significantly different between the two groups (p = 0.0002). This influence of plate-lets diminishes with increasing concentration of either FVIII or FIX. In conclusion, there is a difference in thrombin generation between haemophilia A and B, and this may be attributed to the role of platelets in the assembly of the tenase complex on their surface.