Antithrombotic properties of water-soluble carbon monoxide-releasing molecules.

OBJECTIVE: We compared the antithrombotic effects in vivo of 2 chemically different carbon monoxide-releasing molecules (CORM-A1 and CORM-3) on arterial and venous thrombus formation and on hemostatic parameters such as platelet activation, coagulation, and fibrinolysis. The hypotensive response to CORMs and their effects on whole blood gas analysis and blood cell count were also examined. METHODS AND RESULTS: CORM-A1 (10-30 µmol/kg, i.v.), in a dose-dependent fashion, significantly decreased weight of electrically induced thrombus in rats, whereas CORM-3 inhibited thrombosis only at the highest dose used (30 µmol/kg). CORM-A1 showed a direct and stronger inhibition of platelet aggregation than CORM-3 in healthy rats, both in vitro and in vivo. The antiaggregatory effect of CORM-A1, but not CORM-3, correlated positively with weight of the thrombus. Concentration of active plasminogen activator inhibitor-1 in plasma also decreased in response to CORM-A1, but not to CORM-3. Neither CORM-A1 nor CORM-3 had an effect on plasma concentration of active tissue plasminogen activator. CORM-3, but not CORM-A1, decreased the concentration of fibrinogen, fibrin generation, and prolonged prothrombin time. Similarly, laser-induced venous thrombosis observed intravitally via confocal system in green fluorescent protein mice was significantly decreased by CORMs. Although both CORM-A1 and CORM-3 (30 µmol/kg) decreased platelets accumulation in thrombus, only CORM-A1 (3-30 µmol/kg) inhibited platelet activation to phosphatidylserine on their surface. CONCLUSIONS: CORM-3 and CORM-A1 inhibited thrombosis in vivo, however CORM-A1, which slowly releases carbon monoxide, and displayed a relatively weak hypotensive effect had a more pronounced antithrombotic effect associated with a stronger inhibition of platelet aggregation associated with a decrease in active plasminogen activator inhibitor-1 concentration. In contrast, the fast CO releaser CORM-3 that displayed a more pronounced hypotensive effect inhibited thrombosis primarily through a decrease in fibrin generation, but had no direct influence on platelet aggregation and fibrynolysis.

[Tryptophan and its metabolites in renal allograft recipients]. / Tryptofan i jego metabolity u pacjentów po transplantacji nerki.

AIM: Disturbances in amino acids metabolism are common in chronic renal failure and subside partially after renal tranplantation. Tryptophan (TRP) is one of the most important exogenous amino acids. Its main derivative is L-kynurenine (KYN). Disorders in the TRP metabolism via kynurenine pathway may lead to neurologic disturbances, hypertension and anaemia. The aim of this study was to assess peripheral kynurenine pathway in 28 renal allograft recipients (RAR) (12 women and 16 men) in comparison to 10 hemodialysed patients with chronic renal failure (CRF) and 10 healthy subjects. METHODS: TRP, KYN, 3-hydroxykynurenine (3-HKYN), kyn-urenic acid (KYNA) and quinolinic acid (QA) were determined in plasma using high performance liquid chromatography technique. RESULTS: The plasma concentration of tryptophan in RAR was not significantly different from the control group, but it was almost twice higher than in CRF. There was an increase in most of kynurenine metabolites in RAR as compared to healthy subjects, although not as high as in CRF. We found a significant negative correlation between TRP and serum concentration of urea and creatinine as well as a positive correlation with GFR. CONCLUSIONS: Kidney transplantation tends to normalize TRP level and markedly lowers blood concentration of kynurenine and its main derivatives. A significant negative correlation was found between TRP level and the concentration of urea and creatinine. Lowering of the KYN metabolites level may play a role in lessening uremic neurological symptomes as well as hypertension and anemia in kidney graft recipients.

Experimental asthma in rats.

Animal models of asthma have been used for over 100 years. The accuracy of extrapolations from animal models to human asthmatics is highly dependent on the species of animal selected. The rat, in comparison with other animals, demonstrates many features of airway allergy and allergic asthma that are similar to the human conditions. The following features of human asthma can be effectively investigated in a rat model of the disease: cellular infiltration of the lung, antigen-specific IgE production, and a predominant Th2 response. The majority of available models of asthma are restricted to the acute inflammatory response following a short period of allergen exposure. The frequently used model of ovalbumin (OVA) sensitization and challenge replicates the inflammatory process in the airways.

Pharmacokinetics and pharmacodynamics of aliskiren, an oral direct renin inhibitor.

Intensive efforts have been spent to discover therapeutic, non-peptide and orally effective hypertensive drugs. One drug that emerged from this effort is aliskiren, a direct human renin inhibitor that blocks the conversion of angiotensinogen to angiotensin I (Ang I). In contrast to other antihypertensive agents, aliskiren decreases plasma renin activity (PRA). In healthy human subjects, doses of between 40 and 640 mg of aliskiren exert a dose-dependent reduction in PRA and Ang I and Ang II levels. The bioavailability of aliskiren is low (2%), peak plasma concentrations are reached within one to three hours and the binding with plasma proteins achieves approximately 47-51%. Aliskiren is slightly metabolized (20%) by CYP3A4. The most common adverse events include diarrhea, headache, back pain and gastrointestinal disorders. Aliskiren is well tolerated, and may be used alone or in combination with other antihypertensive agents. Aliskiren belongs to a new class of agents that effectively and specifically inhibit the RAS. This drug functions through a novel mechanism of action and has the potential to become a true alternative to angiotensin converting enzyme inhibitors and angiotensin receptor blockers in the therapy of hypertension and other cardiovascular and renal disorders.

N-methylnicotinamide failed to induce endothelial prostacyclin release in perfused rat hindquarters.

N-methylnicotinamide, a nicotinamide derivative, possesses anti-thrombotic activity, although the mechanism of its action is unclear. Using a rat model of isolated perfused hindlimb, we tested whether this metabolite of nicotinamide is able to inhibit the vasoconstrictive effects of epinephrine, norepinephrine, and angiotensin II, thereby releasing prostacyclin from the endothelium. We found that N-methylnicotinamide administration by infusion or bolus injection did not change the course of perfusion pressure and did not inhibit the vasoconstrictive action of epinephrine, norepinephrine, or angiotensin II. In contrast, prazosin was able to completely abolish the constriction induced by epinephrine. Moreover, we did not find any changes in the level of a stable prostacyclin analog measured in the collected perfusate samples. Thus, we did not observe any endothelial prostacyclin-releasing properties of N-methylnicotinamide in the perfused rat hindquarters model.

A new recombinant thrombolytic and antithrombotic agent with higher fibrin affinity - a staphylokinase variant. An in-vivo study.

The recombinant protein SAK-RGD-K2-Hir is characterized by its fibrin-specific properties of plasminogen activation combined with antithrombin and antiplatelet activities. It was previously shown in our in-vitro studies to be a more potent and faster-acting thrombolytic agent compared with standard r-SAK. In order to document the effects of the thrombolytic potential of SAK-RGD-K2-Hir we examined this protein in an electrically induced carotid artery thrombosis model and stasis-induced venous model in rats. In the arterial thrombosis model, a bolus injection of SAK-RGD-K2-Hir was less effective than rt-PA and r-SAK. However, the most effective in the improvement and maintenance of carotid patency and in arterial thrombus mass reduction was SAK-RGD-K2. In contrast, all r-SAK derivatives reduced venous thrombus weight significantly in comparison to r-SAK and r-Hir. However, the most observable decrease in thrombus weight was obtained after application of recombinant proteins containing the r-Hir. The bleeding time was significantly prolonged in the animals treated with proteins containing r-Hir at a dose of 1.0 mg/kg. There were no observable changes in plasma fibrinogen concentration. In conclusion, our findings show thrombolytic activity in intravenous bolus injection of the novel thrombolytic agent SAK-RGD-K2-Hir in rats. Although this protein compares favourably with r-SAK in rat venous thrombolysis, we were unable to confirm the beneficial effects of SAK-RGD-K2-Hir over r-SAK and rt-PA in the carotid artery thrombolysis model. Furthermore, our results also suggest that SAK-RGD-K2-Hir bears a risk of bleeding, but this may be true for higher doses.

Are the endothelial mechanisms of ACE-Is already established?

The endothelial mechanism of ACE-Is action is multifaceted. On the one hand, by inhibiting ACE, ACE-Is diminish Ang II synthesis, one of the best known active peptides. On the other hand, they modify synthesis and release of PGI(2) and NO via increasing production of other biologically important peptides like bradykinin, Ang-(1-7) or Ang-(1-9). Thus, ACE-Is play a crucial role in the function of endothelium and are effective and important tool for therapy of range of cardiovascular system disorders. Moreover, they are sensitive pharmacological instrument to elucidate and expand our knowledge about the role of RAS in human patophysiology.

Angiotensin II enhances thrombosis development in renovascular hypertensive rats.

There is an increased number of in vitro evidence that angiotensin II (Ang II) may promote thrombosis. However there are no in vivo experiments exploring the effect of Ang II on thrombus formation. In the present study we have investigated the influence of Ang II on venous thrombosis in renovascular hypertensive rats. Furthermore, we examined the role of AT(1) receptor and Ang II metabolites: angiotensin III (Ang III) and angiotensin IV (Ang IV) in the mechanisms of Ang II action. The contribution of coagulation and fibrinolytic systems in the mode of Ang II action was also determined. Venous thrombosis was induced by ligation of vena cava. Ang II infused into rats developing venous thrombosis caused dose-dependent increase in thrombus weight, which was partially reversed by losartan, selective AT(1) antagonist. Ang III did not influence the thrombus formation in hypertensive rats, while Ang IV caused a marked increase in thrombus weight only in one of the used doses. Our study shows that Ang II via AT(1) receptor enhances thrombosis development. The prothrombotic effect of Ang II may partially depend on enhanced leukocytes adhesion to endothelial cells accompanied by accelerated fibrin formation and increased plasma level of PAI-1. Moreover, Ang II action is partially mediated by one of its metabolites - Ang IV.

Tissue distribution of a menthyl-conjugated oligodeoxyribonucleotide antisense to PAI-1 mRNA.

The inhibitory effect of numerous analogues of PO-16, an hexadecadeoxyribonucleotide antisense to sequences -22 to -17 of PAI-1 mRNA coding for a fragment of the signal peptide, on the expression of PAI-1 in endothelial cells, and physiological consequences of the subsequently reduced PAI-1 activity tested in vitro and in vivo, were described in our previous studies. Of particular interest was PO-16 5'-O-conjugated with menthyl phosphorothioate (MPO-16R). In this work, tissue localisation of MPO-16R labelled with [(35)S] phosphorothioate at the 3'-end, was determined. [(35)S]MPO-16R and control [(35)S]MPO-16R-SENSE oligonucleotides were administered intravenously into 22 rats and organ distribution of the labelled bioconjugates was assessed after 24 and 48 h. For this purpose, tissue sections were subjected to autoradiography, and quantitated by liquid scintillation after solubilisation. Overall clearance of radioactivity was already seen after 24 h, with the radioactivity recovered mainly in the kidney and liver. A smaller fraction of radioactivity was also retained in the spleen and heart. The kidney concentration of the labelled probe was higher than that of liver by 50%. The distribution of PAI-1 mRNA in untreated rat kidney, liver, spleen and heart established by two independent techniques: Ribonuclease Protection Assay and Real-Time PCR, shows the same pattern as that observed for [(35)S]MPO-16R antisense.

L-arginine and cardiovascular system.

L-arginine is a basic endogenous amino acid. Its significant metabolic role as the product of ammonia detoxification, the urea cycle metabolite, the precursor of proteins, ornithine, urea and creatinine, and the amino acid involved in the formation of active enzyme centers was very well established. The current interest in this amino acid refers mainly to its close relation with an important signal molecule nitric oxide (NO). Literature review demonstrates that L-arginine, the only substrate of the NO production, affects cardiovascular system (blood vessels and heart). The majority of experimental and clinical studies clearly show a beneficial effect of L-arginine on endothelium in conditions associated with its hypofunction and thus with reduced NO synthesis. Some clinical studies involving healthy volunteers or patients suffering from hypertension and diabetes indicate that it may also regulate vascular hemostasis. Moreover, experiments performed on animals and in vitro data also suggest that L-arginine may have a complex antiaggregatory, anticoagulatory and profibrinolytic effect. Therefore, a novel therapeutic potential of L-arginine should be taken into consideration.

Antithrombotic effect of captopril and enalapril in old rats.

In the present study, we have shown considerably accelerated thrombosis in old rats in comparison with adult rats, which may be related to the impaired hemostatic balance in these animals. In old rats, captopril and enalapril caused a marked reduction of venous thrombus weight. The mechanism of antithrombotic action of these drugs seems to be dependent on the suppression of coagulation cascade and the enhancement of the fibrinolytic processes.

[Metabolism of tryptophan via the kynurenine pathway in saliva]. / Metabolizm tryptofanu w slinie szlakiem kinureninowym.

In this review, the role of the kynurenine pathway enzymes TDO (tryptophan 2,3 dioxygenase) and IDO (indoleamine 2,3 -dioxygenase) as well as the properties of the metabolites of kynurenine degradation present in human saliva described. The implications of these metabolites in the pathomechanism of tissue and organs dysfunction are demonstrated. The authors also describe at length the role of saliva kynurenine derivatives in the onset and development of periodontal disease in humans.

Pharmacological modulation of fibrinolytic response - In vivo and in vitro studies.

Fibrinolysis is an action of converting plasminogen by its activators, like tissue- or urokinase-type plasminogen activators (t-PA, u-PA), to plasmin, which in turn cleaves fibrin, thereby causing clot dissolution and restoration of blood flow. Endothelial cells release t-PA, prostacyclin (PGI2) and nitric oxide (NO), the potent factors playing a crucial role in regulation of the fibrinolytic system. Since blood platelets can release not only prothrombotic, but also antifibrinolytic factors, like plasminogen activator inhibitor type-1 (PAI-1), they are involved in fibrynolysis regulation. Therefore agents enhancing fibrinolysis can be preferred pharmacologicals in many cardiovascular diseases. This review describes mechanisms by which major cardiovascular drugs (renin-angiotensin-aldosterone system inhibitors, statins, adrenergic receptors and calcium channel blockers, aspirin and 1-methylnicotinamide) influence fibrinolysis. The presented data indicate, that the influence of these drugs on endothelium-blood platelets interactions via NO/PGI2 pathway is fundamental for its antithrombotic and profibrinolytic action. We also described new approaches for intravital confocal real-time imaging as a tool useful to investigate mechanisms of thrombus formation and the effects of drugs affecting haemostasis and mechanisms of their action in the circulation.

Impact of aliskiren on some hemostatic parameters in experimental arterial thrombosis in rats.

BACKGROUND: Aliskiren is the first orally active inhibitor of renin to be approved for clinical use as an antihypertensive agent. A number of studies show a link between aliskiren and intravascular thrombosis. MATERIALS AND METHODS: The goal of the present study was to investigate the impact of aliskiren on arterial thrombosis in normotensive and renovascular hypertensive rats. The contribution of each coagulation and fibrinolytic parameters in the mode of aliskiren action was determined. Six weeks after clipping of the left renal artery rats developed hypertension which was confirmed by the "tail cuff" method. Animals were treated with aliskiren (10, 30 and 100mg/kg/day) per os for 10 days. Arterial thrombosis was induced by electrical stimulation of the common carotid artery. RESULTS: It was found that aliskiren in a dose-dependent manner decreased weight of the arterial thrombus in normotensive and hypertensive rats. It has been shown that this result was not associated with the effects on blood pressure, TF, PT, APTT, fibrinogen and hematological parameters. It was found that aliskiren caused increase of t-PA activity and decrease of its inhibitor activity. CONCLUSIONS: The presented results indicate that aliskiren inhibits hemostasis in the arterial thrombosis in rats. The antithrombotic effect is related with improvement of the fibrinolytic balance, and also depends on antiplatelet action.

Proteasome inhibitor prevents experimental arterial thrombosis in renovascular hypertensive rats.

Recent studies indicate that highly selective proteasome inhibitors can be useful in prevention of some cardiovascular events. Here we demonstrate that proteasome inhibitor, Z-Ile-Glu (Ot-Bu) Ala-Leucinal (PSI), is active in the prevention of platelet-dependent arterial thrombosis induced in renovascular hypertensive rats (two-kidney, one clip Goldblatt model, and 2K1C, n=5). The administration of PSI intravenously at a single dose of 0.3 mg/kg before induction of arterial thrombosis markedly increased carotid final flow rate, as compared to control (vehicle) group (10.36 +/- 1.8 ml/min and 1.2 +/- 1.2 ml/min, respectively), significantly decreased the wet (1.23 +/- 0.23 mg and 4.1 +/- 0.94 mg, respectively), and dry (0.46 +/- 0.145 mg and 1.46 +/- 0.39, respectively) thrombus weight, and completely prevented arterial occlusion. Moreover, platelets from PSI - treated thrombotic 2K1C rats, showed in response to collagen a significant inhibition of aggregation in the whole blood (10.26 +/- 0.6 ohms vs. 15.51 +/- 0.91 ohms in the control group). In contrast, collagen-induced platelet aggregation was not inhibited in vitro, after pre-treatment of the blood with PSI at the concentration of 10 microM that effectively inhibited the 20S proteasome activity in platelets, indicating that ex vivo anti-aggregatory effect of PSI proceeds through an indirect mechanism not associated with suppression of 20S proteasome activity in platelets. In conclusion, our in vivo findings demonstrate that proteasome inhibitor, Z-Ile-Glu(Ot-Bu)Ala-Leucinal, prevents the development of arterial thrombosis in renovascular hypertensive rats and effectively suppresses platelet aggregation by an indirect mechanism. Thus the data provide a new insight into the potential role for the proteasome-dependent pathway in cardiovascular events.

Ethanol-induced neurotoxicity is counterbalanced by increased cell proliferation in mouse dentate gyrus.

Chronic ethanol abuse leads to degenerative changes in the hippocampus, which may result in subsequent cognitive impairment. Since the hippocampus retains the ability to produce neurons through adulthood, in the present study we examined if ethanol-induced neuronal loss could be counterbalanced by cell proliferation in mouse dentate gyrus (DG). A total of 14 days of ethanol administration resulted in marked increase in cells positive for TdT-mediated dUTP nick-end labeling in all hippocampal regions studied, indicating that neurons die throughout the hippocampus by apoptotic mechanism. However, cresyl violet staining revealed approximately 20% neuronal loss following ethanol administration in CA1 and CA2 fields (P<0.01 and P<0.05, respectively), but not in DG. At the same time ethanol caused 2-fold increase in the number of proliferating cells in subgranular zone of DG. Thus, long-term ethanol intoxication causes permanent damage to CA1 and CA2, but not to DG which can be counterbalanced by ongoing neurogenesis.

Aspirin and the fibrinolytic response.

Apart from the anti-inflammatory action, aspirin (ASA) by inhibiting thromboxane A2 synthesis, decreases platelets activity and possesses the antithrombotic action. However, an ASA effect on fibrinolysis has not been yet finally established. Menon [Lancet 1 (1970) 364] reported increased fibrinolytic response in patients treated with high doses of ASA and this observation started a series of studies to find the relation between aspirin and fibrinolysis. This review comprises the results of those studies, divided into in vitro and in vivo, animal and human experiments. The results of our animal studies are also included. Data survey shows that the ASA effect on fibrinolysis depends on experimental conditions, the dose and the time of drug administration. The results of our study indicate the essential role of plasma components in the fibrinolysis regulation by ASA.

The plasmin system in airway remodeling.

Recent studies suggest that the plasmin system plays an active role in tissue remodeling. Plasmin degrades the extracellular matrix (ECM), either directly removing glycoproteins from ECM or by activating matrix metalloproteinases (MMPs). PAI-1 blocking MMPs may prevent ECM degradation, but inhibiting fibrinolysis leads to fibrin accumulation and fibrosis. Components of the plasmin system including tissue plasminogen activator (t-PA), urokinase plasminogen activator (u-PA), and plasminogen activator inhibitors PAI-1 and PAI-2 are synthesised by airway cells, and inflammatory mediators affect their expression. The plasmin system, in turn, actively influences the production of inflammatory mediators and growth factors, extending pathological structural changes in the airway. Modulation of the plasmin system might be a new pharmacological strategy that could inhibit the development of airway remodeling.

Increased levels of 3-hydroxykynurenine in different brain regions of rats with chronic renal insufficiency.

Tryptophan (TRP) metabolism via the kynurenine pathway leads to formations of neuroactive substances like kynurenine (KYN) and 3-hydroxykynurenine (3-HK), which may be involved in the pathogenesis of several human brain diseases. 3-Hydroxykynurenine especially is known to have strong neurotoxic properties. The generation of reactive oxygen species (ROS) leads to neuronal cell death with apoptotic features. Because the chronic renal insufficiency (CRI) results in disturbances in the functioning of the central nervous system (CNS), it is conceivable that the metabolism of some kynurenines may be altered and could play an important role in uremic encephalopathy. The levels of TRP, KYN and 3-HK were measured in the plasma and in different brain regions of uremic rats. The total plasma concentration of TRP as well as in all the studied brain samples was significantly diminished during uremia. Surprisingly, the level of KYN and 3-HK were elevated both in the plasma and different brain regions of CRI animals. KYN concentrations were approximately two times higher in the cerebellum, midbrain and cortex compared to the control group. The changes of 3-HK levels were more pronounced in the striatum and medulla than in other structures. This data suggests that CRI results in deep disturbances on the kynurenine pathway in CNS, which could be responsible for neurological abnormalities seen in uremia.

Kynurenine metabolism in central nervous system in experimental chronic renal failure.

Tryptophan metablolism via kynureninc pathway leads to the formation of several neuroactive substances including kynurenine, anthranilic acid and quinolinic acid, which are involved in numerous neurodegenerative diseases. Also chronic renal insufficiency is associated with neurological disturbances but it is still not clear which substances are responsible for those disorders. Thus, the aim of our study was to evaluate the concentration of tryptophan, kynurenine and anthranilic acid in plasma as well as in different brain regions in uremic rats. We have shown that tryptophan concentrations in plasma and in brain were decreased, whereas kynurenine and anthranilic acid levels were elevated, both in plasma and in central nervous system. Only in cerebellum and hippocampus were no difference in concentration of antranilic acid between control and uremic rats. Accumulation of tryptophan metabolites in nervous tissue may be involved in pathogenesis of several neurological disorders in uremia.