Andrzej Mogielnicki

1-Methylnicotinamide (MNA), a primary metabolite of nicotinamide, exerts anti-thrombotic activity mediated by a cyclooxygenase-2/prostacyclin pathway

1‐methylnicotinamide (MNA) has been considered to be an inactive metabolite of nicotinamide. Here we assessed the anti‐thrombotic activity of MNA in vivo.

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.

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. Conclusion—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.

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.

Cationic derivative of dextran reverses anticoagulant activity of unfractionated heparin in animal models of arterial and venous thrombosis.

Heparin is a natural polymer widely used in medicine especially during the treatment of cardiovascular diseases since it is a potent blood anticoagulant. In case of emergency, e.g., massive hemorrhage, the anticoagulant activity of heparin has to be quickly stopped by the administration of a heparin reversing agent. Currently protamine sulfate, an allergenic protein, is used for this purpose. We are reporting the studies on a new polymeric substance, a cationic dextran derivative, which is able to form complexes with heparin. Dextran is a blood compatible polymer which is also frequently applied in medicine. By substituting dextran with glycidyltrimethylammonium chloride a cationic polymer was obtained that in vitro binds to heparin with an efficiency similar to that of protamine. To investigate the influence of modified dextran on the reversal of conventional heparin we used the models of experimental arterial thrombosis induced by electrical stimulation and chemically induced venous thrombosis. A decrease in bleeding time and activated partial thromboplastin time after administration of the cationic dextran to heparinized rats was found. Moreover, other routinely measured blood parameters are significantly affected. Modified dextran, in contrast to protamine sulfate, significantly increases red blood cell counts, hemoglobin level, and hematocrit value. The data we obtained show that the modified dextran may reduce anticoagulative heparin activity both under in vivo and in vitro conditions. Further clinical studies are needed to estimate whether modified dextran could replace protamine sulfate, especially in dialyzed patients with the end-stage renal disease associated with anemia.

Antithrombotic Effects of Pyridinium Compounds Formed from Trigonelline upon Coffee Roasting

Coffee may exert a preventive effect on arterial thrombosis. Trigonelline is one of the most abundant compounds in coffee that undergoes pyrolysis upon roasting of coffee beans. The aim of the present study was to identify pyridinium compounds formed upon trigonelline pyrolysis and coffee roasting and to investigate the effect of three of them, i.e., 1-methylpyridine and 1,3- and 1,4-dimethylpyridine, on experimentally induced arterial thrombosis in rats. 1,3- and 1,4-dimethylpyridine but not 1-methylpyridine inhibited arterial thrombus formation. 1,3-Dimethylpyridine inhibited platelet aggregation and reduced fibrin formation in platelet-rich plasma, whereas 1,4-dimethylpyridine increased the plasma level of 6-keto-PGF1α. 1,4-Dimethylpyridine slightly increased rat tissue plasminogen activator plasma activity. In summary, we demonstrated that pyridinium compounds display mild antithrombotic properties due to stimulation by prostacyclin release (1,4-dimethylpyridine) and inhibition of platelet aggregation (1,3-dimethylpyridine). Those pyridinium compounds may, to some extent, be responsible for the beneficial effects of coffee drinking.

The toxicology of heparin reversal with protamine: past, present and future.

ABSTRACT Introduction: Unfractionated heparin is a strongly anionic anticoagulant used extensively in medicine to prevent blood clotting. In the case of an emergency bleeding in response to heparin, the protamine sulfate is administered. Despite its extensive clinical use, protamine may produce life-threatening side effects such as systemic hypotension, catastrophic pulmonary vasoconstriction or allergic reactions. Recent studies have demonstrated new organ-specific complications of the heparin reversal with protamine. Areas covered: Past and present knowledge of the mechanisms responsible for the toxicity of protamine and the most promising potential replacements of protamine in the different phases of development. Expert opinion: Despite of the low therapeutic index, protamine is the only registered antidote of heparins. The toxicology of protamine depends on a complex interaction of the high molecular weight, a cationic peptide with the surfaces of the vasculature and blood cells. The mechanisms involve membrane receptors and ion channels targeted by different vasoactive compounds, such as nitric oxide, bradykinin or histamine. Unacceptable side effects of protamine have led to a search for new alternatives: UHRA, LMWP, and Dex40-GTMAC3 are in the preclinical stage; the two other agents (andexanet alfa and PER977) are already in the advanced clinical phases.

Nonclinical Evaluation of Novel Cationically Modified Polysaccharide Antidotes for Unfractionated Heparin

Protamine, the only registered antidote of unfractionated heparin (UFH), may produce a number of adverse effects, such as anaphylactic shock or serious hypotension. We aimed to develop an alternative UFH antidote as efficient as protamine, but safer and easier to produce. As a starting material, we have chosen generally non-toxic, biocompatible, widely available, inexpensive, and easy to functionalize polysaccharides. Our approach was to synthesize, purify and characterize cationic derivatives of dextran, hydroxypropylcellulose, pullulan and γ-cyclodextrin, then to screen them for potential heparin-reversal activity using an in vitro assay and finally examine efficacy and safety of the most active polymers in Wistar rat and BALB/c mouse models of experimentally induced arterial and venous thrombosis. Efficacy studies included the measurement of thrombus formation, activated partial thromboplastin time, bleeding time, and anti-factor Xa activity; safety studies included the measurement of hemodynamic, hematologic and immunologic parameters. Linear, high molecular weight dextran substituted with glycidyltrimethylammonium chloride groups at a ratio of 0.65 per glucose unit (Dex40-GTMAC3) is the most potent and the safest UFH inhibitor showing activity comparable to that of protamine while possessing lower immunogenicity. Cationic polysaccharides of various structures neutralize UFH. Dex40-GTMAC3 is a promising and potentially better UFH antidote than protamine.

Angiotensin-(1-9) enhances stasis-induced venous thrombosis in the rat because of the impairment of fibrinolysis

Introduction: ACE2 alternatively converts angiotensin (Ang) II into Ang-(1–7) and Ang I into Ang-(1–9). There is little information in the literature with respect to Ang-(1–9) properties. A number of studies show a link between peptides of the renin-angiotensin system and thrombosis. Materials and methods: We have investigated the influence of Ang-(1–9) on stasis-induced venous thrombosis in the rat. The contribution of coagulation and fibrinolytic systems, angiotensin receptor type 1 (AT1) and MAS receptor in the mode of Ang-(1–9) action was also determined. Results: Ang-(1–9) enhanced thrombosis development, decreased plasma concentration of tissue plasminogen activator and increased the level of its inhibitor (PAI-1). The action of Ang-(1–9) was reversed by selective antagonist of AT1 receptor, but not Ang-(1–7) antagonist. Ang-(1–9) did not bind to the AT1 receptor. Conclusions: Ang-(1–9) enhances venous thrombosis in the rat because of the impairment of fibrinolysis. The prothrombotic effect of Ang-(1–9) is mediated by Ang II acting via the AT1 receptor.

New arginine substituted derivative of poly(allylamine hydrochloride) for heparin reversal

New derivatives of polyallylamine containing arginine moieties (PAH-ARG) were synthesized. The in vitro tests performed in heparinized blood plasma showed that the complexation of heparin by PAH-ARG polymers allowed the reduction of the activated partial thromboplastin time (aPTT) values to the normal level. The dose of PAH-ARG required for complete reversal of aPTT (prolonged by 1 U of heparin) was half of that required for protamine sulfate, the currently used heparin antagonist. The efficacy of these polymers in the neutralization of heparin was confirmed by in vivo tests using a rat model. PAH-ARG polymers were nontoxic to the fibroblast cells.