The methods for removal of direct oral anticoagulants and heparins to improve the monitoring of hemostasis: a narrative literature review.

The assessment of hemostasis is necessary to make suitable decisions on the management of patients with thrombotic disorders. In some clinical situations, for example, during thrombophilia screening, the presence of anticoagulants in sample makes diagnosis impossible. Various elimination methods may overcome anticoagulant interference. DOAC-Stop, DOAC-Remove and DOAC Filter are available methods to remove direct oral anticoagulants in diagnostic tests, although there are still reports on their incomplete efficacy in several assays. The new antidotes for direct oral anticoagulants - idarucizumab and andexanet alfa - could be potentially useful, but have their drawbacks. The necessity to remove heparins is also arising as heparin contamination from central venous catheter or therapy with heparin disturbs the appropriate hemostasis assessment. Heparinase and polybrene are already present in commercial reagents but a fully-effective neutralizer is still a challenge for researchers, thus promising candidates remain in the research phase.

Biological Properties of Heparins Modified with an Arylazopyrazole-Based Photoswitch.

Unfractionated heparin (UFH) and enoxaparin (Enox) were substituted with a photoswitch (PS) showing quantitative trans-cis and cis-trans photoisomerizations. Long half-life of the cis photoisomer enabled comparison of the properties of heparins substituted with both PS photoisomers. Hydrodynamic diameter, Dh, of UFH-PS decreased upon trans-cis photoisomerization, the change being more pronounced for UFH-PS with a higher degree of substitution (DS), while Dh of Enox-PS did not significantly change. The anticoagulative properties of substituted heparins were significantly attenuated compared to non-substituted compounds. The interaction of UFH-PS with HSA, lysozyme, and protamine was studied with ITC. Under serum-free conditions, UFH-PS-trans with a high DS stimulated proliferation of murine fibroblasts, while UFH-PS-cis decreased the viability of these cells. Under serum conditions, both UFH-PS-cis and UFH-PS-trans decreased cell viability, the reduction for UFH-PS-cis being higher than that for UFH-PS-trans. Neither Enox-PS-trans nor Enox-PS-cis influenced the viability at concentrations prolonging aPTT, while at higher concentrations their cytotoxicity did not differ.

Monitoring of Anticoagulant Activity of Dabigatran and Rivaroxaban in the Presence of Heparins.

The routine monitoring of direct oral anticoagulants (DOACs) may be considered in patients with renal impairment, patients who are heavily obese, or patients requiring elective surgery. Using the heparin-binding copolymer (HBC) and polybrene, we aimed to develop a solution for monitoring the anticoagulant activity of DOACs in human plasma in the interfering presence of unfractionated heparin (UFH) and enoxaparin. The thrombin time (TT) and anti-factor Xa activity were monitored in pooled plasma from healthy volunteers. In these tests, plasma with dabigatran or rivaroxaban was mixed with UFH or enoxaparin and then incubated with HBC or polybrene, respectively. HBC and polybrene neutralized heparins and enabled monitoring of anticoagulant activity of dabigatran in the TT test. Both agents allowed for accurate measurement of anti-factor Xa activity in the plasma containing rivaroxaban and heparins in the concentration range reached in patients' blood. Here, we present diagnostic tools that may improve the control of anticoagulation by eliminating the contamination of blood samples with heparins and enabling the monitoring of DOACs' activity.

The effect of ChAdOx1 nCov-19 vaccine on arterial thrombosis development and platelet aggregation in female rats.

ChAdOx1 nCoV-19 adenoviral vector vaccine (ChAd) against coronavirus disease 2019 has been associated with vaccine-induced thrombosis and thrombocytopenia (VITT), especially in young women who have presented with unusual localized thrombosis after receiving the vaccine. The pathogenesis of VITT remains incompletely understood. We tried to provide new insights into mechanisms underlying this phenomenon in the model of arterial thrombosis electrically induced in the carotid artery of female rats. At 28 days post-vaccination, ChAd induced SARS-CoV-2-specific neutralizing antibody responses in all animals. The analysis of the blood vessel/thrombus area showed slight luminal narrowing of the carotid artery with extravasation of blood in vaccinated rats. These small changes were not accompanied by differences in thrombus weight and composition. The vaccinated animals presented a slight increase (by around 14-24%) in platelet aggregation. ChAd did not significantly affect blood coagulation, platelet counts, and their activation markers. Unaffected thrombus formation, the lack of thrombocytopenia and all the measured blood and hemostasis parameters that predominantly stayed unchanged, indicate that the ChAd does not increase the risk of arterial thrombosis development in female rats.

Monitoring of Cardiorespiratory Parameters in Rats-Validation Based on Pharmacological Stimulation.

The methods used in preclinical studies should minimize the suffering and the number of animals but still provide precise and consistent results enabling the introduction of drug candidates into the phase of clinical trials. Thus, we aimed to develop a method allowing us to perform preliminary safety and toxicity studies of candidates for human medicines, while reducing the number of animals. We have devised a method based on a combination of two devices: Plugsys (Transonics System Inc., Ithaca, NY, USA) and PhysioSuite (Kent Scientific Corporation, Torrington, CT, USA), which allow simultaneous registration of nine circulatory and respiratory parameters, and body temperature. Vehicle and adrenaline, or nitroglycerin, as reference substances were administered into the right femoral vein of Wistar rats. Physiological conditions were registered over 60 min after drug administration by measuring systolic, diastolic and mean blood pressure, heart rate (HR), blood perfusion of paw vessels, blood oxygen saturation, respiratory rate, average and peak exhaled CO2, and body temperature. Blood pressure was measured by cannula placed in the left common carotid artery and connected to the pressure transducer (Plugsys). The other parameters were measured by the PhysioSuite. Adrenaline-induced immediate dose-related hypertension and nitroglycerin hypotension were correlated with the change in blood perfusion. They both increased HR. Adrenaline decreased blood oxygen saturation and slightly affected respiratory parameters, while nitroglycerin caused a progressive increase in respiratory rate and a decrease in the peak of exhaled CO2. Our method may become an inseparable part of the preliminary safety and toxicity studies of tested drugs, while being an important step towards improving animal welfare.

Reversal Activity and Toxicity of Heparin-Binding Copolymer after Subcutaneous Administration of Enoxaparin in Mice.

Uncontrolled bleeding after enoxaparin (ENX) is rare but may be life-threatening. The only registered antidote for ENX, protamine sulfate (PS), has 60% efficacy and can cause severe adverse side effects. We developed a diblock copolymer, heparin-binding copolymer (HBC), that reverses intravenously administered heparins. Here, we focused on the HBC inhibitory activity against subcutaneously administered ENX in healthy mice. BALB/c mice were subcutaneously injected with ENX at the dose of 5 mg/kg. After 110 min, vehicle, HBC (6.25 and 12.5 mg/kg), or PS (5 and 10 mg/kg) were administered into the tail vein. The blood was collected after 3, 10, 60, 120, 360, and 600 min after vehicle, HBC, or PS administration. The activities of antifactors Xa and IIa and biochemical parameters were measured. The main organs were collected for histological analysis. HBC at the lower dose reversed the effect of ENX on antifactor Xa activity for 10 min after antidote administration, whereas at the higher dose, HBC reversed the effect on antifactor Xa activity throughout the course of the experiment. Both doses of HBC completely reversed the effect of ENX on antifactor IIa activity. PS did not reverse antifactor Xa activity and partially reversed antifactor IIa activity. HBC modulated biochemical parameters. Histopathological analysis showed changes in the liver, lungs, and spleen of mice treated with HBC and in the lungs and heart of mice treated with PS. HBC administered in an appropriate dose might be an efficient substitute for PS to reverse significantly increased anticoagulant activity that may be connected with major bleeding in patients receiving ENX subcutaneously.

Cardiovascular and Respiratory Toxicity of Protamine Sulfate in Zebrafish and Rodent Models.

Protamine sulfate (PS) is the only available option to reverse the anticoagulant activity of unfractionated heparin (UFH), however it can cause cardiovascular and respiratory complications. We explored the toxicity of PS and its complexes with UFH in zebrafish, rats, and mice. The involvement of nitric oxide (NO) in the above effects was investigated. Concentration-dependent lethality, morphological defects, and decrease in heart rate (HR) were observed in zebrafish larvae. PS affected HR, blood pressure, respiratory rate, peak exhaled CO2, and blood oxygen saturation in rats. We observed hypotension, increase of HR, perfusion of paw vessels, and enhanced respiratory disturbances with increases doses of PS. We found no effects of PS on human hERG channels or signs of heart damage in mice. The hypotension in rats and bradycardia in zebrafish were partially attenuated by the inhibitor of endothelial NO synthase. The disturbances in cardiovascular and respiratory parameters were reduced or delayed when PS was administered together with UFH. The cardiorespiratory toxicity of PS seems to be charge-dependent and involves enhanced release of NO. PS administered at appropriate doses and ratios with UFH should not cause permanent damage of heart tissue, although careful monitoring of cardiorespiratory parameters is necessary.

Heparin-Binding Copolymer as a Complete Antidote for Low-Molecular-Weight Heparins in Rats.

Bleeding resulting from the application of low-molecular-weight heparins (LMWHs) may be treated with protamine sulfate, but this treatment lacks efficiency; its action against antifactor Xa activity is limited to ∼60%. Moreover, protamine sulfate can cause life-threatening hypersensitivity reactions. We developed diblock heparin-binding copolymer (HBC), which can neutralize the anticoagulant activity of parenteral anticoagulants. In the present study, we explored the safety profile of HBC and its potential to reverse enoxaparin, nadroparin, dalteparin, and tinzaparin in human plasma and at in vivo conditions. HBC-LMWH complexes were characterized using zeta potential, isothermal titration calorimetry, and dynamic light scattering. The rat cardiomyocytes and human endothelial cells were used for the assessment of in vitro toxicity. Male Wistar rats were observed for up to 4 days after HBC administration for clinical evaluation, gross necropsy, and biochemistry and histopathological analysis. Rats were treated with LMWHs alone or followed by short-time intravenous infusion of HBC, and bleeding time and antifactor Xa activity were measured. HBC completely reversed antifactor Xa activity prolonged in vitro by all LMWHs with an optimal weight ratio of 2.5:1. The complexes of HBC-LMWHs were below 5 µm. We observed no effects on the viability of cardiovascular cells treated with HBC at concentrations up to 0.05 mg/ml. Single doses up to 20 mg/kg of HBC were well tolerated by rats. HBC completely reversed the effects of LMWHs on bleeding time and antifactor Xa activity in vivo after 20 minutes and retained ∼80% and ∼60% of reversal activity after 1 and 2 hours, respectively. Well-documented efficacy and safety of HBC both in vitro and in vivo make this polymer a promising candidate for LMWHs reversal. SIGNIFICANCE STATEMENT: Over the last decade, there has been significant progress in developing antidotes for the reversal of anticoagulants. Until now, there has been no effective and safe treatment for patients with severe bleeding under low-molecular-weight heparin therapy. Based on our in vitro and in vivo studies, heparin-binding copolymer seems to be a promising candidate for neutralizing all clinically relevant low-molecular-weight heparins.

The Inhibitory Effect of Protamine on Platelets is Attenuated by Heparin without Inducing Thrombocytopenia in Rodents.

Protamine sulfate (PS) is a polycationic protein drug obtained from the sperm of fish, and is used to reverse the anticoagulant effect of unfractionated heparin (UFH). However, the interactions between PS, UFH, and platelets are still not clear. We measured the platelet numbers and collagen-induced aggregation, P-selectin, platelet factor 4, ß-thromboglobulin, prostacyclin metabolite, D-dimers, activated partial thromboplastin time, prothrombin time, anti-factor Xa, fibrinogen, thrombus weight and megakaryocytopoiesis in blood collected from mice and rats in different time points.. All of the groups were treated intravenously with vehicle, UFH, PS, or UFH with PS. We found a short-term antiplatelet activity of PS in mice and rats, and long-term platelet-independent antithrombotic activity in rats with electrically-induced thrombosis. The antiplatelet and antithrombotic potential of PS may contribute to bleeding risk in PS-overdosed patients. The inhibitory effect of PS on the platelets was attenuated by UFH without inducing thrombocytopenia. Treatment with UFH and PS did not affect the formation, number, or activation of platelets, or the thrombosis development in rodents.

The neutralization of heparan sulfate by heparin-binding copolymer as a potential therapeutic target.

Besides regulating ligand-receptor and cell-cell interactions, heparan sulfate (HS) may participate in the development of many diseases, such as cancer, bacterial or viral infections, and their complications, like bleeding or inflammation. In these cases, the neutralization of HS could be a potential therapeutic target. The heparin-binding copolymer (HBC, PEG41-PMAPTAC53) was previously reported by us as a fully synthetic compound for efficient and safe neutralization of heparins and synthetic anticoagulants. In a search for molecular antagonists of HS, we examined the activity of HBC as an HS inhibitor both in vitro and in vivo and characterized HBC/HS complexes. Using a colorimetric Azure A method, isothermal titration calorimetry and dynamic light scattering techniques we found that HBC binds HS by forming complexes below 200 nm with less than 1 : 1 stoichiometry. We confirmed the HBC inhibitory effect in rats by measuring activated partial thromboplastin time, prothrombin time, anti-factor Xa activity, anti-factor IIa activity, and platelet aggregation. HBC reversed the enhancement of all tested parameters caused by HS demonstrating that cationic synthetic block copolymers may have a therapeutic value in various disorders involving overproduction of HS.

Anticoagulant Properties of Poly(sodium 2-(acrylamido)-2-methylpropanesulfonate)-Based Di- and Triblock Polymers.

Di- and triblock copolymers with low dispersity of molecular weight were synthesized using radical addition-fragmentation chain transfer polymerization. The copolymers contained anionic poly(sodium 2-acrylamido-2-methylpropanesulfonate) (PAMPS) block as an anticoagulant component. The block added to lower the toxicity was either poly(ethylene glycol) (PEG) or poly(2-(methacryloyloxy)ethyl phosphorylcholine) (PMPC). The polymers prolonged clotting times both in vitro and in vivo. The influence of the polymer architecture and composition on the efficacy of anticoagulation and safety parameters was evaluated. The polymer with the optimal safety/efficacy profile was PEG47- b-PAMPS108, i.e., a block copolymer with the degrees of polymerization of PEG and PAMPS blocks equal to 47 and 108, respectively. The anticoagulant action of copolymers is probably mediated by antithrombin, but it differs from that of unfractionated heparin. PEG47- b-PAMPS108 also inhibited platelet aggregation in vitro and increased the prostacyclin production but had no antiplatelet properties in vivo. PEG47- b-PAMPS108 anticoagulant activity can be efficiently reversed with a copolymer of PEG and poly((3-(methacryloylamino)propyl)trimethylammonium chloride) (PMAPTAC) (PEG41- b-PMAPTAC53, HBC), which may be attributed to the formation of polyelectrolyte complexes with PEG shells without anticoagulant properties.

Heparin-binding copolymer reverses effects of unfractionated heparin, enoxaparin, and fondaparinux in rats and mice.

The parenteral anticoagulants may cause uncontrolled and life-threatening bleeding. Protamine, the only registered heparin antidote, is partially effective against low-molecular weight heparins, completely ineffective against fondaparinux and may cause unacceptable toxicity. Therefore, we aimed to develop a synthetic compound for safe and efficient neutralization of all parenteral anticoagulants. We synthesized pegylated PMAPTAC block copolymers, and then, we selected a lead heparin-binding copolymer (HBC). We assessed the effectiveness of HBC in the model of arterial thrombosis electrically induced in the carotid artery of rats by measuring thrombus weight, bleeding time, activated partial thromboplastin time, activated clotting time, and anti-factor Xa activity. The intravital tissue distribution, the cardiorespiratory, and organ toxicity were monitored. HBC diminished antithrombotic and anticoagulant effects of unfractionated heparin. Moreover, it stopped bleeding and completely reversed the enhancement of clotting times and anti-factor Xa activity caused by enoxaparin or fondaparinux. We observed slight pulmonary congestion and cell infiltration, but the cardiorespiratory parameters remained unchanged. We found a strong signal of fluorescently-labeled HBC in the urine, and a weaker in the liver and in the kidney. No signs of hepatic or nephrotoxicity were observed in the blood biochemistry or histopathologic examination. We developed a copolymer efficiently neutralizing effects of heparins in the living organism, which shows a very promising efficacy/safety profile and may help in the management of uncontrolled bleeding resulting from an anticoagulant injection. HBC could enable the safe replacement of unfractionated heparin with low-molecular weight heparins in patients undergoing cardiac surgery and complex vascular procedures.

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

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.

The Toxicokinetic Profile of Dex40-GTMAC3-a Novel Polysaccharide Candidate for Reversal of Unfractionated Heparin.

Though protamine sulfate is the only approved antidote of unfractionated heparin (UFH), yet may produce life threatening side effects such as systemic hypotension, catastrophic pulmonary vasoconstriction or allergic reactions. We have described 40 kDa dextrans (Dex40) substituted with glycidyltrimethylammonium chloride (GTMAC) as effective, immunogenically and hemodynamically neutral inhibitors of UFH. The aim of the present study was to evaluate in mice and rats toxicokinetic profile of the most promising polymer-Dex40-GTMAC3. Polymer was rapidly eliminated with a half-time of 12.5 ± 3.0 min in Wistar rats, and was mainly distributed to the kidneys and liver in mice. The safety studies included the measurement of blood count and blood biochemistry, erythrocyte osmotic fragility and the evaluation of the histological alterations in kidneys, liver and lungs of mice and rats in acute and chronic experiments. We found that Dex40-GTMAC3 is not only effective but also very well tolerated. Additionally, we found that protamine may cause overt hemolysis with appearance of permanent changes in the liver and kidneys. In summary, fast renal clearance behavior and generally low tissue accumulation of Dex40-GTMAC3 is likely to contribute to its superior to protamine biocompatibility. Intravenous administration of therapeutic doses to living animals does not result in the immunogenic, hemodynamic, blood, and organ toxicity. Dex40-GTMAC3 seems to be a promising effective and safe candidate for further clinical development as new UFH reversal agent.

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.

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.

Aliskiren inhibits experimental venous thrombosis in two-kidney one- clip hypertensive rats.

A substantial amount of evidence links the renin-angiotensin system with thrombosis. For example, ACE inhibitors and angiotensin receptor blockers possess independent of the hemodynamic changes, antithrombotic activity. Aliskiren direct renin inhibitor belongs to a new very promising antihypertensive drug that effectively inhibits the renin-angiotensin system. The aim of study was to determine the influence of aliskiren on stasis-induced venous thrombosis in renovascular hypertensive and normotensive rats. The involvement of nitric oxide and prostacyclin in the potential antithrombotic action was also elucidated. Six weeks after clipping of the left renal artery rats developed hypertension which was confirmed by the "tail cuff" method. Hypertensive and normotensive rats were treated with aliskiren (10, 30 and 100mg/kg/day) per os for 10days. Venous thrombosis was induced by stasis of vena cava inferior. Aliskiren at the highest dose induced a significant decrease in systolic blood pressure in hypertensive, but did not change this parameter in normotensive rats. Oral administration of aliskiren resulted in dose-dependent decrease of venous thrombus weight in hypertensive and normotensive rats. The antithrombotic activity of aliskiren was abolished both by NO synthase inhibitor and prostacyclin synthesis inhibitor. Aliskiren decreased collagen-induced platelet aggregation, increased plasma level of tissue plasminogen activator activity whereas no changes in plasminogen activator inhibitor activity and coagulation parameters were found. We showed that aliskiren prevents the development of venous thrombosis by enhanced fibrinolysis and the blood platelet inhibition via nitric oxide and/or prostacyclin-dependent mechanism.

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.

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.

Inhibition of platelet aggregation by carbon monoxide-releasing molecules (CO-RMs): comparison with NO donors.

Carbon monoxide (CO) and CO-releasing molecules (CO-RMs) inhibit platelet aggregation in vitro. Herein, we compare the anti-platelet action of CORM-3, which releases CO rapidly (t (½) 1 min), and CORM-A1, which slowly releases CO (t(½) = 21 min). The anti-platelet effects of NO donors with various kinetics of NO release were studied for comparison. The effects of CO-RMs and NO donors were analyzed in washed human platelets (WP), platelets rich plasma (PRP), or whole blood (WB) using aggregometry technique. CORM-3 and CORM-A1 inhibited platelet aggregation in human PRP, WP, or WB, in a concentration-dependent manner. In all three preparations, CORM-A1 was more potent than CORM-3. Inhibition of platelets aggregation by CORM-A1 was not significantly affected by a guanylate cyclase inhibitor (ODQ) and a phosphodiesterase-5 inhibitor, sildenafil. In contrast, inhibition of platelet aggregation by NO donors was more potent with a fast NO releaser (DEA-NO, t (½) = 2 min) than slow NO releasers such as PAPA-NO (t (½) = 15 min) or other slow NO donors. Predictably, the anti-platelet effect of DEA-NO and other NO donors was reversed by ODQ while potentiated by sildenafil. In contrast to NO donors which inhibit platelets proportionally to the kinetics of NO released via activation of soluble guanylate cyclase (sGC), the slow CO-releaser CORM-A1 is a superior anti-platelet agent as compared to CORM-3 which releases CO instantly. The anti-platelet action of CO-RMs does not involve sGC activation. Importantly, CORM-A1 or its derivatives representing the class of slow CO releasers display promising pharmacological profile as anti-platelet agents.