M. Petríková

On the inhibitory effect of chloroquine on blood platelet aggregation

Chloroquine inhibited aggregation of rat blood platelets in a dose-dependent way. The inhibitory effect decreased, depending on the aggregation stimulus used, in the rank order of potency: ADP > thrombin > A23187. Effect of 1 millimolar chloroquine on A23187-stimulated aggregation was significantly decreased or completely abolished by addition of Ca2+ ions in the concentration of 0.1 or 1 mmol/l, respectively. For thrombin-stimulated aggregation the effect of chloroquine was partially decreased by corresponding isomolar calcium; platelet aggregation induced by ADP was not changed in the presence of extracellular Ca2+ ions. Addition of chloroquine during aggregation resulted in disaggregation of ADP-stimulated platelets and inhibition of thrombin- and A23187-induced aggregation.

Mechanism of anti-inflammatory action of liquorice extract and glycyrrhizin

The antiradical activity, protective effect against lipid peroxidation of liposomal membrane, and inhibitory effect on whole blood reactive oxygen species (ROS) liberation of Glycyrrhiza glabra crude extract and glycyrrhizin, its major compound, were assessed. The liquorice extract showed significant activity in all the three assay systems used in a dose dependent manner. It displayed remarkable reactivity with free stable 1,1′-diphenyl-2-picrylhydrazyl (DPPH) radical, inhibitory efficacy in peroxidatively damaged unilamellar dioleoyl phosphatidylcholine (DOPC) liposomes, and inhibition of ROS chemiluminescence, generated by whole blood, induced by both receptor-bypassing stimuli (PMA) and receptor operating stimuli (Opz) in the ranking order of stimuli PMA> Opz. These activities may be attributed to phenolic antioxidants involving isoflavan derivatives, coumarins and chalcones. Nonetheless, triterpene saponin glycyrrhizin exhibited no efficacy in the system of DPPH reaction and peroxidation of liposomal membrane, and negligible inhibition of chemiluminescence generated by inflammatory cells. These results indicate that the mechanism of anti-inflammatory effect of glycyrrhizin most probably does not involve ROS and this major constituent is not responsible for the inhibition effects of liquorice extract on neutrophil functions.

Chloroquine inhibits stimulated platelets at the arachidonic acid pathway

Chloroquine inhibited arachidonic acid liberation from membrane phospholipids of thrombin- and A23187- stimulated platelets. In addition, it dose-dependently inhibited stimulated malondialdehyde formation and thromboxane B2 generation in the same platelets. The linear correlation between the inhibition of arachidonic acid liberation and malondialdehyde formation indicated that chloroquine inhibited activated phospholipase A2 in thrombin-stimulated platelets, similarly as it does in different cells and tissues. Yet, the nonlinear relationship between arachidonic acid liberation along with malondialdehyde formation and thromboxane generation as well as aggregation suggest that phospholipase A2 does not seem to be the only site of chloroquine action. Rather, it may affect platelets either at other levels of the arachidonic acid cascade too, or at some different stimulatory pathways, like intraplatelet calcium mobilisation, phosphoinositide cycle, calmodulin and protein kinase C activation.

An inhibitor of thrombin-stimulated blood platelet aggregation from the salivary glands of the hard tick Amblyomma variegatum (Acari: Ixodidae)

The tropical bont tick. Amblyomma variegation can cause intense skin irritation and inflammation and bites that often develop into septic wounds or abscess in their host. Crude salivary gland extract (SGE) of partially engorged A. variegatum females as well as SGE protein fractions purified by three-step reverse phase HPLC procedure were tested for their antiaggregatory effect on isolated human blood platelets stimulated with thrombin and compared with the effect of recombinant hirudin. At concentrations 10−3 and 5 × 10−3 μg protein/ml the following rank order of antiplatelet activity was detected: AV 16/3 (inhibitor purified from AV-III, third purification) > SGE > AV-II (fraction from first purification) > AV-III (fraction from first purification) > hirudin. The effect of all fractions tested was dose-dependent. For fraction AV 16/3, the inhibitory effect was 49 and 61% for 10−3 and 5 × 10−3 μg protein/ml, respectively. The results suggest that protein fractions from A. variegatum SGE possess an antithrombin effect on human blood platelets with hirudin-like activity.

Serotonin modulates the oxidative burst of human phagocytes via various mechanisms.

Serotonin, the major secretory product of activated platelets, has been widely reported as regulating various constituents of the immune system and immune functions. This modulation is complex and the data available are rather controversial. The aim of the present study was to clarify the mechanisms of serotonin action on human phagocytes. The effect of serotonin in a concentration range of 10−7 M–10−3 M on various parameters of oxidative burst of phagocytes was studied using various luminol-enhanced chemiluminescence methods. Serotonin inhibited the chemiluminescence response of the cells in a dose dependent manner. The effect of serotonin on the activity of myeloperoxidase was studied in further experiments. In this case, serotonin again exerted a dose dependent inhibition of the myeloperoxidase activity. The hypothesis that the inhibitory activity of serotonin might be also receptor mediated was evaluated using various serotonin receptor agonists and antagonists. None of the agonists studied exerted any direct antioxidative properties. Only (±)-DOI hydrochloride, a selective 5-HTR2 agonist, exerted similar effects on phagocytic cells as serotonin. It can be concluded that serotonin could affect the oxidative burst of phagocytes. Responsibility for its inhibitory effects lies with both the decrease in the generation of reactive oxygen species (due to the inhibition of myeloperoxidase activity) and with direct scavenging of reactive oxygen species. The effect of serotonin on phagocytes is also partially mediated by 5-HTR2 receptor.

Antiplatelet activity of carvedilol in comparison to propranolol

The non-selective vasodilating g -blocker carvedilol was found to inhibit platelet aggregation as well as thromboxane B 2 formation more effectively than propranolol. The antiaggregatory activity of carvedilol decreased, depending on the stimulus used, in the following rank order of potency (in parentheses the respective mean inhibitory concentrations of carvedilol and propranolol are given in w mol/l): PMA (19 and 34) > thrombin (55 and 77) > Ca 2+ -ionophore A23187 (58 and 81) > epinephrine (86 and 118). However, aggregation of platelets activated with ADP was not affected by carvedilol in concentrations up to 100 w mol/l. In platelets stimulated with thrombin, carvedilol (10 w mol/l) reduced thromboxane B 2 formation by 64%, whereas propranolol was ineffective at this concentration. Moreover, A23187-induced formation of thromboxane B 2 , not affected by propranolol, was completely blocked by 100 w mol/l carvedilol. In comparison to propranolol, the molecule of carvedilol is more lipophilic and possesses lower dipole moment and higher molar refractivity, thus penetrating into platelet membranes readily and in large quantities. The antiplatelet effect was assumed to result from interactions of carvedilol with membrane macromolecules (phospholipids, ion channels, enzymes, etc.) rather than from blockade of f - and g -adrenergic receptors.

Antiplatelet and antiphagocyte activity of H1-antihistamines

No Abstract..

Antiaggregatory Properties of Beta-adrenoceptor Blocking Drugs are Related to their Physico-chemical Properties

The effects of the beta-adrenoceptor blockers (BAB) alprenolol, atenolol, metipranolol, oxprenolol, practolol and propranolol on platelet aggregation stimulated by ADP, thrombin and collagen were investigated. The BAB were divided into three groups according to their liposolubility and antiaggregatory activity. The first group consists of the highly active drugs alprenolol and propranolol; metipranolol and oxprenolol are in the second group; the least active atenolol and practolol are in the third group. Collagen induced platelet aggregation was inhibited by BAB to the highest extent, followed by inhibition of thrombin stimulated aggregation. ADP-induced aggregation was inhibited to the lowest extent. Consistent with the rank order of their partition coefficients, BAB inhibited aggregation with the following order of potency: propranolol > alprenolol > metipranolol > oxprenolol > practolol > atenolol. A positive correlation was found between inhibition of thrombin and collagen stimulated platelet aggregation and the partition coefficients of the individual drugs. A significant time-dependent inhibition of platelet aggregation developed within 30 s of exposure of blood platelets to BAB.

Inhibition of FMLP-stimulated neutrophil chemiluminescence by blood platelets increased in the presence of the serotonin-liberating drug chloroquine

INTRODUCTION Previously, we reported that human blood platelets significantly decreased the concentration of reactive oxygen species (chemiluminescence) produced by Ca(2+)-ionophore-stimulated neutrophils and that the reduction was partially mediated by serotonin liberated from platelets during their activation. The aim of the present study was to investigate whether platelet inhibition can occur independently of serotonin liberation and whether it can be pharmacologically enhanced. MATERIALS AND METHODS Chemiluminescence was measured after stimulation of human neutrophils with N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) in the presence of luminophore luminol. Concentration of platelet serotonin was estimated fluorometrically. RESULTS Platelets, added to neutrophils in the physiological cell ratio 50:1, decreased neutrophil chemiluminescence by 47%. The inhibition was not accompanied with liberation of platelet serotonin and rose after addition of chloroquine to platelet-neutrophil samples. In the absence of platelets, this drug did not affect neutrophil chemiluminescence. Chloroquine actively liberated serotonin; amine concentrations found in platelet supernatants were sufficient to inhibit neutrophil chemiluminescence. CONCLUSIONS The presented results indicate that unstimulated platelets decreased neutrophil chemiluminescence by a serotonin-independent mechanism, yet their inhibitory effect could be enhanced pharmacologically through chloroquine-induced serotonin liberation.

Antihistaminic drug bromadryl and stimulated platelet aggregation

The H1-receptor antagonist bromadryl dosedependently inhibited stimulated rat platelet aggregationin vitro. Bromadryl was 10 times more effective on the secondary aggregation of thrombin-stimulated platelets compared with its inhibition of ADP-stimulated primary platelet aggregation in plasma. The inhibition of aggregation was accompanied by a dose-dependent inhibition of thrombin-stimulated malondialdehyde formation and thromboxane B2 production. The results indicate that bromadryl may interfere intracellularly with membrane phospholipid peroxidation and the arachidonic acid metabolism of stimulated platelets. Bromadryl, like other cationic amphiphilic drugs, may inhibit stimulated platelet functions by decreasing the stimulus-induced activation of phospholipase A2. Our results support the possible interference of bromadryl with histamine as an intraplatelet messenger responsible for aggregation.