Benoit Xhonneux

Evidence for functional 5-HT2 receptor sites on human blood platelets.

The aggregation of normal human platelets by 5-hydroxytryptamine (5-HT) is the result of a specific interaction of the monoamine with a platelet receptor since it is not influenced by adrenergic receptor blockade, inhibition of fatty acid cyclo-oxygenase or ADP-scavenging. The 5-HT induced platelet reaction is inhibited in a concentration-dependent way by various serotonergic antagonists; the potency of these compounds in this respect correlates strongly with their potential to inhibit the specific binding of [3H] ketanserin, a selective label for 5-HT2 binding sites, to rat prefrontal cortex and striatum and to cat platelet membranes. This study thus provides evidence for a functional role as true receptor initiating a physiological response of the 5-HT2 receptor on human platelets.

The involvement of 5-HT2-receptor sites in the activation of cat platelets.

5-Hydroxytryptamine (5-HT) induces a concentration-dependent aggregation/release of/by cat platelets (Km = 6.2 x 10(-7) M); this activation is inhibited (Ki = 5.24 x 10(-9) M) or reversed by ketanserin, a selective 5-HT2 receptor antagonist. Comparison of the inhibition of specific [3H] ketanserin binding to cat platelet membranes and rat pre-frontal cortex membranes with that of 5-HT-induced aggregation of cat platelets obtained with various drugs, displaying various receptor binding profiles, reveals a highly significant correlation between the ligand binding and the physiological response (Spearman correlation coefficient r = 0.92 and r = 0.91 respectively, p less than 0.0001; n = 14); inhibition of platelet activation by 5-HT and of uptake of 5-HT are not correlated. Secondary aggregation induced by ADP as well as collagen-induced aggregation are inhibited by the 5-HT receptor antagonists suggesting a primary role of 5-HT in the secondary platelet recruitment subsequent to a release reaction. This study demonstrates a functional role for the 5-HT2 receptors in the primary activation of the platelets by 5-HT and in the secondary aggregation induced by other agonists, especially in platelets superreactive to 5-HT2 receptor activation.

Functional expression of the amplification reaction between serotonin and epinephrine on platelets.

Human platelet reactions are enhanced when a combination of serotonin and epinephrine is used as a stimulus. The amplification of the response operates through S2-serotonergic and alpha 2-adrenergic receptor subtypes. The first wave of platelet aggregation, which is independent of platelet release products, is enhanced while the latency period for the occurrence of release reaction and prostaglandin biosynthesis is shortened. Synergism between serotonin and epinephrine on the platelet occurs at low concentration of agonists and may contribute to the formation of an arterial thrombus in vivo.

Enhanced platelet turnover and prostaglandin production in spontaneously hypertensive rats

The anti-hypertensive effect of ketanserin, a selective 5-HT2 antagonist, both in experimental and human pathology (1,2) could implicate the blood platelets as a source for peripherally available 5-hydroxytryptamine (5-HT), acting to increase peripheral vascular resistance (3). Therefore we examined various blood platelet parameters in spontaneously hypertensive rats in comparison with normotensive Wistar rats.

Oral itraconazole versus topical bifonazole treatment in experimental dermatophytosis

Summary. Guinea pigs, infected with either Trichophyton mentagrophytes or Microsporum canis, were treated orally or topically with azole antifungals daily for two weeks. Fungi located in the stratum corneum were affected similarly by both treatment schedules, showing typical cell wall changes after azole exposure and necrosis of internal organelles. Fungi located in the hair sheaths were affected only by the oral treatment, which not only prevented invasion of the inner hair structures and inflammatory responses but also led to a complete clearance of the infection within 7 days. Topically applied azole treatment was not able to injure fungi in the hair sheaths and did not suppress invasion into the hair shafts. These observations are in favour of oral antifungal medication with azoles for the treatment of der‐matophyte infections involving hairy skin.

Effects of ketanserin, a selective 5-HT2 serotonergic antagonist, on the secondary recruitment of human platelets in vitro.

Ketanserin, a selective 5-HT2 serotonergic receptor antagonist, reducesin vitro the release-associated human platelet aggregation induced by threshold concentrations of collagen and curtails the second wave of aggregation/release induced by critical concentrations of ADP in particular and, to a lesser extent, of 1-epinephrine. Its inhibitory effect on the second waves becomes more pronounced when the reaction is already attenuated by moderate cyclo-oxygenase inhibition with esculetin, by yohimbine or by propranolol. The first wave of aggregation induced by ADP or 1-epinephrine is not affected.Such an inhibition of secondary platelet recruitment by ketanserinin vitro may be due to an inhibition of the 5-HT2 receptor-mediated amplifying effects of platelet-released 5-HT or to a non-specific interference with the platelet membrane, reducing the release of mediators from the platelets.Reduction of the increased plasma BTG levels in patients after ketanserin may result from such release-inhibiting mechanisms.

Biochemical mechanisms in 5-hydroxytryptamine-induced human platelet aggregation

The activation of human platelets by 5-hydroxytryptamine (5-HT) is not accompanied by detectable release of ATP or TXB2. The process is unaffected by cyclooxygenase, thromboxane synthetase or combined cyclooxygenase/lipoxygenase inhibition (suprofen, indomethacin, R19 091, dazoxiben, N.D.G.A., BW755C, esculetin), indicating the absence of involvement of arachidonic acid metabolites. Transmembrane Ca2+-entry blockers (flunarizine, nifedipine, nimodipine) have no effect either, indicating that the activator calcium released by 5-HT comes from intracellular stores. The 5-HT-induced platelet activation is inhibited by stimulators of adenylate cyclase (PGE1, PGE2, isoprenaline, adenosine) and inhibitors of cAMP phosphodiesterase (papaverine, anagrelide, RA233), indicating that also for this type of platelet activation cAMP behaves as a unidirectional, inhibitory regulator.

Platelet activation by endogenous 5-hydroxytryptamine and histamine released by mast cell degranulation with compound 48/80 in the rat

The intravenous injection of the mast cell degranulator C 48/80 (1 mg/kg) in rats did not produce thrombocytopenia nor circulating platelet aggregates but sensitized the platelets to aggregate upon turbulence challenge. Such turbulence-induced platelet aggregation was not accompanied by formation of thromboxane B2. Electron microscopy revealed absence of platelet degranulation. Turbulence-induced platelet aggregation was completely prevented by pre-treatment of the rats with cyproheptadine, dipyridamole and VK 774, partially with ketanserin (5HT2-receptor antagonist), but not with methysergide (anti-serotonergic drug), pyrilamine (antihistaminic drug), suprofen, aspirin (cyclo-oxygenase inhibitors), phentolamine, propranolol, flunarizine, lidoflazine, oxycoumarin or Trasylol®. Combined treatment with the anti-histaminic drug pyrilamine and the 5HT2-receptor antagonist ketanserin resulted in a dose-related inhibition for ketanserin of the turbulence-induced platelet aggregation. These experiments point to an interaction between histamine and 5-hydroxytryptamine in the platelet activation by mast cell released mediators.

Dependence of the antagonism at human platelet 5-HT2 receptors by ketanserin on the reaction pH

The potency for the inhibition of 5-hydroxytryptamine (5-HT)-induced human platelet aggregation by ketanserin, pipamperone, spiperone, cyproheptadine and BW 501 in vitro decreased as the reaction pH increased progressively from 7.4 to 8.6, the largest shift (X 1125) in IC50 value (pH 7.4: 4 X 10(-9) M; pH 8.6: 4.5 X 10(-6) M) being found for ketanserin. With such an alkaline pH-shift, the fraction of the ionized form of the drugs decreased, reduction of the inhibitory capacity and of the ionized fraction being strongly correlated. Ketanserin (40 mg orally, - 15 h) in human volunteers, completely inhibited the 5-HT-induced platelet aggregation measured ex vivo, when tested at a reaction pH of 7.4; without gassing with CO2 5% -O2 95%, the plasma pH became alkaline and the inhibitory potency of the drug was strongly reduced (-60%). This study demonstrates the importance of the reaction pH for the aggregation of human platelets induced by 5-HT and its inhibition by ketanserin.

The effects of saperconazole on the morphology of Candida albicans, Pityrosporum ovale and Trichophyton rubrum in vitro.

Fungal cultures were incubated for various periods of time with saperconazole at concentrations ranging from 10(-10) to 10(-5) M: Candida albicans (4 and 24 h), Pityrosporum ovale (2 and 7 days), and Trichophyton rubrum (1, 2, 3, 7 and 14 days). At the end of the incubation period, fungal morphology was compared with that of control cultures by transmission and scanning electron microscopy. In all three species, major ultrastructural changes were seen from concentrations of 10(-8) to 10(-7) M saperconazole onwards, depending on the species and time of incubation. The main changes were inhibition of hyphal outgrowth (C. albicans), abortive hyphal outgrowth (T. rubrum) and deposition of electron-dense vesicles in a thickened cell wall (C. albicans, T. rubrum). In P. ovale, a direct, necrotizing action was seen without concomitant wall changes.