Maria Gabriella Doni

Platelet activation by diacylglycerol or ionomycin is inhibited by nitroprusside.

Experiments were performed to elucidate the role of cyclic guanosine monophosphate (cGMP) on platelet activation induced by protein kinase C (PKC) activators and calcium ionophore. Human platelets were pretreated with acetylsalicylic acid and with hirudin and apyrase. Aggregation and ATP secretion in response to the PKC activators 4 beta-phorbol 12-myristate 13-acetate (PMA) and 1-oleoyl 2-acetylglycerol (OAG) were inhibited by the nitrovasodilator sodium nitroprusside (SNP), an activator of guanylate cyclase, and by 8-bromo-cyclic GMP (8-Br-cGMP). The experiments were performed in the presence of M&B 22948, an inhibitor of cGMP phosphodiesterase. SNP and 8-Br-cGMP also inhibited platelet aggregation and secretion evoked by the ionophore ionomycin. In fura-2 loaded platelets SNP did not affect basal cytosolic Ca2+ level nor the rise induced by low concentrations of ionomycin, both in the presence and absence of extracellular Ca2+. The phosphorylation of the 47 and 20 kDa protein induced by ionomycin or PMA were not significantly decreased by SNP or 8-Br-cGMP. The present results suggest that cGMP is able to inhibit both the PKC and the Ca(2+)-dependent pathways leading to platelet activation by interfering, similarly to cAMP, with processes following protein phosphorylation, close to the effector systems.

Salicylate fails to prevent the inhibitory effect of 5,8,11,14-eicosatetraynoic acid on human platelet cyclo-oxygenase and lipoxygenase activities

Sodium salicylate is inactive both on cyclo-oxygenase and lipoxygenase prepared from human platelets. It prevents the inhibition of cyclo-oxygenase induced by aspirin, but does not counteract the inhibitory effect of 5,8,11,14-eicosatetraynoic acid on both enzymes. It also fails to interfere with the inhibitory activity of nordihydroguaiaretic acid on lipoxygenase. These data indicate that, unlike eicosatetraynoic acid, non-steroidal anti-inflammatory drugs interact with a site on cyclo-oxygenase distinct from the catalytic site, although related to it. Such a supplementary binding site is lacking on lipoxygenase.

Glutathione Peroxidase Blockage Inhibits Prostaglandin Biosynthesis in Rat Platelets and Aorta

In the present study we demonstrated that the compound 3-amino-1,2,4-triazole (AMT) is a strong inhibitor of erythrocyte glutathione peroxidase (GSH-Px) activity. Moreover, AMT inhibits arachidonic-induced malondialdehyde formation in platelet-rich plasma and prostacyclin-like activity generation in aorta rings. These results give new lines of evidence on the connection between GSH-Px activity and prostaglandin synthesis in rat platelets and arterial vessel wall.

High Glutathione Peroxidase and Prostacyclin-Like Activity Generation in Rat Aorta

Glutathione peroxidase (GSH-Px), together with catalase, superoxide dismutase and vitamin E, is involved in the antioxidant defense of living organisms. It reduces toxic lipid hydroperoxides to harmle

Platelet activation and modulation of the induction of nitric oxide synthase in the conscious rat.

Injection of lipopolysaccharide (LPS) (Salmonella W. Typhosa i.v. bolus) into conscious rats, induced a rapid drop of circulating platelets analogous to that induced by ADP. The animals showed a small fall in mean arterial blood pressure (MABP), an increase in heart rate and a significant increase in plasma nitrite and nitrate level. This result is consistent with the stimulation of an inducible NO synthase (i-NOS). The administration of the stable prostacyclin analogue, iloprost plus ADP or LPS, significantly protected against the decrease in free platelet number induced by ADP or LPS. The plasma nitrite and nitrate level stimulated by LPS was significantly reduced by iloprost and also by prostacyclin. These results are consistent with an inhibition of i-NOS by agents that increase the intracellular level of cAMP. The administration of the NO donor S-Nitroso-N-acyl-D-penicillamine (SNAP) plus ADP or LPS, significantly prevented thrombocytopenia induced by ADP and by LPS. SNAP did not decrease the plasma nitrite and nitrate level stimulated by LPS; furthermore it induced a significant increase of heart rate, without affecting MABP, suggesting a direct accelerating effect of NO on the sino-atrial node. The administration of S-nitroso-glutathione (GSNO), a stable nitrosothiol, plus ADP or LPS, significantly prevented thrombocytopenia induced by ADP but not by LPS. GSNO significantly reduced the plasma nitrite and nitrate level stimulated by LPS. These data demonstrate that the L-Arginine: NO pathway in vivo may be modulated by prostanoids and that compounds which increase cAMP, such as iloprost, are able to protect against LPS-induced early thrombocytopenia.

ADP-induced platelet aggregation in vivo after exclusion of different circulatory districts.

The effect of the ADP infusion on the basal platelet count was studied in controls and in rats submitted to the exclusion of the following circulatory districts: splenic, renal, cerebral and of a hind-limb. After these exclusions the ADP-induced thrombocytopenia was less marked than the controls

Adenosine Uptake and Deamination by Blood Platelets in Different Mammalian Species

Citrated, platelet-rich plasma from human, guinea-pig and rat blood was incubated at 37 degrees C with (8-14C)-adenosine at different concentrations (1, 3, 30, 100 microM) with continuous stirring. Nucleotides were separated by high voltage electrophoresis for 1 h in 0.05 M citrate buffer pH 4.5. The spots were eluted and radioactivity was counted. Labelled adenosine was taken up by platelets within a few seconds and radioactivity increased with time of incubation. Guinea-pig platelets incorporated labelled adenosine more slowly than the platelets of other species: after 3 min of incubation with 1 microM (8-14C)-adenosine, 43% of the total radioactivity was found, whereas human and rat platelets incorporated 73 and 72% of the total radioactivity, respectively. the kinetics of incorporation and of nucleotide formation by the enzyme adenosine kinase indicated that the Km was 72.46 microM and the Vmax was 0.85 microM/min in guinea-pig platelets; the Km was 5.06 microM and the Vmax was 0.14 microM/min in human platelets; the Km was 13.72 microM and the Vmax was 0.74 microM/min in rat platelets. The kinetics of deamination and nucleoside formation by adenosine deaminase indicated that the Km was 28.21 microM and Vmax was 3.22 microM/min in guinea-pig platelets; the Km was 26.40 microM and the Vmax was 1.41 microM/min in human platelets, but the Km was 60.25 microM and the Vmax was 1.49 microM/min in rat platelets. It was concluded that there is no correlation between the inhibitory activity of adenosine on ADP-induced aggregation in the various species and the rare of labelled adenosine incorporation or deamination.

What’s Provoking Different Aggregation between Arterial and Venous Platelets in the Rat?

The present study was designed to clarify the reason why rat platelets obtained from arterial blood show a less marked aggregation than those obtained from venous blood, and to investigate the contribution of the vessel wall to this phenomenon. Incubation of arterial or venous platelet-rich plasma (PRP) with papaverine, a phosphodiesterase blocker, resulted in a more marked inhibition of the aggregation parameters for arterial than for venous PRP, indicating that a cAMP-dependent mechanism is involved. Incubation of PRP in vitro with adenosine deaminase did not significantly modify aggregation. Rats treated in vivo with different doses of acetylsalicylic acid or of tranylcypromine, two cyclo-oxygenase inhibitors, abolished the aggregation differences between arterial and venous PRP. It is suggested that this difference in platelet behavior may be due to a mechanism dependent on a PGI2-like, probably cAMP-related activity in which the heart and/or the lungs may play an important role.

Importance of the Liver in the Regulation of Platelet Activity: Effect of Its Functional Exclusion (Eck Fistula)

The functional exclusion of the liver, obtained with end-to-side anastomosis of the porta into the cava vein results in impairment of platelet activity. In fact the maximal amplitude and velocity of platelet aggregation in vitro are reduced in animals which undergo the operation and are examined 1 month later. Adding fibrinogen to the platelet-rich plasma after portacal anastomosis restores a normal type of aggregation indicating that the lack of this factor is involved in the phenomenon, even if alterations occurring to platelets may be implied, too.

Effect of catecholamines on ADP aggregation of rat platelets in vivo

The influence of catecholamines on the platelet count was studied in an in vivo experimental model obtained with cannulation of the carotid and of the femoral vein. The i.v. infusion of epinephrine and l-norepinephrine induces a low drop in the platelet count and also potentiate aggregation by ADP.