Adenylic dinucleotides produced by CD38 are negative endogenous modulators of platelet aggregation.

Diadenosine 5',5'''-P1,P2-diphosphate (Ap2A) is one of the adenylic dinucleotides stored in platelet granules. Along with proaggregant ADP, it is released upon platelet activation and is known to stimulate myocyte proliferation. We have previously demonstrated synthesis of Ap2A and of two isomers thereof, called P18 and P24, from their high pressure liquid chromatography retention time, by the ADP-ribosyl cyclase CD38 in mammalian cells. Here we show that Ap2A and its isomers are present in resting human platelets and are released during thrombin-induced platelet activation. The three adenylic dinucleotides were identified by high pressure liquid chromatography through a comparison with the retention times and the absorption spectra of purified standards. Ap2A, P18, and P24 had no direct effect on platelet aggregation, but they inhibited platelet aggregation induced by physiological agonists (thrombin, ADP, and collagen), with mean IC50 values ranging between 5 and 15 microm. Moreover, the three dinucleotides did not modify the intracellular calcium concentration in resting platelets, whereas they significantly reduced the thrombin-induced intracellular calcium increase. Through binding to the purinergic receptor P2Y11, exogenously applied Ap2A, P18, and P24 increased the intracellular cAMP concentration and stimulated platelet production of nitric oxide, the most important endogenous antiaggregant. The presence of Ap2A, P18, and P24 in resting platelets and their release during thrombin-induced platelet activation at concentrations equal to or higher than the respective IC50 value on platelet aggregation suggest a role of these dinucleotides as endogenous negative modulators of aggregation.

Platelet activation by collagen is increased in retinal vein occlusion.

Retinal vein occlusion (RVO) is the most common retinal vascular disorder second to diabetic retinopathy. The main risk factors in patients with RVO are hypertension, diabetes, hyperlipidemia, increased blood viscosity and glaucoma. The pathogenesis of RVO has not yet been clarified. In these events platelets could play a very important role. In the present study the platelet response to collagen was deeply investigated. Experiments were carried out on a selected group of RVO patients, which were compared to a group of healthy subjects matched for age, sex, clinical and metabolic characteristics. In resting and activated platelets of both groups of subjects p72syk phosphorylation, phospholipase Cgamma2 phosphorylation, protein kinase C activation, intra-cellular calcium levels and nitric oxide formation were measured. Results show that platelets of patients were more responsive to collagen or ADP than healthy subjects and that the response was significantly different (p < 0.0005) at low concentrations of these agonists. In platelets of patients stimulated with collagen increased phosphorylation of p72syk and phospholipase Cgamma2 was found. Also protein kinase C was more activated in patients. In addition intracellular calcium rise induced by collagen was significantly higher in patients than in healthy subjects. RVO patients showed a lower basal level of nitric oxide both in resting and stimulated platelets compared to healthy subjects. Altogether these results suggest that the platelet hyperaggregability described in patients might be an important factor in the development of RVO contributing to the thrombogenic effects.

A role for PLCgamma2 in platelet activation by homocysteine.

The aim of this study was to examine the homocysteine effect on phospholipase Cgamma2 (PLCgamma2) activation and to investigate the signaling pathway involved. We found that homocysteine stimulated the tyrosine phosphorylation and activation of platelet PLCgamma2. The tyrosine kinases p60src and p72syk appeared to be involved upstream. Reactive oxygen species were increased in homocysteine treated platelets. Likely oxidative stress could prime the non receptor-mediated tyrosine kinase p60src, inducing phosphorylation and activation of p72syk. The antioxidant N-acetyl-L-cysteine prevented the activation of these kinases. The phosphorylation and activation of PLCgamma2 were greatly reduced by the inhibition of p72syk through piceatannol. Moreover indomethacin diminished the homocysteine effect on p60src, p72syk and PLCgamma2, suggesting that thromboxane A(2) could be involved. In addition the treatment of platelets with homocysteine caused intracellular calcium rise and protein kinase C activation. Finally homocysteine induced platelet aggregation, that was partially reduced by indomethacin and by N-acetyl-L-cysteine of 35% or 50% respectively, while the PLCgamma2 specific inhibitor U73122 diminished platelet response to homocysteine of 70%. Altogether the data indicate that PLCgamma2 plays an important role in platelet activation by homocysteine and that the stimulation of this pathway requires signals through oxygen free radicals and thromboxane A(2).

Mechanisms involved in the antiplatelet activity of 8-methyl-4-(1-piperazinyl)-7-(3-pyridinylmethoxy)-2H-1-benzopyran-2-one (RC414).

The effect on human platelets of 8-methyl-4-(1-piperazinyl)-7-(3-pyridinylmethoxy)-2H-1-benzopyran-2-one (RC414) was tested in vitro by measuring aggregation induced by several agonists, cAMP and cGMP levels, cAMP phosphodiesterase and PKC activities and [Ca2+]i. The RC414 effect on nitric oxide production was also evaluated. RC414 in a dose-dependent manner inhibited aggregation both in platelet rich plasma and in washed platelets. It was particularly effective in platelets challenged by collagen, ADP and thrombin: IC50 values are 0.51 +/- 0.12 microM, 0.98 +/- 0.36 microM and 1.00 +/- 0.15 microM, respectively. RC414 increased cAMP levels, through the specific inhibition of the cAMP high affinity phosphodiesterase (IC50 = 1.73 +/- 0.35 microM). RC414 reduced [Ca2+]i transients and PKC activation induced by thrombin. In addition RC414 was able to increase nitric oxide formation involving the stimulation of constitutive nitric oxide synthase enzyme. In conclusion, RC414 exerts its powerful anti-platelet activity by increasing cAMP intracellular levels and nitric oxide formation.