Ticlopidine and clopidogrel (SR 25990C) selectively neutralize ADP inhibition of PGE1-activated platelet adenylate cyclase in rats and rabbits.

Ticlopidine and its potent analogue, clopidogrel, are powerful inhibitors of ADP-induced platelet aggregation. In order to improve the understanding of this ADP-selectivity, we studied the effect of these compounds on PGE1-stimulated adenylate cyclase and on the inhibition of this enzyme by ADP, epinephrine and thrombin. Neither drug changed the basal cAMP levels nor the kinetics of cAMP accumulation upon PGE1-stimulation in rat or rabbit platelets, which excludes any direct effect on adenylate cyclase or on cyclic nucleotide phosphodiesterase. However, the drop in cAMP levels observed after addition of ADP to PGE1-stimulated control platelets was inhibited in platelets from treated animals. In contrast, the drop in cAMP levels produced by epinephrine was not prevented by either drug in rabbit platelets. In rat platelets, thrombin inhibited the PGE1-induced cAMP elevation but this effects seems to be entirely mediated by the released ADP. Under these conditions, it was not surprising to find that clopidogrel also potently inhibited that effect of thrombin on platelet adenylate cyclase. In conclusion, ticlopidine and clopidogrel selectively neutralize the ADP inhibition of PGE1-activated platelet adenylate cyclase in rats and rabbits.

The thienopyridine ticlopidine selectively prevents the inhibitory effects of ADP but not of adrenaline on cAMP levels raised by stimulation of the adenylate cyclase of human platelets by PGE1.

After oral administration, ticlopidine specifically inhibits ADP-induced platelet aggregation, prolongs the bleeding time and prevents thrombosis in man. Its mechanism of action is not well known. Ticlopidine inhibits ADP-induced binding of fibrinogen to platelet glycoprotein GP IIb-IIIa but not shape change and increases deaggregation. Ticlopidine has no direct effect on the GP IIb-IIIa complex. We studied the effects of ticlopidine (500 mg/day for 8 days) in four healthy male volunteers on washed platelet aggregation induced by 5 microM ADP or thrombin (0.1 units/mL) and potentiated by 1 microM adrenaline (Adr), on basal and 1 microM PGE1-stimulated cAMP levels and on elevation of cytosolic free Ca2+ concentration ([Ca2+]i). We found that: (i) ticlopidine inhibits aggregation by ADP but not the potentiation by Adr of ADP-induced aggregation; (ii) ADP, Adr or thrombin decreases cAMP levels raised by PGE1, an effect inhibited by ticlopidine only for ADP and not for Adr or thrombin; and (iii) Ca2+ influx and Ca2+ mobilization from internal stores were not affected. These results suggested that ticlopidine or a metabolite impairs the coupling mechanism of the ADP aggregation pathway at an unknown level.

Behavior of confluent endothelial cells after irradiation. Modulation of wound repair by heparin and acidic fibroblast growth factor.

Image analysis was used to study the repair process of a circular mechanical lesion of confluent human endothelial cells in culture after irradiation (10 Gy) prior to wounding. Coverage of denuded areas 48 and 96 h after injury of endothelial cells was identical in control and irradiated cultures, although the labeling index was lowered by 80 to 95% in irradiated cultures. The cell density of non damaged irradiated areas was decreased by 50%. When cultures were submitted to increasing doses of radiation (5.0-30 Gy), the labeling index of the cells diminished rapidly between 0 and 5.0 Gy and reached a plateau at 10 Gy. The decrease in cell density (50% of control at 96 h) was identical at each dose of radiation. Thus cell migration alone could be sufficient for the repair of the lesion, while cell proliferation would mainly maintain the original cell density. The addition of heparin to the culture medium slowed down cell migration and proliferation, but the speed of repair was identical in irradiated and non-irradiated cultures. Acidic fibroblast growth factor plus heparin accelerated equally the repair process whether the cultures were irradiated or not. In irradiated cultures the presence of acidic fibroblast growth factor and heparin maintained cell density in confluent areas at a level similar to that in non-irradiated damaged control cultures without addition of mitogens. Thus heparin and acidic fibroblast growth factor play a role in cell proliferation, in the maintenance of the cell monolayer integrity and in restoring a continuous layer by rapid cell migration and elongation after irradiation.

Sulfated polysaccharides modulate effects of acidic and basic fibroblast growth factors on repair of injured confluent human vascular endothelium.

Semi-automatic analysis of the repair process of a circular mechanical lesion of confluent human vascular endothelial cells in vitro was used to evaluate the contributions of cell migration and cell proliferation. Standard heparin added to culture medium that contained 30% human serum induced an inhibition of cell migration at the lesion margin during the first day after injury. Several sulfated polysaccharides were tested in the presence of 5% human serum. Standard heparin, low molecular weight heparin, or pentosan polysulfate markedly reduced the rate of lesion regeneration. Cell proliferation, measured by 3H-thymidine incorporation at the lesion margin, and cell migration were both decreased. In contrast, the combination of acidic fibroblast growth factor with a sulfated polysaccharide accelerated the repair process. Basic fibroblast growth factor combined with a sulfated polysaccharide gave a regeneration rate similar to that of the control; however, at 4 days after injury, the residual lesion was the same when basic fibroblast growth factor was used alone or when it was combined with sulfated polysaccharides. Acidic fibroblast growth factor totally reversed the effects of sulfated polysaccharides on the repair process by enhancing endothelial cell proliferation and allowing endothelial cell migration.

Activity of pentosan polysulphate and derived compounds on vascular endothelial cell proliferation and migration induced by acidic and basic FGF in vitro.

Pentosan polysulphate (PPS, SP 54, HEMOCLAR), a highly sulphated semi-synthetic polysaccharide of MW 4700 Daltons is as efficient as heparin in potentiating the mitogenic activity of acidic FGF (aFGF) on human umbilical vein endothelial cells (HUVEC). When added to basic FGF (bFGF), no effect was observed on these cells. However, PPS had a strong inhibitory effect on the growth of bovine aortic endothelial cells (BAEC), as did heparin. PPS was fractionated according to molecular weight and the activities of these fractions were compared. A PPS fraction of MW = 3200 Daltons represented the critical size required to affect cell proliferation induced by FGFs. We also report that acidic and basic FGFs are both chemotactic for BAEC and HUVEC. PPS and heparin, which were chemotactic alone on BAEC, potentiated acidic FGF-induced migration but inhibited the chemotactic response of basic FGF. These data suggest that PPS, although having a different structure, can mimic the in vitro activity of heparin on FGF-induced proliferation and migration of endothelial cells and thus the possibility of a specific heparin sequence being involved in the interactions with FGFs can be questioned.

Plasma testosterone levels in intact and hemicastrated growing cockerels.

Changes in the levels of testosterone in plasma were measured by radioimmunoassay in blood samples taken at frequent intervals between 2 and 26 weeks of age from entire cockerels and cockerels hemicastrated before 2 weeks of age. In both groups the pattern of testosterone secretion could be divided into three clearly defined phases. In young birds, the levels of testosterone in plasma were low (0.3 ng/ml) but in the prepubertal period, at 11 weeks of age, they started to rise and continued to rise until 22 weeks of age when adult levels, which fluctuated between 2.5 and 3.5 ng/ml, were reached. In the immediate period after hemicastration, the concentration of testosterone decreased temporarily. From 11 weeks of age the levels of testosterone in the hemicastrated birds were approximately 75% of those in intact birds. These results are discussed in relation to the compensatory testicular hypertrophy which occurs in growing cockerels hemicastrated at an early age.