Summary. This study aimed to determine the binding characteristics of [

Summary.Phosphorylations induced by 2-MeS-ADP, a potent agonist of platelet ADP receptors, have been studied in rat platelets, and the effect of clopidogrel, a compound which inhibits platelet aggregation by selectively reducing the binding of ADP to its low affinity receptors on platelets, has been determined. 2-MeS-ADP induced platelet activation (shape change and aggregation) simultaneously with the phosphorylation of myosin light chain (P 20 ) and plekstrin (P 47 ). Phosphorylation of P 20 and P 47 was transient, a maximum being observed 10 s after addition of the agonist when shape change reached its maximum. P 20 and P 47 phosphorylations were not strongly affected by clopidogrel treatment. Following stimulation of platelets with 2-MeS-ADP, several proteins were phosphorylated at tyrosine residues. Clopidogrel treatment inhibited the increase in phosphorylation of P 140 , P 100 , P 80=85 , P 66 and P 55 concomitantly with the inhibition of platelet aggregation. However, clopidogrel did not interfere with the early phosphorylation of the P 80=85 kD doublet which occurs at the time of the shape change. P 80=85 , identified by immunodetection as cortactin, could be involved in the reorganization of the cytoskeleton necessary for morphological changes.Thus, by using clopidogrel-treated rat platelets, we were able to determine some of the phosphorylations coupled either to clopidogrel-resistant high-affinity ADP receptors leading to shape change or to clopidogrel sensitive low-affinity ADP receptors coupled to the aggregation process.Keywords: clopidogrel, ADP, phosphorylations, Gp IIb-IIIa, platelets.It is now well established that ADP causes aggregation of human blood platelets, but the ways in which platelets respond to ADP is still relatively obscure. Furthermore, it is not even clear whether ADP causes its effect by interacting with only one or via multiple receptors (Hourani & Hall, 1994). The identity of ADP receptor(s) on platelets therefore remains unsolved and controversial. We have previously reported that ADP activates platelets by at least two types of receptors (Savi et al, 1994a), one being a high-affinity binding site involved in the ADP-induced shape change, the other a low-affinity binding site linked to adenylyl cyclase via Gi proteins (Ohlmann et al, 1995).Clopidogrel is a thienopyridine compound which demonstrates a potent antiaggregating activity in several animal species and in humans (Herbert et al, 1993). Clopidogrel activity has been found to be due to the inhibition of binding to platelet ADP receptors (Mills et al, 1992;Savi et al, 1994b). We demonstrated that this effect was selective for the low-affinity binding site linked to the inhibition of adenylyl cyclase, the Gp IIb-IIIa activation and the aggregation, whereas the high affinity sites, linked to shape change, were not affected by clopidogrel treatment (Savi et al, 1994a). The intracellular events that trigger these platelet responses are known to involve signalling pathways that regulate the activation of polyphosphoinositide...

Nerve growth factor (NGF), the prototypic member of the neurotrophin family of growth factors, exerts its action via two receptors, P75 NTR and TrkA, the expression of which varies at the cell surface of neuroblastoma cells (SH-SY5Y cells) in a cycle phase-specific manner. NGF was pro-apoptotic on growing cells expressing preferentially P75 NTR and exhibited a potent anti-apoptotic effect on quiescent cells, when TrkA was prevalent at the cell surface, showing that NGF can have a dual action on SH-SY5Y cells depending on the relative cell surface expression of TrkA and P75 NTR . The pro-apoptotic activity of NGF but not its anti-apoptotic activity was abrogated by an antibody against the extracellular domain of P75 NTR and in cells isolated from P75 NTR knock-out mice indicating that NGF exhibits a proapoptotic activity via P75 NTR exclusively. On the other hand, we showed that the anti-apoptotic activity of NGF was specifically mediated by an interaction with TrkA with no contribution of P75 NTR , as demonstrated on SK-N-BE cells transfected with TrkA in which NGF was a potent anti-apoptotic compound but did not exhibit any pro-apoptotic activity. These results support the hypothesis that the survival response to NGF depends on its binding to TrkA without any involvement of P75 NTR which in turn selectively mediates the pro-apoptotic activity of NGF with no contribution of TrkA and show that, depending on the growth state of the cells, NGF exhibits dual pro-or anti-apoptotic properties via P75 NTR and TrkA, respectively. z 1999 Federation of European Biochemical Societies.

SummaryThe aim of this study was to describe the pharmacological properties of SR 121787, a new antiaggregating drug which is metabolized in vivo into SR 121566, a potent non-peptide antagonist of Gp IIb/IIIa. In vitro, SR 121566 antagonized the binding of [125I]-fibrinogen (IC50 = 19.8 ± 6.3 nM) and of [125I]-L-692,884, an RGD-containing peptide (IC50 = 291 ± 96 nM) to activated human platelets. SR 121566 inhibited the aggregation of human platelets induced by ADP, collagen, thrombin, arachidonic acid and PAF at concentrations lower than 0.1 μM. Adhesion of human platelets to adhesive proteins was inhibited by SR 121566 (IC50 = 40.3 ± 2.5 nM) only when Gp IIb/IIIa and fibrinogen were involved. No effect was found with regard to other adhesive proteins and/or other integrins. SR 121787 demonstrated a potent and sustained antiaggregating effect when administered intravenously to baboons at a dose 50 μg/kg, and eight hours after the administration of 100 μg/kg, ADP-induced aggregation was still strongly inhibited (more than 80%). A single oral administration of 2 mg/kg of SR 121787 produced a nearly complete inhibition of platelet aggregation for up to 8 h (ED50 at 8 h = 193 ± 20 μg/kg), a significant residual antiaggregating activity being still observed 24h after the administration. When administered orally to rabbits, SR 121787 exhibited a potent antiaggregating (ED50 = 2.3 ± 0.3 mg/kg) and antithrombotic activity in an arterio-venous shunt thrombosis model (ED50 = 10.4 ± 0.8 mg/kg). After oral and IV administration, SR 121787 was well tolerated suggesting that SR 121787, the most potent and long lasting orally active Gp IIb/IIIa antagonist described to date, is a promising antithrombotic compound.