Effect of clopidogrel treatment on ADP‐induced phosphorylations in rat platelets

Abstract

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...

Functional characterization of platelet ADP receptors

“…4 9,7 3 These compounds also do not affect ADP-induced phosph orylation of myosin light chain and pleckstrin in platelets. 76 Clopidog rel selectively blocks ADP-mediated G protein activation in rat platelet mem branes. 7 7 These compounds also reduce the number of binding sites for 2M eSA DP to 30%.…”

Molecular Basis for ADP-induced Platelet Activation

“…Second, the thienopyridine analogs ticlopidine and clopidogrel when used in vivo are potent and specific inhibitors of ADP-induced platelet aggregation and antagonize ADP inhibition of prostaglandin E 1 -activated adenylyl cyclase in intact platelets (21). However, these compounds do not block ADP-induced shape change or intracellular calcium mobilization (32) nor do they affect ADP-induced phosphorylation of myosin light chain and pleckstrin in platelets (33). Third, a congenital defect in platelet function attributed to an ADP receptor defect has been described by Nurden et al (34).…”

ADP Receptors and Clinical Bleeding Disorders

“…26 Similarities between these patients and humans or animals that were treated with clopidogrel have been reported in terms of the morphology of ADP-induced platelet aggregates 29 as well as in signal transduction studies. 30 The defect is probably inherited as an autosomal recessive trait, because all patients so far described were born of consanguineous parents. The son of 1 of them, who is considered to be an obligate heterozygote, bound intermediate levels of 2-MeSADP.…”