Tyrosine phosphorylation of a number of platelet proteins is dependent on platelet integrin αIIbβ3 (also termed GPIIb-IIIa) and its engagement in aggregation. For instance, in type I thrombasthenic platelets, which lack αIIbβ3 and do not aggregate, several substrates are either poorly or not phosphorylated. We have compared thrombasthenic platelets of type I, type II (15% αIIbβ3, functional), and variant type (50% αIIbβ3, no fibrinogen binding). The platelets from the three patients exhibited the same low tyrosine phosphorylation profiles, confirming the key role of functional αIIbβ3 in initiating protein tyrosine phosphorylation. We noted that in addition to the characteristic absence of the 100 to 105 kD doublet, a 77 to 80 kD doublet and to a lesser extent a 64-kD band, exhibited low phosphorylation kinetics, but with normal initial phosphorylation rates (up to 60 seconds). Similar results were obtained by inhibition of thrombin aggregation of control platelets by αIIbβ3 antagonists (the RGDS peptide or the monoclonal antibody 10E5), or in the absence of stirring (fibrinogen binding, but no aggregation). These results suggest that tyrosine phosphorylation of the 77 to 80 kD doublet, identified by immunoprecipitation as the cytoskeletal protein cortactin, and the 64 kD band are dependent both on thrombin activation during early steps and on the late steps of αIIbβ3 engagement in aggregation. Implications as to involvement of step-specific kinase and/or phosphatase activities are discussed.

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...

Platelet signal transduction involves not only reversible phosphorylation of proteins on both tyrosine and serine/threonine residues, but also mechanisms of cross-talk to coordinate different pathways. We have, therefore, investigated the effect of okadaic acid, a potent inhibitor of serine/threonine protein phosphatases type 1 and type 2A (PP1 and PP2A), to better understand the interplay that must exist between serine/threonine and tyrosine phosphorylations during platelet activation.Okadaic acid drastically inhibits thrombin-induced platelet aggregation, secretion, and thromboxane synthesis. The inhibition is accompanied by a marked increase in the phosphorylation of at least 5 proteins (230, 210, 74, 57, and 50 to 52 kDa). However, protein kinase C activity is not modified because thrombinand phorbol-12-myristate-13-acetate-induced phosphorylation of pleckstrin is still occurring, although slightly decreased.Inhibition of platelet function and extent of the phosphorylation of the 5 substrates in the presence of okadaic acid are concentration and time dependent, suggesting a relation between the accumulation of one or more phosphoproteins and the inhibitory effect of okadaic acid.Okadaic acid inhibits thrombin-induced tyrosine phosphorylation in a concentration-dependent manner. According to Brautigan and Pinault, the inhibition of protein phosphatases in kidney cells resulted in the activation of a 55-kDa-tyrosine phosphatase and the tyrosine phosphatase activity was synergistically increased when okadaic acid acted in concert with prostaglandin I 2 (PGI 2 ). Interestingly, in agreement with these results, the okadaic acid-induced phosphorylation of the 50-kDa substrate, which occurs without a cyclic adenosine monophosphate increase in platelets, has the same molecular weight as the platelet membrane tyrosine phosphatase isolated by Dawicki and Steiner.Furthermore, we also found that thrombin-induced tyrosine phosphorylation was markedly inhibited in the presence of low concentrations of both okadaic acid and PGI 2 , therefore explaining the synergistic inhibition of platelet aggregation and secretion. The results greatly support the notion of a cross-talk between stimulation of serine/threonine kinases (in response to inhibition of serine/threonine PP) and inhibition of tyrosine phosphorylations and emphasize the role of the 50-kDa substrate in regulating platelet activation. Keywords: Serine/threonine phosphatase, okadaic acid, tyrosine phosphorylation, platelet aggregation, thrombinHuman blood platelet morphology is rapidly altered upon activation by various agonists-shape change, granule secretion or pseudopod formation-resulting in aggregation and finally coagulation. 1 Biochemical events occur in parallel with these changes in morphology. 2 Several signal transduction pathways regulate platelet activation. An early event in thrombin-induced platelet activation is hydrolysis of phosphatidylinositides by phospholipase C-β (PLC-β), stimulated by a heterotrimeric G protein coupled to the seven-transmembrane...

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