ABSTRACT
Tyrosine phosphorylation of a number of platelet proteins is dependent on platelet integrin alphaIIb beta3 (also termed GPIIb-IIIa) and its engagement in aggregation. For instance, in type I thrombasthenic platelets, which lack alphaIIb beta3 and do not aggregate, several substrates are either poorly or not phosphorylated. We have compared thrombasthenic platelets of type I, type II (15% alphaIIb beta3, functional), and variant type (50% alphaIIb beta3, no fibrinogen binding). The platelets from the three patients exhibited the same low tyrosine phosphorylation profiles, confirming the key role of functional alphaIIb beta3 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 alphaIIb beta3 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 alphaIIb beta3 engagement in aggregation. Implications as to involvement of step-specific kinase and/or phosphatase activities are discussed.
Subject(s)
Blood Platelets/metabolism , Microfilament Proteins/metabolism , Phosphoproteins/metabolism , Platelet Aggregation/physiology , Platelet Glycoprotein GPIIb-IIIa Complex/physiology , Protein Processing, Post-Translational , Thrombasthenia/metabolism , Thrombin/physiology , Cortactin , Enzyme Activation , Fibrinogen/metabolism , Humans , Kinetics , Oligopeptides/pharmacology , PhosphorylationABSTRACT
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 (P20) and plekstrin (P47). Phosphorylation of P20 and P47 was transient, a maximum being observed 10 s after addition of the agonist when shape change reached its maximum. P20 and P47 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 P140, P100, P80/85, P66 and P55 concomitantly with the inhibition of platelet aggregation. However, clopidogrel did not interfere with the early phosphorylation of the P80/85 kD doublet which occurs at the time of the shape change. P80/85, 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.
Subject(s)
Blood Platelets/metabolism , Platelet Aggregation Inhibitors/pharmacology , Ticlopidine/analogs & derivatives , Adenosine Diphosphate/analogs & derivatives , Adenosine Diphosphate/metabolism , Animals , Clopidogrel , Female , Phosphorylation , Platelet Activation , Platelet Aggregation/drug effects , Rats , Rats, Sprague-Dawley , Thionucleotides/metabolism , Ticlopidine/pharmacologyABSTRACT
The strong inhibition of thrombin-induced platelet functions induced by okadaic acid is not correlated with the partial modification of pleckstrin phosphorylation, which remains still phosphorylated two min after stimulation, indicating that protein kinase C is not affected by okadaic acid. We then investigated the effect of okadaic acid on platelet lipid metabolism. Our data indicate that inhibition indeed strongly affects phosphatidic acid as well as phosphatidylinositol 3,4-bisphosphate synthesis at low concentrations of okadaic acid, and phosphatidylinositol 4,5-bisphosphate at higher concentrations. Since thrombin-induced tyrosine phosphorylations were completely inhibited in the presence of okadaic acid, as a consequence, phosphatidylinositol 3-kinase was no longer detected in antiphosphotyrosine immunoprecipitates, thus explaining the absence of phosphatidylinositol, 3,4-bisphosphate synthesis. Finally, okadaic acid inhibited thrombin-induced fibrinogen binding, indicating that serine/threonine phosphatases may affect the inside-out signalling which regulates the alpha 11bb3 integrin, downstream protein kinase C activation.
Subject(s)
Blood Platelets/metabolism , Enzyme Inhibitors/pharmacology , Mitogens/pharmacology , Okadaic Acid/pharmacology , Phosphoproteins , Phosphotransferases (Alcohol Group Acceptor)/antagonists & inhibitors , Thrombin/pharmacology , Type C Phospholipases/antagonists & inhibitors , Blood Platelets/drug effects , Blood Proteins/metabolism , Fibrinogen/metabolism , Humans , Phosphatidylinositol 3-Kinases , Phosphatidylinositol Phosphates/metabolism , Phosphorylation , Phosphotyrosine/metabolism , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Precipitin Tests , Tyrosine/metabolismABSTRACT
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 thrombin-and 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 I2 (PGI2). 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 PGI2, 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.