Functional implications of tyrosine protein phosphorylation in platelets. Simultaneous studies with different agonists and inhibitors

Abstract

During activation of platelets by agonists, a number of proteins become phosphorylated at tyrosine residues. Using immunoblotting with a monoclonal anti-phosphotyrosine antibody, we have compared the different phosphotyrosine-protein (PTP) profiles of platelets stimulated with thrombin, collagen, ADP, arachidonic acid, phorbol myristate acetate and P256, an anti-glycoprotein-IIb-IIIa (GPIIb-IIIa) monoclonal antibody (mAb). Only a few PTPs were observed in resting platelets, of molecular masses 130, 64, 56-60 and 36 kDa. After stimulation by different agonists these proteins were more intensely phosphorylated and additional PTPs appeared with molecular masses of 170, 150, 140, 120, 105/97 (doublet), 85, 80, 75 and 45 kDa. The kinetics of phosphorylation differed from one agonist to another, but no significant differences in the overall patterns were detected, except in presence of ADP and P256-F(ab')2, which induced only the additional tyrosine phosphorylation of the 64 kDa protein and to a lesser extent that of a 75 kDa protein. The use of various agonists and the inhibitors (staurosporine, ajoene and RGDS) permitted a better characterization of the relationship between the different steps of activation and phosphorylation on tyrosine residues. The studies suggest the following conclusions: (i) stimulation of tyrosine phosphorylation occurs after activation of protein kinase C; (ii) there is a relationship between ligand binding to GPIIb-IIIa and the tyrosine phosphorylation of the 64 kDa protein; and (iii) there is a close relationship between PTP formation and the intensity of platelet activation and aggregation.

Association of the protein tyrosine phosphatase PTP1C with the protein tyrosine kinase c‐Src in human platelets

“…The migration in SDS-PAGE of PTP1C corresponded to the migration of the prominent tyrosine protein of 64 kDa [29]. No dephosphorylation of PTP1C was observed ~ith thrombin, even after 10 min of activation (data not shown).…”

Inhibition of Platelet Integrin α IIb β 3 by Peptides That Interfere With Protein Kinases and the β 3 Tail

“…12 The tyrosine phosphorylation of proteins with molecular masses of 54, 60, 64, 75, and 130 kDa during binding site exposure suggests that they may contribute to the affinity regulation of ␣ IIb ␤ 3 . [13][14][15] Only a few agonists signal to ␣ IIb ␤ 3 via PTK and protein kinase C (PKC, a Ser/Thr kinase), as illustrated by the inhibition by bisindolylmaleimide I and II and calphostin C and also by the fact that phorbol 12-myrisate 13-acetate (an activator of the PKC family) and 12-deoxyphorbol-13-phenylacetate-20-acetate (an activator of the PKC␤ subtype) trigger the sustained exposure of ligand binding sites. Activated PKC contributes to PTK activation and preserves the high-affinity state of the integrin, probably via stoichiometric phosphorylation of the ␤ 3 subunit.…”

Trombosi̇t Akti̇vasyonu

“…İP döngüsü, agregasyon ve sekresyon fonksiyonları ile bağlantılı olduğu belirlenmiştir. Erbstatin gibi tirozin kinaz inhibitör-lerinin trombin ile indüklenen agregasyon ve sekresyonu inhibe ettiği gösterilmiştir (51,56,57). Ancak trombositler yüksek doz trombin ile uyarıldıklarında inhibisyon olmadığı (49) ve granül sekresyonunun tirozin fosforilasyonundan bağımsız olduğuna dair bulgular da vardır (51).…”

“…Fibrinojenin GPIIb-llla'ya bağlanmasını inhibe eden peptitler tirozin fosforilasyonunu da inhibe etmektedir (50,51,54,56).…”