Thromboxane A 2 (TxA 2 ) is a potent vasoconstrictor and platelet agonist. Pharmacological studies have defined two classes of thromboxane receptors (TPs) in human platelets; sites that bind the agonist 1S-(1,2(5Z),3-(1E,3S),4)-7-3-(3-hydroxy-4-(4-iodophenoxy)-1-butenyl)-7-oxabicyclo-2.2.1-heptan-2-yl-5-heptenoic acid (I-BOP) with high affinity support platelet shape change, whereas low affinity sites that bind irreversibly the antagonist GR 32191 transduce platelet aggregation. As the mechanisms of signal transduction involved in platelet aggregation begin to be elucidated, few results concern those involved in platelet shape change, which is independent of the engagement of GPIIb/IIIa. To elucidate the respective role of the two classes of pharmacological binding sites of TPs in shape change, platelets were incubated with I-BOP at low concentrations or stimulated by I-BOP at high concentrations after pretreatment with GR 32191 or activated with low concentrations of 8-epi-prostaglandin F 2 ␣. Under these three conditions, there is a rapid stimulation of protein tyrosine phosphorylation of the 80/85-kDa doublet identified as the cytoskeletal protein cortactin. Tyrosine phosphorylation of cortactin is kinetically correlated with the occurrence of shape change. These biochemical and morphological events are both inhibited by SQ 29548, a TP antagonist, indicating the specificity of the signal.Since tyrosine kinase Syk was activated early during platelet activation, we examined the possibility that cortactin is a potential substrate of Syk in TxA 2 -induced platelet shape change. p72 Syk phosphorylation and kinase activity took place during the period when platelets were changing shape upon low concentrations of I-BOP stimulation. Furthermore, cortactin was associated with Syk, and this association increases along with the level of phosphorylation. These data suggest a novel pathway for a G protein-coupled TxA 2 high affinity receptor to the protein-tyrosine kinase Syk, which is associated with cortactin in the very early steps of platelet activation.
and pleckstrin @7 b,Da) (12). The hydrolysis of Pi Pz results also from the stimulation of Pl-Cywhich is independent of G protein and requires tyrosine kinase activity. 2) Thrombin Eeatment causes also a dramatic increase in the level of phosphotyrosines on multiple proteins which occur in three temporal waves (3,4), the third one being dependent on aIIbBr integrin engagement and plateletag gregation, since agents that block fibrinogen binding inhibit the tyrosine phosphorylation of the last wave. The increase in tyrosine phosphorylation is associated with the activation and subcellular relocation of a number of non receptor protein tyrosine kinases (PTKs) such as Src family kinases (60 kDa), Syk (72 lDa) and FAI((125 kDa). In addition to their catalytic domain shared by all PTK, Syk contains two additional src homology (SH2) and one SH3 domains. SH2 and SH3 domains play critical roles in regulating infra and intermolecular proteinprotein interactions (5,6). 3) Following agonist occupation of a diverse range of cell surface rcceptors, a phosphoinositide 3-kinase (Pi3 kinase) cataly zes the pho sphorylati on of ino sitolphosph olipids on the 3 position of the inositol ring (7). In platelets Pi3 kinase activation gives rise to phophatidylinositol 3, 4 Pz (Pr 3APz) andphophatidylinositol 3,4,5 P3 (Pi3,4,5P3) which are considered as second messengers (8). 4) The Mitogen Activated Protein Kinase (MAPK) is a conseryed eukaryotic signaling molecule that converts receptor signals into a variety of outputs. MAPK patways are extensively used for transcytoplasmic signaling in the nucleus where transcription of specific genes is induced through phosphorylation and activation of transcription factors (9). Platelet stimulation also leads to stimulation of the MAP kinase pathway (10, 11) in which low molecular weight GTP binding proteins such as 'tas" could participate (12). Once platelets are activated, unidentified intracellular events (PKC ?) acton oIIbFr to induce binding of fibrinogen to the exEacellular domain of the receptor (13). As a result, this integrin transmits signals. The outside-in integrin signaling through oIIbFr is critical for platelet function. It causes increased tyrosine phosphorylation of signaling proteins such as Syk and EAK, increased Ca2* mobilization, calpain activation, and increased cytoskeletal organization.
The signalling pathways that link G-protein-coupled receptors to mitogen-activated protein kinases involve receptor and non-receptor tyrosine kinases and protein kinase C (PKC). We explored the pathways that are implicated in the thromboxane (TX) A(2)-dependent activation of extracellular-signal-regulated protein kinase (ERK) and the role of the two TX receptor (TP) isoforms, TP alpha and TP beta. ERK activation by IBOP, a TX analogue, was dependent on epidermal-growth-factor receptor (EGFR) in TP alpha- or TP beta-transfected cells and in human aortic smooth muscle cells (hASMCs), since AG1478, a selective inhibitor of tyrosine phosphorylation of the EGFR, strongly blocked ERK and EGFR phosphorylation. In addition, EGFR transactivation leading to ERK activation involved matrix metalloproteinases (MMPs), since BB2516, an inhibitor of MMP, decreased ERK and EGFR phosphorylation in TP alpha- or TP beta-transfected cells. Moreover, we showed that both isoforms activate ERK phosphorylation in an Src-kinase-dependent manner, whereas PKC was mainly implicated in ERK activation and EGFR phosphorylation by TP beta. In hASMCs, we showed that ERK activation depended on both pertussis-sensitive and -insensitive G alpha-proteins. We demonstrated further that EGFRs, PKC, Src kinase and MMPs are involved in ERK activation by TX. The results of the present study highlight a role for MMPs and PKC in EGFR transactivation triggered by the TPs and demonstrate this mechanism for the first time in primary cells, i.e. hASMCs.
SummaryADP, a primary stimulus of platelets, binds to one or more populations of receptors on the platelet surface. These receptors are linked to discrete activation pathways. Both G proteins and tyrosine kinases have been implicated in the cellular responses to this agonist. We have studied a patient with a congenital abnormality of ADP-induced platelet aggregation in an effort to gain information on the signalling pathways used by ADP. Immunoblotting with a broadly reactive rabbit antibody recognizing the GTP-binding domain of G protein α-subunits, and with rabbit antibodies specific for Giαl-3, and Gα12 all showed normal reactivity when tested against the patient‘s platelets. The phosphorylation of proteins was studied using an anti-phosphotyrosine MoAb (4G10) and platelets stimulated in a platelet aggregometer with ADP, a thromboxane A2 mimetic (IBOP), TRAP-14-mer peptide and α-thrombin. With normal platelets, a time-dependent phosphorylation of several bands in the 60 to 130 kDa mol. wt. range was observed with all agonists. For the patient, minimal aggregation and little or no phosphorylation of proteins of 80-85 kDa (cortactin), 100-105 kDa and 125-130 kDa were seen in response to ADP. The aggregation and phosphorylation responses were slightly modified in the presence of low doses of thrombin but were normal with high doses. Aggregation and tyrosine phosphorylation were virtually absent with IBOP, a finding reproduced when normal platelets were incubated with IBOP and the CP/CPK ADP scavenging system, thereby underlining the role of ADP in the response to IBOP. Our results show that the ADP receptor pathway deficient in the patient is linked to a selective tyrosine phosphorylation response.
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