Clustering of integrin alphaIIb-beta3 differently regulates tyrosine phosphorylation of pp72syk, PLCgamma2 and pp125FAK in concanavalin A-stimulated platelets.

Tyrosine phosphorylation of the non-receptor tyrosine kinases pp72syk and pp125FAK and of the gamma2 isoform of phospholipase C (PLCgamma2) in human platelets stimulated with the lectin Concanavalin A was investigated. Concanavalin A induced the rapid tyrosine phosphorylation of pp72syk and PLCgamma2 with a similar kinetics, while tyrosine phosphorylation of pp125FAK occurred in a later phase of platelet activation. When compared with other platelet agonists, Concanavalin A revealed to be at least as potent as collagen in inducing tyrosine phosphorylation of PLCgamma2 and pp125FAK, while tyrosine phosphorylation of pp72syk induced by the lectin was much stronger than that induced by thrombin or collagen. Concanavalin A-induced tyrosine phosphorylation of pp72syk, PLCgamma2 and pp125FAK was not dependent on platelet aggregation as it occurred normally even in the absence of sample stirring and when fibrinogen binding to integrin alphaIIb-beta3 was inhibited by the peptide RGDS. Tyrosine phosphorylation of pp72syk, PLCgamma2 and pp125FAK required the binding of the lectin to the platelet surface, but was not observed in platelets treated with succinyl-Concanavalin A, a derivative of the lectin that interacts with the same receptors but does not promote clustering of membrane glycoproteins. Moreover, the aggregation-independent tyrosine phosphorylation of pp125FAK and pp72syk induced by Concanavalin A required the expression of integrin alphaIIb-beta3 on the platelet surface as it was strongly inhibited in platelets from patients affected by Glanzmann thrombasthenia. By contrast, tyrosine phosphorylation of PLCalpha2 occurred normally also in thrombasthenic platelets stimulated with Concanavalin A. These results demonstrate that, even in the absence of aggregation, the clustering of integrin alphaIIb-beta3 induced by Concanavalin A on the platelet surface directly promotes tyrosine phosphorylation of pp72syk and pp125FAK and provide further evidence that the oligomerization of the fibrinogen receptor promoted by its natural ligand during platelet aggregation may be responsible for the tyrosine phosphorylation of these proteins induced by physiological agonists.

Thrombin induces the association of cyclic ADP-ribose-synthesizing CD38 with the platelet cytoskeleton.

The effect of platelet stimulation on the subcellular localization of CD38, a membrane glycoprotein that catalyses the synthesis of cyclic ADP-ribose from beta-NAD+ was investigated. Treatment of human platelets with thrombin caused the association of about 40% of the total ADP-ribosyl cyclase activity with the cytoskeleton, through the translocation of the CD38 molecule from the Triton X-100-soluble to the insoluble fraction. The interaction of CD38 with the cytoskeleton was a specific and reversible process, mediated by the binding to the actin-rich filaments and was inhibited by treatment of platelets with cytochalasin D. This event was regulated by integrin alphaIIb beta3 and platelet aggregation as it was prevented by the inhibition of fibrinogen binding and was not observed in platelets from a patient affected by Glanzmann thrombasthenia. These results demonstrate that the subcellular localization of CD38 can be influenced by platelet stimulation with physiological agonists, and that membrane CD38 can interact with intracellular proteins.

Lysophosphatidic acid induces protein tyrosine phosphorylation in the absence of phospholipase C activation in human platelets.

Lysophosphatidic acid is a biologically active phospholipid able to induce cell proliferation and platelet aggregation. In this study we investigated the biochemical mechanisms of platelet activation by lysophosphatidic acid. We found that lysophosphatidic acid stimulated the binding of the photoreactive GTP-analog 4-azidoanilido-[alpha(32)P]GTP to a 40-kDa protein on platelet membranes. Moreover, lysophosphatidic acid induced the rapid decrease of the intracellular concentration of cAMP in intact platelets, indicating that this lipid activates platelets by binding to a membrane receptor coupled to the inhibitory GTP-binding protein Gi. In agreement with a receptor-mediated action, we found that platelet activation by lysophosphatidic acid underwent homologous desensitization. In the absence of extracellular CaCl(2), lysophosphatidic acid did not induce platelet aggregation, and did not stimulate phospholipase C. However, under the same conditions, lysophosphatidic acid produced the rapid tyrosine phosphorylation of several platelet proteins. This effect was not mediated by the formation of thromboxane A(2). Our results demonstrate that, in lysophosphatidic acid-stimulated platelets, activation of protein-tyrosine kinases occurs in the absence of phospholipase C activation and platelet aggregation, and may be directly related to the activation of the G-protein-coupled lysophosphatidic acid-receptor.

Agonist-induced actin polymerization is required for the irreversibility of platelet aggregation.

Cytochalasin D was used to investigate the role of intracellular cytoskeleton in the stabilization of platelet aggregation induced by strong platelet agonists. Incubation of gel-filtered platelets with increasing concentrations of cytochalasin D resulted in a dose-dependent inhibition of actin polymerization and association of actin-binding proteins with the Triton X-100-insoluble material induced by the thromboxane analogue, U46619, and the thrombin receptor activating peptide, TRAP. The same concentrations of cytochalasin D did not significantly inhibit platelet aggregation promoted by the two agonists. The addition of the chelating agent EDTA to fully aggregated platelets, that had been treated with cytochalasin D, resulted in the rapid and almost complete disaggregation. EDTA did not cause disaggregation of control, solvent-treated, aggregated platelets. The degree of platelet disaggregation induced by EDTA was dependent on the dose of cytochalasin D used, and was correlated with the inhibition of the cytoskeletal reorganization. Aggregation of cytochalasin D-treated platelets stimulated with U46619 or TRAP was also reverted by the addition of the tetrapeptide RGDS or the fibrinogen gamma-chain dodecapeptide, which competitively interfere with fibrinogen binding to the glycoprotein IIb-IIIa complex. These results indicate that the intracellular cytoskeleton plays an essential role in the stabilization of the fibrinogen-platelet interaction, and is necessary for the irreversibility of platelet aggregation induced by strong agonists.

Expression of cyclic ADP-ribose-synthetizing CD38 molecule on human platelet membrane.

CD38 is a cell surface molecule widely used as a marker for immature and activated lymphocytes. It has been recently shown that CD38 displays three enzymatic activities: hydrolysis of NAD+ to ADP-ribose, synthesis of cyclic ADP-ribose from NAD+, and hydrolysis of cyclic ADP-ribose to ADP-ribose. Thus, CD38 plays a key role in the synthesis of cyclic ADP-ribose, a calcium-mobilizing compound. We investigate here the expression and cellular localization of CD38 in human platelets using a specific monoclonal antibody. Results showed that CD38 is expressed by human platelet membranes. Moreover, we show that platelet CD38 possesses NAD glycohydrolase, ADP-ribose cyclase, and cyclic ADP-ribose hydrolase activities. This finding indicates that the calcium-mobilizing agent cyclic ADP-ribose can be synthetized by human platelets and raises the question about the possible role of CD38 expression and enzymatic activities in the signal transduction pathways leading to platelet activation.