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We have recently shown that ADP-induced activation of protein kinase C (PKC) requires the co-stimulation of both P2Y1 and P2Y12 receptors. In this work, we show that inhibition of ADP-mediated phosphorylation of pleckstrin, the main PKC substrate, caused by antagonists of the P2Y12 receptor can be reversed by stimulation of the α2-adrenergic receptor by epinephrine. However, we also observed that addition of epinephrine alone caused a marked phosphorylation of pleckstrin. This effect occurred in the absence of Gq stimulation, as it was not associated to intracellular Ca2+ release. Epinephrine-induced pleckstrin phosphorylation was time- and dose-dependent, and was inhibited by the α2-adrenergic antagonist yohimbin. Phosphorylation of pleckstrin did not occur when platelet stimulation with epinephrine was performed in the presence of the ADP scavenger apyrase, and was suppressed by antagonists of both P2Y1 and P2Y12 ADP receptors. Importantly, no release of dense granules was measured in epinephrine-treated platelets. Addition of epinephrine to platelets was also able to stimulate Rap1b activation. Similarly to pleckstrin phosphorylation, however, this effect was prevented in the presence of apyrase or upon pharmacologic blockade of either P2Y1 or P2Y12 receptors. These results indicate that sub-threshold amounts of ADP in the medium are essential to allow epinephrine stimulation of α2-adrenergic receptor to elicit platelet responses, and reveal a novel synergism among strong stimulation of Gz and sub-threshold stimulation of both Gq and Gi, able to dissociate PKC activation from intracellular Ca2+ mobilisation.

Platelet activation induced by von Willebrand factor (VWF) binding to the membrane GPIb-IX-V receptor involves multiple signal transduction pathways. Among these, recruitment and activation of the FCgammaRIIA and stimulation of phospholipase A2 represent independent events equally essential to support a complete platelet response. Phospholipase A2 is activated by calcium and by phosphorylation through MAP kinases. In this work, we found that VWF stimulated the rapid and sustained phosphorylation of p38 MAP kinase (p38MAPK). In vitro kinase assay revealed that VWF-stimulated phosphorylation of p38MAPK was associated with increased kinase activity. Binding of VWF to GPIb-IX-V, but not to integrin alphaIibbeta3, was required to support phosphorylation of p38MAPK. Neither the blockade of the membrane FCgammaRIIA by a specific monoclonal antibody or the prevention of thromboxane A(2) synthesis by cyclooxygenase inhibitors affected VWF-induced p38MAPK activation. How-ever, phosphorylation of p38MAPK was prevented by the tyro-sine kinase Syk inhibitor piceatannol. Treatment of platelets with the p38MAPK inhibitor SB203580 totally prevented VWF-stimulated platelet aggregation. Moreover, release of arachidonic acid induced by VWF was strongly impaired by inhibition of p38MAPK. We also found that VWF induced phosphorylation of cytosolic phospholipase A(2), and that this process was prevented by the p38MAPK inhibitor SB203580. These results demon-strate that p38MAPK is a key element in the FCgammaRIIA-independent pathway for VWF-induced platelet activation, and is involved in the stimulation of phospholipase A(2) and arachidonic acid release.

Stimulation of human platelets with von Willebrand factor (vWF) induces the rapid tyrosine phosphorylation of several proteins, but very little is known on the tyrosine kinases involved in this process. In the present work, we investigated and compared the activation of two related tyrosine kinases expressed in platelets: the proline-rich tyrosine kinase 2 (Pyk2) and the focal adhesion kinase (FAK). Both kinases were tyrosine phosphorylated upon vWF interaction with glycoprotein Ib-IX-V complex, but with different mechanisms. Tyrosine phosphorylation of FAK was totally dependent on thromboxane A2 production, and was inhibited by the integrin alphaIIbeta3 antagonist RGDS peptide. Moreover, chelation of intracellular calcium or inhibition of protein kinase C (PKC) totally blocked vWF-induced tyrosine phosphorylation of FAK, indicating that this event is downstream phospholipase A2 and phospholipase C activation. By contrast, tyrosine phosphorylation of Pyk2 was only partially reduced by aspirin and RGDS, and was not affected by either calcium chelation or PKC inhibition, suggesting that activation of this kinase does not require phospholipase-mediated signalling. Both FAK and Pyk2 translocated to the cytoskeleton upon vWF stimulation of human platelets by a mechanism depending on agonist-induced actin polymerisation. Prevention of cytoskeletal relocation of Pyk2 and FAK by cytochalasin D totally blocked vWF-induced tyrosine phosphorylation of both kinases. Finally, phosphorylation of Pyk2 induced by vWF, but not by thrombin, was inhibited by piceatannol, suggesting that this kinase lies downstream Syk. These results demonstrate that both Pyk2 and FAK are involved in platelet stimulation by vWF, but indicate that only Pyk2 may play a role in the early signal transduction events activated by ligand binding to glycoprotein Ib-IX-V.

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.

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.

We found that intracellular Ca2+ concentration ([Ca2+]i) increased during ristocetin-induced agglutination of aequorin loaded platelets resuspended in plasma. Chelation of extracellular Ca2+ had no effect on platelet clumping, but delayed and greatly reduced Ca2+ increase, indicating that it derived for the most part from Ca2+ influx. Nine monoclonal antibodies (MA) against glycoprotein (GP) Ib largely prevented ristocetin-induced platelet clumping and [Ca2+]i increase, while three anti-GPIb MA with no effect on platelet clumping did not interfere with Ca2+ movement. In unstirred samples platelet agglutination was greatly reduced and [Ca2+]i increase was abolished, suggesting that close platelet-to-platelet contact, in addition to von Willebrand factor (vWF) binding to GPIb, is necessary for Ca2+ transient. Nine MA against GPIIb/IIIa, the gly-arg-gly-asp-ser (GRGDS) peptide and GPIIb/IIIa complex dissociation had no effect on platelet agglutination, but significantly reduced Ca2+ increase. Our results suggest that platelet clumping induced by vWF binding to GPIb is responsible for GPIIb-IIIa dependent Ca2+ influx.

A young patient developed chronic idiopathic thrombocytopenic purpura. Prednisone therapy normalized platelet number, but bleeding symptoms did not disappear. Platelet function was severely impaired, since platelet aggregation, ATP release and adhesion to collagen and subendothelial matrix were significantly reduced. Plasma and purified immunoglobulins of the patient reproduced the functional defects in normal platelets. Immunoblotting revealed that patient's plasma contained an antibody reacting with a component of platelets with the same electrophoretic mobility of glycoproteins IIIa of normal platelets. Moreover, patient's plasma inhibited the binding of an anti-GPIIb/IIIa monoclonal antibody to platelet surface. Additional immunosuppressive therapy with prednisone and azathioprine normalized platelet function and induced the disappearance of bleeding symptoms.