Paolo Lova

A Gi-dependent pathway is required for activation of the small GTPase Rap1B in human platelets.

Stimulation of human platelets by cross-linking of the low affinity receptor for immunoglobulin, FcγRIIA, caused the rapid activation of the small GTPase Rap1B, as monitored by accumulation of the GTP-bound form of the protein. This process was totally dependent on the action of secreted ADP since it was completely prevented in the presence of either apyrase or creatine phosphate and creatine phosphokinase. Dose-dependent experiments revealed that the inhibitory effect of ADP scavengers was not related to the reduced increase of cytosolic Ca2+ concentration in stimulated platelets. Activation of Rap1B induced by clustering of FcγRIIA was totally suppressed by AR-C69931MX, a specific antagonist of the Gi-coupled ADP receptor P2Y12, but was not affected by blockade of the Gq-coupled receptor, P2Y1. Similarly, direct stimulation of platelets with ADP induced the rapid activation of Rap1B. Pharmacological blockade of the P2Y1 receptor totally prevented ADP-induced Ca2+ mobilization but did not affect activation of Rap1B. By contrast, prevention of ADP binding to the P2Y12 receptor totally suppressed activation of Rap1B without affecting Ca2+ signaling. In platelets stimulated by cross-linking of FcγRIIA, inhibition of Rap1B activation by ADP scavengers could be overcome by the simultaneous recruitment of the Gi-coupled α2A-adrenergic receptor by epinephrine. By contrast, serotonin, which binds to a Gq-coupled receptor, could not restore activation of Rap1B. When tested alone, epinephrine was found to be able to induce GTP binding to Rap1B, whereas serotonin produced only a slight effect. Finally, activation of Rap1B induced by stimulation of the Gq-coupled thromboxane A2receptor by U46619 was completely inhibited by ADP scavengers under conditions in which intracellular Ca2+ mobilization was unaffected. Inhibition of U46619-induced Rap1B activation was also observed upon blockade of the P2Y12 but not of the P2Y1 receptor for ADP. These results demonstrate that stimulation of a Gi-dependent signaling pathway by either ADP of epinephrine is necessary and sufficient to activate the small GTPase Rap1B.

Adhesive receptors, extracellular proteins and myosin IIA orchestrate proplatelet formation by human megakaryocytes

Summary.  Background: Megakaryocytes release platelets from the tips of cytoplasmic extensions, called proplatelets. In humans, the regulation of this process is still poorly characterized. Objective: To analyse the regulation of proplatelet formation by megakaryocyte adhesion to extracellular adhesive proteins through different membrane receptors. Methods: Human megakaryocytes were obtained by differentiation of cord blood‐derived CD34+ cells, and proplatelet formation was evaluated by phase contrast and fluorescence microscopy. Results: We found that human megakaryocytes extended proplatelets in a time‐dependent manner. Adhesion to fibrinogen, fibronectin or von Willebrand factor (VWF) anticipated the development of proplatelets, but dramatically limited both amplitude and duration of the process. Type I, but not type III or type IV, collagen totally suppressed proplatelet extension, and this effect was overcome by the myosin IIA antagonist blebbistatin. Integrin αIIbβ3 was essential for megakaryocyte spreading on fibrinogen or VWF, but was not required for proplatelet formation. In contrast, proplatelet formation was prevented by blockade of GPIb‐IX‐V, or upon cleavage of GPIbα by the metalloproteinase mocarhagin. Membrane‐associated VWF was detected exclusively on proplatelet‐forming megakaryocytes, but not on round mature cells that do not extend proplatelets. Conclusions: Our findings show that proplatelet formation in human megakaryocytes undergoes a complex spatio‐temporal regulation orchestrated by adhesive proteins, GPIb‐IX‐V and myosin IIA.

The Gi-coupled P2Y12 Receptor Regulates Diacylglycerol-mediated Signaling in Human Platelets

Stimulation of Gq-coupled receptors activates phospholipase C and is supposed to promote both intracellular Ca2+ mobilization and protein kinase C (PKC) activation. We found that ADP-induced phosphorylation of pleckstrin, the main platelet substrate for PKC, was completely inhibited not only by an antagonist of the Gq-coupled P2Y1 receptor but also upon blockade of the Gi-coupled P2Y12 receptor. The role of Gi on PKC regulation required stimulation of phosphatidylinositol 3-kinase rather than inhibition of adenylyl cyclase. P2Y12 antagonists also inhibited pleckstrin phosphorylation, Rap1b activation, and platelet aggregation induced upon Gq stimulation by the thromboxane A2 analogue U46619. Importantly, activation of phospholipase C and intracellular Ca2+ mobilization occurred normally. Phorbol 12-myristate 13-acetate overcame the inhibitory effect of P2Y12 receptor blockade on PKC activation but not on Rap1b activation and platelet aggregation. By contrast, inhibition of diacylglycerol kinase restored both PKC and Rap1b activity and caused platelet aggregation. Stimulation of P2Y12 receptor or direct inhibition of diacylglycerol kinase potentiated the effect of membrane-permeable sn-1,2-dioctanoylglycerol on platelet aggregation and pleckstrin phosphorylation, in association with inhibition of its phosphorylation to phosphatidic acid. These results reveal a novel and unexpected role of the Gi-coupled P2Y12 receptor in the regulation of diacylglycerol-mediated events in activated platelets.

Thrombin induces platelet activation in the absence of functional protease activated receptors 1 and 4 and glycoprotein Ib-IX-V

Three different surface receptors mediate thrombin-induced activation and aggregation of human blood platelets: the protease activated receptors 1 and 4 (PAR1 and PAR4), and the glycoprotein (GP) Ibalpha of the GPIb-IX-V complex. However, their relative contribution in the stimulation of specific intracellular signaling pathways by thrombin remains largely controversial. In this work, we have shown that activation of PAR1 and PAR4 by thrombin or by selective activating peptides stimulated phospholipase C, tyrosine kinases, as well as the small GTPase Rap1b, promoted actin polymerization and cytoskeleton reorganization. When platelets were desensitized for both PAR1 and PAR4, high doses of thrombin, were unable to activate Rap1b, but produced a still evident stimulation of phospholipase C, as documented by the measurement of intracellular Ca(2+) mobilization and protein kinase C activation. These events were abrogated upon proteolysis of GPIbalpha by the metalloproteinase mocarhagin. In PAR1- and PAR4-desensitized platelets, thrombin also induced tyrosine phosphorylation of some substrates, but, surprisingly, this event was largely independent of GPIbalpha binding, as it persisted upon platelet treatment with mocarhagin. Similarly, thrombin-induced actin polymerization and cytoskeleton reorganization were only minimally altered upon PAR1 and PAR4 inactivation and GPIbalpha proteolysis. Interestingly, none of these events were elicited by enzymatically inactive thrombin. Finally we found that GPIbalpha cleavage reduced, but did not abrogate, platelet aggregation in PAR1- and PAR4-desensitized platelets. These results identify a novel pathway for platelet activation operated by thrombin independently of PAR1, PAR4 and GPIbalpha.

Epinephrine-mediated protein kinase C and Rap1b activation requires the co-stimulation of Gz-, Gq-, and Gi-coupled receptors

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.

Epinephrine induces intracellular Ca2+ mobilization in thrombin-desensitized platelets: a role for GPIb-IX-V

In this work we have investigated the ability of epinephrine to trigger the release of intracellular Ca2+ in thrombin-desensitized platelets. Addition of thrombin to platelets in the presence of extracellular EGTA caused a rapid and transient release of Ca2+ from intracellular stores and rendered platelets unresponsive to a second addition of the same agonist. Although epinephrine alone had no effect on intracellular Ca2+ mobilization, its addition to thrombin-desensitized platelets was associated to a rapid and evident secondary release of intracellular Ca2+. This effect of epinephrine was not observed when platelets were desensitized with other agonists able to induce phospholipase C activation, including convulxin, U46619, and ADP. Although the platelet receptor for epinephrine is coupled to the Gi family member Gz, no secondary Ca2+ release was seen in thrombin-desensitized platelets upon stimulation of other Gi-coupled receptors, including the P2Y12 receptor and the CXCR4. Addition of hirudin to thrombin-desensitized platelets prevented epinephrine-promoted secondary release of Ca2+, indicating that thrombin, rather than epinephrine itself, is actually responsible for this event as a consequence of thrombin receptors resensitization. Studies with platelets stimulated with specific PAR1- and PAR4- activating peptides proved that neither one of these thrombin receptors were involved in the secondary epinephrine-assisted Ca2+ release. Moreover, we found that thrombin was still able to induce a reduced, but evident release of Ca2+ from internal stores in PAR1- and PAR4-desensitized platelets, which could be followed by a secondary Ca2+ release upon subsequent addition of epinephrine. Importantly, both the primary and the secondary Ca2+ release induced by thrombin and epinephrine in PAR1- and PAR4-desensitized platelets were abrogated upon cleavage of GPIbα by the metalloproteinase mocarhagin. These results demonstrate a direct role of thrombin binding to GPIb-IX-V in the mobilization of Ca2+ from intracellular stores, and reveal that epinephrine can restore this process in desensitized platelets, thus prolonging the effect of thrombin stimulation.