Mauro Torti

E-cadherin endocytosis regulates the activity of Rap1: a traffic light GTPase at the crossroads between cadherin and integrin function

The coordinate modulation of cadherin and integrin functions plays an essential role in fundamental physiological and pathological processes, including morphogenesis and cancer. However, the molecular mechanisms underlying the functional crosstalk between cadherins and integrins are still elusive. Here, we demonstrate that the small GTPase Rap1, a crucial regulator of the inside-out activation of integrins, is a target for E-cadherin-mediated outside-in signaling. In particular, we show that a strong activation of Rap1 occurs upon adherens junction disassembly that is triggered by E-cadherin internalization and trafficking along the endocytic pathway. By contrast, Rap1 activity is not influenced by integrin outside-in signaling. Furthermore, we demonstrate that the E-cadherin endocytosis-dependent activation of Rap1 is associated with and controlled by an increased Src kinase activity, and is paralleled by the colocalization of Rap1 and E-cadherin at the perinuclear Rab11-positive recycling endosome compartment, and the association of Rap1 with a subset of E-cadherin-catenin complexes that does not contain p120ctn. Conversely, Rap1 activity is suppressed by the formation of E-cadherin-dependent cell-cell junctions as well as by agents that inhibit either Src activity or E-cadherin internalization and intracellular trafficking. Finally, we demonstrate that the E-cadherin endocytosis-dependent activation of Rap1 is associated with and is required for the formation of integrin-based focal adhesions. Our findings provide the first evidence of an E-cadherin-modulated endosomal signaling pathway involving Rap1, and suggest that cadherins may have a novel modulatory role in integrin adhesive functions by fine-tuning Rap1 activation.

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.

Megakaryocytes of patients with MYH9-related thrombocytopenia present an altered proplatelet formation

MYH9-related disease (MYH9-RD) is an autosomal-dominant thrombocytopenia caused by mutations of MYH9, the gene for the heavy chain of myosin-IIA. Pathogenesis of thrombocytopenia of MYH9-RD is unknown. Recent studies in mice demonstrated that myosin-IIA is an inhibitor of proplatelet formation (PPF), and suggested that it could be involved in the suppression of PPF exerted by megakaryocyte adhesion to type I collagen, which regulates the timing of platelet release within bone marrow. However, the consequences on PPF of the heterozygous mutations causative of the MYH9-RD have never been investigated. We studied the in-vitro PPF by megakaryocytes obtained from four patients carrying the p.D1424N or the p.R1933X mutations. We demonstrated that MYH9-RD megakaryocytes completely lose the physiologic suppression of proplatelet extension exerted by interaction with type I collagen, thus supporting the hypothesis that a premature platelet release within bone marrow contributes to pathogenesis of MYH9-related thrombocytopenia. Moreover, proplatelets extended by MYH9-RD megakaryocytes presented a significant defect in branching in secondary processes (p=0.001) and formed a significantly lower number of proplatelet tips (p=0.005). Since platelets are assembled at the level of proplatelet tips, this defect could further contribute to pathogenesis of thrombocytopenia of MYH9-RD patients.

Intraplatelet signaling mechanisms of the priming effect of matrix metalloproteinase‐2 on platelet aggregation

Summary.  Objective: Platelets contain and release some matrix metalloproteinases (MMPs), enzymes involved in the degradation of extracellular matrix, and one of these (MMP‐2) exerts a proaggregatory effect. We explored the signal transduction mechanisms activated by MMP‐2 in human blood platelets. Methods and results:Recombinant, human MMP‐2, added before stimulation with subthreshold doses of different agonists, potentiated platelet activation, calcium influx, IP3 formation, and pleckstrin phosphorylation. Wortmannin and LY29400, two PI3‐K inhibitors, suppressed the potentiating effects of MMP‐2 and preincubation with MMP‐2 enhanced the thrombin‐induced association of the p85α PI3‐K subunit with the cytoskeleton and increased the phosphorylation of PKB. Protein tyrosine kinase inhibitors, MAP kinase inhibitors, PLA2 inhibitors, cyclooxygenase inhibitors and antagonists of the P2Y1 and P2Y12 receptors did not affect the potentiating activity of MMP‐2 on platelets. Conclusion: Our data show that MMP‐2, at a concentration released by activated platelets, facilitates platelet activation acting at the level of a second messenger system common to different agonists and related to the activation of PI3‐K. Platelet‐released MMP‐2 may contribute to platelet activation in vivo.

Proplatelet formation in heterozygous Bernard‐Soulier syndrome type Bolzano

Summary.  Background: Although mutations of GPIbα are among the most frequent causes of inherited platelet disorders, the mechanisms for the onset of thrombocytopenia and platelet macrocytosis are still poorly defined. Objective: In this work we analyzed in vitro megakaryocyte differentiation and proplatelet formation in six subjects heterozygous for the Ala156Val mutation in the GPIbα (Bolzano mutation). Methods: Human megakaryocytes were obtained by differentiation of patient cord blood‐derived CD34+ cells and peripheral blood‐derived CD45+ cells. Proplatelet formation was evaluated by phase contrast and fluorescence microscopy. Results: Megakaryocyte differentiation from both cord blood (one patient) and peripheral blood (five patients) was comparable to controls. However, proplatelet formation was reduced by about 50% with respect to controls. An identical defect of proplatelet formation was observed when megakaryocytes were plated on fibrinogen, von Willebrand factor or grown in suspension. Morphological evaluation of proplatelet formation revealed an increased size of proplatelet tips, which was consistent with the increased diameters of patients’ blood platelets. Moreover, α‐tubulin distribution within proplatelets was severely deranged. Conclusions: Megakaryocytes from patients carrying a Bolzano allele of GPIbα display both quantitative and qualitative abnormalities of proplatelet formation in vitro. These results suggest that a defect of platelet formation contributes to macrothrombocytopenia associated to the Bolzano mutation, and indicate a key role for GPIbα in proplatelet formation.

A role for p38 MAP kinase in platelet activation by von Willebrand Factor

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.

Alzheimer disease and platelets: how’s that relevant

Alzheimer Disease (AD) is the most common neurodegenerative disorder worldwide, and account for 60% to 70% of all cases of progressive cognitive impairment in elderly patients. At the microscopic level distinctive features of AD are neurons and synapses degeneration, together with extensive amounts of senile plaques and neurofibrillars tangles. The degenerative process probably starts 20–30 years before the clinical onset of the disease. Senile plaques are composed of a central core of amyloid β peptide, Aβ, derived from the metabolism of the larger amyloid precursor protein, APP, which is expressed not only in the brain, but even in non neuronal tissues. More than 30 years ago, some studies reported that human platelets express APP and all the enzymatic activities necessary to process this protein through the same pathways described in the brain. Since then a large number of evidence has been accumulated to suggest that platelets may be a good peripheral model to study the metabolism of APP, and the pathophysiology of the onset of AD. In this review, we will summarize the current knowledge on the involvement of platelets in Alzheimer Disease. Although platelets are generally accepted as a suitable model for AD, the current scientific interest on this model is very high, because many concepts still remain debated and controversial. At the same time, however, these still unsolved divergences mirror a difficulty to establish constant parameters to better defined the role of platelets in AD.

Altered cytoskeleton organization in platelets from patients with MYH9-related disease

Summary.  MYH9‐related disease (MYH9‐RD) is an autosomal dominant disorder deriving from mutations in the MYH9 gene encoding for the heavy chain of non‐muscle myosin IIA, and characterized by thrombocytopenia and giant platelets. Isoform IIA of myosin is the only one expressed in platelets, but the possibility that MYH9 mutations affect the organization of contractile structures in these blood elements has never been investigated. In this work we have analyzed the composition and the agonist‐induced reorganization of the platelet cytoskeleton from seven MYH9‐RD patients belonging to four different families. We found that an increased amount of myosin was constitutively associated with actin in the cytoskeleton of resting MYH9‐RD platelets. Upon platelet stimulation, an impaired increase in the total cytoskeletal proteins was observed. Moreover, selected membrane glycoproteins, tyrosine kinases, and small GTPases failed to interact with the cytoskeleton in agonist‐stimulated MYH9‐RD platelets. These results demonstrate for the first time that mutations of MYH9 result in an alteration of the composition and agonist‐induced reorganization of the platelet cytoskeleton. We suggest that these abnormalities may represent the biochemical basis for the previously reported functional alterations of MYH9‐RD platelets, and for the abnormal platelet formation from megakaryocytes, resulting in thrombocytopenia and giant platelets.

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.