Cesare Balduini

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.

Megakaryocyte-matrix interaction within bone marrow: new roles for fibronectin and factor XIII-A

The mechanisms by which megakaryocytes (MKs) differentiate and release platelets into the circulation are not well understood. However, growing evidence indicates that a complex regulatory mechanism involving MK-matrix interactions may contribute to the quiescent or permissive microenvironment related to platelet release within bone marrow. To address this hypothesis, in this study we demonstrate that human MKs express and synthesize cellular fibronectin (cFN) and transglutaminase factor XIII-A (FXIII-A). We proposed that these 2 molecules are involved in a new regulatory mechanism of MK-type I collagen interaction in the osteoblastic niche. In particular, we demonstrate that MK adhesion to type I collagen promotes MK spreading and inhibits pro-platelet formation through the release and relocation to the plasma membrane of cFN. This regulatory mechanism is dependent on the engagement of FN receptors at the MK plasma membrane and on transglutaminase FXIII-A activity. Consistently, the same mechanism regulated the assembly of plasma FN (pFN) by adherent MKs to type I collagen. In conclusion, our data extend the knowledge of the mechanisms that regulate MK-matrix interactions within the bone marrow environment and could serve as an important step for inquiring into the origins of diseases such as myelofibrosis and congenital thrombocytopenias that are still poorly understood.

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.

Role of decorin on in vitro fibrillogenesis of type I collagen.

Tendon and corneal decorins are differently iduronated dermatan sulphate/proteoglycan (DS/PG) and the biochemical parameter that differentiates type I collagens is the hydroxylysine glycoside content. We have examined the effect of tendon and corneal decorins on the individual phases (tlag, dA/dt) of differently glycosylated type I collagens fibril formation, at molar ratios PG:collagen monomer ranging from 0.15 : 1 to 0.45 : 1. The results obtained indicate that decorins exert a different effect on the individual phases of fibril formation, correlated to the degree of glycosylation of collagen: at the same PG:collagen ratio the fibril formation of highly glycosylated corneal collagen is more efficiently inhibited than that of the poorly glycosylated one (tendon). Moreover tendon and corneal decorins exert a higher control on the fibrillogenesis of homologous collagen with respect to the heterologous one. These data suggest a possible tissue-specificity of the interaction decorin/type I collagen correlated to the structure of the PG and collagen present in extracellular matrices.

Detergent-resistant membranes in human erythrocytes and their connection to the membrane-skeleton.

In cell membranes, local inhomogeneity in the lateral distribution of lipids and proteins is thought to existin vivo in the form of lipid ‘rafts’, microdomains enriched in cholesterol and sphingolipids, and in specific classes of proteins, that appear to play specialized roles for signal transduction, cell-cell recognition, parasite or virus infection, and vesicular trafficking. These structures are operationally defined as membranes resistant to solubilization by nonionic detergents at 4°C (detergent-resistant membranes, DRMs). This definition appears to be necessary and sufficient, although additional manoeuvres, not always described with sufficient detail, may be needed to ensure isolation of DRMs, like mechanical homogenization, and changes in the pH and/or ionic strength of the solubilization medium. We show here for the human erythrocyte that the different conditions adopted may lead to the isolation of qualitatively and quantitatively different DRM fractions, thus contributing to the complexity of the notion itself of lipid raft. A significant portion of erythrocyte DRMs enriched in reported lipid raft markers, such as flotillin-1, flotillin-2 and GM1, is anchored to the spectrin membrane-skeleton via electrostatic interactions that can be disrupted by the simultaneous increase in pH and ionic strength of the solubilization medium.

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.

Differential sorting of tyrosine kinases and phosphotyrosine phosphatases acting on band 3 during vesiculation of human erythrocytes.

One of the most intensively studied post-translational modifications of erythrocyte proteins is the phosphorylation of tyrosine residues of band 3, which is strictly regulated in vivo by PTKs (protein-tyrosine kinases) and PTPs (protein-phosphotyrosine phosphatases). Two PTKs (p72(syk) and p56/53(lyn)) and two PTP activities (PTP1B and SHPTP-2) have been immunologically identified so far in mature human erythrocytes. We have shown previously that band 3 undergoes tyrosine phosphorylation upon a decrease in cell volume, as occurs when erythrocytes treated with Ca(2+)/Ca(2+) ionophore (A23187) lose KCl and release microvesicles. Similar levels of band 3 tyrosine phosphorylation in vesicles and in the parent cells are induced by this treatment. However, we have found that tyrosine phosphorylation of band 3 in vesicles is more stable than in whole erythrocytes. Examination of how the identified PTPs and PTKs are partitioned between the vesicles and the remnant cells during vesiculation reveals that PTP1B, unlike the PTKs, is retained entirely in the parent cell compartment. Since a tight association between PTP1B and band 3 has been documented previously, we have investigated the partitioning of PTP1B and band 3 between the membrane and the membrane-skeletal fractions prepared from resting or Ca(2+)/A23187-treated cells. Our results rule out the possibility that the preferential retention of PTP1B within the cell was due to an increase in the amount of membrane-skeleton-associated band 3 (and of PTP1B) during the release of spectrin-free vesicles, suggesting a more complex modality of interaction of PTP1B with band 3 in the erythrocyte membrane. Analysis of erythrocytes of different cell ages revealed that PTP1B, unlike the other enzymes examined, was quantitatively conserved during erythrocyte aging. This suggests important roles for the down-regulation of tyrosine phosphorylation of band 3 in erythrocyte physiology, and for vesiculation as a mechanism of human erythrocyte senescence.

Nanoparticles induce platelet activation in vitro through stimulation of canonical signalling pathways.

UNLABELLED Nanomaterials are attracting growing interest for their potential use in several applications as nanomedicine; therefore, the analysis of their potential toxic effects on various cellular models, including circulating blood cells, is mandatory. This study aimed to investigate the effect of three unrelated nanomaterials, namely nanoscale silica, multiwalled carbon nanotubes, and carbon black, on platelet activation and aggregation. We found that these nanomaterials stimulate some of the typical biochemical pathways involved in canonical platelet activation, such as the stimulation of phospholipase C and Rap1b, resulting in the integrin α(IIb)β3-mediated platelet aggregation, through a mechanism largely dependent on the release of the extracellular second messengers ADP and thromboxane A2. Importantly, we found that doses of nanoparticles unable to trigger appreciable responses can synergize with subthreshold amounts of physiological agonists to mediate platelet aggregation, indicating that even small amounts of nanomaterials in the bloodstream might contribute to the development of thrombosis. FROM THE CLINICAL EDITOR In this study, nanosized particles of three virtually unrelated materials (silica, multi-walled carbon nanotubes and carbon black) were investigated regarding their effects on platelet activation and aggregation. All were found to stimulate some of the typical biochemical pathways involved in canonical platelet activation, and were found to have synergistic effects with physiologic platelet activator agonists.

Role and regulation of phosphatidylinositol 3-kinase β in platelet integrin α2β1 signaling

Integrin α2β1-mediated adhesion of human platelets to monomeric type I collagen or to the GFOGER peptide caused a time-dependent activation of PI3K and Akt phosphorylation. This process was abrogated by pharmacologic inhibition of PI3Kβ, but not of PI3Kγ or PI3Kα. Moreover, Akt phosphorylation was undetectable in murine platelets expressing a kinase-dead mutant of PI3Kβ (PI3Kβ(KD)), but occurred normally in PI3Kγ(KD) platelets. Integrin α2β1 failed to stimulate PI3Kβ in platelets from phospholipase Cγ2 (PLCγ2)-knockout mice, and we found that intracellular Ca(2+) linked PLCγ2 to PI3Kβ activation. Integrin α2β1 also caused a time-dependent stimulation of the focal kinase Pyk2 downstream of PLCγ2 and intracellular Ca(2+). Whereas activation of Pyk2 occurred normally in PI3Kβ(KD) platelets, stimulation of PI3Kβ was strongly reduced in Pyk2-knockout mice. Neither Pyk2 nor PI3Kβ was required for α2β1-mediated adhesion and spreading. However, activation of Rap1b and inside-out stimulation of integrin αIIbβ3 were reduced after inhibition of PI3Kβ and were significantly impaired in Pyk2-deficient platelets. Finally, both PI3Kβ and Pyk2 significantly contributed to thrombus formation under flow. These results demonstrate that Pyk2 regulates PI3Kβ downstream of integrin α2β1, and document a novel role for Pyk2 and PI3Kβ in integrin α2β1 promoted inside-out activation of integrin αIIbβ3 and thrombus formation.