Marie-Pierre Gratacap

Deletion of the p110β isoform of phosphoinositide 3-kinase in platelets reveals its central role in Akt activation and thrombus formation in vitro and in vivo

During platelet activation, phosphoinositide 3-kinases (PI3Ks) produce lipid second messengers participating in the regulation of functional responses. Here, we generated a megakaryocyte-restricted p110beta null mouse model and demonstrated a critical role of PI3Kbeta in platelet activation via an immunoreceptor tyrosine-based activation motif, the glyco-protein VI-Fc receptor gamma-chain complex, and its contribution in response to G-protein-coupled receptors. Interestingly, the production of phosphatidylinositol 3,4,5-trisphosphate and the activation of protein kinase B/Akt were strongly inhibited in p110beta null platelets stimulated either via immunoreceptor tyrosine-based activation motif or G-protein-coupled receptors. Functional studies showed an important delay in fibrin clot retraction and an almost complete inability of these platelets to adhere onto fibrinogen under flow condition, suggesting that PI3Kbeta is also acting downstream of alpha(IIb)beta(3). In vivo studies showed that these mice have a normal bleeding time and are not protected from acute pulmonary thromboembolism but are resistant to thrombosis after FeCl(3) injury of the carotid, suggesting that PI3Kbeta is a potential target for antithrombotic drugs.

The new tyrosine-kinase inhibitor and anticancer drug dasatinib reversibly affects platelet activation in vitro and in vivo

Dasatinib is an oral potent adenosine triphosphate (ATP)-competitive inhibitor of BCR-ABL, cKIT, platelet-derived growth factor receptor, and SRC family kinases (SFKs), which has demonstrated high efficiency in patients with imatinib-resistant chronic myelogenous leukemia. Here, we show that dasatinib weakly affects platelet activation by thrombin or adenosine diphosphate but is a potent inhibitor of platelet signaling and functions initiated by collagen or FcgammaRIIA cross-linking, which require immunoreceptor tyrosine-based activation motif phosphorylation by SFKs. Accordingly, dasatinib treatment rapidly decreases the volume of thrombi formed under arterial flow conditions in whole blood from patients or mice perfused over a matrix of collagen. Moreover, treatment of mice with dasatinib increases the tail bleeding time in a dose-dependent manner. Interestingly, these effects are rapidly reversible after interruption of the treatment. Our data clearly demonstrate that, in contrast to imatinib, dasatinib affects platelet functions in vitro and in vivo, which has important implications in clinic and could explain increased risks of bleeding observed in patients. Moreover, dasatinib efficiently prevents platelet activation mediated by FcgammaRIIA cross-linking and by sera from patients with heparin-induced thrombocytopenia, suggesting that reversible antiplatelet agents acting as ATP-competitive inhibitors of SFKs may be of therapeutic interest in the treatment of this pathology.

Regulation and roles of PI3Kβ, a major actor in platelet signaling and functions.

Phosphoinositide 3-kinases (PI3Ks) are important signaling enzymes involved in the regulation of a number of critical cell functions. Significant progress has been made during the last few years in defining the implication of individual PI3K isoforms. The role of the class IA PI3Kβ in different cell types has only been recently uncovered by the use of isoform-selective inhibitors and the development of mouse models harboring p110β catalytic subunit knock-out or germline knock-in of a kinase-dead allele of p110β. Although it is classically admitted that class IA PI3Ks are activated by receptor tyrosine kinases through recruitment of the regulatory subunits to specific tyrosine phosphorylated motifs via their SH2 domains, PI3Kβ is activated downstream of G protein-coupled receptors, and by co-operation between heterotrimeric G proteins and tyrosine kinases. PI3Kβ has been extensively studied in platelets where it appears to play an important role downstream of ITAM signaling, G protein-coupled receptors and aIIbβ3 integrin. Accordingly, mouse exhibiting p110β inactivation selectively in megakaryocyte/platelets are resistant to thromboembolism induced by carotid injury. The present review summarizes recent data concerning the mechanisms of PI3Kβ regulation and the roles of this PI3K isoform in blood platelet functions and other cell types.

Phosphoinositides: Important lipids in the coordination of cell dynamics.

By interacting specifically with proteins, phosphoinositides organize the spatiotemporal formation of protein complexes involved in the control of intracellular signaling, vesicular trafficking and cytoskeleton dynamics. A set of specific kinases and phosphatases ensures the production, degradation and inter-conversion of phosphoinositides to achieve a high level of precision in the regulation of cellular dynamics coordinated by these lipids. The direct involvement of these enzymes in cancer, genetic or infectious diseases, and the recent arrival of inhibitors targeting specific phosphoinositide kinases in clinic, emphasize the importance of these lipids and their metabolism in the biomedical field.

Platelet PI3Kβ and GSK3 regulate thrombus stability at a high shear rate

Class IA phosphoinositide 3-kinase β (PI3Kβ) is considered a potential drug target in arterial thrombosis, which is a major cause of death worldwide. Here we show that a striking phenotype of mice with selective p110β deletion in the megakaryocyte lineage is thrombus instability at a high shear rate, which is an effect that is not detected in the absence of p110α in platelets. The high shear rate-dependent thrombus instability in the absence of p110β is observed both ex vivo and in vivo with the formation of platelet emboli. Moreover, PI3Kβ is required for the recruitment of new platelets to a growing thrombus when a pathological high shear is applied. Treatment of human blood with AZD6482, a selective PI3Kβ inhibitor, phenocopies p110β deletion in mouse platelets, which highlights the role of the kinase activity of p110β. Within the growing platelet thrombus, p110β inactivation impairs the activating phosphorylations of Akt and the inhibitory phosphorylation of GSK3. In accord with these data, pharmacologic inhibition of GSK3 restores thrombus stability. Thus, platelet PI3Kβ is not essential for thrombus growth and stability at normal arterial shear but has a specific and critical role in maintaining the integrity of the formed thrombus on elevation of shear rate, suggesting a potential risk of embolization on treatment with PI3Kβ inhibitors.

Chronic estradiol treatment reduces platelet responses and protects mice from thromboembolism through the hematopoietic estrogen receptor α

Although estrogens are known to have a deleterious effect on the venous thrombosis risk and a preventive action on the development of arterial atheroma, their effect on platelet function in vivo remains unclear. Here, we demonstrate that a chronic high physiologic level of estradiol (E2) in mice leads to a marked decrease in platelet responsiveness ex vivo and in vivo compared with ovariectomized controls. E2 treatment led to increased bleeding time and a resistance to thromboembolism. Hematopoietic chimera mice harboring a selective deletion of estrogen receptors (ERs) α or β were used to demonstrate that the effects of E2 were exclusively because of hematopoietic ERα. Within ERα the activation function-1 domain was not required for resistance to thromboembolism, as was previously shown for atheroprotection. This domain is mandatory for E2-mediated reproductive function and suggests that this role is controlled independently. Differential proteomics indicated that E2 treatment modulated the expression of platelet proteins including β1 tubulin and a few other proteins that may impact platelet production and activation. Overall, these data demonstrate a previously unrecognized role for E2 in regulating the platelet proteome and platelet function, and point to new potential antithrombotic and vasculoprotective therapeutic strategies.

Polymorphisms of protein tyrosine phosphatase CD148 influence FcγRIIA-dependent platelet activation and the risk of heparin-induced thrombocytopenia

Heparin-induced thrombocytopenia (HIT) is due primarily to IgG antibodies specific to platelet factor 4/heparin complexes (PF4/Hs) that activate platelets via FcγRIIA. CD148 is a protein tyrosine phosphatase that regulates Src kinases and collagen-induced platelet activation. Three polymorphisms affecting CD148 (Q276P, R326Q, and D872E) were studied in HIT patients and 2 control groups, with or without antibodies to PF4/Hs. Heterozygote status for CD148 276P or 326Q alleles was less frequent in HIT patients, suggesting a protective effect of these polymorphisms. Aggregation tests performed with collagen, HIT plasma, and monoclonal antibodies cross-linking FcγRIIA showed consistent hyporesponsiveness of platelets expressing the 276P/326Q alleles. In addition, platelets expressing the 276P/326Q alleles exhibited a greater sensitivity to the Src family kinases inhibitor dasatinib in response to collagen or ALB6 cross-linking FcγRIIA receptors. Moreover, the activatory phosphorylation of Src family kinases was considerably delayed as well as the phosphorylation of Linker for activation of T cells and phospholipase Cγ2, 2 major signaling proteins downstream from FcγRIIA. In conclusion, this study shows that CD148 polymorphisms affect platelet activation and probably exert a protective effect on the risk of HIT in patients with antibodies to PF4/Hs.

A novel mass assay to quantify the bioactive lipid PtdIns3P in various biological samples.

PtdIns3P is recognized as an important player in the control of the endocytotic pathway and in autophagy. Recent data also suggest that PtdIns3P contributes to molecular mechanisms taking place at the plasma membrane and at the midbody during cytokinesis. This lipid is present in low amounts in mammalian cells and remains difficult to quantify either by traditional techniques based on radiolabelling followed by HPLC to separate the different phosphatidylinositol monophosphates, or by high-sensitive liquid chromatography coupled to MS, which is still under development. In the present study, we describe a mass assay to quantify this lipid from various biological samples using the recombinant PtdIns3P 5-kinase, PIKfyve. Using this assay, we show an increase in the mass level of PtdIns3P in mouse and human platelets following stimulation, loss of this lipid in Vps34-deficient yeasts and its relative enrichment in early endosomes isolated from BHK cells.

Essential role of class II PI3K-C2α in platelet membrane morphology

The physiologic roles of the class II phosphoinositide 3-kinases (PI3Ks) and their contributions to phosphatidylinositol 3-monophosphate (PI3P) and PI(3,4)P2 production remain elusive. Here we report that mice heterozygous for a constitutively kinase-dead PI3K-C2α display aberrant platelet morphology with an elevated number of barbell-shaped proplatelets, a recently discovered intermediate stage in the final process of platelet production. Platelets with heterozygous PI3K-C2α inactivation have critical defects in α-granules and membrane structure that are associated with modifications in megakaryocytes. These platelets are more rigid and unable to form filopodia after stimulation. Heterozygous PI3K-C2α inactivation in platelets led to a significant reduction in the basal pool of PI3P and a mislocalization of several membrane skeleton proteins known to control the interactions between the plasma membrane and cytoskeleton. These alterations had repercussions on the performance of platelet responses with delay in the time of arterial occlusion in an in vivo model of thrombosis and defect in thrombus formation in an ex vivo blood flow system. These data uncover a key role for PI3K-C2α activity in the generation of a basal housekeeping PI3P pool and in the control of membrane remodeling, critical for megakaryocytopoiesis and normal platelet production and function.

A confocal-based morphometric analysis shows a functional crosstalk between the actin filament system and microtubules in thrombin-stimulated platelets.

Characteristic to resting platelets, microtubules form an internal circumferential marginal band composed of 7–12 filamentous rings of polymers of ab-tubulin dimers located beneath the plasma membrane that maintain the discoid shape of resting platelets [1]. First insights into marginal band structure have emerged from electron microscopy studies suggesting that one continuous microtubule was forming the concentric rings [2]. Recently, time-lapse fluorescent microscopy studies in living resting platelets favor a model in which a principal component of the marginal band is a single, stable microtubule arranged in a coil associated withmultiple, bipolar short and highly dynamic microtubules [3]. The actin cytoskeleton is strongly developed in platelets (actin is estimated at 15– 20% of the total protein) and following activation a rapid and dramatic increase and rearrangement of F-actin occurs. These cytoskeleton reorganizations are essential for platelet morphological modifications and functions [4]. While many studies have been conducted on microtubule and actin cytoskeleton systems as individual entities in platelets, their interdependence following platelet activation remains ill defined. Here, we used confocal microscopy to investigate the organization of F-actin and microtubules, and to achieve a morphometric analysis of the two systems in parallel. Mouse platelets were stimulated by thrombin in suspension and non-aggregating conditions, in the presence of drugs preventing actin polymerization or disrupting actin microfilaments (cytochalasin D and latrunculin A). Inhibitors of microtubule dynamics such as taxol (microtubule stabilizer) or nocodazole (microtubule disruptor) were also used. The degree of internal platelet contraction was quantified using the linescan function of the LSM Image Browser software (Carl Zeiss, Oberkochen, Germany) to calculate the diameter of individual platelets from thin acquisitions of optical confocal sections. The linescan function displays a two-dimensional graph of the intensities of pixels along a line drawn within each platelet. The x-axis corresponds to the distance along the line, and the y-axis to the pixel intensity measured in the red (Alexa fluor 594 phalloidin) or the green (anti-a-tubulin antibody/Alexa fluor 488 antibody) channels (as exemplified in Fig. 1Aa, B). The distance between the two green peaks (i.e. diameter of microtubule rings) was used to evaluate the degree of platelet internal contraction. In resting platelets, the microtubule band was at the periphery of platelets, whereas F-actin displayed a weak and diffuse staining (Fig. 1Aa–c, B). Following thrombin stimulation, the actin cytoskeleton was reorganized, showing a central concentration of actin bundles with peripheral filopodial extensions (arrows, Fig. 1Ae). In parallel, an extensive reorganization of the microtubule coil occurred as it became rapidly compressed in the cell center (Fig. 1Ad). Consistent with a recent report [3], microtubules were seen within some filopodial extensions together with F-actin (arrows, Fig. 1Ad–f). Measurements of the tubulin ring indicated an average diameter of 2.73 ± 0.04 lm in resting conditions vs. 0.95 ± 0.03 lm in thrombin-stimulated platelets (Fig. 1C), which reflects internal platelet contraction under thrombin stimulation in suspension and non-aggregating conditions. In platelets where F-actin formation or stability was challenged by the two drugs, cytochalasin D (Fig. 1Ag–i) or latrunculin A (Fig. 1Aj–l), actin labeling remained diffuse in thrombin-stimulated platelets (Fig. 1Ah,k). Interestingly, both drugs abrogated microtubule reorganization and internal contraction (Fig. 1Ag,j, B, C), even at a longer time of stimulation (Fig. S1). These data suggest a functional link between the actin cytoskeleton and the microtubule network, actin dynamics being mandatory for microtubule constriction upon platelet activation. To assess whether the Arp2/3 complex [5] was involved in bridging actin to the tubulin cytoskeleton in Correspondence: Sonia Séverin, Inserm U1048, I2MC, 1 Avenue Jean Poulhés, BP 84225, 31432 Toulouse Cedex 04, France. Tel.: +33 5 31 22 41 43; fax: +33 5 61 32 56 21. E-mail: sonia.severin@inserm.fr