Sonia Severin

Deficiency of Src homology 2 domain–containing inositol 5-phosphatase 1 affects platelet responses and thrombus growth

Platelets are critical for normal hemostasis. Their deregulation can lead to bleeding or to arterial thrombosis, a primary cause of heart attack and ischemic stroke. Src homology 2 domain-containing inositol 5-phosphatase 1 (SHIP1) is a 5-phosphatase capable of dephosphorylating the phosphatidylinositol 3,4,5-trisphosphate second messenger into phosphatidylinositol 3,4-bisphosphate. SHIP1 plays a critical role in regulating the level of these 2 lipids in platelets. Using SHIP1-deficient mice, we found that its loss affects platelet aggregation in response to several agonists with minor effects on fibrinogen binding and beta(3) integrin tyrosine phosphorylation. Accordingly, SHIP1-null mice showed defects in arterial thrombus formation in response to a localized laser-induced injury. Moreover, these mice had a prolonged tail bleeding time. Upon stimulation, SHIP1-deficient platelets showed large membrane extensions, abnormalities in the open canalicular system, and a dramatic decrease in close cell-cell contacts. Interestingly, SHIP1 appeared to be required for platelet contractility, thrombus organization, and fibrin clot retraction. These data indicate that SHIP1 is an important element of the platelet signaling machinery to support normal hemostasis. To our knowledge, this is the first report unraveling an important function of SHIP1 in the activation of hematopoietic cells, in contrast to its well-documented role in the negative regulation of lymphocytes.

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.

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

Cdc42-dependent F-actin dynamics drive structuration of the demarcation membrane system in megakaryocytes.

Essentials Information about the formation of the demarcation membrane system (DMS) is still lacking. We investigated the role of the cytoskeleton in DMS structuration in megakaryocytes. Cdc42/Pak‐dependent F‐actin remodeling regulates DMS organization for proper megakaryopoiesis. These data highlight the mandatory role of F‐actin in platelet biogenesis.

A dual role for the class III PI3K, Vps34, in platelet production and thrombus growth

To uncover the role of Vps34, the sole class III phosphoinositide 3-kinase (PI3K), in megakaryocytes (MKs) and platelets, we created a mouse model with Vps34 deletion in the MK/platelet lineage (Pf4-Cre/Vps34lox/lox). Deletion of Vps34 in MKs led to the loss of its regulator protein, Vps15, and was associated with microthrombocytopenia and platelet granule abnormalities. Although Vps34 deficiency did not affect MK polyploidisation or proplatelet formation, it dampened MK granule biogenesis and directional migration toward an SDF1α gradient, leading to ectopic platelet release within the bone marrow. In MKs, the level of phosphatidylinositol 3-monophosphate (PI3P) was significantly reduced by Vps34 deletion, resulting in endocytic/trafficking defects. In platelets, the basal level of PI3P was only slightly affected by Vps34 loss, whereas the stimulation-dependent pool of PI3P was significantly decreased. Accordingly, a significant increase in the specific activity of Vps34 lipid kinase was observed after acute platelet stimulation. Similar to Vps34-deficient platelets, ex vivo treatment of wild-type mouse or human platelets with the Vps34-specific inhibitors, SAR405 and VPS34-IN1, induced abnormal secretion and affected thrombus growth at arterial shear rate, indicating a role for Vps34 kinase activity in platelet activation, independent from its role in MKs. In vivo, Vps34 deficiency had no impact on tail bleeding time, but significantly reduced platelet prothrombotic capacity after carotid injury. This study uncovers a dual role for Vps34 as a regulator of platelet production by MKs and as an unexpected regulator of platelet activation and arterial thrombus formation dynamics.

Targeting kinases in cancer therapies: adverse effects on blood platelets.

The development of targeted therapy drugs acting on tumor growth and progression is greatly expanding these last years. Among them kinase inhibitors have a prominent position and have demonstrated efficacy and clinical benefits in solid and hematologic malignancies. Compared to conventional systemic cytotoxic chemotherapeutic agents, their specific mechanism of action limits the occurrence of adverse events. However, as targeted kinases are shared by normal cells, their inhibition can affect physiological cell function. In this review we will focus on the side effects of kinase inhibitors on blood platelets which actively use kinase-related signalling pathways to prevent haemorrhages following vessel injury. Major functions of platelets are to adhere to the subendothelial matrix and to aggregate to form a haemostatic plug preventing excessive blood loss upon vascular lesion. Several kinase inhibitors including dasatinib and ibrutinib have been reported to affect specific steps of platelet activation process and to increase bleeding risk. This has important clinical implications particularly in patients treated with antithrombotic drugs. We will describe the effect of kinase inhibitors known to affect platelet activation and discuss the potential impact of those under development that may also interfere with platelet functions.

ATP-binding cassette transporter 1 (ABCA1) deficiency decreases platelet reactivity and reduces thromboxane A2 production independently of hematopoietic ABCA1.

Essentials The role of ATP‐binding cassette transporter 1 (ABCA1) in platelet functions is poorly characterized. We studied the impact of ABCA1 deficiency on platelet responses in a mouse model and two Tangier patients. ABCA1‐deficient platelets exhibit reduced positive feedback loop mechanisms. This reduced reactivity is dependent on external environment and independent of hematopoietic ABCA1.