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First published online 25 January 2005
doi: 10.1242/jcs.01648

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Integrin-dependent interaction of lipid rafts with the actin cytoskeleton in activated human platelets

¹ Inserm U.563, Centre de Physiopathologie de Toulouse Purpan, Department of Oncogenesis and Signaling in Haematopoïetic Cells, IFR30, Hôpital Purpan, 31059 Toulouse, France
² Laboratoire d'Hématologie, CHU Purpan, 31059 Toulouse, France
³ Inserm U.311, Etablissement Français du Sang-Alsace, 10 Rue Spielman, BP 36, 67065 Strasbourg, France

View larger version (26K): [in a new window]   Fig. 1. Similarities in the lipid composition of rafts and actin cytoskeleton isolated from activated platelets. (A) Cytoskeleton was isolated from platelets (3x109 cells) stimulated or not with 1 IU/ml thrombin for 3 minutes. The major phospholipid and cholesterol composition of isolated cytoskeleton was analyzed as described in the Methods. Results are expressed as the mean±s.d. of three different experiments. Proteins from the isolated cytoskeleton were separated by 7.5% SDS-PAGE and the relative amounts of the raft protein markers CD36 and LAT were analyzed by western blotting. Results shown in the right panel are representative of two independent experiments. (B) Rafts were isolated from 3x109 resting platelets as previously described (Bodin et al., 2003b) and their lipid composition was analyzed. Results are expressed as the mean±s.d. of four different experiments. To illustrate the validity of the raft isolation procedure, the level (% of control) of CD36, a raft marker and ß1 integrin, a transmembrane non-raft protein, in each fraction of the sucrose gradient is represented after quantification of western blots by densitometric analysis (NIH Image). Results shown are the means of two independent experiments.

View larger version (32K): [in a new window]   Fig. 2. Lipid rafts can be isolated from the actin cytoskeleton of activated platelets. (A) Schematic representation of the isolation procedure of lipid rafts from the cytoskeleton of thrombin-activated platelets. A representative picture of the gradient obtained after centrifugation is shown. (B) Distribution of the raft marker proteins actin, CD36, LAT and in the eight fractions of the sucrose gradient containing the depolymerized actin cytoskeleton isolated from 3x109 activated platelets (as described in A). Results are representative of two independent experiments. In the top panel, the distribution of actin was determined by densitometry analysis of western blots (10-15% of F-actin associated to lipid rafts). (C) Rafts were isolated from both the cytoskeleton fraction pre-treated with KI (0.6 M) and the supernatant (as described in A) prepared from resting or thrombin-activated platelets (3x109 cells). The lipid composition of rafts (pooled fractions 2-4) was analyzed qualitatively and quantitatively. Results are expressed as nmoles of lipids and are the mean of two independent experiments with very similar results. Similar results were obtained in the presence or absence of 0.6 M of KI in the supernatant.

View larger version (25K): [in a new window]   Fig. 3. The association of lipid rafts with the actin cytoskeleton is dependent on IIbß3 integrin engagement. Platelets pretreated or not with 500 µM RGDS for 1 minute (A,B,C) or 400 µM SR-121566 for 10 minutes (B,C) were stimulated at the concentration of 2x109 cells/ml, with 5 µM TRAP for 3 minutes and their actin cytoskeleton was isolated. (A) The effect of RGDS treatment on platelet aggregation was assessed using a chrono-Log dual-channel aggregometer with stirring at 900 rpm (left panel). Proteins from the isolated actin cytoskeleton were separated by SDS-PAGE (7.5%) and stained with Coomassie Blue (right panel). (B) The quantification of the major phospholipids and cholesterol associated with the actin cytoskeleton isolated from 3x109 platelets was performed as described in Fig. 1A. Results shown are the mean±s.d. of four independent experiments. (C) Lipid rafts were isolated from whole platelets (2x109 cells) pretreated or not with RGDS or SR-121566 and stimulated or not with 5 µM TRAP for 3 minutes and their position in the sucrose density gradient is shown. The distance `d', indicated by the gray bars, represents the height in cm from the top of gradient where the light refractive band corresponding to the raft fraction was observed. The relative amount of actin present in rafts (pooled fractions 2-4) was determined by western blotting using an anti-actin antibody. As a loading control, the amount of CD36 was also analyzed by western blotting. Data shown are representative of three independent experiments.

View larger version (26K): [in a new window]   Fig. 4. Lipid rafts do not associate with the actin cytoskeleton of platelets from patients with Glanzmann thrombasthenia. Platelets from healthy volunteer or from patients with type I Glanzmann thrombasthenia were stimulated or not with 1 IU/ml thrombin for 3 minutes in an aggregometer with stirring at 900 rpm (2x109 cells/ml). (A) Proteins from the isolated cytoskeleton were separated by 7.5% SDS-PAGE followed by Coomassie Blue staining (upper panel) or by western blotting (bottom panel) to determine the relative amount of the raft markers CD36 and LAT using specific antibodies. (B) The amount of cholesterol in cytoskeleton isolated from 3x109 platelets was determined as described in Fig. 1. Results shown are representative of three independent experiments. (C) Rafts were isolated from resting or 5 µM TRAP-stimulated control platelets and the relative amount of ß3 subunits in rafts was quantified by western blotting and densitometric analysis and compared to the total amount of ß3 in the same number of platelets. In the western blot shown, rafts isolated from 1.5x109 platelets and a homogenate from 4x108 platelets were analyzed. Results shown are mean±s.d. of three independent experiments.

View larger version (23K): [in a new window]   Fig. 5. The association of lipid rafts with the actin cytoskeleton is reversible. Platelets (3x109 cells) were stimulated by TRAP (5 µM) as in Fig. 4A. In order to reverse platelet aggregation, 50 nM wortmannin (A and B, left panels) or 400 µM SR-121566 (A and B, right panel) was added 2 and 1.5 minutes after addition of the agonist, respectively. (A) Platelet aggregation profiles show disaggregation after the addition of wortmannin (left panel) or SR-121566 (right panel). (B) The actin cytoskeletons were isolated at 0, 3 or 7 minutes and their cholesterol (O) and SM () content was determined as in Fig. 1A. The drop in cholesterol and SM concomitant with platelet disaggregation induced either by wortmannin or by SR-121566 (dotted lines) illustrates a transient association of rafts with the cytoskeleton under these conditions. Results shown are mean±s.d. of two (for cholesterol) or three (for SM) independent experiments.

View larger version (26K): [in a new window]   Fig. 6. IIbß3 integrin-dependent upregulation of PtdIns(4,5)P2 in rafts and concomitant recruitment of cytoskeleton regulatory proteins. (A) [32P]-labeled platelets (1.5x109 cells) preincubated or not with 400 µM SR-121566 for 10 minutes (white bars) or with 500 µM RGDS for 1 minute (gray bars) were stimulated with 10 µM TRAP for the indicated time. Lipids were extracted from isolated rafts or from whole cells and the amount of [32P]PtdIns(4,5)P2 was quantified by HPLC. ([32P]PtdIns(4,5)P2 concentration in rafts and platelets after RGDS treatment and 6.5-minute TRAP stimulation have not been determined.) Results are the mean±s.d. of four independent experiments and are expressed as fold increase compared to the level of [32P]PtdIns(4,5)P2 under resting conditions in the absence of RGDS or SR-121566 treatment (corresponding to 7.9x104 cpm of [32P]PtdIns(4,5)P2 in rafts isolated from 1.5x109 platelets and 1.1x106 cpm in whole cells). *P<0.05; **P<0.01. (B) Rafts were isolated from 3x109 platelets pretreated or not with 500 µM RGDS for 1 minute and stimulated or not with 10 µM TRAP for 3 minutes. Isolated rafts were submitted to an in vitro PtdIns(4)P kinase assay as described in Materials and methods. The level of [32P]PtdIns(4,5)P2 produced was quantified by HPLC. Results are expressed as cpm of [32P]PtdIns(4,5)P2 produced and are the means of two independent experiments with similar results (upper panel). The amount of Rac associated with rafts (isolated from 1.5x109 cells) or present in 4x108 platelets was estimated by western blotting and densitometry analysis (lower panel). (C) Rafts were isolated from platelets pre-incubated or not with 500 µM RGDS for 1 minute and stimulated or not with 5 µM TRAP for 3 minutes. Proteins from rafts (isolated from 1.5x109 cells) and from whole platelets lysate (from 4x108 cells) were analyzed by western blotting. Results are representative of two to three independent experiments. CD36 was used as a loading control. The amount of this raft marker was constant under the different conditions tested as shown in Fig. 3C.

View larger version (48K): [in a new window]   Fig. 7. The integrity of lipid rafts is required for efficient platelet fibrin clot retraction. (A) Control showing raft disruption by cholesterol depletion (left panel). The level of LAT and CD36 in raft (pooled fractions 2-4 of the sucrose gradient) isolated from platelet pre-treated or not with 5 mM MßCD for 10 minutes (leading to 50±2% depletion of total cholesterol and 56±4% depletion of cholesterol in rafts) was analyzed by western blotting. The effect of raft disruption on platelet aggregation induced by low (0.2 IU/µl) or high (0.5 IU/µl) thrombin concentration is shown in the right panel. (B) Effect of raft disruption on fibrin clot retraction. Platelets treated or not with 5 mM MßCD for 10 minutes, were resuspended at 3x108/ml in 2 ml autologous plasma prior to treatment with either 0.5 IU/ml thrombin and 0.1 µg/ml atroxin or 2 IU/ml thrombin alone at 37°C. For the study with 0.5 IU/ml thrombin, atroxin was added to induce clot formation. After 2 hours, the extent of clot retraction was observed.

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