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Blood, Vol. 114, Issue 2, 415-424, July 9, 2009
Unraveling a novel Rac1-mediated signaling pathway that regulates cofilin dephosphorylation and secretion in thrombin-stimulated platelets
Blood Pandey et al.
114: 415
Supplemental materials for: Pandey et al
Isolation of human washed platelets and measurement of ATP-secretion, aggregation and P-selectin expression
Approval was obtained from the Ethic Commission of the Medical Faculty of the University of Munich. Informed consent was provided according to the Declaration of Helsinki Protocol. Platelets from acetylsalicylic acid-treated human blood were isolated by centrifugation in the presence of apyrase and resuspended in a buffer (pH 7.4) containing 20mM HEPES, 138mM NaCl, 2.9mM KCl, 1mM MgCl2 and 0.6 ADPase units/ml apyrase (4 × 108cells/ml) as described previously.1 Platelet ATP-secretion and aggregation induced by thrombin (0.5 U/ml) was measured by recording the luminescence and light transmission, respectively in an lumi-aggregometer as described.1 No external fibrinogen was added to the platelets before stimulation with thrombin. For P-selectin expression, the final platelet resuspension was done in buffer containing 0.35% fatty acid free albumin. Aliquots of stirred platelets (2 × 107) either unstimulated or stimulated with agonists in the presence of RGDS were fixed with equal volume of Dulbecco's phosphate buffered saline (PBS) containing 3.7% formaldehyde and stained with anti-human CD62P-FITC antibody for 1 hr at room temperature. Anti–CD62P-FITC labeled platelets were then 20-fold diluted with PBS and P-selectin positive platelets were quantified by flow cytometry (FACScan, Becton Dickinson, NJ, USA) and the CELLQuest software after collecting 10000 platelet events. Platelet events of isotype matched IgG1-FITC labeled platelets were subtracted during analysis. Platelets were pre-treated with all inhibitors (except NSC23766) with the given concentrations at 37°C for 20 minutes before stimulation. NSC23766 was incubated at 37°C for 5 minutes before stimulation of platelets with thrombin. RGDS (0.5mM) was added 2 minutes before platelet stimulation. Measurement of Rac1 activity
Human washed platelet suspensions (4 × 108/mL, 0.4 mL) were stimulated with thrombin in the presence or absence of the inhibitors. RGDS was added prior to thrombin-stimulation in order to avoid platelet aggregation. Samples were lysed in an equal volume of ice cold 2× Assay/Lysis buffer provided with kit (50mM HEPES, pH 7.5, 300mM NaCl, 2% NP-40, 20mM MgCl2, 2mM EDTA, 4% Glycerol 5mM Na3VO4, phosphatase cocktail 1:100, and complete mini protease inhibitor 1 tablet/8 mL) for 30 minutes on ice. The lysates were clarified by centrifugation at 16,000g for 15 minutes and to the supernatants 40µl of PAK PBD agarose beads were added, and then incubated at 4°C for 1 hour. Beads were then washed once with 1× lysis buffer and twice with PBS, pulled down Rac1 was taken up into SDS-PAGE sample buffer and immunoblotting using Rac1 specific antibody was performed. Immunoblotting and measurement of protein phosphorylation
Aliquots (100µl) of unstimulated and stimulated platelet suspensions were transferred from the aggregometer cuvettes to an equal volume of 2X SDS-PAGE sample buffer. Proteins were separated on 12% SDS-PAGE (cofilin and phospho-cofilin) or 10% SDS-PAGE (phospho-Akt, phospho-MYPT, LIMK-1, phospho-LIMK-1, PAKs, and phospho-PAKs). To separate ADF and cofilin in platelets lysates we used 18% SDS-PAGE. Proteins were blotted to nitrocellulose membranes using the Mini Trans-Blot electrophoresis cell (Bio-Rad, Hercules, CA). Membranes were blocked with 5% (w/v) non-fat milk in TBS (Tris-buffered saline) and incubated with the respective primary and secondary antibodies. The dilutions of the primary antibodies were: anti-cofilin (1:12,000), anti–phospho-MYPT antibody (1:3000), anti–phospho-cofilin (1:2000), and anti–phospho-Akt, anti–LIMK-1, anti–phospho-LIMK1/2, anti-PAK1, anti-PAK2, anti-PAK3, anti-PAK4, anti–phospho-PAK1/2, and anti–phospho-PAK4/5/6 (1:1000). The dilution of horseradish-peroxidase–linked secondary anti-rabbit and anti-mouse antibodies was 1:5000. The membranes were developed and densitometric analysis of the proteins was done as described previously.1 The densitometric values of phosphorylated proteins were divided by the corresponding values of unphosphorylated proteins respectively. Absorption of proteins in unstimulated control samples was set to 100%. Data are presented as mean ± S.D. of individual experiments from different blood donors. REFERENCE 1. Pandey D, Goyal P, Bamburg JR, Siess W. Regulation of LIM-kinase 1 and cofilin in thrombin-stimulated platelets. Blood. 2006;107:575–583.
Files in this Data Supplement:
- Figure S1 (JPG, 183 KB)
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(A) Thrombin-stimulated dense granule release from platelet is independent of integrin αIIBβ3 engagement. Inhibition of secretion by NSC23766. Platelets untreated (left) or pretreated with NSC23766 (right) were stimulated with thrombin (0.5U/ml) in the presence of the integrin αIIBβ3 blocker RGDS (0.5mM). Representative tracings for change in light transmission (LT) and ATP secretion are shown. (B) Inhibition of thrombin-induced aggregation by NSC23766 is through inhibition of dense granule secretion. Left, platelets untreated or pretreated with NSC23766 were stimulated with thrombin (0.5U/ml). Addition of fibrinogen (0.5mg/ml) 15 sec after thrombin-stimulation of NSC23766-treated platelet does not induce aggregation and secretion. Representative tracings for change in light transmission (LT) and ATP secretion are shown. Right, NSC23766 was not able to inhibit platelet aggregation induced by ADP (10µM). Fibrinogen (0.5mg/ml) was added 15 sec prior to stimulation with ADP. Representative tracings for change in light transmission are shown.
- Figure S2. Effect of NSC23766 on MYPT1 (Thr850) phosphorylation during platelet aggregation induced by thrombin (0.5U/ml) (JPG, 88.7 KB)
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Lysates of platelet stimulated with thrombin in the absence (-■-) or presence (-□-) of NSC23766 were immunoblotted with anti–phospho-MYPT1 and changes were estimated densitometrically. Graphic representation of the results. Values are the mean + S.D or mean − S.D for three independent experiments.
- Figure S3 (JPG, 235 KB)
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(A) Specificity of anti-cofilin and anti–phospho-cofilin antibodies. Platelet lysates (lane 1) and ADF immunoprecipitated from platelets (lane 2) were subjected to 18% SDS-PAGE and then immunoblotted with anti-ADF, anti–phospho-cofilin, anti-cofilin, and in combination of antibodies (anti–phospho-cofilin plus ant-ADF, and anti-cofilin plus anti-ADF). (B) Specificity of anti-ADF antibody. Purified recombinant his-tagged cofilin-GFP and his-tagged cofilin were immunoblotted with ant-cofilin and anti-ADF antibodies.
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