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Blood, Vol. 113, Issue 26, 6699-6706, June 25, 2009
PAF-acetylhydrolase expressed during megakaryocyte differentiation inactivates PAF-like lipids
Blood Foulks et al.
113: 6699
Supplemental materials for: Foulks et al
Files in this Data Supplement:
- Figure S1. Megakaryocytes release small amounts of PAF-AH (JPG, 152 KB)
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PAF-AH activity was measured in the supernatants from freshly-isolated CD34+ cells (culture day 0), megakaryocyte precursors (culture day 7), or megakaryocytes (culture day 13) adherent to immobilized fibrinogen. The bars in this graph represent the mean+SD for three independent experiments. The asterisk indicates statistical significance (p<0.05) compared to freshly-isolated CD34+ cells or megakaryocyte precursors. Although PAF-AH activity is increased in the supernatants of megakaryocytes, the amount of extracellular PAF-AH is very small when compared to PAF-AH secreted by human macrophages.42
- Figure S2. PAF-AH1b2 is present in megakaryocyte precursors and megakaryocytes (JPG, 476 KB)
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(A) Megakaryocyte precursors (culture day 7) stained for PAF-AH1b2 (white). (B) High magnification of PAF-AH1b2 staining (white, left panel) in a megakaryocyte (culture day 13) adherent to immobilized fibrinogen. Staining specificity for PAF-AH1b2 antibody was demonstrated by pre-incubation with recombinant human PAF-AH1b2 (right panel).
- Figure S3. Recombinant PAF-AH eliminates PAF-like lipid activity generated by megakaryocytes (JPG, 348 KB)
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(A) Phospholipids from Pefabloc treated megakaryocytes (culture day 13) adherent to immobilized fibrinogen were extracted, treated with 10 µg recombinant PAF-AH and subsequently added (arrow 1) to Fura-2 AM loaded PMNs to measure intracellular calcium release by fluorimetry. PAF responsiveness of the PMNs (arrow 2) was subsequently demonstrated with the addition of exogenous cPAF. (B) PMNs were pretreated with a PAFR blocker (i.e., WEB 2086, 10 µM), then stimulated with lipids from Pefabloc treated megakaryocytes to demonstrate signaling specific to the PAFR (arrow 3). The efficacy of PAFR blockade was tested by adding a high concentration of cPAF (1 µM) to PMNs (arrow 4) that were previously treated with a PAFR blocker (i.e., WEB 2086). This figure is representative of two independent experiments.
- Figure S4. Undifferentiated CD34+ cells, megakaryocyte precursors, and megakaryocytes do not express mRNA for PAFR transcript-2 (JPG, 259 KB)
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(A) RT-PCR for PAFR-2 (PAFR Tr 2) for undifferentiated CD34+ cells, megakaryocyte precursors or megakaryocytes. (B) Expression of PAFR-2 (PAFR Tr 2) in HepG2 cells.
- Figure S5. cPAF induces PAFR-dependent calcium fluxes in megakaryocytes (JPG, 141 KB)
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Megakaryocytes were loaded with Fluo-4 AM in the presence or absence of the PAFR blocker WEB 2086 (10 µM) and subsequently stimulated with Lyso-PAF or cPAF. Intracellular calcium fluxes were measured and are presented as mean peak fluorescence. The bars represent the mean±SD for three independent experiments respectively. The asterisk indicates statistical significance (p<0.05) compared to the other experimental treatment groups.
- Figure S6. Activation of megakaryocytes by thrombin is independent of the PAFR (JPG, 562 KB)
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Megakaryocytes were pretreated or not with the PAFR blocker WEB 2086 (10 µM) and subsequently treated with thrombin (0.5 U/mL) as they adhered to immobilized fibrinogen. After 30 minutes, the cells were fixed and stained for β-tubulin (white). This experiment is representative of three independent experiments.
- Figure S7. Inhibition of endogenous PAF-AH activity induces spreading in megakaryocytes (JPG, 172 KB)
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Megakaryocytes were pretreated with or without the PAFR blocker, WEB 2086 (10 µM), in the presence of 100 µM of Pefabloc SC, a PAF-AH inhibitor, or its inactive analog, AEBSNH2 (100 µM). The megakaryocytes were subsequently placed on immobilized fibrinogen and cell spreading was determined as described in the Material and Methods section. Bars represent the mean+SD (n=3) of cells greater than 30 µm. The asterisk in this figure represents statistical significance (p<0.05) between the treatment groups.
- Figure S8. cPAF induces migration in megakaryocytes (JPG, 165 KB)
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Megakaryocytes were pretreated with or without the PAFR blocker, WEB 2086 (10 µM), and the cells were subsequently loaded into the upper chamber of a transwell micropore filter apparatus. cPAF (10 nM) or its vehicle was added to the lower chamber and migration of megakaryocytes into this chamber was determined after 18 hours. The migrating cells were stained with Calcein AM and compared to a standard dilution of stained megakaryocytes for cell quantification. The bars are the mean±SD of three independent experiments and the asterisk identifies statistical significance (p<0.05) between the groups.
- Figure S9. cPAF enhances adhesion and spreading of megakaryocytes on immobilized fibrinogen, but not vitronectin or laminin (JPG, 368 KB)
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Megakaryocytes were placed on immobilized vitronectin, laminin, or fibrinogen in the presence of 10 nM cPAF or its vehicle for 30 minutes. β-tubulin was localized in the megakaryocytes (white stain). Scale bar = 50 µm.
- Figure S10. A stable PAF analog activates the integrin αIIbβ3 complex in megakaryocytes (JPG, 407 KB)
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Megakaryocytes (culture day 13) left in suspension culture were treated with 10 nM cPAF or 0.5 U/mL thrombin for 5 minutes. Cells positive for PAC-1 and CD41 are shown in the upper right quadrant as a percent of total CD41 cells. Data are representative of five independent experiments.
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