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Blood, Vol. 110, Issue 6, 1879-1886, September 15, 2007
Conversion of platelets from a proaggregatory to a proinflammatory adhesive phenotype: role of PAF in spatially regulating neutrophil adhesion and spreading
Blood Kulkarni et al.
110: 1879
Supplemental materials for Kulkarni et al, Vol. 110, Issue 6, 1879-1886
Files in this Data Supplement:
- Figure S1. Reverse phase LC/MS of platelet lipid extract (JPG, 31.3 KB)
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Platelets in Tyrode buffer (2.5 × 108/mL) were allowed to adhere and spread onto 9 cm glass dishes in the absence of extracellular calcium for 60 minutes at 37°C. In sample 1 (blue line), SCIP formation was induced in 100% of adherent cells by the addition of ionophore A23187 (1 µM) and 1 mM CaCl2 for 10 minutes following the 60-minute adhesion period. Sample 2 (red line) was exposed to buffer containing ionophore A23187 (1 µM) and 1 mM EGTA and 2 mM MgCl2 for the same time period. Following removal of nonadherent platelets, membrane lipids were then extracted from adherent platelets (2.3 × 108/dish) using the method of Bligh and Dyer.31 The organic phases were then dried, subjected to thin layer chromatography (TLC), alongside 1 µg of authentic PAF, using a methanol:chloroform:water (65:35:6) development solution for 70 minutes. Authentic PAF on the TLC plate was visualized using iodine vapor and the areas (1.5 cm2) of the TLC plate containing sample lipids corresponding to the PAF standard were scraped and subjected to reverse phase LC/MS according to the method of Owen et al (J. Lipid Res, 2005;46:372-382). LC/MS profiles showed that using this method Lyso-PC is clearly distinguishable from platelet-derived PAF C:18 and that only PAF C:18 significantly increased in sample 1 where annexin 5+ve platelet formation was induced compared with sample 2, where platelets remained spread and did not convert to annexin 5+ve forms.
- Figure S2. Thin Layer Chromatography of authentic PAF and PAF related compounds (JPG, 19.2 KB)
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PC (200µg, lane 1), Lyso-PC (lane 2), PAF C:16 (lane 3), PAF C:18 (lane 4), and OAG (lane 5) were dissolved in chloroform and spotted onto TLC plates and the lipids were separated using a methanol:chloroform:water (65:35:6) development solution for 70 minutes. Separated lipids were then visualized using iodine vapor and the TLC plate immediately scanned using the GelPro XR Imaging System (Bio-Rad, Hercules, CA). NB: While the two species of PAF are indistinguishable from each other with regards to their migration on the TLC plate, Lyso-PC migrates lower than PAF. Notably, OAG, given the absence of phosphate groups in its composition, fails to bind to the silica in the TLC plate and migrates with the solvent front (dotted arrow).
- Video 1. Neutrophil morphology on the surface of vWf thrombi (MOV, 5.25 MB)
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Citrated whole blood was perfused over a VWF matrix (100 µg/mL) for 5 minutes at 1800 s−1 followed by a 5-minute wash in calcium-free Tyrode buffer. Isolated neutrophils (106) were then perfused over preformed thrombi and neutrophil-thrombus interactions were visualized by high magnification DIC microscopy (63× lens, Leica DMIRB, Leica, Germany). These images are from one experiment representative of >25 independent experiments. Note: Neutrophils adherent to VWF-thrombi are activated but compact in shape and only extend transient polarised lamellae.
- Video 2. Neutrophil morphology on the surface of spread platelet monolayers (MOV, 2.5 MB)
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Spread platelet monolayers were formed as described in “Materials and methods, Perfusion studies, Platelet monolayer studies.” Isolated neutrophils (106/mL) resuspended in calcium-supplemented (1 mM) Tyrode buffer were perfused over monolayers for 5 minutes at 150 s−1 and the resulting neutrophil-monolayer interactions were visualized by high magnification DIC microscopy (63× lens, Leica DMIRB, Leica, Germany). These images are from one experiment representative of over 50 independent experiments. Note: Neutrophils adherent to spread platelets appeared activated and changed shape. Similar to cells adherent to VWF-thrombi, these neutrophils protruded membranous extensions from a particular aspect of the cell.
- Video 3. Neutrophil morphology on the surface of SCIP monolayers (MOV, 8.41 MB)
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SCIP monolayers were formed as described in “Materials and methods, Perfusion studies, Platelet monolayer studies.” Isolated neutrophils (106/mL) resuspended in calcium-supplemented (1 mM) Tyrode buffer were perfused over monolayers for 5 minutes at 150 s−1 and the resulting neutrophil-monolayer interactions were visualized by high magnification DIC microscopy (63× lens, Leica DMIRB, Leica, Germany). These images are from one experiment representative of over 50 independent experiments. Note: Neutrophils adherent to SCIPs platelets exhibited spread morphology, appearing much flatter than those adherent to spread platelets. Similar to cells adherent to collagen-thrombi, these neutrophils protruded lamellae from the entire periphery of the cell membrane.
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