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Blood, Vol. 113, Issue 20, 4942-4954, May 14, 2009
The tyrosine phosphatase CD148 is an essential positive regulator of platelet activation and thrombosis
Blood Senis et al.
113: 4942
Supplemental materials for: Senis et al
Files in this Data Supplement:
- Figure S1. Quantitative flow cytometry of CD148 on the surface of resting human platelets (JPG, 71.1 KB)
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(A) Calibration beads coated with a known number of antibody binding sites (490, 13,000, 38,000 and 96,000) were stained with FITC-conjugated anti-mouse antibody for 15 minutes and analyzed by flow cytometry. (B) A calibration curve was obtained by plotting the geometric mean fluorescence intensity (GMFI) of each peak in panel (A) against the known number of antibody binding sites for each peak. (C) Resting human platelets were incubated with 10 µg/ml of either mouse anti-human CD148 primary antibody (CD148, gray line), that recognizes the extracellular region of CD148, or the same amount of an isotype control antibody (IgG, black line) for 15 minutes, then stained for 15 minutes with a FITC-conjugated anti-mouse IgG antibody. The GMFI of the isotype control antibody was subtracted from the GMFI of the anti-CD148 antibody before the number of surface molecules per platelet was calculated. (D) The mean number of copies of CD148 on the surface of resting human platelets was calculated to be 2,834 ± 90 (± standard error). Each dot represents an individual and the horizontal line represents the mean.
- Figure S2. Surface expression of glycoproteins on platelets from wild-type (WT) and CD148 transmembrane-knockout (CD148 TM-KO) mice (JPG, 72.8 KB)
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Platelets from WT and CD148 TM-KO mice were stained with either: (Ai) rat anti-mouse GPVI primary antibody and FITC-conjugated anti-rat IgG secondary antibody (GPVI, gray line); (Bi) FITC-conjugated rat anti-mouse α2 antibody (α2β1, gray line); or (Ci) FITC-conjugated rat anti-mouse αIIbβ3 antibody (αIIbβ3, gray line). Primary antibodies were replaced with isotype control antibodies, as negative controls (IgG, black lines). Representative histograms are shown (n = 5–9 mice per stain). (Aii, Bii and Cii) Relative levels of surface expression of GPVI, α2β1 and αIIbβ3 were quantified by flow cytometry. Geometric mean fluorescence intensities (MFI) of platelets from CD148 TM-KO mice were compared with WT platelet levels (n = 5–9 mice; mean ± standard deviation).
- Video 1 (Supplement to Fig. 4). Wild-type platelets spread on a fibrinogen-coated surface (MOV, 1.52 MB)
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Wild-type (WT) platelets spreading on a fibrinogen-coated surface was imaged in real-time by differential interference contrast microscopy (Zeiss Axiovert 200M microscope; Hamamatsu Orca 285 cooled digital camera). The time in seconds is indicated in the top left corner. Representative video from three experiments using three WT mice.
- Video 2 (Supplement to Fig. 4). CD148-deficient platelets do not spread on a fibrinogen-coated surface (MOV, 1.53 MB)
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CD148-deficient (CD148 TM-KO) platelets plated on a fibrinogen-coated surface were imaged in real-time by differential interference contrast microscopy. The time in seconds is indicated in the top left corner. Representative video from three experiments using three CD148 TM-KO mice.
- Video 3 (Supplement to Fig. 6). Wild-type megakaryocytes migrate towards a SDF-1α gradient over a fibronectin-coated surface (MOV, 1.11 MB)
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A wild-type (WT) bone marrow-derived megakaryocyte plated on a fibrinogen-coated surface was imaged in real-time migrating towards a SDF-1α gradient. Images were captured by differential interference contrast microscopy (Zeiss Axiovert 200 microscope; Hamamatsu Orca 285 cooled digital camera). The time in minutes is indicated in the top left corner. Representative video from three experiments using three WT mice.
- Video 4 (Supplement to Fig. 6). CD148-deficient megakaryocytes do not migrate towards a SDF-1α gradient over a fibronectin-coated surface (MOV, 988 KB)
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A CD148-deficient (CD148 TM-KO) bone marrow-derived megakaryocyte plated on a fibrinogen-coated surface in a SDF-1α gradient was imaged in real-time by differential interference contrast microscopy. The time in minutes is indicated in the top left corner. Representative video from three experiments using three CD148 TM-KO mice.
- Video 5 (Supplement to Fig. 7). Wild-type platelets formi aggregates on a collagen-coated surface under flow (MOV, 2.79 MB)
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Anti-coagulated blood from a wild-type (WT) mouse was fluorescently labelled with DiOC6 and flowed through a collagen-coated capillary tube at a shear rate of 1000 s−1. The accumulation of DiOC6-labelled platelets was monitored in real-time using fluorescence microscopy (Leica DM IRB microscope; CoolSnap ES digital camera, Photometrics). The time in seconds is indicated in the top left corner. Representative video from three mice.
- Video 6 (Supplement to Fig. 7). CD148-deficient platelets adhere, but do not form aggregates on a collagen-coated surface under flow (MOV, 3.25 MB)
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Anti-coagulated blood from a CD148 transmembrane-knockout (CD148 TM-KO) mouse was fluorescently-labelled with DiOC6 and flowed through a collagen-coated capillary tube at a shear rate of 1000 s−1. The accumulation of DiOC6-labelled platelets was monitored in real-time using fluorescence microscopy. The time in seconds is indicated in the top left corner. Representative video from three mice.
- Video 7 (Supplement to Fig. 7). Laser-induced thrombus formation in an arteriole of a wild-type mouse (MOV, 6.18 MB)
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Fluorescently-labelled platelets (green) are shown accumulating at the site of a laser-induced injury in an arteriole of the cremaster muscle of a wild-type mouse. Platelets were labelled ex vivo with a rat anti-mouse αIIb primary antibody and an Alex488-conjugated secondary antibody, before being reintroduced into the mouse. A timer is show in the top left corner and a 10 µm scale bar in the bottom left corner. Representative video of twenty-five thrombi induced in different wild-type mice.
- Video 8 (Supplement to Fig. 7). Laser-induced thrombus formation in an arteriole of a CD148 transmembrane-knockout mouse (MOV, 6.18 MB)
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Fluorescently-labelled platelets (green) are shown accumulating at the site of a laser-induced injury in an arteriole of the cremaster muscle of a CD148 transmembrane-knockout (CD148 TM-KO) mouse. Platelets were labelled ex vivo with a rat anti-mouse αIIb primary antibody and an Alex488-conjugated secondary antibody, before being reintroduced into the mouse. A timer is show in the top left corner and a 10 µm scale bar in the bottom left corner. Representative video of twenty-five thrombi induced in five CD148 TM-KO mice.
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