Journal: Blood

Article Title: The kinetics of ?IIb?3 activation determines the size and stability of thrombi in mice: implications for antiplatelet therapy

doi: 10.1182/blood-2010-07-297713

Figure Lengend Snippet: Platelet adhesion to fibrillar collagen under physiological flow conditions. (A) Whole blood from WT (WT, black lines and bars), CalDAG-GEFI−/− (knockout [KO], red), clopidogrel-treated WT (WT + clop., blue), or clopidogrel-treated KO (KO + clop., green) mice was perfused over collagen at arterial (2000 s−1, left) or venous (400 s−1, right) shear conditions. Platelets in whole blood were labeled with Alexa488-labeled antibodies to GPIX before perfusion. The top graphs represent time traces of the mean fluorescence intensity ± SEM expressed as a percentage of the maximal fluorescence observed (WT blood, 400 s−1). The bar graphs show the area coverage by fluorescent platelets after 5 minutes of blood perfusion, expressed as percentage of the collagen-coated area. Data are shown as mean ± SEM (n = 4-6, 3 independent experiments). *P < .05, **P < .01, ***P < .001. See supplemental Videos 1 to 4 for a better visualization of the differences in thrombus growth and stability observed in the respective study groups. (B-C) Effect of exogenous ADP and TxA2 (U46619) on the adhesion of CalDAG-GEFI−/− platelets. WT and CalDAG-GEFI−/− (KO) whole blood was perfused over collagen at 400 s−1 or 2000 s−1 in the presence (KO + ADP/U46) or absence (KO) of exogenous ADP (25μM) and U46619 (5μM). (B) Bar graphs for area coverage (top) and fluorescence intensity (bottom) measured after 5 minutes of perfusion with the following blood samples: WT (black bar), KO (red bar), and KO reconstituted with 25μM ADP and 5μM U46619 (KO + ADP/U46, red checkered bar). Data are shown as mean ± SEM (n = 5, 3 independent experiments). *P < .05; **P < .01; ***P < .001. (C) Representative images. Images were obtained after 5 minutes of perfusion on a Nikon Eclipse Ti-U inverted microscope (equipped with a Retiga EXL monochrome camera [QImaging] and Nikon NIS Elements software [NIS-Elements Advanced Research]).

Article Snippet: Adhesion of platelets was monitored continuously with a Nikon Ti-U inverted microscope (Nikon Instruments Inc) equipped with a Retiga EXL monochrome camera (QImaging; objective lenses, Plan fluor (air) 20×/0.45).

Techniques: Knock-Out, Shear, Labeling, Fluorescence, Inverted Microscopy, Software

Journal: Blood

Article Title: The kinetics of ?IIb?3 activation determines the size and stability of thrombi in mice: implications for antiplatelet therapy

doi: 10.1182/blood-2010-07-297713

Figure Lengend Snippet: FeCl3-induced thrombosis in the mesentery. (A) Images of mesenteric venules taken 13 minutes after FeCl3 injury. Platelets were labeled by infusion of fluorophore-labeled antibodies to platelet receptor GPIX. White bar = 100 μm. Dotted lines mark the vessel wall. Representative of 5 independent experiments. (B) Embolizing thrombi in clopidogrel-treated WT animals stained positive for JON/A-PE, a probe that selectively detects the activated form of αIIbβ3 integrin.23 Representative of 3 experiments. (C) Mean occlusion time in FeCl3-injured venules of mice of WT (solid black bar), WT/clopidogrel (WT + clop., solid white), and CalDAG-GEFI−/− (KO, checkered) mice (n = 7-9). Note that none of the WT/clopidogrel or CalDAG-GEFI−/− mice occluded within the 40 minutes observation period. (D) Average time required to form a first thrombus of more than 20 μm in diameter. (E) Number of emboli with a diameter of more than 20 μm forming 8-13 minutes after FeCl3 injury. Mean venule diameter: WT: 254.9 ± 19.5 μm; WT + clopidogrel: 305 ± 28.7 μm; CalDAG-GEFI−/−: 239 ± 13.7 μm; P = NS for all comparisons. All images were obtained on a Nikon Eclipse Ti-U inverted microscope (Nikon) equipped with a Retiga EXL monochrome camera (QImaging) and the Nikon NIS Elements software (NIS-Elements Advanced Research).

Article Snippet: Adhesion of platelets was monitored continuously with a Nikon Ti-U inverted microscope (Nikon Instruments Inc) equipped with a Retiga EXL monochrome camera (QImaging; objective lenses, Plan fluor (air) 20×/0.45).

Techniques: Labeling, Staining, Inverted Microscopy, Software

Journal: Blood

Article Title: The kinetics of ?IIb?3 activation determines the size and stability of thrombi in mice: implications for antiplatelet therapy

doi: 10.1182/blood-2010-07-297713

Figure Lengend Snippet: Thrombosis and hemostasis in mice expressing CalDAG-GEFIΔC1 in circulating blood cells. (A-B) Platelet adhesion to collagen at 400 s−1 (A) or 2000 s−1 (B). (Top) Representative images (KO: CalDAG-GEFI−/−, ΔC1: CalDAG-GEFIΔC1). All images were obtained on a Nikon Eclipse Ti-U inverted microscope (Nikon) equipped with a Retiga EXL monochrome camera (QImaging) and the Nikon NIS Elements software (NIS-Elements Advanced Research). (Bottom)Thrombosis score (fluorescence intensity multiplied with area coverage after 5 minutes of perfusion) for the indicated genotypes (WT: wild-type; HET: CalDAG-GEFI+/−; KO: CalDAG-GEFI−/−; ΔC1: CalDAG-GEFIΔC1 chimera). n = 5-6, 3 independent experiments. (C) Bleeding time and blood loss volume determined in WT/GFP (circle) or CalDAG-GEFIΔC1/GFP (ΔC1/GFP, square) chimeric mice.

Article Snippet: Adhesion of platelets was monitored continuously with a Nikon Ti-U inverted microscope (Nikon Instruments Inc) equipped with a Retiga EXL monochrome camera (QImaging; objective lenses, Plan fluor (air) 20×/0.45).

Techniques: Expressing, Inverted Microscopy, Software, Fluorescence