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Blood, Vol. 107, Issue 10, 3902-3906, May 15, 2006 This Article Abstract Full Text Services Email this article to a friend Alert me to new issues of the journal Rights and Permissions Citing Articles Citing Articles via CrossRef Glycoprotein VI–dependent and –independent pathways of thrombus formation in vivo Blood Dubois et al. 107: 3902 Supplemental materials for: Dubois et al Vessel wall injury is performed while looking through the microscope ocular to produce ablation in the correct focal plane. Typically one or two pulses were required to induce vessel wall injury. The microscope is immediately returned to the camera view to initiate image capture. Over the course of a 2 hour experiment in a single mouse, 6 to 13 thrombi were formed and studied. New thrombi were formed upstream of earlier thrombi to avoid any contribution from thrombi generated earlier in the animal under study. There were no characteristic trends in thrombus size or thrombus composition in sequential thrombi generated in a single mouse. For each thrombus generated, a rectangular mask was defined that includes a portion of the vessel upstream of the site of injury. The maximum fluorescence intensity of the pixels contained in this mask was extracted for all frames (pre and post-injury) for each thrombus. The mean value calculated from the maximal intensity values in the mask for each frame was determined and used as the background value to determine the fluorescence intensity values and area of the signal corresponding to the specific signal at the injury site. Finally, for each frame the Integrated Fluorescence Intensity was calculated by subtracting the background value from the fluorescence intensity values by the following equation: Integrated Fluorescence Intensity = (mean intensity value of the fluorescence signal)(area of the signal) — (mean intensity value of the background)(area of the signal). This calculation was performed for all frames in each thrombus and is plotted versus time in seconds to provide the kinetics of thrombus formation over time. When multiple fluorescence channels are used calculations of background are made independently for each channel. The data from multiple thrombi were used to determine the median value of integrated fluorescence intensity (arbitrary units) at each time point and plotted versus time (s) to account for the variability of thrombus formation at any given set of experimental conditions. When comparing the kinetics of thrombus formation over time between two or more genotypes or phenotypes of mice, all the experiments were done in a limited time frame (generally within a week) to minimize instrument and reagent variability. Files in this Data Supplement: Figure S1. Platelet accumulation and thrombus growth in wild type and FcRγ null mice after a FeCl3 or a laser-induced injury (JPG, 323 KB) - Platelets were labeled by infusion of Alexa 660-conjugated rat anti mouse CD41 antibody (250 ng/g body weight). A and B, Platelet fluorescence, red pseudo color. (A) Representative picture of thrombus formation and platelet accumulation after treatment of the mesentery with a solution of 10% FeCl3 for 5 min in wild type and FcR null mice. (B) Representative pictures of thrombus formation in wild-type and FcR null mice after laser-induced injury. (C) The time to the maximum platelet accumulation into growing thrombi after laser-induced injury to mesenteric arterioles is reported and the calculated median time indicated (8 thrombi in each genotype). Wild-type mice, WT; FcR null mice, FcR—/—. Figure S2. Platelet accumulation and thrombus growth in GP VI depleted mice is normal after laser induced injury (JPG, 116 KB) - Wild type mice were treated with an anti-GPVI or control antibody as described in Experimental Procedures. (A) Left panel: The level of GP VI on the platelet surface of GPVI depleted mice (black line), FcR null mice (light gray line) and wild-type mice (dark gray line) was determined by flow cytometry analysis. Platelets were fluorescently labeled with FITC-conjugated rat anti-mouse GPVI, Right panel: The level of 2 on the platelets of wild type mice treated (dark gray) or untreated (light gray) with the control anti-2 antibody was determined by flow cytometry analysis. Platelets were stained with FITC-conjugated rat anti-mouse 2 or an irrelevant control antibody. (B) The median integrated platelet fluorescence intensity as function of time for multiple thrombi in GP VI depleted or mice treated with anti-2 antibody (Control) (40 thrombi in 3 mice and 35 thrombi in 3 mice respectively) were calculated. This Article Abstract Full Text Services Email this article to a friend Alert me to new issues of the journal Rights and Permissions Citing Articles Citing Articles via CrossRef

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