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. 2014 Jun 1;20(16):2528–2540. doi: 10.1089/ars.2013.5337

FIG. 5.

FIG. 5.

GPx1/Cat double deficiency facilitates platelet aggregation and thrombus formation in a redox-dependent manner. (A, B) Washed platelets from the WT and GPx1−/−Cat−/− mice were incubated with (+) or without (−) convulxin (CVX, 25 or 250 ng/ml) for 5 min in the presence of 0.5 mg/ml FITC-conjugated anti-P-selectin (P-selectin-FITC in A) or 0.5 mg/ml PE-conjugated anti-active-integrin αIIbβ3 (JON/A-PE in B). Binding of anti-P-selectin (A) and active-integrin αIIbβ3 to platelets was analyzed using flow cytometry. The histograms are representative of at least three different experiments and three different mice. The quantitative data are the mean±S.D. (n=3; **p<0.01) for the mean fluorescence intensity. (C) Washed WT and GPx1−/−Cat−/− platelets were stimulated with the indicated concentrations of collagen for 5 min. Platelet aggregation was assessed and expressed as described in Figure 1A. The representative aggregation peaks from three independent experiments are shown. The quantitative data are the mean±S.D. (n=3; *p<0.05; **p<0.01). (D) The left carotid artery of WT and GPx1−/−Cat−/− mice was injured by the topical application of 20% ferric chloride. The blood volume changes in the carotid artery downstream of the site of injury were measured by the photoplethysmography method using a minimized OxiPulse probe in transmission mode. On the left panel, representative photoplethysmography waveforms from independent experiments are shown. The amplitudes, representing the differences between the peaks and valleys of each waveform, were averaged to obtain the mean blood volume changes in the carotid artery. The time to thrombotic occlusion was defined as the time required for >90% loss of the initial blood volume. The quantitative data are the mean±S.D. (n=8–11, p<0.001). (E) DiOC6-labeled platelets from WT and GPx1−/−Cat−/− mice were perfused through a collagen-coated coverslip placed in a parallel plate flow chamber at a shear rate of 150 s−1 for the indicated times. Chamber surface coverage by the thrombi (fluorescence positive) was quantified. Representative images of platelet adhesion from three independent experiments are shown. Scale bar represents 50 μm. The quantitative data are the means±S.D. (n=3; **p<0.01) for DiOC6 fluorescence.