Time-dependent accumulation of Glu-plg-568 on the surface of IMC-treated washed platelets from GFP-mice.

<p>Washed platelets from GFP-mice were treated with IMC (10 μM) in the presence of Glu-plg-568, and accumulation of Glu-plg-568 on the surface of platelets was monitored over time by CLSM either in the absence (A) or in the presence of 100 mM EACA (B) or 15 U/ml CPB (C). Representative CLSM images are shown at the indicated time points after addition of IMC. A decrease in GFP fluorescence intensity in individual platelets indicates the full activation of platelets with PS exposure on the surface, as a result of a sustained elevation of intracellular calcium ion concentration caused by IMC. The decrease in GFP fluorescence intensity was followed by gradual binding of Glu-plg-568 to the platelet surfaces, which was strongly limited by EACA and CPB. The scale bars represent 10 μm. (D) The platelet Glu-plg-568 binding relative ratio was expressed as the integrated fluorescence intensity of Glu-plg-568 bound to the surface of activated platelet 120 minutes after IMC supplementation and divided by the area of the platelet (n = 30 activated platelets from three independent experiments in each column). This assay was analyzed with a <i>t</i>-test for independent samples. Results are normalized to the control sample mean value (mean ± SD).