To further explore the finding that peptides derived from specifically exposed regions of band 3 inhibited sickle cell adhesiveness in vitro, we examined the effects of such peptides with human sickle cells in the mouse microcirculation in vivo. Human red cells were loaded with the fluorescent dye BCECF, and infused into the arterial circulation of anaesthetized mice. The microcirculation of several tissues was examined in a specially designed warm-stage microscope with stroboscopic epi-fluorescent illumination. Adhesive events were video-recorded and quantified after peptide infusion and after treatment of the mice with various biological modifiers. An adhesive index (AI), proportionate to the number of adhesive events per number of cells entering a vessel, was calculated. Sickle cells (SS) were found to be more adhesive than normal cells particularly in small vessels such as post-capillary venules of the cremaster muscle (AI 2.3 vs 0.5). Moreover, endothelium activation substantially increased SS adhesiveness (e.g., AI 8.3 after pretreatment with platelet activating factor (PAF), 5.8 with tumor necrosis factor, and 8.7 with thrombin-activated mouse platelets). A band 3 peptide which blocked SS adhesiveness in vitro inhibited much of the increased adhesiveness of SS cells (e.g., in the PAF-treated mouse AI 2.8 vs 8.3, vs pretreatment control 2.3). No inhibitory effect was seen with a control peptide with the same composition but a scrambled sequence. These preliminary findings suggest that the inhibitory activity of specific band 3 sequences on SS adhesiveness is retained in vivo and extend the previous in vitro demonstration of an adhesive role for these exposed band 3 sequences. In addition, they imply a substantial role for endothelial and/or platelet activation in the adhesiveness of sickle cells in vivo. Finally, they demonstrate the utility of direct visualization of the mouse microcirculation for studying the adhesiveness of human cells.
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