An Ultrasound Contrast Agent Targeted to P-Selectin Detects Activated Platelets at Supra-Arterial Shear Flow Conditions

Objectives: To evaluate targeting of a microbubble contrast agent to platelets under high shear flow using the natural selectin ligand sialyl Lewis(a). Materials and Methods: Biotinylated polyacrylamide Sialyl Lewisa or biotinylated carbohydrate-free polymer (used as a control) were attached to biotinylated microbubbles via a streptavidin linker. Activated human platelets were isolated and attached to fibrinogen-coated culture dishes. Fibrinogen-coated dishes without platelets or platelet dishes blocked by an anti-P-selectin antibody served as negative control substrates. Dishes coated by recombinant P-selectin served as a positive control substrate. Microbubble adhesion was assessed by microscopy in an inverted parallel plate flow chamber, with wall shear stress values of 40, 30, 20, 10, and 5 dynes/cm(2). The ratio of binding and passing microbubbles was defined as capture efficiency. Results: There was no significant difference between the groups regarding the number of microbubbles in the fluid flow at each shear rate. Sialyl Lewisa-targeted microbubbles were binding and slowly rolling on the surface of activated platelets and P-selectin-coated dishes at all the flow conditions including 40 dynes/cm(2). Capture efficiency of targeted microbubbles to activated platelets and recombinant P-selectin decreased with increasing shear flow: at 5 dynes/cm(2), capture efficiency was 16.11% on activated platelets versus 21.83% on P-selectin, and, at 40 dynes/cm(2), adhesion efficiency was still 3.4% in both groups. There was neither significant adhesion of Sialyl Lewisa-targeted microbubbles to control substrates, nor adhesion of control microbubbles to activated platelets or to recombinant P-selectin. Conclusions: Microbubble targeting using sialyl Lewisa, a fast-binding ligand to P-selectin, is a promising strategy for the design of ultrasound contrast binding to activated platelets under high shear stress conditions.