Novel Single-Chain Antibody-Targeted Microbubbles for Molecular Ultrasound Imaging of Thrombosis Validation of a Unique Noninvasive Method for Rapid and Sensitive Detection of Thrombi and Monitoring of Success or Failure of Thrombolysis in Mice

Background —Molecular imaging is a fast emerging technology allowing non-invasive detection of vascular pathologies. However, imaging modalities offering high resolution currently do not allow real-time imaging. We hypothesized that contrast enhanced ultrasound with microbubbles selectively targeted to activated platelets would offer high-resolution, real-time molecular imaging of evolving and dissolving arterial thrombi. Methods and Results —Lipid-shell based gas-filled microbubbles (MB) were conjugated to either a single-chain antibody (scFv) specific for activated GPIIb/IIIa via binding to a Ligand-Induced Binding Site (LIBS-MB), or a non-specific scFv (control-MB). Successful conjugation was assessed in flow cytometry and immunofluorescence double-staining. LIBS-MB, but not control-MB, strongly adhered to both immobilized activated platelets and micro-thrombi under flow. Thrombi, induced in carotid arteries of C57Bl6-mice in vivo by ferric chloride injury, were then assessed with ultrasound before and 20 minutes after MB injection, using grayscale area intensity measurement. Grayscale units converted to decibels demonstrated a significant increase after LIBS-MB but not after control-MB injection (9.55 ± 1.7 versus 1.46 ± 1.3 dB; p<0.01). Further-more after thrombolysis with urokinase, LIBS-MB ultrasound-imaging allows monitoring of the reduction of thrombus size (p<0.001). Conclusions —We demonstrate that GPIIb/IIIa-targeted microbubbles specifically bind to activated platelets in vitro and allow real-time molecular imaging of acute arterial thrombosis as well as monitoring success or failure of pharmacological thrombolysis in vivo .