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Dr. Peter Karlheinz
Karlheinz Peter (Australia)
List of Authors: Peter Karlheinz, Wang Xiaowei, Hagemeyer Christoph, Jia Fu, Leitner Ephraem, Ahrens Ingo
Antibody-coupling and targeting of microbubbles to activated platelets allows molecular imaging of thrombosis and monitoring of success or failure of thrombolysis using non-invasive ultrasound. Most acute cases of myocardial infarction and stroke are caused by atherothrombosis, when platelet adhesion, activation and aggregation lead to thrombus formation and vessel occlusion. The glycoprotein (GP) IIb/IIIa receptor mediates platelet adhesion and aggregation and is the most abundant receptor expressed on the platelet surface. We hypothesized that contrast enhanced ultrasound with microbubbles selectively targeted to the activated conformation of GPIIb/IIIa and thus to activated platelets would offer real-time as well as high resolution molecular imaging of evolving and dissolving arterial thrombi. Lipid-shell based gas-filled microbubbles were conjugated to either a single-chain antibody (scFv) specific for an epitope named ligand-induced binding site (LIBS) on activated GPIIb/IIIa (LIBS-MB), or a non-specific scFv (control-MB). Non-conjugated microbubbles (MB) were used as additional control. Flow-chamber experiments demonstrated strong adhesion of LIBS-MB to immobilized activated platelets compared with control-MB or MB (p<0.001). Similar results were obtained in flow chamber experiments using microthrombi (p<0.01). Microbubble adherence was tested in vivo in C57/Bl6 mice: Platelet-rich thrombi were induced in carotid arteries of mice by application of ferric chloride (6%, 3 min), before microbubbles were injected and then imaged by ultrasound. Greyscale values of the thrombus area was calculated, both before and 20 min after injection. There were no differences for thrombi injected with MB or control-MB but a significant increase was noted for the LIBS-MB injected animals (p<0.001). After thrombolysis with urokinase, ultrasound imaging showed a significant reduction in thrombus size (p<0.001, see Figure). Ultrasound assessment of thrombolysis without contrast enhancing administration of LIBS-MB made the evaluation of changes in the thrombus size difficult due to the lack of contrast provided by the native thrombus. In conclusion, we demonstrate that GPIIb/IIIa-targeted microbubbles specifically bind to activated platelets in vitro and in vivo. Platelet-targeted molecular ultrasound imaging is a cost effective and widely available imaging modality that pro¬vides the unique opportunity to detect thrombi in real time and with high resolution. The translation of this technology towards imaging in patients promises early diagnosis and rapid therapy of thrombosis as well as the ability to identify success or failure of thrombolytic therapy. Figure: Ultrasound imaging of pharmacological thrombolysis in the carotid artery of mice. A) Imaging of thrombolysis with LIBS-MB provides clear images of dissolving thrombi. Thrombus size in the control group, in which saline was administrated, remained the same. B) Imaging of thrombolysis without LIBS-MB shows a lack of contrast of the thrombus and the inability to monitor success/failure of thrombolysis.
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