In Vivo Ultrasound Molecular Imaging of Inflammatory Thrombosis in Arteries With Cyclic Arg-Gly-Asp–Modified Microbubbles Targeted to Glycoprotein IIb/IIIa

Abstract

Ultrasound molecular imaging has the potential to detect activated platelets, thus identifying atherosclerotic plaque instability before onset of serious clinical events. However, it has not been well defined in inflammatory arterial thrombosis. We hypothesized that microbubbles (MBs) target glycoprotein IIb/IIIa (GP IIb/IIIa) could achieve a noninvasive in vivo detection of inflammatory thrombosis in large arteries through contrast-enhanced ultrasound (CEU) imaging. Lipid shell-based gas-filled MBs were modified covalently with a cyclic Arg-Gly-Asp (RGD) peptide (MB-cRGD) targeted to activated GP IIb/IIIa or a negative control peptide (MB-CON) via thiol-maleimide coupling. Adherence of MB-cRGD and MB-CON to GP IIb/IIIa was determined in vitro by using a parallel plate flow chamber at variable shear stress (0.5-8 dynes/cm2). Inflammatory platelet thrombosis was induced by periadvential application of arachidonic acid (AA) to one of the bilateral carotids of C57BL/6 mice (n = 20) and confirmed through intravital fluorescence microscopy. Attachment of MBs was determined in vivo with CEU imaging of bilateral carotids in the AA application mice with (n = 10) or without (n = 10) pretreatment of GP IIb/IIIa antagonist. The expression of integrin GP IIb/IIIa was assessed through immunohistochemistry. Microbubble-cRGD but not MB-CON had excellent affinity to GP IIb/IIIa under all shear stress conditions. Successful inflammatory platelet activation and thrombosis in AA application carotids were noted through intravital fluorescence microscopy. Contrast video intensity from adhered MB-cRGD in the thrombi was significantly higher than that from MB-CON (P < 0.05). Video intensity of MB-cRGD in the thrombi was suppressed significantly by preblocking with GP IIb/IIIa antagonist (P < 0.05) but not for MB-CON. Immunohistochemical finding demonstrates that expression of integrin GP IIb/IIIa in the thrombi was abundant; it was inhibited significantly through pretreatment with GP IIb/IIIa antagonist (P < 0.05). Cyclic RGD-modified MBs targeted to GP IIb/IIIa with CEU are capable of detecting inflammation-activated platelets and thrombosis in large arteries, thus providing a potential tool for identification of vulnerable atherosclerotic plaques.