A molecular intravascular ultrasound contrast agent allows detection of activated platelets on the surface of symptomatic human plaques.

BACKGROUND AND AIMS: Activated platelets are amongst the most attractive imaging targets in atherosclerosis due to their important role in early processes of atherogenesis and thrombus formation. We developed a molecular intravascular ultrasound (IVUS) approach to detect activated platelets ex vivo on the surface of human plaques, using an IVUS system applied in clinical routine. METHODS: Human carotid endarterectomy specimens were obtained directly from the operating room and exposed to artificial arterial flow conditions for incubation with the contrast agent. This consists of microbubbles (MB), which are linked to an antibody against the ligand induced binding site (LIBS) of the activated platelet glycoprotein IIb/IIa, and a sialyl Lewis polymer (SL), which mediates binding to selectins (LIBS-SL-MB). IVUS was performed pre and post incubation with LIBS-SL-MB and after rinsing with PBS. In comparison, IVUS was performed pre and post incubation with MBs linked to an unspecific control antibody and a dysfunctional polymer (control-MB). All imaging results were correlated to histology findings. RESULTS: IVUS imaging showed a high signal enhancement after administration of LIBS-SL-MB. After rinsing with PBS, the signal enhancement remained stable. Immunofluorescence and immunohistochemistry confirmed significant binding of microbubbles to thrombi on the plaque surface. Moreover, thrombus size and number of bound MBs correlated well. CONCLUSIONS: LIBS-SL-MB allows ex vivo IVUS imaging of even small numbers of activated platelets on the surface of human carotid endarterectomy specimens. This diagnostic approach could deliver valuable additional information for risk stratification of atherosclerotic plaques, especially since we apply a clinically well-established IVUS imaging system.

An approach towards molecular imaging of activated platelets allows imaging of symptomatic human carotid plaques in a new model of a tissue flow chamber.

The development of magnetic resonance imaging (MRI) contrast agents targeting epitopes in atherosclerosis is of general interest. In particular, early detection of activated platelets as key players in plaque rupture could provide improved triage of patients. However, so far the efficiency of contrast agents targeting human pathologies can only be examined in animal experiments, which do not necessarily reflect human in vivo conditions. We therefore describe application of a contrast agent targeting activated human platelets in an MRI tissue flow chamber, allowing detection and characterization of contrast agent binding. Microparticles of iron oxide (MPIO) were conjugated to an antibody targeting ligand-induced binding sites (LIBS) on the activated platelet glycoprotein IIb/IIIa-receptor or to control antibody, resulting in LIBS-MPIO or control-MPIO contrast agent. Human endarterectomy specimens from patients with acute stroke or transient ischemic attack were imaged ex vivo before and after contrast agent perfusion using a 9.4 T MRI system. Specimens were measured under static (n = 18) or flow conditions (n = 18) in a specially designed flow chamber setup, simulating physiological conditions in a stenosed vessel. A significant MPIO-induced negative contrast was achieved in MRI by LIBS-MPIO in specimens under static and flow conditions (LIBS-MPIO vs control-MPIO: p < 0.01), and the location of LIBS-MPIO binding corresponded well between histology and MRI (p < 0.05). The number of MPIOs per platelet area on endarterectomy specimens in histology was significantly higher with LIBS-MPIO (p < 0.001). Furthermore, the intensity of contrast agent binding and signal change showed the potential to reflect the severity of clinical symptoms. LIBS-MPIO allows the detection of activated platelets on the surface of symptomatic atherosclerotic human plaques using molecular MRI. Furthermore, the MRI tissue flow chamber setup described could help to evaluate binding properties of contrast agents, and might therefore be an interesting tool for contrast agent development from animal experiments towards clinical application.