Molecular Imaging of Factor XIIIa Activity in Thrombosis Using a Novel, Near-Infrared Fluorescent Contrast Agent That Covalently Links to Thrombi

Abstract

Background-Activated factor XIII (FXIIIa) mediates fibrinolytic resistance and is a hallmark of newly formed thrombi.In vivo imaging of FXIIIa activity could further elucidate the role of this molecule in thrombosis and other biological processes and aid in the clinical detection of acute thrombi. Methods and Results-An FXIIIa-sensitive near-infrared fluorescence imaging agent (A15) was engineered by conjugating a near-infrared fluorochrome to a peptide ligand derived from the amino terminus of ␣ 2 -antiplasmin. To evaluate the molecular specificity of A15 for FXIIIa, a control agent (C15) was also synthesized by modifying a single key glutamine residue in A15. Fluorescence imaging experiments with A15 demonstrated stronger thrombosis enhancement in human plasma clots in vitro (PϽ0.001 versus C15 clots and other controls). A15 was found to be highly specific for the active site of FXIIIa and was covalently bound to fibrin. In vivo murine experiments with A15 demonstrated significant signal enhancement in acute intravascular thrombi (PϽ0.05 versus C15 group). Minimal A15 enhancement was seen in older aged thrombi (Ͼ24 hours), consistent with an expected decline of FXIIIa activity over time. Imaging results were confirmed on correlative histopathology and fluorescence microscopy. Conclusions-A15 is a novel optical imaging agent that is specifically crosslinked to fibrin by FXIIIa, permitting detection of FXIIIa activity in experimental thrombi in vivo. This agent should permit assessment of FXIIIa activity in a broad range of biological processes and could aid in the clinical diagnosis of acute thrombi.

Development of Magnetic Resonance Imaging Contrast Material for In vivo Mapping of Tissue Transglutaminase Activity

“…A small functional peptide, seven amino acids long (NQEQVSP) from the a2 plasmin inhibitor human gene (12), serving as a substrate of transglutaminase, was linked with biotin or dansyl on the N terminus and with lysine-gadolinium diethylenetriaminepentaacetic acid (lysine-GdDTPA) on the C terminus. A similar approach was reported recently for near-IR and MRI of the activity of factor XIII (13,14). We report here detection of significant magnetic resonance and fluorescence contrast changes on transglutaminase-mediated cross-linking of TGS in three-dimensional multicellular tumor spheroids and in fibrin clots.…”

“…Thus, structural modifications might be required and should be tested in preclinical models. A similar approach was reported recently for imaging the activity of factor XIII in atherosclerotic plaques in animal models by near-IR imaging (13,14).…”

Peptides selected for binding to clotted plasma accumulate in tumor stroma and wounds

“…Thus, the CLT peptides may be useful as a general imaging or drug delivery agent for tumors, as well as for tissue injuries. Fibrin-binding peptides and antibodies have been used for imaging, but only in cardiovascular applications (19,20). Our peptides are likely to be different from these earlier reagents in that the probable target is fibronectin rather than fibrin.…”

Noninvasive Imaging of Early Venous Thrombosis by 19 F Magnetic Resonance Imaging With Targeted Perfluorocarbon Nanoemulsions

“…[15][16][17] As control, Q3 was converted to alanine (Q3A), leading to a low-affinity substrate for FXIIIa. 17 Both peptides were further functionalized with a cysteine residue at amino acid position 13 ( Figure I in the online-only Data Supplement) for coupling to the cholesterol-PEG anchor.…”