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Molecular MRI of Early Thrombus Formation Using a Bimodal ₂-Antiplasmin–Based Contrast Agent

Robbert-Jan J.H.M. Miserus, MSc^_*,, M. Veronica Herías, PhD, Lenneke Prinzen, MSc^,, Marc B.I. Lobbes, MD^_*, Robert-Jan Van Suylen, MD, PhD, Anouk Dirksen, PhD^,||, Tilman M. Hackeng, PhD^,||, Johan W.M. Heemskerk, PhD^,||, Jos M.A. van Engelshoven, MD, PhD^_*,, Mat J.A.P. Daemen, MD, PhD^,, Marc A.M.J. van Zandvoort, PhD^,, Sylvia Heeneman, PhD^,, Marianne Eline Kooi, PhD^_*,,_*

^_* Department of Radiology, Maastricht University Medical Centre, Maastricht, the Netherlands
Cardiovascular Research Institute Maastricht (CARIM), Maastricht University Medical Centre, Maastricht, the Netherlands
Department of Pathology, Maastricht University Medical Centre, Maastricht, the Netherlands
Department of Biomedical Engineering, Maastricht University Medical Centre, Maastricht, the Netherlands
^|| Department of Biochemistry, Maastricht University Medical Centre, Maastricht, the Netherlands

^_* Reprint requests and correspondence: Dr. Marianne E. Kooi, Cardiovascular Research Institute Maastricht (CARIM), Department of Radiology, Maastricht University Medical Centre, P.O. Box 5800, 6202 AZ Maastricht, the Netherlands (Email: eline.kooi{at}mumc.nl).

Objectives: We aimed to investigate whether early thrombus formation can be visualized with in vivo magnetic resonance imaging (MRI) by the use of a novel bimodal ₂-antiplasmin–based contrast agent (CA).

Background: Thrombus formation plays a central role in several vascular diseases. During the early phases of thrombus formation, activated factor XIII (FXIIIa) covalently cross-links ₂-antiplasmin to fibrin, indicating the potential of ₂-antiplasmin–based CAs in the detection of early thrombus formation.

Methods: A bimodal CA was synthesized by coupling gadolinium-diethylene triamine pentaacetic acid and rhodamine to an ₂-antiplasmin–based peptide. For the control CA, a glutamine residue essential for cross-linking was replaced by alanine. In vitro-generated thrombi were exposed to both CAs and imaged by MRI and 2-photon laser-scanning microscopy. Immunohistochemistry was performed on human pulmonary thromboemboli sections to determine the presence of ₂-antiplasmin and FXIII in different thrombus remodeling phases. In vivo feasibility of the CA in detecting early thrombus formation specifically was investigated with MRI.

Results: In vitro–generated thrombi exposed to the ₂-antiplasmin–based CA showed hyperintense magnetic resonance signal intensities at the thrombus edge. No hyperintense signal was observed when we used the ₂-antiplasmin–based CA in the presence of FXIII inhibitor dansylcadaverine nor when we used the control CA. Two-photon laser-scanning microscopy demonstrated that the ₂-antiplasmin–based CA bound to fibrin. Immunohistochemistry demonstrated substantial ₂-antiplasmin staining in fresh compared with lytic and organized thrombi. The administration of CA in vivo within seconds after inducing thrombus formation increased contrast-to-noise ratios (CNRs 2.28 ± 0.39, n=6) at the site of thrombus formation compared with the control CA (CNRs –0.14 ± 0.55, p = 0.003, n = 6) and ₂-antiplasmin–based CA administration 24 to 48 h after thrombus formation (CNRs 0.11 ± 0.23, p = 0.006, n = 6).

Conclusions: A bimodal CA was developed, characterized, and validated. Our results showed that this bimodal CA enabled noninvasive in vivo magnetic resonance visualization of early thrombus formation.

Key Words: thrombosis • magnetic resonance imaging • contrast media

Abbreviations and Acronyms   2-AP = 2-antiplasmin   Bi-2AP-CA = specific bimodal 2-antiplasmin–based contrast agent   Bi-con-CA = bimodal control contrast agent   CA = contrast agent   CNR = contrast-to-noise ratio   DTPA = diethylene triamine pentaacetic acid   FXIIIa = activated factor XIII   MALDI-MS = matrix-assisted laser desorption/ionization mass spectrometry   MRI = magnetic resonance imaging   NSA = number of signal averages   PBS = phosphate-buffered saline   TE = echo time   TI = inversion time   TPLSM = 2-photon laser-scanning microscopy   TR = repetition time