Molecular Detection of Venous Thrombosis in Mouse Models Using SPECT/CT
Annemiek Dickhout,
Pieter Van de Vijver,
Nicole Bitsch,
Stefan J. van Hoof,
Stella L. G. D. Thomassen,
Steffen Massberg,
Peter Timmerman,
Frank Verhaegen,
Rory R. Koenen,
Ingrid Dijkgraaf,
Tilman M. Hackeng
Affiliations
Annemiek Dickhout
Department of Biochemistry, CARIM—School for Cardiovascular Diseases, Maastricht University, 6229 ER Maastricht, The Netherlands
Pieter Van de Vijver
Department of Biochemistry, CARIM—School for Cardiovascular Diseases, Maastricht University, 6229 ER Maastricht, The Netherlands
Nicole Bitsch
Muroidean Facility, CARIM—School for Cardiovascular Diseases, Maastricht University, 6229 ER Maastricht, The Netherlands
Stefan J. van Hoof
Department of Radiation Oncology (MAASTRO), GROW—School for Oncology and Developmental Biology, Maastricht University Medical Center, 6229 ER Maastricht, The Netherlands
Stella L. G. D. Thomassen
Department of Biochemistry, CARIM—School for Cardiovascular Diseases, Maastricht University, 6229 ER Maastricht, The Netherlands
Steffen Massberg
Department of Medicine I, University Hospital, LMU Munich, 80336 Munich, Germany
Peter Timmerman
Pepscan Therapeutics B.V., 8243 RC Lelystad, The Netherlands
Frank Verhaegen
Department of Radiology and Nuclear Medicine, Maastricht University Medical Center, 6229 HX Maastricht, The Netherlands
Rory R. Koenen
Department of Biochemistry, CARIM—School for Cardiovascular Diseases, Maastricht University, 6229 ER Maastricht, The Netherlands
Ingrid Dijkgraaf
Department of Biochemistry, CARIM—School for Cardiovascular Diseases, Maastricht University, 6229 ER Maastricht, The Netherlands
Tilman M. Hackeng
Department of Biochemistry, CARIM—School for Cardiovascular Diseases, Maastricht University, 6229 ER Maastricht, The Netherlands
The efficacy of thrombolysis is inversely correlated with thrombus age. During early thrombogenesis, activated factor XIII (FXIIIa) cross-links α2-AP to fibrin to protect it from early lysis. This was exploited to develop an α2-AP-based imaging agent to detect early clot formation likely susceptible to thrombolysis treatment. In this study, this imaging probe was improved and validated using 111In SPECT/CT in a mouse thrombosis model. In vitro fluorescent- and 111In-labelled imaging probe-to-fibrin cross-linking assays were performed. Thrombus formation was induced in C57Bl/6 mice by endothelial damage (FeCl3) or by ligation (stenosis) of the infrarenal vena cava (IVC). Two or six hours post-surgery, mice were injected with 111In-DTPA-A16 and ExiTron Nano 12000, and binding of the imaging tracer to thrombi was assessed by SPECT/CT. Subsequently, ex vivo IVCs were subjected to autoradiography and histochemical analysis for platelets and fibrin. Efficient in vitro cross-linking of A16 imaging probe to fibrin was obtained. In vivo IVC thrombosis models yielded stable platelet-rich thrombi with FeCl3 and fibrin and red cell-rich thrombi with stenosis. In the stenosis model, clot formation in the vena cava corresponded with a SPECT hotspot using an A16 imaging probe as a molecular tracer. The fibrin-targeting A16 probe showed specific binding to mouse thrombi in in vitro assays and the in vivo DVT model. The use of specific and covalent fibrin-binding probes might enable the clinical non-invasive imaging of early and active thrombosis.
