Non-invasive in vivo imaging of acute thrombosis: development of a novel factor XIIIa radiotracer

Abstract

Aims: Cardiovascular thrombosis is responsible a quarter of deaths annually worldwide. Current imaging methods for cardiovascular thrombosis focus on anatomical identification of thrombus but cannot determine thrombus age or activity. Molecular imaging techniques hold promise for identification and quantification of thrombosis in vivo. Our objective was to assess a novel optical and positron-emitting probe targeting Factor XIIIa (ENC2015) as biomarker of active thrombus formation.

Methods and results: Optical and positron-emitting ENC2015 probes were assessed ex vivo using blood drawn from human volunteers and passed through perfusion chambers containing denuded porcine aorta as a model of arterial injury. Specificity of ENC2015 was established with co-infusion of a factor XIIIa inhibitor. In vivo18F-ENC2015 biodistribution, kinetics, radiometabolism, and thrombus binding were characterized in rats. Both Cy5 and fluorine-18 labelled ENC2015 rapidly and specifically bound to thrombi. Thrombus uptake was inhibited by a factor XIIIa inhibitor. 18F-ENC2015 remained unmetabolized over 8 h when incubated in ex vivo human blood. In vivo, 42% of parent radiotracer remained in blood 60 min post-administration. Biodistribution studies demonstrated rapid clearance from tissues with elimination via the urinary system. In vivo,18F-ENC2015 uptake was markedly increased in the thrombosed carotid artery compared to the contralateral patent artery (mean standard uptake value ratio of 2.40 vs. 0.74, P < 0.0001).

Conclusion: ENC2015 rapidly and selectively binds to acute thrombus in both an ex vivo human translational model and an in vivo rodent model of arterial thrombosis. This probe holds promise for the non-invasive identification of thrombus formation in cardiovascular disease.

Keywords: Atherothrombosis; PET/CT; Positron emission tomography; Radiotracer; Thrombosis; Thrombus.

Figure 1

Cy5 and fluorine-18 labelled chemical structure of ENC2015 compound. (A) The chemical structure of NC100668 upon which ENC2015 is based.^, (B) The ENC2015 peptide with the optical Cy5 label. (C) The ENC2015 peptide after chelation with Al¹⁸F.

Figure 2

Demonstration of tracer uptake quantification in each experiment. (A and B) Regions of interest placed over the thrombus on the porcine strip in both optical and PET studies are shown, respectively. (C) A cross-section Cy5-ENC2015-labelled thrombus with a ROI placed over the media exposed to blood. The blue fluorescent stain is 4′, 6-diamidino-2-phenylindole (DAPI), a commonly used nuclear counterstain to improve immunofluorescence contrast. (D) An ¹⁸F-ENC2015 ROI placed over area of uptake within the rat left common carotid artery. ROI, region of interest

Figure 3

Binding of Cy5-ENC2015 to acutely formed human thrombus. (A–F) Histological cross-sections of the porcine aortic strips at ×20 magnification (sectioned at yellow dotted line in panel G). (G–L) Macroscopic fluorescence of the corresponding sections above is demonstrated. Note clear thrombus fluorescence in A and G with corresponding brightfield direct antibody stain to factor XIIIa (A inset). (M–P) Sequence 1 in bar charts demonstrates the tracer binding to both low and high shear thrombi and how this is specifically blocked by the administration of a factor XIII inhibitor (Sequence 2) and how it can clearly be distinguished from the autofluorescence of blood (Sequence 3). Values expressed as mean ± SEM, n = 6 (paired two-way ANOVA). Asterisk shows thrombus enhancement on histological cross-sections.

Figure 4

Binding of ¹⁸F-ENC2015 to acutely formed human thrombus. (A) Avid thrombus binding of ¹⁸F-ENC2015 on Micro-PET CT similar to optical version of ENC2015. (B) Diminished thrombus uptake of the radiotracer in the presence of factor XIIIa inhibitor. (C) An autoradiographic plate showing uptake of ¹⁸F-ENC2015 on sections (5 μm thickness) of freshly formed human thrombus. (D) Corresponding serial sections pre-treated with cold tracer demonstrating specific inhibition of binding sites. Bar charts (E + F) demonstrate ¹⁸F-ENC2015 tracer uptake in both low and high shear thrombi (Sequence 1) and how this is specifically blocked by a factor XIII inhibitor (Sequence 2) (n = 10). (G) Bar chart shows a marked and significant reduction in ¹⁸F-ENC2015 uptake in human thrombus co-incubated to unlabelled tracer (n = 3). Values expressed as mean ± SEM, (paired t-test).

Figure 5

  In vivo metabolism of ¹⁸F-ENC2015 in rat blood. (A) Graph shows the radioactive concentration of ¹⁸F-ENC2015 over time in rat whole blood and plasma (n = 2). (B) HPLC analysis in chromatogram shows a single peak of parent compound after 2 min of in vivo¹⁸F-ENC2015 circulation. (C) After 30 min, a second smaller peak ‘metabolite A’ can be appreciated on chromatogram. (D) At 60 min, chromatogram shows the radioactivity of metabolite A surpassing that of the parent compound.

Figure 6

Biodistribution and thrombus binding of ¹⁸F-ENC2015 in rats. (A) Images of the time-averaged uptake within the thrombus and skin incision compared with the contralateral vessel. (B) A decay corrected time activity curve showing ¹⁸F-ENC2015 kinetics (mean SUV, n = 3) within our in vivo thrombosis model. Note the rapid blood pool clearance of ¹⁸F-ENC2015 with early uptake in the skin incision which tails off to equilibrate similarly to the arterial thrombus between 60 and 90 min (pseudo-equilibrium). (C) The mean change in SUV ratio of the thrombosed vessel compared with contralateral vessel and LV blood pool over time (n = 3) was demonstrated. Thrombus vs. contralateral vessel SUV from 35 to 180 min P-value < 0.0001 (paired t-test).