Activation and retention: a magnetic resonance probe for the detection of acute thrombosis

Abstract

Blood-clot formation that results in the complete occlusion of a blood vessel (thrombosis) often leads to serious life-threatening events, such as strokes and heart attacks. As the composition of a thrombus changes as it matures, new imaging methods that are capable of distinguishing new clots from old clots may yield important diagnostic and prognostic information. To address this need, an activatable magnetic resonance (MR) probe that is responsive to a key biochemical process associated with recently formed clots has been developed.

Keywords: disulfide bonds; enzymatic activation; fibrin; imaging agents; magnetic resonance imaging.

Figure 1

Mechanism of probe activation and retention at the clotting site. PDI activity expressed on the surface of activated platelets in newly formed thrombi catalyzes the conversion of compound 1 to EP-2104R, which then binds neighboring fibrin with high affinity.

Figure 2

Series of HPLC traces showing the extent of conversion of compound 1 (40 μM) to EP-2104R after 15 minutes under four different reaction conditions: untreated (no enzyme or DTT added); with PDI (4 mg/L); with DTT (40 μM); and with both PDI (4 mg/L) and DTT (40 μM).

Figure 3

Binding of probes to DD(E) (2 μM) is detected by measuring the decrease in fluorescence anisotropy that occurs upon competitive displacement of the TRITC-Tn6 probe (0.1 μM). A) EP-2104R (○) exhibits a strong dose-dependent response whereas 1 (●) does not. B) The conversion of 1 (10 μM) to EP-2104R is followed in real-time by observing the displacement of TRITC-Tn6 from DD(E). The reaction was run under four different reaction conditions: untreated (○); 1 mg/L PDI (△); 10 μM DTT (●); and 10 μM DTT plus 1 mg/L PDI (◆).

Figure 3

Binding of probes to DD(E) (2 μM) is detected by measuring the decrease in fluorescence anisotropy that occurs upon competitive displacement of the TRITC-Tn6 probe (0.1 μM). A) EP-2104R (○) exhibits a strong dose-dependent response whereas 1 (●) does not. B) The conversion of 1 (10 μM) to EP-2104R is followed in real-time by observing the displacement of TRITC-Tn6 from DD(E). The reaction was run under four different reaction conditions: untreated (○); 1 mg/L PDI (△); 10 μM DTT (●); and 10 μM DTT plus 1 mg/L PDI (◆).

Figure 4

T1-weighted MR images of phantoms 1-3 obtained at 1.5T. Solutions of 1, EP-2104R, and 2 treated with both PDI and DTT (+) are paired with solutions that were not treated (−). The probe-containing solutions of phantoms 2 and 3 were treated with thrombin and CaCl₂ to clot the fibrinogen. The inserts of phantom 3 were centrifuged to pellet the clots.

Scheme 1

Structures of the fibrin imaging probe EP-2104R and the mixed disulfide 1, which can be converted to EP-2104R through the enzymatic activity of PDI. Compound 2 is a non-binding analogue to 1 that is not a substrate for PDI and serves as a negative control.