Background

[PubMed]

Thrombosis plays a major role in many cardiovascular diseases such as myocardial infarction, pulmonary embolism, deep venous thrombosis or cerebral venous thrombosis (CVT) (1). Unfortunately, the clinical diagnosis of CVT is complex, as most of its symptoms are non-specific and the well-established modalities such as conventional angiography, CT or MR venography, do not offer readily available information on chronicity or activity without serial anatomic testing over time (2).

Thrombosis occurs by an activation process of thrombin, which then converts fibrinogen into fibrin. Thrombin initiates the crosslinking of the polymerized fibrin via the activation of a transaminase called coagulation factor XIII (FXIIIa = activated factor XIII) (3). FXIIIa augments acute thrombus stability by rapidly crosslinking fibrin α- and γ-chains, and by covalently binding to alpha-2 antiplasmin (α2AP) (4) FXIIIa has a catalytic half-life of ~ 20 min, in vivo (5). Optical imaging with a FXIIIa probe may be a useful tool in the diagnostic of thrombosis (including CVT) and may provide valuable information currently unavailable through anatomically-based imaging modalities (6, 7).

A15 is a novel near-infrared fluorescence (NIRF) probe recognized by the enzyme FXIIIa and that covalently binds to fibrin by means of the transglutamase activity of FXIIIa. Preliminary research has shown that A15 is capable of testing the activity of FXIIIa and visualizing thrombus in vivo (8, 9). A15 may find applications as a molecular diagnostic tool for therapy with fibrinolytic or anti-FXIIIa agents, direct or indirect inhibitors, and other drugs for diseases such as CVT, ischemic stroke or myocardial infarction. Additional and more extensive research studies are needed to fully assess the potential and possible clinical benefits of the A15-NIRF imaging modality (9).

Synthesis

[PubMed]

A15 can be prepared by solid-state peptide synthesis, as described by Tung et al. (10). Briefly, an acetyl glycine group is added to the N terminus of the α2AP fragment for capping, and a tryptophan residue and a cysteine residue are then added to the C terminus for quantitation and conjugation purposes. A15 is then labeled with a sulfhydryl reactive fluorochrome (Alexa Fluor 680C2 maleimide, AF680) via the C-terminal cysteine side chain (in sodium acetate buffer, at pH 6.5). After purification by HPLC, the final A15 product shows an excitation wavelength of 679 nM and an emission wavelength of 702 nm (9).

In addition to synthesizing A15, Tung et al. (10) and Jaffer et al (8). also prepared a control probe CA15 by replacing the glutamine residue at position 14 with an alanine residue. The fluorescent signal obtained with CA15 was less than 3 fold higher than the background signal, compared with a 11 fold enhancement for A15.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Tung et al. (10) performed in vitro assays using both A15 and CA15 probes (1µM) to assess the importance of the glutamine residue at position 14 in the crosslinking reaction with FXIII. The experimental protocol involved using mouse blood clots that were homogenized, washed in phosphate buffered saline and incubated with either A15 or CA15. Subsequent imaging of the clots (imaging system with a 630 + 15 nm excitation and a 700 + 20 nm emission) revealed the critical role of the glutamine residue. An intense fluorescence signal (fluorescence intensity ~ 760 AU, n = 3 clots tested) was obtained for the clot incubated with A15 (11 fold higher than the background of the blood), whereas a much weaker signal (fluorescence intensity ~ 200 AU, n = 3) for the clot incubated with the control probe CA15 (less than 3 fold higher than background).

Using human plasma clots, Jaffer et al. (8) investigated the ability of A15 to be crosslinked by human FXIIIa. The experimental protocol involved creating clots by mixing 90 µl of fresh frozen plasma with 3 µl of A15, CA15 (control probe), AF680 (free NIRF fluorochrome) or normal saline. All imaging agents were used at a concentration of 100 µmol/l, based on AF680. After incubation of the plasma clots with the individual imaging agents (90 min, 37°C), NIRF imaging was performed with each group (11). The collected data showed that in the A15 group, thrombi had a 347-fold NIRF signal increase compared with the saline (1562 ± 193.8 arbitrary units (AU) for A15 and 4.5 ± 0.6 AU for saline, P<0.001 A15 vs. each other group) and a 4.8 to 6.3-fold increase over the other control groups (247.5 ± 65.6 AU for CA15 and 328.0 ± 72.9 AU). Pre-incubation of FFP thrombi with IAA, a FXIII inhibitor, substantially reduced the A15 thrombus enhancement by 92.9% (P<0.001), supporting the fact that A15 is ‘FXIIIa-specific’.

The authors also showed that A15 was crosslinked to fibrin by FXIIIa, by performing a Western blot protein analysis (5) that used denaturated clots subjected to immunoblotting, and a polyclonal antibody that bound to the N-terminal peptide of the α₂AP factor forming the backbone of A15. Clots formed with A15 (mass of A15 < 3kDa) showed a band at ~70 kDa that appeared strongly immunoreactive with the antibody to the N- terminus of α₂AP. (No band was detected with CA15).

Animal Studies

Human Studies

[PubMed]

No publication is currently available

References

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