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Past, Present, and Future of Annexin A5: From Protein Discovery to Clinical Applications*

Hendrikus H. Boersma, Bas L.J.H. Kietselaer, Leo M.L. Stolk, Abdelkader Bennaghmouch, Leonard Hofstra, Jagat Narula, Guido A.K. Heidendal and Chris P.M. Reutelingsperger
Journal of Nuclear Medicine December 2005, 46 (12) 2035-2050;

Article Figures & Data

Figures

  • FIGURE 1.
    FIGURE 1.

    X-ray analysis revealed the tertiary structure of anxA5, as depicted here. The protein consists of 319 amino acids. Molecule is arranged in planar cyclic structure of 4 domains, which are indicated by different colors in structure. Also shown are Ca2+ ions (green spheres).

  • FIGURE 2.
    FIGURE 2.

    AnxA5 is able to bind to cells in all stages of cell death program. Shown are Fas (D95) ligand–stimulated Jurkat cells. (Top left) Early stage of cell death. Fluorescent anxA5 (green) binds to outer cell membrane. (Top right) Cell in later phase showing binding of PI (red), which is intercalated with DNA and anxA5. Binding of PI is indicative of disintegration of cell membrane. Also, formation of apoptotic bodies is visible. (Bottom) Final stage of PCD. Here, only anxA5 binding to cellular membrane residues can be observed. Also shown is cell undergoing secondary necrosis after PCD. AnxA5 uptake is still present, but cell structures are different from those of cells undergoing PCD only.

  • FIGURE 3.
    FIGURE 3.

    Transverse section of murine heart after 30 min of ischemia and 12 h of reperfusion. Cells were stained for presence of biotinylated anxA5.

  • FIGURE 4.
    FIGURE 4.

    Views of whole-body scans of male volunteer (35 y) (2 left views), male patient (53 y) (2 middle views), and male volunteer (22 y) (2 right views) taken after administration of 440 MBq of imino-anxA5, 566 MBq of BTAP-anxA5, and 263 MBq of HYNIC-anxA5, respectively. In each set of views, anterior is on left and posterior is on right. These views demonstrate low uptake of HYNIC-anxA5 in abdomen, compared with that of other 2 isotope conjugation types for anxA5. HYNIC-anxA5, however, shows more uptake in kidneys than either of other conjugation types. Adapted from (43,53).

  • FIGURE 5.
    FIGURE 5.

    (A) Methoxyisobutylisonitrile (MIBI) scintigraphy depicting area of perfusion, indicating area at risk. (B) Binding of 99mTc-anxA5 to reperfused myocardium, indicative of presence of PCD. ANT = anterior; LAT = lateral; INF = inferior; Vert LA = vertical long axis; Horz LA = horizontal long axis; RV = right ventricle; LV = left ventricle; LA = left atrium; Ao = aorta; RA = right atrium.

  • FIGURE 6.
    FIGURE 6.

    Example of 99mTc-anxA5 uptake in patient with heart failure. Dual-isotope scan shows multifocal anxA5 uptake in patient with idiopathic dilated cardiomyopathy. (Top rows) 201Tl images for orientation purposes. (Bottom rows) Corresponding anxA5 images showing multifocal uptake (arrows). SEPT = septal; LAT = lateral; INF = inferior; ANT = anterior. Adapted from (74).

  • FIGURE 7.
    FIGURE 7.

    99mTc-AnxA5 imaging in patients with atherosclerosis. (A) Transverse and coronal SPECT views from patient who had had transient ischemic attack 3 d before imaging. Although stenosis was clinically significant in both carotid arteries, uptake of anxA5 was evident only in culprit lesion (arrows). (B) Histopathologic examination of tissues from this patient shows extensive binding of anxA5 detected by rabbit antiannexin antibodies. (C) For another patient, who had had transient ischemic attack 3 mo before imaging, images do not show any uptake of anxA5. (D) Similarly, histopathologic analysis does not show significant binding of anxA5, although lesion within the carotid artery is evident. Adapted from (40).

  • FIGURE 8.
    FIGURE 8.

    (A) Thallium perfusion scintigraphy of malignant tumor showing perfused myocardium of left ventricle in short-axis orientation. (B) Increased uptake of BTAP-anxA5 within contour of left ventricle on short-axis SPECT with orientation similar to that of perfusion scintigram. ANT = anterior; SEPT = septal; LAT = lateral; INF = inferior; Vert LA = vertical long axis; Horz LA = horizontal long axis; RV = right ventricle; LV = left ventricle; LA = left atrium; Ao = aorta; RA = right atrium. Adapted from (41).

  • FIGURE 9.
    FIGURE 9.

    Malignant intracardiac sarcoma (immunohistologic analysis). (A) AnxA5 antibody staining (brown staining, arrows). (B) CM-1 antibody staining, indicating caspase 3 activation (brown staining, arrows). (C) Double staining with both CM-1 antibody (red staining) and anxA5 antibody (brown staining, arrows). Adapted from (41).

  • FIGURE 10.
    FIGURE 10.

    (A) Thallium perfusion scintigraphy of benign tumor shows perfused left ventricle of myocardium in short-axis orientation. (B) No increased uptake of BTAP-anxA5 can be seen within contour of left ventricle on short-axis SPECT with orientation similar to that of perfusion scintigram. (C) Upon histologic examination, tissue was found to have features of myxoma, with clustering of nuclei and large cellular matrices. No significant anxA5 antibody uptake and no caspase 3 activation could be demonstrated. ANT = anterior; SEPT = septal; LAT = lateral; INF = inferior; Vert LA = vertical long axis; Horz LA = horizontal long axis; RV = right ventricle; LV = left ventricle; LA = left atrium; Ao = aorta; RA = right atrium.

  • FIGURE 11.
    FIGURE 11.

    SPECT image of non–small cell lung carcinoma. Increased uptake of HYNIC-anxA5 can be seen within thorax (arrows). ANT = anterior; POST = posterior; 4 h PI = 4 h after injection of HYNIC-anxA5; PRE CHEMO = before chemotherapy.

  • FIGURE 12.
    FIGURE 12.

    Planar images of patient with sarcoma of right leg at 16 h after injection (p.i.) of BTAP-anxA5. There was increased uptake of BTAP-anxA5 in major part of upper right leg, with central zone of decreased activity. Almost no uptake was seen in left leg. Adapted from (43).

Tables

  • TABLE 1

    Features, Advantages, and Disadvantages of Isotopes and Radiopharmaceuticals

    RadiopharmaceuticalEmissions and energyPhysical half-lifeAdvantages (Pro) or Disadvantages (Con)
    99mTc-AnxA5 (general)γ, 140.5 keV6.02 hPro: 99mTc can be obtained easily at low cost by use of 99Mo/99mTc generators; excellent imaging properties
    Con: difficult radiochemistry for technetium; biodistribution influenced by conjugation method
    99mTc-Imino-anxA5γ, 140.5 keV6.02 hPro: labeling can be performed easily
    Con: RCP ≈ 80%; high uptake of radiopharmaceutical in liver, kidneys, and spleen; long biologic half-life
    99mTc-BTAP-anxA5γ, 140.5 keV6.02 hPro: RCP > 93%; short biologic half-life
    Con: labeling method very laborious; high radioactivity uptake in liver, kidneys, spleen, and abdomen; low radiochemical yield
    99mTc-HYNIC-anxA5γ, 140.5 keV6.02 hPro: labeling method well established; prefabricated kit, so labeling can be performed easily
    Con: high uptake of radiopharmaceutical in kidneys and liver; long biologic half-life
    99mTc-EC-anxA5γ, 140.5 keV6.02 hPro: prefabricated kit, so labeling can be performed easily
    Con: to establish this method, more research is required
    94mTc-AnxA5 (general)β+, 2.5 MeV53 minPro: easiest way to transform SPECT into PET; when HYNIC-anxA5 is used, labeling can be performed easily
    Con: difficult radiochemistry for technetium; biodistribution influenced by conjugation method; expensive and difficult to obtain
    123I-AnxA5γ, 160 keV13 hPro: no uptake in liver and kidneys after 12 h; good imaging expected in abdominal region, compared to that with known 99mTc compounds; good radiochemical purity
    Con: laborious labeling method; more expensive than 99mTc labeling
    124I-AnxA5β+, E = 1.53, 2.14 MeV, γ (complex decay under emission of several high-energy γ-photons)4.2 dPro: long-lived PET tracer; suitable for animal studies and studies in humans with terminal disease; labeled compound with long shelf-life (4 d without detectable deiodination); RCP > 95%
    Con: high radiation burden and therefore less suitable for patient imaging; laborious labeling method
    125I-AnxA5EC, 35 keV60 dPro: long-lived isotope; very useful for research purposes to test iodine labeling techniques in animals; low radiation burden for research workers
    Con: no imaging possibility
    111In-AnxA5γ, 173 keV, 247 keV (ratio of both energies is 1:1), electron capture gives x-ray energy of 23 keV2.8 dPro: longer-lived isotope; may be suitable for imaging of tumor response in patients
    Con: difficult radiochemistry for indium; biodistribution influenced by conjugation method; biodistribution of pegylated In-conjugated anxA5 appears to be poor; rather high radiation burden in patients
    18F-AnxA5β+, 633 keV110 minPro: PET tracer with optimal half-life for imaging; promising agent for patient imaging
    Con: more research is required to establish its use in patients; labeling method is not yet standardized
  • TABLE 2

    Summary of Studies of Clinical Imaging

    Cell death imaging targets in studies supporting clinical evidenceReference(s)
    Cardiovascular disease
        Myocardial infarction2,64
        Cardiac allograft rejection39
        Heart failure74
        Carotid atherosclerosis40
    Oncology
        Intracardiac tumors (malignant: 99mTc-anxA5 uptake; benign: so far no 99mTc-anxA5 uptake)40,79
        Head and neck tumors42
        Non-Hodgkin’s lymphoma43,85
        Recurrent follicular lymphoma86
        Leukemia87
        Non–small cell lung cancer85
        Sarcoma43
        Breast cancer43,85
        Cancer therapy: positive correlation between 99mTc-anxA5 uptake and therapy efficacy85,87
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