Evaluation of annexin V as a platelet-directed thrombus targeting agent

doi:10.1016/0049-3848(94)90224-0

Thrombosis Research

Volume 75, Issue 5, 1 September 1994, Pages 491-501

https://doi.org/10.1016/0049-3848(94)90224-0 Get rights and content

Abstract

Annexin V is a human phospholipid binding protein (M_r 36,000) that binds with high affinity to activated platelets in vitro. We studied the biodistribution and thrombus binding of annexin V in rabbit and swine models of fully occlusive arterial thrombi formed 1–2 h prior to injection of annexin V. Iodinated annexin V was cleared from blood in a rapid early phase ( $t_{12} = 6.4$ min, 76% of radioactivity) and a slower late phase ( $t_{12} = 71$ min, 24% of radioactivity). Organ uptake was highest in the kidney and spleen and lowest in heart and skeletal muscle. Thrombus/blood uptake ratios were (mean ± SEM): 6.39 ± 1.80 for rabbit iliac artery, 6.97 ± 1.45 for swine carotid artery, and 7.68 ± 1.70 for swine femoral artery (all p values < 0.01 versus control artery); a control protein, ovalbumin, showed an uptake ratio of 0.59 ± 0.08 in swine femoral artery thrombi. These results indicate that annexin V is useful as an agent for selective targeting of platelet-containing thrombi.

References (18)

J. Tait et al.
Phospholipid binding of annexin V: effects of calcium and membrane phosphatidyl serine content
Arch. Biochem. Biophys.
(1992)
P. Thiagarajan et al.
Binding of annexin V/placental anticoagulant protein I to platelets
P. Thiagarajan et al.
Collagen-induced exposure of anionic phospholipid in platelets and platelet-derived microparticles
J. Biol. Chem.
(1991)
J. Dachary-Prigent et al.
Annexin V as a probe of aminophospholipid exposure and platelet membrane vesiculation: a flow cytometry study showing a role for free sulfhydryl groups
Blood
(1993)
J. Romisch et al.
In vivo antithrombotic potency of placenta protein 4 (annexin V)
Thromb. Res.
(1991)
P. Fraker et al.
Protein and cell membrane iodinations with a sparingly soluble chloroamide, 1, 3, 4, 6-tetrachloro-3a, 6a- diphenylglycoluril
Biochem. Biophys. Res. Commun.
(1978)
K. Kojima et al.
Carbohydrate-binding proteins in bovine kidney have consensus amino acid sequences of annexin family proteins
J. Biol. Chem.
(1992)
J. Romisch et al.
Annexins: calcium-binding proteins of multi-functional importance?
Med. Microbiol. Immunol.
(1991)
C. Creutz
The annexins and exocytosis
Science
(1992)

There are more references available in the full text version of this article.

Cited by (70)

Nanomedicine progress in thrombolytic therapy
2020, Biomaterials
Citation Excerpt :
Two years earlier, the same team published a work similarly dealing with LM-adsorbed micelles from triblock polymer – polycaprolactone-block-poly(2-(dimethylamino) ethyl methacrylate)-block-poly(2-hydroxyethyl methacrylate) (PCL-PDMAEMA-PHEMA) [110]. In a less common targeting strategy, Annexin V, a human phospholipid-binding protein that binds phosphatidylserine on the surface of activated platelets in a calcium-dependent manner and is proposed to play a role in the inhibition of blood coagulation [111], was conjugated to the micelles to target thrombi in a murine FeCl3-induced model and perform the in vivo thrombolytic activity. Poly(2-oxazoline) (POx).
Thrombotic occlusions of blood vessels are responsible for life-threatening cardiovascular disorders such as myocardial infarction, ischemic stroke, and venous thromboembolism. Current thrombolytic therapy, the injection of Plasminogen Activators (PA), is yet limited by a narrow therapeutic window, rapid drug elimination, and risks of hemorrhagic complications. Nanomedicine-based vectorization of PA protects the drug from the enzymatic degradation, improves the therapeutic outcomes, and diminishes adverse effects in preclinical models. Herein, we review the pathophysiology of arterial and venous thrombosis and summarize clinically approved PA for the treatment of acute thrombotic diseases. We examine current challenges and perspectives in the recent key research on various (lipid, polymeric, inorganic, biological) targeted nanocarriers intended for the site-specific delivery of PA. Microbubbles and ultrasound-assisted sonothrombolysis that demonstrate thrombolysis enhancement in clinical trials are further discussed. Moreover, this review features strategies for the rational design of nanocarriers for targeted thrombolysis and effective PA encapsulation in view of interactions between nanomaterials and biological systems. Overall, nanomedicine represents a valued approach for the precise treatment of acute thrombotic pathologies.
An α<inf>IIb</inf>β<inf>3</inf>- And phosphatidylserine (PS)-binding recombinant fusion protein promotes PS-dependent anticoagulation and integrin-dependent antithrombosis
2019, Journal of Biological Chemistry
Citation Excerpt :
Cell surface–expressed PS is potentially an attractive target for TDD, especially for the prevention and treatment of platelet-derived thrombosis. ANV is a human protein that binds with high affinity and specificity to the abundant PS molecules exposed on activated platelets and accumulates selectively in thrombi after intravenous administration in animal models of arterial thrombosis (28). ANV is often used as a guiding molecule for targeting PS to construct potential recombinant protein drug candidates for the treatment of thrombotic diseases.
Blood platelets are required for normal wound healing, but they are also involved in thrombotic diseases, which are usually managed with anticoagulant drugs. Here, using genetic engineering, we coupled the disintegrin protein echistatin, which specifically binds to the platelet integrin α_IIbβ₃ receptor, to annexin V, which binds platelet membrane-associated phosphatidylserine (PS), to create the bifunctional antithrombotic molecule recombinant echistatin–annexin V fusion protein (r-EchAV). Lipid binding and plasma coagulation studies revealed that r-EchAV dose-dependently binds PS and delays plasma clotting time. Moreover, r-EchAV inhibited ADP-induced platelet aggregation in a dose-dependent manner and exhibited potent antiplatelet aggregation effects. r-EchAV significantly prolonged activated partial thromboplastin time, suggesting that it primarily affects the in vivo coagulation pathway. Flow cytometry results indicated that r-EchAV could effectively bind to the platelet α_IIbβ₃ receptor, indicating that r-EchAV retains echistatin’s receptor-recognition region. In vivo experiments in mice disclosed that r-EchAV significantly prolongs bleeding time, indicating a significant anticoagulant effect in vivo resulting from the joint binding of r-EchAV to both PS and the α_IIbβ₃ receptor. We also report optimization of the r-EchAV production steps and its purification for high purity and yield. Our findings indicate that r-EchAV retains the active structural regions of echistatin and annexin V and that the whole molecule exhibits multitarget-binding ability arising from the dual functions of echistatin and annexin V. Therefore, r-EchAV represents a new class of anticoagulant that specifically targets the anionic membrane-associated coagulation enzyme complexes at thrombogenesis sites and may be a potentially useful antithrombotic agent.
Multiplexed invivo fluorescence optical imaging of the therapeutic efficacy of photodynamic therapy
2013, Biomaterials
Citation Excerpt :
It also implies a rapid blood clearance. The half-life of annexin V after intravenous injection is known to be less than 7 min [23,39,40]. Due to the high affinity (KD = 20 nm) and high specificity (reduction of fluorescence around 89% in a blocking experiment), our DY-734-annexin V probe was still able to efficiently and specifically detect the therapeutic efficacy of PDT at 2 days after treatment as our in vivo imaging data show.
In our study we wanted to elucidate a time frame for in vivo optical imaging of the therapeutic efficacy of photodynamic therapy (PDT) by using a multiplexed imaging approach for detecting apoptosis and vascularization. The internalization of the photosensitizer Foslip^® into tongue-squamous epithelium carcinoma cells (CAL-27) was examined in vitro and in vivo. For detecting apoptosis, annexin V was covalently coupled to the near-infrared dye DY-734 and the spectroscopic properties and binding affinity to apoptotic CAL-27 cells were elucidated. CAL-27 tumor bearing mice were treated with PDT and injected 2 days and 2 weeks thereafter with DY-734-annexin V. PDT-induced changes in tumor vascularization were detected with the contrast agent IRDye^® 800CW RGD up to 3 weeks after PDT. A perinuclear enrichment of Foslip^® could be seen in vitro which was reflected in an accumulation in CAL-27 tumors in vivo. The DY-734-annexin V (coupling efficiency 30–50%) revealed a high binding affinity to apoptotic compared to non-apoptotic cells (17.2% vs. 1.2%) with a K_D-value of 20 nm. After PDT-treatment, the probe showed a significantly higher (p <0.05) contrast in tumors at 2 days compared to 2 weeks after therapy (2–8 h post injection). A reduction of the vascularization could be detected after PDT especially in the central tumor areas. To detect the therapeutic efficacy of PDT, a multiplexed imaging approach is necessary. A detection of apoptotic cells is possible just shortly after therapy, whereas at later time points the efficacy can be verified by investigating the vascularization.
In vitro and in vivo evaluation of [<sup>99m</sup>Tc]-labeled tricarbonyl His-annexin A5 as an imaging agent for the detection of phosphatidylserine-expressing cells
2010, Nuclear Medicine and Biology
Apoptosis is one of the mechanisms behind successful chemotherapy and radiation treatment. Radiolabeled annexin A5 has been demonstrated to be a successful tool in the detection of apoptosis following chemotherapy in vivo.
His-tagged annexin A5 was labeled with [^99mTc]-tricarbonyl and evaluated as apoptosis imaging radiotracer in vitro and in vivo. The binding of the radiotracer was evaluated in Colo205 cells stimulated with 5-FU (1 mM) for 4 and 24 h, and confirmed by flow cytometry. Biodistribution and dosimetric studies were performed in healthy nude mice (n=5) via planar scintigraphy. [^99mTc]-(CO)₃ His-annexin A5 was also evaluated for in vivo imaging of spontaneous apoptosis in Colo205-bearing mice (n=12).
The labeling procedure yielded a compound with 95-99% radiochemical purity and good in vitro stability. In vitro binding experiments indicated that the radiotracer retained its PS-binding activity. [^99mTc]-(CO)₃ His-annexin A5 rapidly cleared from the blood and predominantly accumulated in the kidneys. Absorbed dose (per organ) was found to be 116±64 μGy/MBq for the kidneys and 10.38±0.50 μGy/MBq for the liver. The effective dose was 7.00±0.28 μSv/MBq. Spontaneous apoptosis in Colo205-bearing mice was visualised by [^99mTc]-(CO)₃ His-annexin A5 SPECT and correlated well with caspase-3 immunostaining (R=0.867, P<.01).
[^99mTc]-(CO)₃ His-annexin A5 may be a useful novel radioligand for the in vivo detection of cell death associated with PS expression. A simple, noninvasive way of detecting apoptosis in vivo could have many applications including a better understanding of the extent and timing of apoptosis in response to cancer therapies and assessment of early tumor response.
Functional Imaging of Atherosclerosis to Advance Vascular Biology
2009, European Journal of Vascular and Endovascular Surgery
Citation Excerpt :
Annexin V specifically binds, with nanomolar affinity, to phosphatidyl serine (PS), which is exposed on the surface of activated platelets35 and apoptotic cells.36 Therefore, radiolabelled 99mTc-annexin-V has been used for in vivo scintigraphic imaging of both apoptotic cells in animals and humans37 and acute or chronic platelet-rich thrombi in animals.38,39 In a recent study, we have shown the ability of 99mTc-annexin-V to assess the renewal activity of chronic aseptic intra-luminal thrombi, at the interface between circulating blood and thrombus, in an in vivo experimental model of AAA, and ex vivo in human ILT.37
Preliminary events leading to the rupture of atherosclerotic plaques or aneurysmal wall expansion undoubtedly are linked to altered and increased metabolism of cells in the vascular wall. To allow in vivo identification of this local activity, imaging techniques such as positron emission tomography (PET) and contrast ultrasonography may be used. However, the use of complementary multimodal imaging methods, such as computed tomography (CT), magnetic resonance imaging (MRI), single photon emission computed tomography (SPECT), etc., can inform about other processes, including vascular wall calcification, haemosiderin deposits, apoptosis and accumulation of activated platelets in the arterial wall. Such techniques may be used as an adjunct in following the evolution of the disease, as well as having crucial roles as molecular and cellular probes of arterial disease. Therefore, functional imaging techniques may be able to help us take more reliable decisions on the need for medical or surgical treatment of arterial disease.
Molecular Imaging and Targeted Therapy: Radiopharmaceuticals and Clinical Applications, Second Edition
2023, Molecular Imaging and Targeted Therapy: Radiopharmaceuticals and Clinical Applications, Second Edition

View all citing articles on Scopus

View full text

PaperEvaluation of annexin V as a platelet-directed thrombus targeting agent

Abstract

Arch. Biochem. Biophys.

J. Biol. Chem.

Blood

Thromb. Res.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Annexins: calcium-binding proteins of multi-functional importance?

Med. Microbiol. Immunol.

The annexins and exocytosis

Science

Paper
Evaluation of annexin V as a platelet-directed thrombus targeting agent