ANXA5 Human

What is the molecular structure of human ANXA5 and how does it contribute to its function?

Human Annexin A5 (hAnxA5) is a 35.8 kDa protein (theoretical molecular weight of recombinant protein is 37.8 kDa) consisting of 320 amino acids. It is the smallest member of the annexin family . The protein contains four homologous domains arranged in a cyclic array, creating a slightly curved disc with a convex surface that interacts with membrane phospholipids and a concave surface facing away from the membrane.

The molecular structure of hAnxA5 enables it to bind phosphatidylserine (PS) with high affinity in a calcium-dependent manner. This binding is facilitated by calcium ions that serve as bridges between the protein and the negatively charged phospholipid headgroups. When bound to membranes, hAnxA5 does not exist as monomers but rather self-assembles into trimers and ordered arrays of trimers, forming two-dimensional crystals with either p6 or p3 symmetry . This self-assembly property is crucial for many of its biological functions, including its anticoagulant activity.

What are the primary cellular locations and expression patterns of ANXA5?

ANXA5 is abundantly expressed in almost all human cells except neurons, making it the most widely distributed annexin in the body . It is particularly abundant in placenta, where it was first discovered in 1979, as well as in endothelial cells and platelets .

Despite lacking a signal peptide and a precursor form, ANXA5 is found both intracellularly and extracellularly. Intracellularly, it is involved in membrane transport, ion channel regulation, cell cycle control, and apoptosis . Extracellularly, it is present in plasma (typically less than 5 ng/mL in healthy individuals), amniotic fluid, and cell culture medium .

The protein is released into the extracellular space through unconventional secretory pathways that are independent of the endoplasmic reticulum and Golgi apparatus. This release can occur during cell damage or apoptosis, particularly from cardiomyocytes and macrophages . Once in circulation, ANXA5 can bind to PS-exposing membranes and influence various biochemical and cellular processes.

How does ANXA5 function in hemostasis and coagulation?

ANXA5 plays a significant role in hemostasis by binding to PS exposed on cell membranes, particularly activated platelets. In the coagulation cascade, negatively charged phospholipids like PS serve as essential cofactors for the assembly of coagulation complexes . When ANXA5 binds to PS, it forms two-dimensional crystalline arrays that shield these phospholipids from participating in coagulation reactions, thereby exerting an anticoagulant effect .

The anticoagulant property of ANXA5 stems from its ability to indirectly inhibit the thromboplastin-specific complex involved in the blood coagulation cascade . By covering PS-rich surfaces, ANXA5 prevents the binding and activation of coagulation factors that require PS as a cofactor, effectively blocking both the intrinsic and extrinsic coagulation pathways.

In pathological conditions such as sickle cell disease (SCD), plasma levels of ANXA5 and PS-exposed blood cell microparticles are significantly increased . The high-affinity binding between ANXA5 and PS may prevent interactions between PS and other plasma proteins, inhibiting the adhesion of sickle red blood cells to the endothelium and regulating exposed PS-induced pathological processes .

What is the significance of ANXA5 in cardiovascular diseases?

ANXA5 demonstrates significant relevance to cardiovascular pathologies, particularly in atherosclerosis and myocardial infarction. In atherosclerosis, ANXA5 has shown protective effects by reducing vascular inflammation and improving endothelial function . Systemic injection of ANXA5 in mice with damaged blood vessels effectively reduces vascular inflammation and remodeling while improving vascular function, suggesting therapeutic potential for atherosclerosis treatment .

During myocardial infarction, cardiomyocytes undergo apoptosis and expose PS on their cell surface. Radiolabeled ANXA5 can bind to these apoptotic cardiomyocytes, allowing for imaging and quantification of cell death in the myocardium . This application has clinical value for diagnosis and assessment of therapeutic efficacy in cardiac pathologies.

Furthermore, ANXA5 plays a role in a unique endocytic pathway that exists in cardiomyocytes expressing PS due to ischemia/reperfusion stress . This pathway, driven by the 2D crystallization of ANXA5, results in membrane invagination and vesicle formation, potentially serving as a mechanism for targeted drug delivery to rescue ischemic cardiomyocytes .

How is ANXA5 involved in autoimmune disorders such as antiphospholipid syndrome?

Antiphospholipid syndrome (APS) is an autoimmune disorder characterized by thrombosis and/or pregnancy loss associated with persistent antiphospholipid antibody (aPL) positivity . ANXA5 plays a central role in the pathophysiology of APS through several mechanisms:

• The binding of ANXA5 to apical membranes of placental villi is reduced in APS patients, compromising its protective anticoagulant shield .

• Antiphospholipid antibodies accelerate coagulation partly by reducing ANXA5's anticoagulant activity . These antibodies disrupt the ordered array structure of ANXA5 on phospholipid surfaces, creating spaces for coagulation factors to bind.

• The competing mechanisms between ANXA5 and aPL antibodies for binding to phospholipids are related to topographical differences in their binding patterns. While ANXA5 self-assembles on the phospholipid surface to form a protective shield, aPL antibodies disrupt this arrangement, allowing coagulation factors like prothrombin to access the phospholipid surface .

This interplay between ANXA5 and aPL antibodies has significant implications for pregnancy complications in APS, as the disruption of ANXA5's protective effect may contribute to placental thrombosis and subsequent pregnancy loss.

How can researchers optimize ANXA5 expression and purification for laboratory studies?

For optimal expression and purification of recombinant Human ANXA5 in laboratory settings:

What are the methodological considerations for using ANXA5 as an apoptosis marker?

ANXA5 is widely used as a marker for apoptosis due to its ability to bind phosphatidylserine exposed on the outer leaflet of apoptotic cell membranes . When designing apoptosis detection experiments using ANXA5:

• Labeling Strategies: Choose appropriate labels based on experimental needs:

  • Fluorescent labels (e.g., FITC, Alexa Fluor) for flow cytometry and microscopy

  • Radioactive isotopes for in vivo imaging and quantification

  • Enzyme conjugates for biochemical assays

• Calcium Requirements: Ensure sufficient calcium concentration (typically 1-2 mM) in binding buffers, as ANXA5-PS binding is strictly calcium-dependent .

• Controls: Include appropriate controls:

  • Positive controls (cells treated with apoptosis inducers)

  • Negative controls (healthy cells)

  • Calcium-free conditions to demonstrate specificity

• Distinguishing Apoptosis from Necrosis: Combine ANXA5 with membrane impermeant nuclear dyes (e.g., propidium iodide) to distinguish early apoptotic cells (ANXA5+/PI-) from late apoptotic or necrotic cells (ANXA5+/PI+).

• Timing Considerations: ANXA5 detects PS externalization, which occurs relatively early in apoptosis. For comprehensive apoptosis analysis, consider combining with markers for other apoptotic events (e.g., caspase activation, DNA fragmentation).

• Quantification: Develop standardized protocols for quantification to ensure reproducibility and comparability across experiments.

What are the current approaches for radiolabeling ANXA5 for in vivo imaging applications?

Radiolabeled ANXA5 has emerged as a valuable tool for visualizing cell death in vivo, particularly in conditions such as myocardial infarction, atherosclerosis, and cancer . Current approaches for radiolabeling include:

• Direct Labeling Methods:

  • Iodination with 123I, 124I, or 125I through chloramine-T or Iodogen methods

  • Technetium-99m (99mTc) labeling using various chelating agents

• Indirect Labeling via Bifunctional Chelators:

  • HYNIC (hydrazinonicotinamide) conjugation for 99mTc labeling

  • DTPA (diethylenetriaminepentaacetic acid) or DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) for labeling with 111In, 67Ga, or 68Ga

• Site-Specific Labeling:

  • Engineering of ANXA5 with specific sites for conjugation away from the PS-binding region

  • Use of click chemistry for bioorthogonal conjugation

• Methodological Considerations:

  • Maintain protein functionality after labeling by avoiding modification of amino acids in the PS-binding domain

  • Optimize specific activity to achieve sufficient signal-to-noise ratio

  • Validate binding affinity of labeled ANXA5 compared to unlabeled protein

• Quality Control:

  • Radiochemical purity assessment by radio-HPLC or TLC

  • In vitro binding assays to confirm PS-binding capacity

  • Biodistribution studies to characterize pharmacokinetics

These methods have enabled the development of ANXA5 as a promising radiopharmaceutical with potential clinical applications in cardiology and oncology, allowing for non-invasive monitoring of therapeutic interventions targeting cell death pathways .

How does the self-assembly of ANXA5 on phospholipid membranes influence its biological functions?

The self-assembly of ANXA5 on phospholipid membranes is a complex process that significantly impacts its biological activities:

• Two-Dimensional Crystallization: Upon calcium-dependent binding to PS-containing membranes, ANXA5 molecules organize into trimers that further arrange into two-dimensional crystals with either p6 or p3 symmetry . The type of crystal formed depends on PS content in the membrane: p6 symmetry occurs at 5-20% PS, while p3 symmetry forms at >40% PS content .

• Membrane Remodeling: The convex shape of phospholipid-bound ANXA5 trimers induces membrane invagination. This property enables ANXA5 to bend membranes inward, resulting in vesicle formation and initiating a novel endocytic pathway . The energy required for this membrane deformation comes from the 2D crystallization process itself.

• Anticoagulant Effect: The 2D crystalline arrays of ANXA5 form a protective shield over PS-exposing surfaces, preventing access of coagulation factors to PS and thereby inhibiting coagulation reactions . This mechanism explains ANXA5's anticoagulant properties and its role in preventing inappropriate thrombosis.

• Functional Implications: Research suggests that ANXA5's biological functions are carried out by trimers or 2D arrays rather than monomers . This has important implications for experimental design, as monomeric ANXA5 may not fully recapitulate the protein's natural activity.

• Therapeutic Applications: Understanding the self-assembly process has led to novel approaches for targeted drug delivery, leveraging ANXA5's ability to induce endocytosis specifically in PS-exposing cells such as tumor cells or stressed cardiomyocytes .

The distinct p6 and p3 crystal forms and the reversible transitions between them suggest a dynamic regulation of ANXA5 function that may be tailored to specific physiological contexts and PS exposure levels.

What are the emerging applications of ANXA5 in theranostic approaches?

ANXA5 is increasingly being explored as a platform for theranostic approaches that combine diagnostic imaging with therapeutic interventions:

• Targeted Drug Delivery: The ability of ANXA5 to specifically bind PS-exposing cells and induce endocytosis makes it an attractive vehicle for delivering therapeutic payloads to diseased tissues. ANXA5 has been described as a potential biological "cruise missile" for drug targeting .

• Combined Imaging and Therapy: Modified ANXA5 molecules can carry both imaging agents and therapeutic compounds, allowing for simultaneous visualization and treatment of PS-exposing tissues. This is particularly valuable in oncology, where tumor cells often externalize PS.

• Monitoring Treatment Response: Radiolabeled ANXA5 can be used to monitor the efficacy of cell death-inducing therapies by quantifying changes in apoptosis levels before and after treatment . This provides valuable information for personalized medicine approaches.

• Cardiovascular Applications: In the context of cardiovascular disease, ANXA5-based theranostics could simultaneously visualize vulnerable atherosclerotic plaques and deliver anti-inflammatory or plaque-stabilizing agents .

• Immune Modulation: ANXA5's ability to shield PS can prevent the immunosuppressive effects of PS exposure, potentially enhancing anti-tumor immune responses when combined with immunotherapy.

These emerging applications leverage the unique biological properties of ANXA5, particularly its specific binding to PS and its ability to induce membrane invagination and endocytosis. The development of ANXA5 as a theranostic agent represents a promising frontier in personalized medicine.

How do genetic polymorphisms in the ANXA5 gene correlate with disease susceptibility?

Genetic variations in the ANXA5 gene have been associated with several pathological conditions, particularly those involving coagulation disorders and pregnancy complications:

• Promoter Polymorphisms: Several single nucleotide polymorphisms (SNPs) in the ANXA5 gene promoter region have been identified. The most studied is the M2 haplotype, characterized by four consecutive nucleotide substitutions that reduce ANXA5 expression levels.

• Association with Recurrent Pregnancy Loss: Women carrying the M2 haplotype show reduced placental ANXA5 expression, which may compromise the protein's anticoagulant function at the maternal-fetal interface. This has been associated with an increased risk of recurrent pregnancy loss and pregnancy complications.

• Antiphospholipid Syndrome: Polymorphisms affecting ANXA5 expression or function may exacerbate the effects of antiphospholipid antibodies in APS patients . Reduced ANXA5 levels due to genetic variations could impair the formation of the protective anticoagulant shield on placental villi.

• Cardiovascular Disease Risk: Some studies suggest that ANXA5 polymorphisms may influence susceptibility to atherosclerosis and thrombotic events. Given ANXA5's role in reducing vascular inflammation and improving endothelial function in atherosclerosis animal models , genetic variations affecting its function could modify cardiovascular risk.

• Methodological Considerations for Genetic Studies:

  • SNP genotyping using PCR-based methods

  • Functional studies to assess the impact of polymorphisms on protein expression and activity

  • Integration of genetic data with clinical outcomes and biomarker levels

Understanding the relationship between ANXA5 polymorphisms and disease susceptibility not only provides insights into pathophysiological mechanisms but also opens avenues for personalized risk assessment and targeted interventions.

What are the latest developments in using ANXA5 for cancer research and therapy?

ANXA5 has gained significant attention in cancer research due to its ability to detect apoptotic cells and potential applications in both diagnostics and therapeutics:

• Apoptosis Monitoring in Tumors: Radiolabeled ANXA5 serves as a valuable tool for visualizing and quantifying apoptosis in tumors, allowing for:

  • Assessment of tumor response to chemotherapy or radiotherapy

  • Prediction of treatment outcomes based on early apoptotic response

  • Non-invasive monitoring of treatment efficacy in longitudinal studies

• PS-Targeting Strategies: Leveraging ANXA5's high affinity for PS exposed on tumor cells and tumor vasculature for:

  • Development of ANXA5-drug conjugates for targeted delivery

  • Enhancement of tumor immunogenicity by blocking immunosuppressive PS signaling

  • Disruption of tumor microenvironment by targeting PS-exposed endothelial cells

• Novel Endocytic Pathway Exploitation: The unique endocytosis mechanism induced by ANXA5's 2D crystallization on PS-exposing membranes is being investigated for:

  • Selective internalization of therapeutic payloads into cancer cells

  • Bypassing conventional drug resistance mechanisms

  • Enhancing intracellular drug concentrations in target cells

• Combination with Immunotherapy: Emerging research explores combining ANXA5-based approaches with immunotherapy to:

  • Block PS-mediated immune suppression in the tumor microenvironment

  • Enhance recognition of tumor cells by immune effectors

  • Improve efficacy of checkpoint inhibitors through modulation of tumor immunogenicity

• Methodological Innovations: Technical advances include:

  • Development of multimodal imaging probes based on ANXA5

  • Engineering of ANXA5 variants with optimized pharmacokinetics

  • Creation of ANXA5 fusion proteins with enhanced therapeutic properties

These developments highlight ANXA5's versatility as both a diagnostic tool and a therapeutic platform in cancer research, with potential for clinical translation in oncology.

How can researchers address challenges in interpreting ANXA5 binding data in complex biological systems?

Interpreting ANXA5 binding data in complex biological systems presents several challenges that require careful methodological considerations:

• PS Exposure in Different Cell Death Modes:

  • PS externalization occurs in apoptosis but can also appear in other forms of cell death like necroptosis and pyroptosis

  • Use complementary markers to distinguish between different cell death modes

  • Employ kinetic studies to capture the dynamics of PS exposure in different contexts

• Calcium Dependency and Competing Ions:

  • Maintain consistent calcium concentrations across experiments

  • Consider the influence of other divalent cations that may compete with calcium

  • Account for local variations in calcium concentration in different microenvironments

• Binding Competition with Endogenous Molecules:

  • Endogenous PS-binding proteins may compete with exogenous ANXA5

  • Plasma proteins can interfere with ANXA5 binding in vivo

  • Develop strategies to account for or minimize competitive binding effects

• Quantification Challenges:

  • Establish standardized methods for quantifying ANXA5 binding

  • Use appropriate internal standards and controls

  • Develop mathematical models to correlate binding intensity with biological parameters

• Validation Approaches:

  • Implement multiple complementary techniques (flow cytometry, microscopy, radiolabeling)

  • Use ANXA5 mutants with altered binding properties as controls

  • Perform competition assays with unlabeled ANXA5 to demonstrate specificity