Visualization of macro-immune complexes in the antiphospholipid syndrome by multi-modal microscopy imaging.

The antiphospholipid syndrome (APS) is an autoimmune thrombotic condition that is marked by autoantibodies against phospholipid-binding proteins. The mechanism(s) of thrombogenesis has (have) resisted elucidation since its description over thirty years ago. Nevertheless, a defining aspect of the disorder is positivity for clinical laboratory tests that confirm antibody binding to anionic phospholipids.

Atomic force microscopy: High resolution dynamic imaging of cellular and molecular structure in health and disease.

The atomic force microscope (AFM), invented in 1986, and a member of the scanning probe family of microscopes, offers the unprecedented ability to image biological samples unfixed and in a hydrated environment at high resolution. This opens the possibility to investigate biological mechanisms temporally in a heretofore unattainable resolution. We have used AFM to investigate: (1) fundamental issues in cell biology (secretion) and, (2) the pathological basis of a human thrombotic disease, the antiphospholipid syndrome (APS).

Viewing dynamic interactions of proteins and a model lipid membrane with atomic force microscopy.

The information covered in this chapter will present a model homogenous membrane preparation technique and dynamic imaging procedure that can be successfully applied to more than one type of lipid study and atomic force microscope (AFM) instrument setup. The basic procedural steps have been used with an Asylum Research MFP-3D BIO and the Bruker (formerly, Veeco) BioScope. The AFM imaging protocol has been supplemented by procedures (not to be presented in this chapter) of ellipsometry, standardized western blotting, and dot-blots to verify appropriate purity and activity of all experimental molecular components; excellent purity and activity level of the lipids, proteins, and drug(s) greatly influence the success of imaging experiments in the scanning probe microscopy field.

Insights into the pathophysiology of the antiphospholipid syndrome provided by atomic force microscopy.

The antiphospholipid syndrome (APS) is an enigmatic autoimmune disorder in which patients present with thrombosis and/or recurrent pregnancy losses together with laboratory evidence for the presence of autoantibodies in the blood that recognize proteins that bind to anionic phospholipids - the most important of which is β(2)-glycoprotein I (β(2)GPI). Earlier, we hypothesized that the clinical manifestations arise from antibody-induced disruption of a two-dimensional anticoagulant crystal shield, composed of annexin A5, present on placental trophoblast plasma membranes. Accordingly, we reasoned that a high resolution imaging technology, such as atomic force microscopy could be used to investigate such molecular interactions at high resolution in a non-fixed hydrated environment.

Hydroxychloroquine protects the annexin A5 anticoagulant shield from disruption by antiphospholipid antibodies: evidence for a novel effect for an old antimalarial drug.

Annexin A5 (AnxA5) is a potent anticoagulant protein that crystallizes over phospholipid bilayers (PLBs), blocking their availability for coagulation reactions. Antiphospholipid antibodies disrupt AnxA5 binding, thereby accelerating coagulation reactions. This disruption may contribute to thrombosis and miscarriages in the antiphospholipid syndrome (APS).

Hydroxychloroquine directly reduces the binding of antiphospholipid antibody-beta2-glycoprotein I complexes to phospholipid bilayers.

Treatment with the antimalarial drug hydroxychloroquine (HCQ) has been associated with reduced risk of thrombosis in the antiphospholipid (aPL) syndrome (APS) and, in an animal model of APS, with reduction of experimentally induced thrombosis. Recognition of beta2-glycoprotein I (beta2GPI) by aPL antibodies appears to play a major role in the disease process. We therefore used the techniques of ellipsometry and atomic force microscopy (AFM) to investigate whether HCQ directly affects the formation of aPL IgG-beta2GPI complexes on phospholipid bilayers.

Imaging aspects of cardiovascular disease at the cell and molecular level.

Cell and molecular imaging has a long and distinguished history. Erythrocytes were visualized microscopically by van Leeuwenhoek in 1674, and microscope technology has evolved mightily since the first single-lens instruments, and now incorporates many types that do not use photons of light for image formation. The combination of these instruments with preparations stained with histochemical and immunohistochemical markers has revolutionized imaging by allowing the biochemical identification of components at subcellular resolution.

Energy-dependent disassembly of self-assembled SNARE complex: observation at nanometer resolution using atomic force microscopy.

Full-length v-SNARE protein reconstituted in lipid vesicles, when exposed to t-SNARE-reconstituted lipid membrane, results in the self-assembly of a t-/v-SNARE complex in a ring pattern, forming pores and the establishment of continuity between the opposing bilayers. In contrast, when v-SNARE protein alone (without liposomes) is exposed to t-SNARE-reconstituted lipid membrane, they also self-assemble to form t-/v-SNARE complexes, although such complexes fail to possess the characteristic ring pattern, nor do they help in the establishment of continuity between the opposing bilayers. Hence, t-SNAREs and v-SNARE need to be membrane-associated to interact in a circular array to form conducting pores in the presence of calcium.

Human monoclonal antiphospholipid antibodies disrupt the annexin A5 anticoagulant crystal shield on phospholipid bilayers: evidence from atomic force microscopy and functional assay.

The antiphospholipid (aPL) syndrome is an autoimmune condition that is marked by recurrent pregnancy losses and/or systemic vascular thrombosis in patients who have antibodies against phospholipid/co-factor complexes. The mechanism(s) for pregnancy losses and thrombosis in this condition is (are) not known. Annexin A5 is a potent anticoagulant protein, expressed by placental trophoblasts and endothelial cells, that crystallizes over anionic phospholipids, shielding them from availability for coagulation reactions.

Structure and dynamics of the fusion pore in live cells.

Atomic force microscopy reveal pit-like structures typically containing three or four, approximately 150 nm in diameter depressions at the apical plasma membrane in live pancreatic acinar cells. Stimulation of secretion causes these depressions to dilate and return to their resting size following completion of the process. Exposure of acinar cells to cytochalasin B results in decreased depression size and a loss in stimulable secretion.