Anthony S. Quinn

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. We previously presented data supporting the hypothesis that aPL antibody-mediated disruption of the anticoagulant annexin A5 shield could be a thrombogenic mechanism in the aPL syndrome. However, this has remained a subject of controversy. We therefore used atomic force microscopy, a method previously used to study the crystallization of annexin A5, to image the effects of monoclonal human aPL antibodies on the crystal structure of the protein over phospholipid bilayers. In the presence of the aPL monoclonal antibodies (mAbs) and beta(2)-GPI, the major aPL co-factor, structures presumed to be aPL mAb-antigen complexes were associated with varying degrees of disruption to the annexin A5 crystallization pattern over the bilayer. In addition, measurements of prothrombinase activity on the phospholipid bilayers showed that the aPL mAbs reduced the anti-coagulant effect of annexin A5 and promoted thrombin generation. These data provide morphological evidence that support the hypothesis that aPL antibodies can disrupt annexin A5 binding to phospholipid membranes and permit increased generation of thrombin. The aPL antibody-mediated disruption of the annexin A5 anticoagulant shield may be an important prothrombotic mechanism in the aPL syndrome.

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). We investigated whether the antimalarial drug, hydroxychloroquine (HCQ), might affect this prothrombotic mechanism. Binding of AnxA5 to PLBs was measured with labeled AnxA5 and also imaged with atomic force microscopy. Immunoglobulin G levels, AnxA5, and plasma coagulation times were measured on cultured human umbilical vein endothelial cells and a syncytialized trophoblast cell line. AnxA5 anticoagulant activities of APS patient plasmas were also determined. HCQ reversed the effect of antiphospholipid antibodies on AnxA5 and restored AnxA5 binding to PLBs, an effect corroborated by atomic force microscopy. Similar reversals of antiphospholipid-induced abnormalities were measured on the surfaces of human umbilical vein endothelial cells and syncytialized trophoblast cell lines, wherein HCQ reduced the binding of antiphospholipid antibodies, increased cell-surface AnxA5 concentrations, and prolonged plasma coagulation to control levels. In addition, HCQ increased the AnxA5 anticoagulant activities of APS patient plasmas. In conclusion, HCQ reversed antiphospholipid-mediated disruptions of AnxA5 on PLBs and cultured cells, and in APS patient plasmas. These results support the concept of novel therapeutic approaches that address specific APS disease mechanisms.

The annexin A5-mediated pathogenic mechanism in the antiphospholipid syndrome: role in pregnancy losses and thrombosis.

Annexin A5 (AnxA5) binds to phospholipid bilayers, forming two-dimensional crystals that block the phospholipids from availability for coagulation enzyme reactions. Antiphospholipid (aPL) antibodies cause gaps in the ordered crystallization of AnxA5 which expose phospholipids and thereby accelerate blood coagulation reactions. The aPL antibody-mediated disruption of AnxA5 crystallization has been confirmed on artificial phospholipid bilayers and on cell membranes including endothelial cells, placental trophoblasts and platelets. Recently, we reported that hydroxychloroquine, a synthetic antimalarial drug, can reverse this antibody-mediated process through two mechanisms: (1) by inhibiting the formation of aPL IgG-β2glycoprotein I complexes; and (2) by promoting the formation of a second layer of AnxA5 crystal ‘patches’ over areas where the immune complexes had disrupted AnxA5 crystallization. In another translational application, we have developed a mechanistic assay that reports resistance to AnxA5 anticoagulant activity in plasmas of patients with aPL antibodies. AnxA5 resistance may identify a subset of aPL syndrome patients for whom this is a mechanism for pregnancy losses and thrombosis. The elucidation of aPL-mediated mechanisms for thrombosis and pregnancy complications may open new paths towards addressing this disorder with targeted treatments and mechanistic assays.

STRUCTURE AND DYNAMICS OF THE FUSION PORE IN LIVE CELLS

Atomic force microscopy reveal pit‐like structures typically containing three or four, ∼150nm 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. It is hypothesized that depressions are the fusion pores, where membrane‐bound secretory vesicles dock and fuse to release vesicular contents. Zymogen granules, the membrane‐bound secretory vesicles in exocrine pancreas, contain the starch digesting enzyme, amylase. Using amylase‐specific immunogold labeling, localization of amylase at depressions following stimulation of secretion is demonstrated. This study confirms depressions to be the fusion pores in pancreatic acinar cells. High‐resolution images of the fusion pore in live pancreatic acinar cells reveal the structure in much greater detail than has previously been observed.

Resistance to annexin A5 anticoagulant activity: a thrombogenic mechanism for the antiphospholipid syndrome

The phospholipid binding protein, annexin A5 (AnxA5), has potent anticoagulant properties that result from its forming 2-dimensional crystals over phospholipids, blocking the availability of the phospholipids for critical coagulation enzyme reactions. This article reviews the evidence that antiphospholipid antibodies can disrupt this anticoagulant shield and unmask thrombogenic anionic phospholipids, which may thereby contribute to thrombosis in patients with the antiphospholipid syndrome (APS). This mechanism for thrombosis in APS can be monitored with coagulation assays for resistance to anticoagulant activity of AnxA5.

TERTIARY STRUCTURE OF THE HEPATIC CELL PROTEIN FIBRINOGEN IN FLUID REVEALED BY ATOMIC FORCE MICROSCOPY

Fibrinogen participates in important cellular physiological processes, such as cell adhesion and blood clotting. Although the primary and secondary structures of fibrinogen are known, its tertiary structure is yet to be determined. In attempts to understand the tertiary structure of this important hydrated cellular and plasma membrane protein, the present study using atomic force microscopy was carried out. The techniques presented in this manuscript may also be applicable to enhance the imaging of live cells as well as their subcellular components. The authors have imaged fibrinogen by Tapping Mode atomic force microscopy in fluid. Purified human fibrinogen, together with 15‐nm colloidal gold particles serving as an internal calibration standard, were adhered to a poly‐l‐lysine substrate on freshly cleaved mica. Atomic force microscopy images were obtained using oxide‐sharpened silicon nitride probes, either unaltered or with an electron beam deposited extended tip. Although various structures were observed, the predominant forms consisted of a bi‐ or trinodular slightly curved linear shape. Approximately 300 of these structures were observed with six different tips (1 unaltered and 5 electron beam deposited) and their lengths and heights were analyzed. The mean length of the fibrinogen molecules was 65.8nm and the mean height was 3.4nm. The quantitative measurements were little influenced by the shape of the tip, whereas the sharper electron beam deposited tips seemed to produce qualitatively superior images.

QUALITY ASSESSMENT OF ATOMIC FORCE MICROSCOPY PROBES BY SCANNING ELECTRON MICROSCOPY : CORRELATION OF TIP STRUCTURE WITH RENDERED IMAGES

While image quality from instruments such as electron microscopes, light microscopes, and confocal laser scanning microscopes is mostly influenced by the alignment of optical train components, the atomic force microscope differs in that image quality is highly dependent upon a consumable component, the scanning probe. Although many types of scanning probes are commercially available, specific configurations and styles are generally recommended for specific applications. For instance, in our area of interest, tapping mode imaging of biological constituents in fluid, double ended, oxide‐sharpened pyramidal silicon nitride probes are most often employed. These cantilevers contain four differently sized probes; thick‐ and thin‐legged 100 μm long and thick‐ and thin‐legged 200 μm long, with only one probe used per cantilever. In a recent investigation [Taatjes et al. (1997) Cell Biol. Int. 21:715–726], we used the scanning electron microscope to modify the oxide‐sharpened pyramidal probe by creating an electron beam deposited tip with a higher aspect ratio than unmodified tips. Placing the probes in the scanning electron microscope for modification prompted us to begin to examine the probes for defects both before and after use with the atomic force microscope. The most frequently encountered defect was a mis‐centered probe, or a probe hanging off the end of the cantilever. If we had difficulty imaging with a probe, we would examine the probe in the scanning electron microscope to determine if any defects were present, or if the tip had become contaminated during scanning. Moreover, we observed that electron beam deposited tips were blunted by the act of scanning a hard specimen, such as colloidal gold with the atomic force microscope. We also present a mathematical geometric model for deducing the interaction between an electron beam deposited tip and either a spherical or elliptical specimen. Examination of probes in the scanning electron microscope may assist in interpreting images generated by the atomic force microscope. Microsc. Res. Tech. 44:312–326, 1999.

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). These studies have incorporated the imaging of live cells at nanometer resolution, leading to discovery of the “porosome,” the universal secretory portal in cells, and a molecular understanding of membrane fusion from imaging the interaction and assembly of proteins between opposing lipid membranes. Similarly, the development of an in vitro simulacrum for investigating the molecular interactions between proteins and lipids has helped define an etiological explanation for APS. The prime importance of AFM in the success of these investigations will be presented in this manuscript, as well as a discussion of the limitations of this technique for the study of biomedical samples. J. Cell. Physiol. 228: 1949–1955, 2013.

Tailoring the Chain Packing in Ultrathin Polyelectrolyte Films Formed by Sequential Adsorption: Nanoscale Probing by Positron Annihilation Spectroscopy

Depth profiling experiments by positron annihilation spectroscopy have been used to investigate the free volume element size and concentration in films assembled using the layer-by-layer (LbL) adsorption method. Films prepared from strong polyelectrolytes, weak polyelectrolytes, hydrogen-bonding polymers, and blended polyelectrolyte multilayers have different chain packing that is reflected in the free volume characteristics. The influence of various parameters on free volume, such as number of bilayers, salt concentration, solution pH, and molecular weight, has been systematically studied. The free volume cavity diameters vary from 4 to 6 Å, and the free volume concentrations vary from (1.1-4.3) × 10(20) cm(-3), depending on the choice of assembly polymers and conditions. Films assembled from strong polyelectrolytes have fewer free volume cavities with a larger average size than films prepared from weak polyelectrolytes. Blending the weak polyanion poly(acrylic acid), PAA, with the strong polyanion poly(styrene sulfonate), PSS, to layer alternately with the polycation poly(allyamine hydrochloride), PAH, is shown to be a viable method to achieve intermediate free volume characteristics in these LbL films. An increase in salt concentration of the adsorption solutions for films prepared from strong polyelectrolytes makes these films tend toward weaker polyelectrolyte free volume characteristics. Hydrogen-bonded layered films show larger free volume element size and concentration than do their electrostatically bonded counterparts, while reducing the molecular weight of these hydrogen-bonded polymers results in slightly reduced free volume size and concentration. A study of the effect of solution pH on films prepared from weak polyelectrolytes shows that when both polyelectrolytes are substantially charged in solution (assembly pH = 7.5), the chains pack similarly to strong polyelectrolytes (i.e., lower free volume concentration), but with smaller average cavity sizes. These results give, for the first time, a clear indication of how the free volume profile develops in LbL thin films, offering numerous methods to tailor the Ångström-scale free volume properties by judicious selection of the assembly polymers and conditions. These findings can be potentially exploited to tailor the properties of thin polymer films for applications spanning membranes, sensing, and drug delivery.

Imaging of collagen type III in fluid by atomic force microscopy

Type III collagen is a component of the basement membrane of endothelial cells, and may play a role in the interaction between hemostatic system proteins and the basement membrane of blood vessels. To begin to investigate these structural interactions, we have imaged type III collagen in solution by atomic force microscopy. A 20 μg/ml solution of type III collagen in bicarbonate buffer (pH 9.5) from calf skin was deposited onto a freshly cleaved mica substrate. Atomic force microscopy images were acquired using a fluid cell and tapping mode with oxide‐sharpened silicon nitride probes 2, 3, and 4 hours after deposition of the collagen onto the mica. Two‐hour preparations displayed fibrillar networks with well‐defined sites of nucleation and lateral growth. At 3 and 4 hour polymerizations, more mature fibrils of increasing lengths, diameters, and complexity were observed. Fibrils appeared to be aligning and twisting (helical formation) to form a mature fibril with a higher mass per unit area. Interestingly, the mature fibrils appeared larger centrally with tapered ends displaying declining slopes. These observations compare favorably with those previously published on collagen type I assembly [Gale et al. (1995) Biophys. J. 68:2124–2128]. High resolution atomic force microscopy images of type III collagen in solution should provide a template for observation of the interactions between basement membrane components and hemostatic system proteins present in cardiovascular disease. Microsc. Res. Tech. 44:347–352, 1999.