Fibrinolysis at Rest and after Exercise in Hepatic Cirrhosis

S ummary . A study of the fibrinolytic enzyme system in 37 cirrhotic patients at rest and 15 cirrhotic patients following a treadmill exercise procedure is reported. An outstanding feature of the resting studies was a significant elevation in the levels of plasma plasminogen activator and serum fibrin/fibrinogen degradation products, when compared to age and sex-matched healthy controls. Moreover, there was a significant positive correlation between those two fibrinolytic parameters. No significant difference was demonstrated between the control and cirrhotic groups in relation to plasma fibrinogen, euglobulin plasminogen and plasma urokinase inhibitor activity. The exercise studies showed that following an entirely physiological stimulus the plasminogen activator response in some cirrhotics was excessive in terms of either the initial response and/or prolongation of the recovery period. However, an exaggerated initial response was not necessarily followed by a prolongation recovery period nor was a relationship established between the resting levels of plasminogen and an abnormal exercise response. Furthermore, there was no correlation between the increases in plasminogen activator and serum fibrin/ fibrinogen degradation products following exercise.     

Packing at the protein-water interface.

We have determined the packing efficiency at the protein-water interface by calculating the volumes of atoms on the protein surface and nearby water molecules in 22 crystal structures. We find that an atom on the protein surface occupies, on average, a volume approximately 7% larger than an atom of equivalent chemical type in the protein core. In these calculations, larger volumes result from voids between atoms and thus imply a looser or less efficient packing. We further find that the volumes of individual atoms are not related to their chemical type but rather to their structural location. More exposed atoms have larger volumes. Moreover, the packing around atoms in locally concave, grooved regions of protein surfaces is looser than that around atoms in locally convex, ridge regions. This as a direct manifestation of surface curvature-dependent hydration. The net volume increase for atoms on the protein surface is compensated by volume decreases in water molecules near the surface. These waters occupy volumes smaller than those in the bulk solvent by up to 20%; the precise amount of this decrease is directly related to the extent of contact with the protein.     

TLR signaling at the intestinal epithelial interface

The intestinal epithelium provides a critical interface between lumenal bacteria and the mucosal immune system. Whereas normal commensal flora do not trigger acute inflammation, pathogenic bacteria trigger a potent inflammatory response. Our studies emanate from the hypothesis that the intestinal epithelium is normally hyporesponsive to commensal pathogen-associated molecular patterns (PAMPs) such as LPS. Our data demonstrate that normal human colonic epithelial cells and lamina propria cells express low levels of TLR4 and its co-receptor MD-2. This expression pattern is mirrored by intestinal epithelial cell (IEC) lines. Co-expression of TLR4 and MD-2 is necessary and sufficient for LPS responsiveness in IEC. Moreover, LPS sensing occurs along the basolateral membrane of polarized IEC in culture. Expression of MD-2 is regulated by IFN-gamma. Cloning of the MD-2 promoter demonstrates that promoter activity is increased by IFN-gamma and blocked by the STAT inhibitor SOCS3. We conclude from our studies that the intestinal epithelium down-regulates expression of TLR4 and MD-2 and is LPS unresponsive. The Th1 cytokine IFN-gamma up-regulates expression of MD-2 in a STAT-dependent fashion. The results of our studies have important implications for understanding human inflammatory bowel diseases.     

Ordered polyelectrolyte assembly at the oil-water interface

Polyelectrolytes (PEs) are widely used in applications such as water purification, wastewater treatment, and mineral recovery. Although much has been learned in past decades about the behavior of PEs in bulk aqueous solutions, their molecular behavior at a surface, and particularly an oil-water interface where many of their applications are most relevant, is largely unknown. From these surface spectroscopic and thermodynamics studies we report the unique molecular characteristics that several common polyelectrolytes, poly(acrylic acid) and poly(methylacrylic acid), exhibit when they adsorb at a fluid interface between water and a simple insoluble organic oil. These PEs are found to adsorb to the interface from aqueous solution in a multistepped process with a very thin initial layer of oriented polymer followed by multiple layers of randomly oriented polymer. This additional layering is thwarted when the PE conformation is constrained. The adsorption/desorption process is highly pH dependent and distinctly different than what might be expected from bulk aqueous phase behavior.     

Fibrinolysis and the biliary tree.

AIMS: To investigate the fibrinolytic activity of normal and calculous human bile. METHODS: Fibrinolytic properties of the biliary tract were studied in patients with gall bladder stones (n = 7) compared with acalculous gall bladders (n = 8). RESULTS: Bile plasminogen activating activity was detected in a wide range in both groups (calculous bile median 0.35 IU/ml; range: 0.06-6.59, versus normal bile 0.70 IU/ml; 0.19-3.56). There was no difference in the bile concentration of tissue plasminogen activator between the two groups (calculous bile median 21.5 ng/ml versus normal bile 9.5 ng/ml), which was present in much greater concentrations than urokinase (calculous bile median 0.10 ng/ml versus normal bile 0.36 ng/ml). Both plasminogen activators were detected in low concentrations in gall bladder mucosa. Plasminogen activator inhibitors-1 and 2 were detected in bile in significantly greater concentrations in patients with gall bladder stones (plasminogen activator inhibitor-1: calculous bile median 15 ng/ml versus normal bile < 2 ng/ml, plasminogen activator inhibitor-2: 157 ng/ml versus < 6 ng/ml, p < 0.05). CONCLUSIONS: Human bile possesses fibrinolytic activity and the principal plasminogen activator in bile seems to be tissue plasminogen activator. Plasminogen activator inhibitors were present in greater concentrations in stone bile and may be a factor in the pathogenesis of gall stone formation.     

Characterization of the structure-function relationship at the ligament-to-bone interface

Soft tissues such as ligaments and tendons integrate with bone through a fibrocartilaginous interface divided into noncalcified and calcified regions. This junction between distinct tissue types is frequently injured and not reestablished after surgical repair. Its regeneration is also limited by a lack of understanding of the structure-function relationship inherent at this complex interface. Therefore, focusing on the insertion site between the anterior cruciate ligament (ACL) and bone, the objectives of this study are: (i) to determine interface compressive mechanical properties, (ii) to characterize interface mineral presence and distribution, and (iii) to evaluate insertion site-dependent changes in mechanical properties and matrix mineral content. Interface mechanical properties were determined by coupling microcompression with optimized digital image correlation analysis, whereas mineral presence and distribution were characterized by energy dispersive x-ray analysis and backscattered scanning electron microscopy. Both region- and insertion-dependent changes in mechanical properties were found, with the calcified interface region exhibiting significantly greater compressive mechanical properties than the noncalcified region. Mineral presence was only detectable within the calcified interface and bone regions, and its distribution corresponds to region-dependent mechanical inhomogeneity. Additionally, the compressive mechanical properties of the tibial insertion were greater than those of the femoral. The interface structure-function relationship elucidated in this study provides critical insight for interface regeneration and the formation of complex tissue systems.     

Mural thrombi in the heart

Of 771 consecutive, adult, autopsied patients who died of heart disease, 265, or 34.4 per cent, had one or more mural thrombi. Coronary artery disease, with myocardial infarction, was the type of heart disease most often associated with mural thrombi; in two-thirds of these cases this complication was present. Mural thrombi were associated with coronary artery disease without myocardial infarction, hypertensive heart disease, and rheumatic heart disease, in one-third of the cases; they were present in one-fifth of the cases of syphilitic heart disease, but were uncommonly found in cases of cor pulmonale and bacterial endocarditis.     

Impaired Fibrinolysis in the Antiphospholipid Syndrome

The antiphospholipid syndrome (APS) is characterized by venous and/or arterial thrombosis, or recurrent fetal loss, in the presence of antiphospholipid antibodies (APL). The pathogenesis of APS is multifaceted and involves numerous mechanisms including activation of endothelial cells, monocytes, and/or platelets; inhibition of natural anticoagulant pathways such as protein C, tissue factor inhibitor, and annexin A5; activation of the complement system; and impairment of the fibrinolytic system. Fibrinolysis—the process by which fibrin thrombi are remodeled and degraded—involves the conversion of plasminogen to plasmin by tissue plasminogen activator (tPA) or urokinase-type plasminogen activator, and is tightly regulated. Although the role of altered fibrinolysis in patients with APS is relatively understudied, several reports suggest that deficient fibrinolytic activity may contribute to the pathogenesis of disease in these patients. This article discusses the function of the fibrinolytic system and reviews studies that have reported alterations in fibrinolytic pathways that may contribute to thrombosis in patients with APL. Some of these mechanisms include elevations in plasminogen activator inhibitor-1 levels, inhibitory antibodies against tPA or other components of the fibrinolytic system, antibodies against annexin A2, and finally, antibodies to β₂-glycoprotein-I (β₂GPI) that block the ability of β₂GPI to stimulate tPA-mediated plasminogen activation.     

Serum Inhibitors of Fibrinolysis

Four protease inhibitors have been identified in human serum and methods for their isolation are described. After removal of the euglobulin fraction, serum was submitted to ion exchange chromatography and gel filtration, and fractions were tested for inhibition of the lysis of plasminogen-deficient fibrin clots by plasmin, trypsin and elastase. In addition inhibitors of plasminogen activation were sought by studying the effects of separated fractions on the lysis of plasminogen-rich fibrin clots by urokinase. Examination by immunophoresis showed that three of the separated inhibitors were alpha2-macroglobulin, alpha1-antitrypsin and inter-alpha-trypsin inhibitor. The fourth antiprotease was a powerful inhibitor of both urokinase-induced and plasmin-induced clot lysis, and was identified as an inter-alpha-globulin from its electrophoretic mobility in agarose gels.     

Tissue transglutaminase at embryo-maternal interface

CONTEXT: Tissue transglutaminase (tTG) has a high affinity for fibronectin (FN) and is a coreceptor of both beta1 and beta3 integrin subunits. Considering the notion that FN and integrins have critical roles during the implantation process, this study was undertaken to elucidate the expression pattern and the potential physiological function of tTG at the embryo-maternal interface. METHODS: The primary cultures of human placentas from 15 legal elective abortions at the first trimester of normal pregnancies and endometrial biopsies of 12 female patients in the midluteal phase as well as normal trophoblastic cell lines (CRL) were employed to address these issues using several approaches, such as scanning and transmission electron microscopies, immunostaining for light and electron microscopies, western blotting, and function assays using GRGDSP hexapeptide and an antibody against tTG. RESULTS: The results demonstrated tTG expression on uterine pinopodes and lamellipodia of extravillous trophoblasts. The colocalization of tTG with beta1 and beta3 integrins and its interaction with alpha(v)beta3 integrin and integrin-associated proteins at focal adhesions of the extravillous trophoblasts were illustrated in the results of immunofluorescence, immunoblot, and coimmunoprecipitation studies. Furthermore, function assays revealed that tTG mediated the adhesion and spread of the placental cells on intact FN-coated and 42- and 110-kDa FN fragment-coated wells. CONCLUSION: In conclusion, our findings demonstrated for the first time that tTG actively participates in adhesion events at the embryo-maternal interface through its interaction with FN, at least in part, by activating integrin-signaling pathways.     

Localized proton microcircuits at the biological membrane-water interface

Cellular processes such as nerve conduction, energy metabolism, and import of nutrients into cells all depend on transport of ions across biological membranes through specialized membrane-spanning proteins. Understanding these processes at a molecular level requires mechanistic insights into the interaction between these proteins and the membrane itself. To explore the role of the membrane in ion translocation we used an approach based on fluorescence correlation spectroscopy. Specifically, we investigated exchange of protons between the water phase and the membrane surface, as well as diffusion of protons along membrane surfaces, at a single-molecule level. We show that the lipid head groups collectively act as a proton-collecting antenna, dramatically accelerating proton uptake from water to a membrane-anchored proton acceptor. Furthermore, the results show that proton transfer along the surface can be significantly faster than that between the lipid head groups and the surrounding water phase. Thus, ion translocation across membranes and between the different membrane protein components is a complex interplay between the proteins and the membrane itself, where the membrane acts as a proton-conducting link between membrane-spanning proton transporters.     

Novel therapeutic targets at the platelet vascular interface

Platelet activation in vivo can be part of the hemostatic response to injury or a pathological response to disease. In either setting, platelets adhere to the vessel wall and to each other, forming a closely packed mass interspersed with fibrin. Recent studies have identified new molecules on the platelet surface and within platelets that support and regulate thrombus growth and stability, ensuring that platelet accumulation after injury is sufficient to stop bleeding, but not so exuberant that vascular occlusion occurs. An understanding of how this balance is achieved helps to illuminate the events of platelet activation and, at the same time, provides potential targets for new classes of antiplatelet agents.     

Stereodynamics in state-resolved scattering at the gas-liquid interface

Stereodynamics at the gas-liquid interface provides insight into the important physical interactions that directly influence heterogeneous chemistry at the surface and within the bulk liquid. We investigate molecular beam scattering of CO(2) from a liquid perfluoropolyether (PFPE) surface in vacuum [incident energy E(inc) = 10.6(8) kcal/mol, incident angle theta(inc) = 60 degrees] to specifically reveal rotational angular-momentum directions for scattered molecules. Experimentally, internal quantum state populations and M(J) distributions are probed by high-resolution polarization-modulated infrared laser spectroscopy. Analysis of J-state populations reveals dual-channel scattering dynamics characterized by a two-temperature Boltzmann distribution for trapping-desorption and impulsive scattering. In addition, molecular dynamics simulations of CO(2) + fluorinated self-assembled monolayers have been used to model CO(2) + PFPE dynamics. Experimental results and molecular dynamics simulations reveal highly oriented CO(2) distributions that preferentially scatter with "top spin" as a strongly increasing function of J state.     

Viral and bacterial pathogens at the maternal-fetal interface

We studied the incidence of pathogenic bacteria and concurrent infections with human cytomegalovirus (CMV) and herpes simplex virus (HSV) type 1 and 2 in biopsy samples from the placenta and decidua of women with healthy pregnancies. By polymerase chain reaction analysis, we found that 38% of placental samples were positive for selected bacteria and viruses. CMV, HSV-1, and HSV-2 were detected in isolation or with bacteria in first- and second-trimester samples. Certain bacteria were detected more often during the second trimester than during the first--Ureaplasma urealyticum, Mycoplasma hominis, and Gardnerella/Bifidobacterium species. In paired samples from first-trimester tissues, the detection rate for viruses, compared with most bacteria, was higher in the decidua than in the adjacent placenta. In contrast, bacteria were more frequently detected in placenta. Analyses of immunoglobulin G isolated from the placenta support the hypothesis that immune responses suppress CMV reactivation in the presence of pathogenic bacteria at the maternal-fetal interface.     

Nanoparticle self-assembly at the interface of liquid crystal droplets

Nanoparticles adsorbed at the interface of nematic liquid crystals are known to form ordered structures whose morphology depends on the orientation of the underlying nematic field. The origin of such structures is believed to result from an interplay between the liquid crystal orientation at the particles’ surface, the orientation at the liquid crystal’s air interface, and the bulk elasticity of the underlying liquid crystal. In this work, we consider nanoparticle assembly at the interface of nematic droplets. We present a systematic study of the free energy of nanoparticle-laden droplets in terms of experiments and a Landau–de Gennes formalism. The results of that study indicate that, even for conditions under which particles interact only weakly at flat interfaces, particles aggregate at the poles of bipolar droplets and assemble into robust, quantized arrangements that can be mapped onto hexagonal lattices. The contributions of elasticity and interfacial energy corresponding to different arrangements are used to explain the resulting morphologies, and the predictions of the model are shown to be consistent with experimental observations. The findings presented here suggest that particle-laden liquid crystal droplets could provide a unique and versatile route toward building blocks for hierarchical materials assembly.A growing body of theoretical and experimental work has sought to direct the assembly of molecules and nanoparticles at interfaces by exploiting the elastic forces that arise in liquid crystals (LCs) (1–5). Nematic LCs possess orientational order along a unit vector, the so-called nematic director. They also exhibit defects—regions of low order whose morphology and position depends on a delicate balance between elastic, enthalpic, and interfacial contributions to the free energy. The orientation of nematic LCs and any corresponding defects can be perturbed by introducing particles. The symmetry and structure of the director field around a particle also depends on the interaction between the LC and the particle, often referred to as anchoring. Particles with perpendicular (homeotropic) anchoring induce either dipolar or quadrupolar symmetry in the LC, leading to formation of point defects or Saturn-ring defects, respectively (6). Particles with planar anchoring induce quadrupolar symmetry, which is accompanied by two surface defects, generally referred to as boojums (6). Distortions of the nematic field cost elastic energy and therefore give rise to anisotropic, long-range interactions between particles. Indeed, particles in nematic LCs aggregate and “bind,” thereby minimizing the volume of defects and the large free energy that is associated with their elastic strain. Equilibrium particle arrangements in nematic LCs depend strongly on the topology of the underlying defects. Homeotropic particles with point, dipolar defects form chains along the nematic director, whereas quadrupolar, Saturn-ring defects form kinked chains that are perpendicular to the nematic director (7). Particles with planar anchoring form chains whose main axis forms a 30° angle with the nematic director (8). Recent work has also shown that particles can be trapped in topological defects (9, 10) and in chiral defects (11, 12). Particles localized at a planar LC interface also exhibit LC-induced interactions. For the particular case of perpendicular anchoring, it has been shown that particles aggregate into ordered structures whose morphology can be controlled by addition of surfactants (13). Recently, a robust mechanism has been reported to direct assembly of homeotropic particles trapped at the LC interface into reconfigurable structures by controlling surface anchoring and bulk defect structure (14).This work considers the aggregation of nanoparticles at LC droplet interfaces. Past studies from our own groups have shown that LCs confined in small droplets can be used to induce formation of intriguing surfactant nanophases at their interfaces (15). Experiments and simulations have also shown that the defects that arise in LC droplets can be used to localize individual nanoparticles or pairs of nanoparticles with considerable precision (16, 17). More generally, droplets offer an effective, yet simple means for confining LCs, thereby controlling the balance of interfacial and elastic contributions to the free energy and the response of LCs to external cues (18, 19). Depending on surface anchoring, LC droplets can exhibit two primary morphologies (20). Homeotropic anchoring leads to radial LC droplets, with a single ring or point defect in the center, whereas planar anchoring leads to bipolar droplets having two surface point (or boojum) defects. The localization of particles at boojums reduces the splay elastic free energy significantly. Remarkably, the trapping of particles into the boojums is independent of the type of anchoring of LC at the particle surfaces (16). A recent study examined the self-assembly of homeotropic particles at the surface of a bipolar droplet and observed formation of star-like patterns (21). Dipole–dipole interactions between particles led to formation of linear chains along the longitudinal orientation of the director field that, upon finding the boojums, organized into stars.Our focus is to examine and understand the behavior of planar particles located at the surface of micrometer-sized bipolar droplets. We restrict our attention to degenerate planar anchoring and planar droplets because such conditions are much more permissive than homeotropic anchoring, and it is therefore difficult to anticipate the types of arrangements that may arise on the basis of symmetry arguments. By examining the structures and LC morphologies that arise as a function of particle number, we are able to provide a systematic view of nanoparticle assembly in such systems. Our past experiments have shown that a single polystyrene (PS) particle with planar anchoring and radius of 0.5 µm adsorbed at the surface of a bipolar 5CB droplet diffuses into a boojum defect (16). Here, we show that when more particles are adsorbed at the droplet surface, they migrate to the poles and assemble into arrangements that can be mapped into hexagonal arrays around the boojum. The predictions of our theoretical calculations are shown to be consistent with experimental observations (22).