Maria P. McGee

Chondroitin Sulfate Anticoagulant Activity Is Linked to Water Transfer Relevance to Proteoglycan Structure in Atherosclerosis

Objective—Changes in chondroitin sulfate (CS) proteoglycan (PG) during atherosclerosis are associated with chronic inflammatory changes and increased incidence of thrombosis. To explore how glycosaminoglycan changes could influence the thrombogenicity of atherosclerotic lesions, water-transfer reactions were examined during activation of antithrombin by CS. Methods and Results—Advanced type IV atherosclerotic lesions prone to thrombosis contained CSPG (versican) with undersulfated CS relative to CS of the adjacent healthy aorta. Approximately 11% of the CS disaccharide in versican from healthy arteries was oversulfated, but this proportion decreased markedly to 3% in atherosclerotic lesions. Oversulfated CS functionally bound antithrombin with a dissociation constant of 3.3±1.9 &mgr;mol/L. Measured by osmotic stress (OS) techniques with an ≈26-Å probe, the reaction was linked to transfer of ≈2500 mol water per mole of coagulation factor Xa inhibited. Under OS, the anticoagulant efficiency of CS was 1.3 (&mgr;mol/L)−1 · s−1, ≈5- and 15-fold higher than heparan sulfate efficiency measured under OS and standard conditions, respectively. Conclusions—Decreased sulfation of high molecular weight CSPG in the advancing atherosclerotic lesions may predispose the lesions to thrombosis by disrupting osmotic regulation, limiting avidity for antithrombin and decreasing activation efficiency.

Specific Regulation of Procoagulant Activity on Monocytes INTRINSIC PATHWAY INHIBITION BY CHONDROITIN 4,6-DISULFATE

Hypercoagulability of blood, monocytic infiltration, and changes in pericellular and extracellular matrix glycosaminoglycans (GAGs) are observed in atherosclerosis, inflammation, and neoplasia. In the present studies, monocyte procoagulants and different GAGs including chondroitin sulfate (CS) A, CSB, CSC, CSD, CSE, and heparan sulfate, were tested either in clotting assays with whole plasma or in chromogenic assays with purified coagulation proteases. Procoagulant activity in plasma was inhibited by three of the seven GAGs, including heparan sulfate, CSE, and CSB. In contrast, activity of purified coagulation protease was inhibited only by CSE, and the inhibition was observed with intrinsic (factor VIIIa/IXa) but not extrinsic (tissue factor/factor VII) components. Reciprocal titration experiments with enzyme and substrate and Scatchard type analyses were consistent with concentration-dependent inhibitory interactions between CSE and sites on both factor VIIIa and IXa. On purified phospholipids, CSE concentration resulting in half-maximal inhibition (K) was 5 ng/ml for interaction with factor IXa and >500 ng/ml for interaction with factor VIIIa. The K values were lower for reactions on purified lipid than for reactions on monocyte surfaces and for reactions on resting than on endotoxin-stimulated monocytes. Experiments with CSE oligosaccharides of defined size indicated that the smallest CSE fragment capable of inhibitory activity was composed of 12-18 monosaccharide units. Collectively, these results indicate that factor X-activating reactions are inhibited by GAGs expressed on monocyte membranes. Inhibition is specific with respect to the structure of both the GAG and the activating protease. Lack of inhibition by added CSA, CSB, and CSC in contrast to CSE strongly suggests a direct role of 4,6-di-O-sulfated N-acetylgalactosamine GAG structures in the inhibition of intrinsic pathway protease. These findings also suggest potential pharmacologic use of CSE as specific anticoagulant in the management of prothrombotic states mediated by intrinsic pathway coagulation reactions.

Swelling and pressure-volume relationships in the dermis measured by osmotic-stress technique

Water transfer across the extracellular matrix (ECM) involves interstitial osmotic forces in as yet unclear ways. In particular, the traditional values of Starling forces cannot adequately explain fluid transfer rates. Here, we reassess these forces by analyzing fluid transfer in live pig and human dermal explants. Pressure potentials were controlled with inert polymers adjusted by membrane osmometry (range = 3-219 mmHg), and fluid transfer in and out of the explants was followed by sequential precision weighing. Water motional freedom in the dermis was examined by NMR. In pigs, mean hydration pressure (HP; the pressure at which volume did not change) was 107 +/- 22 and 47 +/- 12 (SE) mmHg at 4 degrees C and 37 degrees C (P = 0.012, paired t-test, n = 7). Volume changes observed in response to pressure potential were reversible. The equation, Volume change = V(max)/[1+(time/T(1/2))(d)], where V(max) is maximal volume change; T(1/2), time at volume = 1/2 V(max); and d, a rate parameter, was fitted to experimental progression curves (r(2) > 0.9), yielding V(max) values linearly related to pressure, with mean slopes -3.5 +/- 0.28 and -2.6 +/- 0.21(SE) mul.g(-1).mmHg(-1) at 4 degrees C and 37 degrees C. NMR spin-spin relaxation times (T(2)) varied within 200- to 400-mum distances in directions perpendicular to the epidermis, with slopes reaching 0.03 ms/mum. Results support a mechanism in which fluid transport across the ECM is locally regulated at micrometer scales by cell- and fiber-gel-dependent osmomechanical forces. The large HP helps to explain the fast interstitial in/out flow rates observed clinically.

Unidirectional flux of phenylalanine into Vero cells. Measurement using paired tracers in perfused cultures

The uptake of phenylalanine by Vero cells in perfused culture was measured using a double-label technique. Cells were anchored in microcarrier beads and maintained in a column perfused at a constant rate. The extracellular tracer [14C]mannitol and the test tracer [3H]phenylalanine were injected as a bolus, and the column effluent was sampled at 10-s intervals. The proportion of the test tracer retained by the cells was calculated by analysis of time-dilution curves of test and reference tracers. Uptake measurements were specific and highly reproducible. Uptake of [3H]phenylalanine was inhibited by unlabelled phenylalanine and by other amino acids that utilize transport system L. This new approach proved useful for rapid measurement of unidirectional uptake, and for determination of kinetics parameters of uptake under steady state conditions. This rapid technique obviates some of the limitations associated with uptake measurements in whole organs and with measurements in conventional cell cultures.

Hydration Structure of Antithrombin Conformers and Water Transfer during Reactive Loop Insertion

The serine protease inhibitor antithrombin undergoes extensive conformational changes during functional interaction with its target proteases. Changes include insertion of the reactive loop region into a beta-sheet structure in the protein core. We explore the possibility that these changes are linked to water transfer. Volumes of water transferred during inhibition of coagulation factor Xa are compared to water-permeable volumes in the x-ray structure of two different antithrombin conformers. In one conformer, the reactive loop is largely exposed to solvent, and in the other, the loop is inserted. Hydration fingerprints of antithrombin (that is, water-permeable pockets) are analyzed to determine their location, volume, and size of access pores, using alpha shape-based methods from computational geometry. Water transfer during reactions is calculated from changes in rate with osmotic pressure. Hydration fingerprints prove markedly different in the two conformers. There is an excess of 61-76 water molecules in loop-exposed as compared to loop-inserted conformers. Quantitatively, rate increases with osmotic pressure are consistent with the transfer of 73 +/- 7 water molecules. This study demonstrates that conformational changes of antithrombin, including loop insertion, are linked to water transfer from antithrombin to bulk solution. It also illustrates the combined use of osmotic stress and analytical geometry as a new and effective tool for structure/function studies.

A study of the inability of subcellular fractions to elicit primary anti-h-2 cytotoxic t lymphocytes.

Abstract An analysis was undertaken to understand the inability of H-2 containing plasma membranes and partially purified H-2 antigens incorporated into lipid vesicles to elicit primary anti-H-2 CTLs. It was found that in the presence of supernatants from concanavalin A stimulated spleen cells H-2 containing-subcellular fractions could elicit anti-H-2 CTLs. The result suggests that cytotoxic T lymphocyte precursor cells are available for interaction with alloantigens within the subcellular fractions and that the defect is the inability of these H-2 antigen-containing subcellular fractions to stimulate T helper activity.

Collagen Unfolding Accelerates Water Influx, Determining Hydration in the Interstitial Matrix

In the interstitial matrix, collagen unfolding at physiologic temperatures is thought to facilitate interactions with enzymes and scaffold molecules during inflammation, tissue remodeling, and wound healing. We tested the hypothesis that it also plays a role in modulating flows and matrix hydration potential. After progressively unfolding dermal collagen in situ, we measured the hydration parameters by osmotic stress techniques and modeled them as linear functions of unfolded collagen, quantified by differential scanning calorimetry after timed heat treatment. Consistent with the hypothetical model, the thermodynamic and flow parameters obtained experimentally were related linearly to the unfolded collagen fraction. The increases in relative humidity and intensity of T(2) maps were also consistent with interfacial energy contributions to the hydration potential and the hydrophobic character of the newly formed protein/water interfaces. As a plausible explanation, we propose that increased tension at interfaces formed during collagen unfolding generate local gradients in the matrix that accelerate water transfer in the dermis. This mechanism adds a convective component to interstitial transfer of biological fluids that, unlike diffusion, can speed the dispersion of water and large solutes within the matrix.

Electrostatic interactions during activation of coagulation factor IX via the tissue factor pathway: effect of univalent salts

Interaction between the Gla-domain of coagulation proteins and negatively charged phospholipid membranes is essential for blood coagulation reactions. The interaction is calcium-dependent and mediated both by electrostatic and hydrophobic forces. This report focuses on the electrostatic component of factor IX activation via the extrinsic pathway. Effective charges during the reaction are measured by ionic titration of activity, according to the Debye-Huckel and Gouy-Chapman models. Rates of activation decrease with ionic strength independently of the type of monovalent salt used to control ionic strength. Moreover, the effect of ionic strength decreases at concentrations of charged phospholipid approaching saturation levels, indicating that membrane charges participate directly in the ionic interaction measured. The effective charge on calcium-bound factor IX during activation on phospholipid membranes is 0.95+/-0.1. Possible sites mediating contacts between the Gla-domain and membranes are selected by geometrical criteria in several metal-bound Gla-domain structures. A pocket with a solvent opening-pore of area 24-38 A2 is found in the Gla-domain of factors IX, VII, and prothrombin. The pocket contains atoms with negative partial charges, including carboxylate oxygens from Gla residues, and has a volume of 57-114 A3, sufficient to accommodate additional calcium atoms. These studies demonstrate that electrostatic forces modify the activity coefficient of factor IX during functional interactions and suggest a conserved pocket motif as the contact site between the calcium-bound Gla-domain and charged membranes.

The local pathology of interstitial edema: Surface tension increases hydration potential in heat-damaged skin

The local pathogenesis of interstitial edema in burns is incompletely understood. This ex vivo study investigates the forces mediating water‐transfer in and out of heat‐denatured interstitial matrix. Experimentally, full‐thickness dermal samples are heated progressively to disrupt glycosaminoglycans, kill cells, and denature collagen under conditions that prevent water loss/gain; subsequently, a battery of complementary techniques including among others, high‐resolution magnetic resonance imaging, equilibrium vapor pressure and osmotic stress are used to compare water‐potential parameters of nonheated and heated dermis. The hydration potential (HP) determined by osmotic stress is a measure of the total water‐potential defined empirically as the pressure at which no net water influx/efflux into/from the dermis is detected. Results show that after heat denaturation, the HP, the intensity of T2‐weighed magnetic resonance images, and the vapor pressure increase indicating higher water activity and necessarily, smaller contributions from colloidosmotic forces to fluid influx in burned relative to healthy dermis. Concomitant increases in HP and in water activity implicate local changes in interfacial and metabolic energy as the source of excess fluid‐transfer potential. These ex vivo findings also show that these additional forces contributing to abnormal fluid‐transfer in burned skin develop independently of inflammatory and systemic hydrodynamic responses.

Surface-dependent Coagulation Enzymes FLOW KINETICS OF FACTOR Xa GENERATION ON LIVE CELL MEMBRANES

The initial surface reactions of the extrinsic coagulation pathway on live cell membranes were examined under flow conditions. Generation of activated coagulation factor X (fXa) was measured on spherical monolayers of epithelial cells with a total surface area of 41–47 cm2 expressing tissue factor (TF) at >25 fmol/cm2. Concentrations of reactants and product were monitored as a function of time with radiolabeled proteins and a chromogenic substrate at resolutions of 2–8 s. At physiological concentrations of fVIIa and fX, the reaction rate was 3.05 ± 0.75 fmol fXa/s/cm2, independent of flux, and 10 times slower than that expected for collision-limited reactions. Rates were also independent of surface fVIIa concentrations within the range 0.6–25 fmol/cm2. The transit time of fX activated on the reaction chamber was prolonged relative to transit times of nonreacting tracers or preformed fXa. Membrane reactions were modeled using a set of nonlinear kinetic equations and a lagged normal density curve to track the expected surface concentration of reactants for various hypothetical reaction mechanisms. The experimental results were theoretically predicted only when the models used a slow intermediate reaction step, consistent with surface diffusion. These results provide evidence that the transfer of substrate within the membrane is rate-limiting in the kinetic mechanisms leading to initiation of blood coagulation by the TF pathway.