Inhibitory effect of nitrite on coagulation processes demonstrated by thrombelastography.

Nitric oxide (NO) can be generated by two-step reduction pathway in which nitrate is converted first into nitrite and then into NO via several mechanisms, as well as from arginine by endogenous nitric oxide synthase (NOS). We have recently shown that nitrite ions in the presence of erythrocytes inhibit platelet aggregation and activation, as measured by aggregometry and flow cytometric analysis of P-selectin, through its reduction to NO under partially deoxygenated conditions. In the current study, we investigated how nitrite may affect overall clotting processes via modulating platelet function using thrombelastography (TEG). We measured three major TEG parameters, reaction time (R, time to initial fibrin formation), α angle (velocity of clot growth) and maximum amplitude (MA, maximum clot strength) using blood from healthy volunteers. An NO donor (DEANONOate) showed inhibitory effects on all TEG parameters in platelet rich plasma (PRP) and whole blood, resulting in delayed R, decreased angle, and reduced MA in a dose dependent manner. Nitrite ions also exhibited inhibitory effects in whole blood at 20% hematocrit, and this was greatly enhanced under hypoxic conditions, being demonstrable at 0.1 µM concentration. Neither compound changed any TEG parameters in plasma. Our results suggest that nitrite affects overall blood clotting and that TEG may be used to follow this process. Further the physiological effects of factors which determine NO bioavailability, such as endogenous levels of blood and tissue nitrite, may be useful as biomarkers for predicting hemostatic potential.

Discrepancy between phase behavior of lung surfactant phospholipids and the classical model of surfactant function.

The studies reported here used fluorescence microscopy and Brewster angle microscopy to test the classical model of how pulmonary surfactant forms films that are metastable at high surface pressures in the lungs. The model predicts that the functional film is liquid-condensed (LC) and greatly enriched in dipalmitoyl phosphatidylcholine (DPPC). Both microscopic methods show that, in monolayers containing the complete set of phospholipids from calf surfactant, an expanded phase persists in coexistence with condensed domains at surface pressures approaching 70 mN/m. Constituents collapsed from the interface above 45 mN/m, but the relative area of the two phases changed little, and the LC phase never occupied more than 30% of the interface. Calculations based on these findings and on isotherms obtained on the continuous interface of a captive bubble estimated that collapse of other constituents increased the mol fraction of DPPC to no higher than 0.37. We conclude that monolayers containing the complete set of phospholipids achieve high surface pressures without forming a homogeneous LC film and with a mixed composition that falls far short of the nearly pure DPPC predicted previously. These findings contradict the classical model.

Fluorescence quenching and electron spin resonance study of percolation in a two-phase lipid bilayer containing bacteriorhodopsin.

The effect of bacteriorhodopsin (BR) on the percolation properties of dimyristoylphosphatidylcholine/distearoylphosphatidylcholine bilayers was examined by studying the quenching of a lipid-bound fluorophore by a lipid-bound quencher, and by spin-spin interactions of a nitroxide-labeled lipid using electron spin resonance (ESR). At the low concentrations of BR used, differential scanning calorimetry showed that although the transition enthalpy was reduced in a concentration-dependent manner by incorporation of BR, the solidus and fluidus phase boundaries and overall shape of the heat capacity profiles were essentially unchanged. However, fluorescence quenching and spin-label ESR data showed that the domain topology, as reflected in the percolation properties, is strongly affected by the protein. In contrast to our previous fluorescence data for the pure lipid mixtures, quenching in the coexistence region is independent of the fluid phase fraction when BR is present. In addition, the percolation threshold estimated by spin-label ESR is shifted in the presence of BR to a higher gel phase fraction at a given lipid composition. Both the fluorescence quenching and spin-label ESR data, together with the results of earlier simulations, strongly suggest that the fluid phase domains are substantially larger and/or less ramified in the presence of BR than in its absence. We have previously reported a similar effect of a transmembrane peptide, pOmpA (Escherichia coli outer membrane protein A signal peptide), on fluid domain connectivity in binary phosphatidylcholine mixtures.

Fluorescence-quenching study of percolation and compartmentalization in two-phase lipid bilayers.

Fluorescence quenching of a lipid-labeled fluorophore by a lipid spin-labeled quencher has been studied experimentally in two-component, two-phase phosphatidylcholine bilayers to examine the effect of phase connection and disconnection on quenching. Both fluorophore and quencher prefer the fluid phase. At the percolation threshold, the point at which the fluid phase becomes subdivided into may small disconnected domains, the quenching drops abruptly. This decrease in quenching is a function of the fluid-phase fraction and is due to the heterogeneous distribution of fluorophores and quenchers over the fluid-phase domains. Computer simulations of the system were carried out with a triangular lattice divided into closed compartments of variable size and reactant occupancy. The simulations demonstrate that the degree of quenching is reduced in the disconnected systems and that the reduction is correlated with the size of the disconnected domains. The combination of experimental data with simulations leads to the conclusion that at constant temperature the size of fluid-phase domains, nfluid, in the region of the coexistence of the fluid and gel phases is proportional to the fluid fraction, Xfluid. This is in a qualitative agreement with a previous electron spin resonance study of interlipid spin-spin interactions in the same two-component, two-phase bilayer system.

Can a single bacteriorhodopsin molecule change the structural state of one liposome?

Using ultrasonic velocity measurements the interaction of bacteriorhodopsin (BR) with large unilamellar liposomes of dipalmitoylphosphatidylcholine (DPPC) was studied in gel (25 degrees C) and in liquid crystalline state (50 degrees C) of lipid bilayer. We could show that with the increasing BR concentration the increment of ultrasonic velocity increases and a saturation occur at a BR/Liposomes of ratio approximately 0.5 mol/mol. BR incorporation into the lipid bilayer in gel state leads to an increase of the increment of the ultrasonic velocity of the lipid to 9.51 +/- 1.47 ml/mol. This could be mainly attributed to a decrease in membrane compressibility or an increase in membrane volume or both. No changes of ultrasonic velocity increment were observed with the membrane in liquid crystalline state. In this case, BR probably is not able to change the mechanical properties of a considerably disordered membrane.

Lipid domains and lipid/protein interactions in biological membranes.

In the fluid mosaic model of membranes, lipids are organized in the form of a bilayer supporting peripheral and integral proteins. This model considers the lipid bilayer as a two-dimensional fluid in which lipids and proteins are free to diffuse. As a direct consequence, both types of molecules would be expected to be randomly distributed within the membrane. In fact, evidences are accumulating to indicate the occurrence of both a transverse and lateral regionalization of membranes which can be described in terms of micro- and macrodomains, including the two leaflets of the lipid bilayer. The nature of the interactions responsible for the formation of domains, the way they develop and the time- and space-scale over which they exist represent today as many challenging problems in membranology. In this report, we will first consider some of the basic observations which point to the role of proteins in the transverse and lateral regionalization of membranes. Then, we will discuss some of the possible mechanisms which, in particular in terms of lipid/protein interactions, can explain lateral heterogenities in membranes and which have the merit of providing a thermodynamic support to the existence of lipid domains in membranes.

Hydrophobic mismatch and long-range protein/lipid interactions in bacteriorhodopsin/phosphatidylcholine vesicles.

Mismatch between the hydrophobic thicknesses of transmembrane proteins and the supporting lipid bilayer and its consequences on the lateral organization of lipids have been investigated with bacteriorhodopsin and phosphatidylcholine species with a variety of acyl-chain lengths. The purple membrane, from the bacterium Halobacterium halobium, was used and reconstituted with dilauroyl-(Lau2GroPCho), dimyristoyl- (Myr2GroPCho), dipalmitoyl- (Pam2GroPCho) and distearoyl- (Ste2GroPCho) glycerophosphocholine. The phase behaviour of the lipids was investigated at different temperatures and different protein/lipid molar ratios, by analyzing the fluorescence excitation spectra of the 1-acyl-2-[8-(2-anthroyl)-octanoyl]-sn-glycero-3-phosphocholine probe, and by measuring the fluorescence depolarization of the 1,6-diphenyl-1,3,5-hexatriene probe. Data obtained with 1-acyl-2-[8-(2-anthroyl)-octanoyl]-sn-glycero-3-phosphocholine shows that bacteriorhodopsin produced positive or negative shifts in the phase transition temperature of the host lipids depending on the strength and sign of the mismatch between the lipid and protein hydrophobic thicknesses and also on the protein concentration and aggregation state in the lipid bilayer. In the region of high protein concentration (bacteriorhodopsin/phosphatidylcholine molar ratios approximately 1:50) and despite the presence of the endogenous lipids, bacteriorhodopsin (hydrophobic length dP approximately 3.0-3.1 nm) brought about a large upward shift in the phase-transition temperature of Lau2GroPCho (delta T approximately 40 K, mean hydrophobic thickness d approximately 2.4 nm), and to a lesser extent of Myr2GroPCho (delta T approximately 23 K, d approximately 2.8 nm), accounting for a strong rigidifying effect of the protein on these short-chain lipids. Bacteriorhodopsin had no influence on the phase properties of Pam2GroPCho (delta T approximately 0 K, d approximately 3.2 nm), a lipid whose mean hydrophobic thickness is similar to that of the protein. In contrast, the transition temperature of Ste2GroPCho was decreased (delta T approximately -13 K, d approximately 3.7 nm), indicating a fluidifying effect of the protein on this long-chain lipid. Similar effects on the lipid acyl-chain order were observed in the region of high-protein dilution (bacteriorhodopsin/phosphatidylcholine molar ratios < 1:500). In this region and for Lau2GroPCho, both the spectroscopic data and circular-dichroism spectra indicated that the protein was in the monomeric form. Phase diagrams, in temperature versus bacteriorhodopsin concentration, were constructed for Lau2GroPCho and Ste2GroPCho. On account of microscopic theoretical models and of the relative values of dP and d, these diagrams indicate a preference of the protein for those lipid molecules which are in the gel-ordered state in Lau2GroPCho but in the liquid disordered state in Ste2GroPCho.(ABSTRACT TRUNCATED AT 400 WORDS)

Thermodynamical characteristics and volume compressibility of dipalmitoylphosphatidylcholine liposomes containing bacteriorhodopsin.

The effects of bacteriorhodopsin (BR) interaction with large dipalmitoylphosphatidylcholine (DPPC) liposomes (approx. 100 nm in diameter) were examined at various BR/DPPC ratios, using differential scanning calorimetry (DSC) and ultrasonic velocimetry (USV). On DSC, the lipid phase transition temperature, Tc, and the half-width of the phase transition peak, delta T1/2, showed significant non-monotonic changes with the increasing BR concentration. Two exponential segments could be distinguished in the dependence of the transition enthalpy change per mol of lipid (delta H/nL) on the BR/DPPC ratio: one corresponding to ratios between 0:1 and 1:64, and another corresponding to ratios between 1:44 and 1:16. A maximal value of delta H/nL was observed for BR/DPPC ratio 1:44, probably corresponding to maximal BR-lipid ordering with each BR molecule being surrounded by two layers of lipid molecules. The nonmonotonic changes of thermodynamical parameters suggest long-distance interactions between regions of altered bilayer structure which form around each BR molecule. The results obtained with USV provided support for the above conclusions. The dependence of ultrasound velocity increment A on BR concentration supplies information on relative changes of membrane volume compressibility. Decreasing volume compressibility is reflected in increasing values of parameter A. Within T less than Tc, the values of A increased with the increasing BR concentration; saturation was observed at BR/DPPC ratio 1:500 (A = A(BR/DPPC]. No significant BR-concentration dependent changes of A were observed at T greater than Tc. From these values the average diameter of the distorted region of lipid bilayer was estimated to be approximately 20 nm.