Aggregation, fusion, and vesicle formation of modified low density lipoprotein particles: molecular mechanisms and effects on matrix interactions

Initiation of atherosclerosis is characterized by accumulation of aggregates of small lipid droplets and vesicles in the extracellular matrix of the arterial intima. The droplets and vesicles have features that suggest that they are formed from modified plasma-derived low density lipoprotein (LDL) particles. A variety of hydrolytic enzymes and prooxidative agents that could lead to extracellular assembly of LDL-derived droplets and vesicles are present in the arterial intima. In fact, in vitro studies have demonstrated that extensive oxidation of LDL and treatment of LDL with either proteolytic or lipolytic enzymes will induce LDL aggregation and fusion and treatment of LDL with cholesterol esterase will cause formation of vesicles. Fusion of LDL particles proceeds faster in vitro when they are bound to components of the extracellular matrix derived from the arterial intima, such as proteoglycans, and, depending on the type of modification, the strength of binding of modified LDL to the matrix components may either increase or decrease. In the present article, we discuss molecular mechanisms that provide clues as to how aggregated lipid droplets and vesicles may be derived from modified LDL particles. We also describe how these modified forms of LDL, by means of their trapping to the extracellular matrix, may lead to extracellular lipid accumulation in the arterial intima.     

Altered phospholipid-apoB-100 interactions and generation of extra membrane material in proteolysis-induced fusion of LDL particles

Lipid droplets and membrane material are produced in the extracellular matrix of the arterial intima during atherogenesis. Both in vitro and in vivo experimentation suggests that fusion of modified LDL particles leads to formation of such lipid droplets. Here we applied proton NMR spectroscopy to probe surface phospholipids phosphatidylcholine (PC) and sphingomyelin (SM) of LDL particles during proteolytic degradation of apolipoprotein B-100 (apoB-100). Initiation of apoB-100 degradation was accompanied by the abruptly increased intensity of the choline -N(CH(3))(3) resonance of PC molecules, indicating disruption of their interactions with apoB-100. However, subsequent particle fusion was accompanied by a steady decrease in the intensity of the choline resonances of both PC and SM. Electron microscopy of the proteolyzed LDL revealed irregularly shaped multilamellar membranes attached to aggregates of fused particles. This suggests formation of membrane material with low hydration, in which some of the atomic motions are hindered. Characterization of the behavior of the surface lipids of LDL particles during apoB-100 degradation and other types of LDL modification will aid in understanding molecular mechanisms leading to fusion and generation of multilamellar membrane material in the arterial intima during atherogenesis.     

Generation in Human Plasma of Misfolded, Aggregation-Prone Electronegative Low Density Lipoprotein

Human plasma contains small amounts of a low density lipoprotein in which apoprotein is misfolded. Originally identified and isolated by means of anion-exchange chromatography, this component was subsequently described as electronegative low density lipoprotein (LDL)(−), with increased concentrations associated with elevated cardiovascular disease risk. It has been recognized recently as the trigger of LDL amyloidogenesis, which produces aggregates similar to subendothelial droplets observed in vivo in early atherogenesis. Although LDL(−) has been produced in vitro through various manipulations, the mechanisms involved in its generation in vivo remain obscure. By using a more physiological model, we demonstrate spontaneous, sustained and noticeable production of LDL(−) during incubation of unprocessed human plasma at 37°C. In addition to a higher fraction of amyloidogenic LDL(−), LDL purified from incubated plasma contains an increased level of lysophospholipids and free fatty acids; analysis of LDL lipids packing shows their loosening. As a result, during plasma incubation, lipid destabilization and protein misfolding take place, and aggregation-prone particles are generated. All these phenomena can be prevented by inhibiting calcium-dependent secretory phospholipases A2. Our plasma incubation model, without removal of reaction products, effectively shows a lipid-protein interplay in LDL, where lipid destabilization after lipolysis threatens the apoprotein's structure, which misfolds and becomes aggregation-prone.     

Effects of oxidation on the structure and stability of human low-density lipoprotein

Oxidation of low-density lipoprotein (LDL), the major cholesterol carrier in plasma, is thought to promote atherogenesis via several mechanisms. One proposed mechanism involves fusion of oxidized LDL in the arterial wall; another involves oxidation-induced amyloid formation by LDL apolipoprotein B. To test these mechanisms and to determine the effects of oxidation on the protein secondary structure and lipoprotein fusion in vitro, we analyzed LDL oxidized by nonenzymatic (Cu2+, H2O2, and HOCl) or enzymatic methods (myeloperoxidase/H2O2/Cl- and myeloperoxidase/H2O2/NO2-). Far-UV circular dichroism spectra showed that LDL oxidation induces partial unfolding of the secondary structure rather than folding into cross-beta amyloid conformation. This unfolding correlates with increased negative charge of oxidized LDL and with a moderate increase in thioflavin T fluorescence that may result from electrostatic attraction between the cationic dye and electronegative LDL rather than from dye binding to amyloid. These and other spectroscopic studies of low- and high-density lipoproteins, which encompass amyloid-promoting conditions (high protein concentrations, high temperatures, acidic pH), demonstrate that in vitro lipoprotein oxidation does not induce amyloid formation. Surprisingly, turbidity, near-UV circular dichroism, and electron microscopic data demonstrate that advanced oxidation inhibits heat-induced LDL fusion that is characteristic of native lipoproteins. Such fusion inhibition may result from the accumulation of anionic lipids and lysophospholipids on the particle surface and/or from protein cross-linking upon advanced lipoprotein oxidation. Consequently, oxidation alone may prevent rather than promote LDL fusion, suggesting that additional factors, such as albumin-mediated removal of lipid peroxidation products and/or LDL binding to arterial proteoglycans, facilitate fusion of oxidized LDL in vivo.     

Structural basis for thermal stability of human low-density lipoprotein

The stability of human low-density lipoprotein (LDL), the major cholesterol carrier in plasma, was analyzed by heating samples of different concentrations at a rate from 11 to 90 K/h. Correlation of the calorimetric, circular dichroism, fluorescence, turbidity, and electron microscopic data shows that thermal disruption of LDL involves irreversible changes in the particle morphology and protein conformation but no global protein unfolding. Heating to 85 degrees C induces LDL conversion into smaller and larger particles and apparent partial dissociation, but not unfolding, of its sole protein, apoB. Further heating leads to partial unfolding of the beta-sheets in apoB and to fusion of the protein-depleted LDL into large aggregated lipid droplets, resulting in a previously unidentified high-temperature calorimetric peak. These lipid droplets resemble in size and morphology the extracellular lipid deposits formed in the arterial wall in early atherosclerosis. The strong concentration dependence of LDL fusion revealed by near-UV/visible CD, turbidity, and calorimetry indicates high reaction order, and the heating rate dependence suggests high activation energy that arises from transient disruption of lipid and/or protein packing interactions in the course of particle fusion and apparent apoB dissociation. Consequently, thermal stability of LDL is modulated by kinetic barriers. Similar barriers may confer structural integrity to LDL subclasses in vivo.     

Effects of phospholipase A(2) and its products on structural stability of human LDL: relevance to formation of LDL-derived lipid droplets

Hydrolysis and oxidation of LDL stimulate LDL entrapment in the arterial wall and promote inflammation and atherosclerosis via various mechanisms including lipoprotein fusion and lipid droplet formation. To determine the effects of FFA on these transitions, we hydrolyzed LDL by phospholipase A(2) (PLA(2)), removed FFA by albumin, and analyzed structural stability of the modified lipoproteins. Earlier, we showed that heating induces LDL remodeling, rupture, and coalescence into lipid droplets resembling those found in atherosclerotic lesions. Here, we report how FFA affect these transitions. Circular dichroism showed that mild LDL lipolysis induces partial β-sheet unfolding in apolipoprotein B. Electron microscopy, turbidity, and differential scanning calorimetry showed that mild lipolysis promotes LDL coalescence into lipid droplets. FFA removal by albumin restores LDL stability but not the protein conformation. Consequently, FFA enhance LDL coalescence into lipid droplets. Similar effects of FFA were observed in minimally oxidized LDL, in LDL enriched with exogenous FFA, and in HDL and VLDL. Our results imply that FFA promote lipoprotein coalescence into lipid droplets and explain why LDL oxidation enhances such coalescence in vivo but hampers it in vitro. Such lipid droplet formation potentially contributes to the pro-atherogenic effects of FFA.     

Cysteine protease cathepsin F is expressed in human atherosclerotic lesions, is secreted by cultured macrophages, and modifies low density lipoprotein particles in vitro

During atherogenesis, low density lipoprotein (LDL) particles in the arterial intima become modified and fuse to form extracellular lipid droplets. Proteolytic modification of apolipoprotein (apo) B-100 may be one mechanism of droplet formation from LDL. Here we studied whether the newly described acid protease cathepsin F can generate LDL-derived lipid droplets in vitro. Treatment of LDL particles with human recombinant cathepsin F led to extensive degradation of apoB-100, which, as determined by rate zonal flotation, electron microscopy, and NMR spectroscopy, triggered both aggregation and fusion of the LDL particles. Two other acid cysteine proteases, cathepsins S and K, which have been shown to be present in the arterial intima, were also capable of degrading apoB-100, albeit less efficiently. Cathepsin F treatment resulted also in enhanced retention of LDL to human arterial proteoglycans in vitro. Cultured monocyte-derived macrophages were found to secrete active cathepsin F. In addition, similarly with cathepsins S and K, cathepsin F was found to be localized mainly within the macrophage-rich areas of the human coronary atherosclerotic plaques. These results suggest that proteolytic modification of LDL by cathepsin F may be one mechanism leading to the extracellular accumulation of LDL-derived lipid droplets within the proteoglycan-rich extracellular matrix of the arterial intima during atherogenesis.     

Oxidative cross-linking of ApoB100 and hemoglobin results in low density lipoprotein modification in blood. Relevance to atherogenesis caused by hemodialysis.

Human blood contains a form of minimally modified low density lipoprotein (LDL), termed LDL-, whose origin remains unknown. Exploring the mechanism of formation, we found that LDL- can be produced in plasma in the absence of oxygen following LDL incubation with oxidized hemoglobin species. A high degree of apolipoprotein B100 modification results from covalent association of hemoglobin with LDL involving dityrosine formation but not due to the malonaldehyde epitope formation. This was evidenced by the cross-reactivity of oxidized LDL with antibodies against hemoglobin that was accompanied by a 60-fold increase in dityrosine levels. In this study we found significantly higher LDL- levels in the blood of hemodialysis patients, perhaps contributing to their greatly increased risk of atherosclerosis. The mechanism of LDL- formation was studied during ex vivo blood circulation using a model system resembling clinical hemodialysis in terms of the induction of inflammatory responses. This circulation increased free hemoglobin and LDL- levels compared with non-circulated blood without appreciable lipid peroxidation. Pronounced increases in LDL- were found also during circulation of plasma supplemented with nanomolar hemoglobin levels. The increase in dityrosine content and presence of heme in LDL after blood circulation suggest that LDL is modified, in part, by hemoglobin-LDL conjugates containing heme. Thus, hemoglobin-mediated reactions leading to LDL oxidation in plasma can account for high LDL- levels in hemodialysis patients.     

Effects of triacylglycerol on the structural remodeling of human plasma very low- and low-density lipoproteins

Very low-density lipoprotein (VLDL) is the main plasma carrier of triacylglycerol that is elevated in pathological conditions such as diabetes, metabolic syndrome, obesity and dyslipidemia. How variations in triacylglycerol levels influence structural stability and remodeling of VLDL and its metabolic product, low-density lipoproteins (LDL), is unknown. We applied a biochemical and biophysical approach using lipoprotein remodeling by lipoprotein lipase and cholesterol ester transfer protein, along with thermal denaturation that mimics key aspects of lipoprotein remodeling in vivo. The results revealed that increasing the triacylglycerol content in VLDL promotes changes in the lipoprotein size and release of the exchangeable apolipoproteins. Similarly, increased triacylglycerol content in LDL promotes lipoprotein remodeling and fusion. These effects were observed in single-donor lipoproteins from healthy subjects enriched in exogenous triolein, in single-donor lipoproteins from healthy subjects with naturally occurring differences in endogenous triacylglycerol, and in LDL and VLDL from pooled plasma of diabetic and normolipidemic patients. Consequently, triacylglycerol-induced destabilization is a general property of plasma lipoproteins. This destabilization reflects a direct effect of triacylglycerol on lipoproteins. Moreover, we show that TG can act indirectly by increasing lipoprotein susceptibility to oxidation and lipolysis and thereby promoting the generation of free fatty acids that augment fusion. These in vitro findings are relevant to lipoprotein remodeling and fusion in vivo. In fact, fusion of LDL and VLDL enhances their retention in the arterial wall and, according to the response-to-retention hypothesis, triggers atherosclerosis. Therefore, enhanced fusion of triacylglycerol-rich lipoproteins suggests a new causative link between elevated plasma triacylglycerol and atherosclerosis.     

LDL phospholipid hydrolysis produces modified electronegative particles with an unfolded apoB-100 protein

Electronegative low density lipoprotein (LDL(-)) formation that structurally resembles LDL(-) isolated from plasma was evaluated after LDL treatment with snake venom phospholipase A(2) (PLA(2)). PLA(2) treatment of LDL increased its electrophoretic mobility in proportion to the amount of LDL(-) formed without evidence of lipid peroxidation. These changes dose-dependently correlated with the degree of phospholipid hydrolysis. Strong immunoreactivity of LDL(-) subfraction from plasma and PLA(2)-treated LDL (PLA(2)-LDL) to amyloid oligomer-specific antibody was observed. Higher beta-strand structural content and unfolding proportionate to the loss of alpha-helical structure of apolipoprotein B-100 (apoB-100) of LDL(-) isolated from both native and PLA(2)-LDLs was demonstrated by circular dichroism (CD) spectropolarimetry. These structural changes resembled the characteristics of some oxidatively modified LDLs and soluble oligomeric aggregates of amyloidogenic proteins. PLA(2)-LDL was also more susceptible to nitration by peroxynitrite, likely because of exposure of otherwise inaccessible hydrophilic and hydrophobic domains arising from apoB-100 unfolding. This was also demonstrated for plasma LDL(-). In contrast, PLA(2)-LDL was more resistant to copper-mediated oxidation that was reversed upon the addition of small amounts of unsaturated fatty acids. The observed similarities between PLA(2)-LDL(-)-derived LDL(-) and plasma LDL(-) implicate a role for secretory PLA(2) in producing modified LDL(-) that is facilitated by unfolding of apoB-100.     

Altered ultrastructural morphology of self-aggregated low density lipoproteins: coalescence of lipid domains forming droplets and vesicles

Lipid droplets and vesicles can presumably be formed directly from lipoproteins in the extracellular space in atherosclerosis, but an in vitro demonstration of the phenomenon in the absence of cellular pathways has been lacking. Low density lipoproteins (LDL) are known to undergo self-aggregation after brief vortexing in vitro. In the present study, LDL aggregates were examined by electron microscopy, using new mordant techniques for lipid visualization, and by chemical analysis. Aggregation of LDL by vortexing is regularly accompanied by the formation of comparatively large lipid droplets (up to 600 nm diameter) and vesicles. Aggregates containing droplets and vesicles were formed after as little as 5 sec of vortexing, and LDL protein and cholesteryl ester were almost completely (95%) incorporated into aggregates after 4 min vortexing. Substantial fractions of phospholipid and unesterified cholesterol from the original LDL remained in solution even after 4 min vortexing, forming large multilamellar vesicles that did not adhere to the aggregated material. Spontaneous aggregates retrieved from LDL solutions after prolonged storage were also examined by electron microscopy, revealing similar lipid droplets and vesicles. The ultrastructural appearance of LDL aggregated in vitro is remarkably similar to the appearance of extracellular lipid deposits in atherosclerosis, lending credence to the hypothesis of direct extracellular formation of these deposits from lipoproteins.     

Postprandial oxidative stress

Consumption of a meal containing oxidized and oxidizable lipids gives rise to an increased plasma concentration of lipid hydroperoxides, detectable by a sensitive chemiluminescence procedure. This is associated with increased susceptibility of LDL to oxidation, apparently due a structural perturbation at the particle surface brought about by lipid oxidation products. The postprandial modification of LDL is at least partially accounted for by an increase of LDL-, a subfraction containing lipid oxidation products where apoprotein-B-100 (apoB-100) is denatured. Consuming the meal with a suitable source of antioxidants, such as those found in red wine, minimizes this postprandial oxidative stress. The inhibition of peroxidation of lipids present in the meal during digestion is a possible mechanism for the observed protection of LDL. The in vivo oxidatively modified LDL- has numerous features that correspond to the atherogenic minimally modified LDL produced in vitro. These modified particles could account for a relevant link between nutrition and early biological processes that foster the development of atherosclerosis.     

Lipoprotein aggregation as an essential condition of intracellular lipid accumulation caused by modified low density lipoproteins

We have tested a hypothesis that aggregates of modified low density lipoproteins (LDL) play the key role in the accumulation of lipids by cells of unaffected aortic intima. It was demonstrated using analysis of relative dispersion of light transmission fluctuations as well as gel filtration on Sepharose CL-2B that LDL modified by oxidation, glycosylation, desialylation and malondialdehyde treatment form aggregates under the conditions of culture. Native LDL failed to aggregate under the same conditions. It was demonstrated that modified LDL, unlike native LDL, bring about a 2- to 3-fold rise in cholesteryl ester levels of cultured cells. Moreover, direct and strong correlation (r = 0.86) was observed between the degree of lipoprotein aggregation and the amount of cholesteryl esters accumulated. Removal of modified LDL aggregates by filtration through a 0.1 micron filter or gel filtration completely prevented the intracellular accumulation of cholesteryl esters. These findings indicate that LDL aggregates play an essential, if not the decisive, role in the intracellular accumulation of lipids in vitro.     

Macrophages actively accumulate malonyldialdehyde-modified but not enzymatically oxidized low density lipoprotein

In this study, we show that low density lipoproteins (LDL) from human blood plasma which was oxidized by animal C-15 lipoxygenase is taken up by cultivated human macrophages with the same effectiveness as with non-oxidized (native) LDL. At the same time malonyldialdehyde-modified LDL is captured by cultivated macrophages very actively. Based on differences in catabolism of LDL with various levels of primary and secondary products of free-radical oxidation, it was offered to discriminate between the oxidized LDL itself (lipohydroperoxide-rich LDL) and the LDL that was chemically modified by free-radical oxidation secondary products of aldehyde nature. In this respect, aldehyde-modified but not oxidized (lipohydroperoxide-containing) LDL is atherogenic.     

Effects of hypochlorite-modified low-density and high-density lipoproteins on intracellular Ca2+ and plasma membrane Ca(2+)-ATPase activity of human platelets

The presence of hypochlorite-modified lipoproteins in atherosclerotic lesions suggests that HOCl, a naturally occurring oxidant formed by the myeloperoxidase-catalyzed reaction of H2O2 and Cl-, is a candidate for generation of modified lipoproteins in vivo. We have previously demonstrated that Cu(2+)-oxidized LDL inhibits platelet plasma membrane Ca(2+)-ATPase (PMCA) in isolated membranes and causes an increase in cytosolic Ca2+ in resting whole platelets. However, Cu(2+)-oxidized LDL may not be identical in structure and function to the physiologically modified lipoprotein. Since platelet function may be affected by native and modified lipoproteins, the effect of HOCl-modified LDL and HDL3 on platelet PMCA and on the free intracellular Ca2+ concentration ([Ca2+]i) of whole platelets has been investigated. We demonstrate that in contrast to Cu(2+)-oxidized LDL, HOCl-modified LDL and HDL3 stimulate platelet PMCA activity in isolated membranes and that this effect results in a decrease of [Ca2+]i in vivo. Thus, HOCl-oxidation produces modified lipoproteins with the potential for altering platelet function and with properties different from those of the Cu(2+)-oxidized counterparts.     

Characterization of low density lipoprotein binding to human adipocytes and adipocyte membranes

125I-labeled low density lipoprotein (LDL) binding to purified plasma membranes prepared from freshly isolated human adipocytes was saturable, specific, and displaceable by unlabeled ligand. The maximum specific binding capacity measured at saturating concentrations of 125I-LDL was 1.95 +/- 1.17 micrograms of LDL bound/mg of membrane protein (mean +/- S.D., n = 16). In contrast to cultured fibroblasts, specific binding of LDL to adipocyte membranes was calcium-independent, was not affected by EDTA or NaCl, and was not destroyed by pronase. Plasma membranes purified directly from homogenized adipose tissue also showed calcium-independent LDL specific binding (0.58 +/- 0.33 micrograms of LDL bound/mg of membrane protein, mean +/- S.D. n = 11). Specific binding, internalization, and degradation of 125I-methylated LDL was demonstrated in isolated adipocytes and competition experiments showed that native and methylated LDL interacted with adipocytes through some common recognition mechanism(s). Compared to native LDL, specific binding of methylated LDL to adipocyte membranes was significantly reduced (43%), indicating that interaction of LDL with adipocyte was dependent in part on the lysine residues of apolipoprotein B. LDL binding to adipocyte plasma membranes was also competitively inhibited by human high density lipoprotein subfractions HDL2 and HDL3. Thus, LDL metabolism in mature adipocytes appears to be regulated by mechanisms distinctly different from a variety of cultured mesenchymal cells. In addition, the ability of adipocytes to bind, internalize, and degrade significant amounts of methylated LDL supports the view that adipose tissue is involved in the metabolism of modified lipoproteins in vivo.     

Atherosclerosis: another protein misfolding disease?

The secondary structure and conformation of apo-B 100 in low-density lipoproteins (LDL) are imposed by lipid-protein interactions and dynamics, and affected by the introduction or removal of lipids during the course of lipoprotein metabolism. Following an alteration of the water-lipid interface as a result of, for example, oxidation of lipids, the supramolecular structure becomes destabilized and apoB can misfold. These events have been observed in LDL(-), a fraction of oxidatively modified LDL isolated in vivo. This modified lipoprotein possesses several atherogenic properties and represents an in vivo counterpart of in vitro modified LDL that is implicated in atherosclerosis. The misfolding of apoB, its aggregation, resistance to proteolysis, and cytotoxicity are common motifs shared by LDL(-) and amyloidogenic proteins. Based on these analogies, we propose that atherogenesis could be considered as a disease produced by the accumulation of cytotoxic and pro-inflammatory misfolded lipoproteins.     

Lipoproteins of the extravascular space: enhanced macrophage degradation of low density lipoproteins from interstitial inflammatory fluid

Current evidence has demonstrated that cholesteryl ester-loaded macrophages are important components of the atherosclerotic lesion. Additional studies have implicated low density lipoproteins (LDL) and circulating monocytes as central to the origin of lipid-laden foam cells found in the arterial wall. This is a result of the finding of accelerated macrophage uptake of LDL chemically modified by reaction with malondialdehyde (MDA-LDL), acetic anhydride (Ac-LDL), or incubation with arterial cells in vitro. In concert with these chemical modifications, we have previously demonstrated selective in vivo modification of LDL isolated from interstitial inflammatory fluid (IF) of the rabbit. Utilizing the polyvinyl sponge implant model, we reported that IF-LDL had an altered chemical composition, electrophoretic mobility, and particle size distribution when compared to LDL isolated from homologous plasma (WP-LDL). In this study reported herein, we examined the metabolism of IF-LDL by resident mouse peritoneal macrophages (MPM) in culture. IF-LDL was degraded substantially faster by MPM, and resulted in a substantial increase in cellular cholesteryl ester when compared to cells incubated with WP-LDL. IF-LDL binding to MPM was inhibited by Ac-LDL derived from WP-LDL, but only minimally by unmodified WP-LDL. Transmission electron microscopy of MPM revealed extensive lipid deposition in cells incubated with Ac-LDL and IF-LDL. These results implicate LDL from interstitial inflammatory fluid as an in vivo modified lipoprotein that can enhance uptake via the acetyl LDL receptor pathway in resident macrophages.     

Plasma very-low-density lipoprotein, low-density lipoprotein, and high-density lipoprotein oxidative modification induces procoagulant profiles in endogenous hypertriglyceridemia

This study was to investigate whether oxidatively modified lipoproteins were associated with changes of pro- and anticoagulant profiles in hypertriglyceridemic subjects. Plasma VLDL, LDL, and HDL were isolated with the one-step density gradient ultracentrifugation method. The oxidation of the lipoproteins was identified. Prothrombin time (PT) and activated partial thrombplastin time (APTT), tissue plasminogen activator and plasminogen activator inhibitor-1, and platelet aggregation rate were determined with a reaction system consisting of mixed fresh normal plasma, in endogenous hypertriglyceridemic (HTG) patients, in in vitro modified lipoproteins from a normolipidemic donor, and in experimental rats. The results indicated that oxVLDL, oxLDL, and oxHDL occurred in the plasma of HTG patients. Compared with the control group, PT and APTT, incubated with plasma VLDL, LDL, or HDL from HTG patients, respectively, were significantly reduced, while platelet maximal aggregation rates were significantly higher (P < 0.05-0.01). Similar procoagulant profiles were observed in in vitro modified lipoprotein components and in rats with intrinsic hypertriglyceridemia as well. These results support our previous finding that LDL, VLDL, and HDL were all oxidatively modified in vivo in the subjects with HTG, and suggest that procoagulation state may result from the abnormal plasma lipoprotein oxidative modification in vivo.     

Proteins in membrane mimetic systems. Insertion of myelin basic protein into microemulsion droplets.

The insertion of myelin basic protein into microemulsion droplets of sodium bis (2-ethylhexyl) sulfosuccinate (AOT) has been studied by quasi-elastic light scattering. Measurements were made at both low and high molar ratios of water to surfactant, as a function of protein occupancy. The hydrodynamic radii of filled and empty droplets were experimentally evaluated. These were compared to values calculated using a water shell model of protein encapsulation, and excellent agreement was obtained. At low molar ratio of water to surfactant (w0 = 5.6), the hydrodynamic radius of filled droplets is significantly larger than the radius of empty ones. Under these conditions, about three empty (water-filled) droplets are required to build up a droplet of sufficient size to accommodate a single protein molecule. At maximum solubilization, which occurs at w0 = 5.6, a small fraction of droplets are found containing protein aggregates. In contrast, results at high values of w0 (22.4) reveal radii for empty and occupied droplets of comparable dimension, and the absence of aggregates. The results are discussed in terms of the model and the mechanism of interaction of this protein with the aqueous interfaces provided by these membrane-mimetic systems.