Lupus antibody bivalency is required to enhance prothrombin binding to phospholipid

Lupus anticoagulants (LA) are a family of autoantibodies that are associated with in vitro anticoagulant activity but a strong predisposition to in vivo thrombosis. They are directed against plasma phospholipid-binding proteins including prothrombin. We have proposed that LA propagates coagulation in flowing blood by facilitating prothrombin interaction with the damaged blood vessel wall. A murine monoclonal anti-prothrombin Ab and three of three LA IgGs enhanced prothrombin binding to 75:25 phosphatidyl choline:phosphatidyl serine vesicles measured by either ultracentrifugation or right-angle light scattering. The assembly of prothrombin and LA IgG on phospholipid vesicles was estimated by surface plasmon resonance. The on rates for prothrombin and LA IgG were approximately the same as the on rate for prothrombin alone. In contrast, the off rates for prothrombin and LA IgG were 2- to 3-fold slower than the off rate for prothrombin. LA IgG bivalency was required for enhanced prothrombin binding to phospholipid vesicles, as Fab of the LA IgGs did not influence prothrombin binding at concentrations up to 40 microM. Modeling of the interactions of prothrombin, LA IgG and phospholipid vesicles indicated that augmentation of prothrombin binding to phospholipid vesicles by LA IgG could be accounted for by the bivalency of the LA IgG and the elevated microenvironmental concentration of prothrombin on the surface of phospholipid vesicles.     

Binding of lipopolysaccharide and complexes of lipopolysaccharide to serum low density lipoproteins to liver macrophages

Binding of [³H]-lipopolysaccharide toxin (LPS) and complexes of LPS with serum [¹²⁵I]-labeled low density lipoproteins (LDL) to primary culture of rat liver macrophages (Kupffer cells) has been studied. Total, specific and nonspecific binding was determined. The receptor interaction was shown to dominate for both LPS and LDL-LPS complexes, representing 70–77% and 80–85%, respectively. The Scatchard plot was essentially non-linear for LPS binding but linear for the LDL-LPS complexes. At the Scatchard graph of LPS binding, however, two regions approximately fitting the linear regression could be identified. These regions correspond to two different types of specific binding sites: the first is for lower toxin concentrations of 0.25–0.50 μg/ml with K _d = 0.75 μg/ml; while the second is for higher LPS concentrations of 7.5–15 μg/ml with K _d = 5.39 μg/ml. For LDL-LPS complexes only K _d of 2.80 μg/ml was obtained. The LDL-LPS complexes significantly blocked the LPS binding (?40%) while acetylated or oxidized LDLs exerted a less pronounced effect. LPS inhibited binding of LDL-LPS complexes (?60%), while acetylated or oxidized LDLs suppressed interaction of LDL-LPS complexes with Kupffer cells insignificantly. It is suggested that, while binding to the Kupffer cell surface, a substantial portion of both LPS and LDL-LPS complexes share the same scavenger receptors with which, however, modified LDLs interact weakly. The LDL-LPS complexes can interact, apart from receptors common with LPS, with other receptors exhibiting similar binding parameters, with the apo-B/E receptors playing an inessential role.     

Morphological studies on the binding of low-density lipoproteins and acetylated low-density lipoproteins to the plasma membrane of cultured monocytes

Binding of low density lipoproteins (LDL) and acetyl-LDL to the plasma membrane of cultured swine monocytes was investigated by immunofluorescent and immunoelectron microscopy. Binding sites for native LDL, visualized on both the light microscopical and the ultrastructural level, were found to be comparable to those of cultured human fibroblasts. These techniques, however, failed to reveal binding of acetyl-LDL to the cell surface. Biochemical experiments showed that both LDL and acetyl-LDL have specific receptors, the acetyl-LDL receptor being distinctly different from the LDL receptor. It is concluded that there are morphological differences in the binding of LDL and acetyl-LDL to cultured monocytes. These differences are supported by biochemical data.     

Triacylglycerol and phospholipid hydrolysis in human plasma lipoproteins: role of lipoprotein and hepatic lipase.

To explore the interactions of triacylglycerol and phospholipid hydrolysis in lipoprotein conversions and remodeling, we compared the activities of lipoprotein and hepatic lipases on human VLDL, IDL, LDL, and HDL2. Triacylglycerol and phospholipid hydrolysis by each enzyme were measured concomitantly in each lipoprotein class by measuring hydrolysis of [14C]triolein and [3H]dipalmitoylphosphatidylcholine incorporated into each lipoprotein by lipid transfer processes. Hepatic lipase was 2-3 times more efficient than lipoprotein lipase at hydrolyzing phospholipid both in absolute terms and in relation to triacylglycerol hydrolysis in all lipoproteins. The relationship between phospholipid hydrolysis and triacylglycerol hydrolysis was generally linear until half of particle triacylglycerol was hydrolyzed. For either enzyme acting on a single lipoprotein fraction, the degree of phosphohydrolysis closely correlated with triacylglycerol hydrolysis and was largely independent of the kinetics of hydrolysis, suggesting that triacylglycerol removed from a lipoprotein core is an important determinant of phospholipid removal via hydrolysis by the lipase. Phospholipid hydrolysis relative to triacylglycerol hydrolysis was most efficient in VLDL followed in descending order by IDL, HDL, and LDL. Even with hepatic lipase, phospholipid hydrolysis could not deplete VLDL and IDL of sufficient phospholipid molecules to account for the loss of surface phospholipid that accompanies triacylglycerol hydrolysis and decreasing core volume as LDL is formed (or for conversion of HDL2 to HDL3). Thus, shedding of whole phospholipid molecules, presumably in liposomal-like particles, must be a major mechanism for losing excess surface lipid as large lipoprotein particles are converted to smaller particles. Also, this shedding phenomenon, like phospholipid hydrolysis, is closely related to the hydrolysis of lipoprotein triacylglycerol.     

Altered thyroid hormone binding to plasma lipoproteins in the syndrome of resistance to thyroid hormones.

Lipoproteins, especially HDL, are carriers of a small fraction of the thyroid hormones in plasma and participate in the intracellular transport of T4. In previous work we showed that a brief period of hypothyroidism alters the hormone distribution among the lipoproteins, causing a decrease in VLDL and LDL binding despite a relative increase in VLDL and LDL cholesterol, an increase in HDL binding, and a reversal of T4 and T3 binding to the smallest HDL size subgroup. The present study of three patients with thyroid hormone resistance and largely compensated hypothyroidism showed thyroid hormone distribution that differed markedly from both normal and hypothyroid subjects. The most striking difference was a much lower binding of both T4 and T3 to HDL and a much higher binding to LDL. If confirmed in a larger group of patients, this might serve as a marker for thyroid hormone resistance.     

Acipimox reduces circulating levels of insulin and associated neutrophilic inflammation in metabolic syndrome

Metabolic syndrome is a proatherosclerotic condition clustering cardiovascular risk factors, including glucose and lipid profile alterations. The pathophysiological mechanisms favoring atherosclerotic inflammation in the metabolic syndrome remain elusive. Here, we investigated the potential role of the antilipolytic drug acipimox on neutrophil- and monocyte-mediated inflammation in the metabolic syndrome. Acipimox (500 mg) was orally administered to metabolic syndrome patients (n = 11) or healthy controls (n = 8). Serum and plasma was collected before acipimox administration (time 0) as well as 2-5 h afterward to assess metabolic and hematologic parameters. In vitro, the effects of the incubation with metabolic syndrome serum were assessed on human neutrophil and monocyte migration toward the proatherosclerotic chemokine CCL3. Two to five hours after acipimox administration, a significant reduction in circulating levels of insulin and nonesterified fatty acid (NEFA) was shown in metabolic syndrome patients. At time 0 and 2 h after acipimox administration, metabolic syndrome serum increased neutrophil migration to CCL3 compared with healthy controls. No effect was shown in human monocytes. At these time points, serum-induced neutrophil migration positively correlated with serum levels of insulin and NEFA. Metabolic syndrome serum or recombinant insulin did not upregulate CCR5 expression on neutrophil surface membrane, but it increased intracellular JNK1/2 phosphorylation. Insulin immunodepletion blocked serum-induced neutrophil migration and associated JNK1/2 phosphorylation. Although mRNA expression of acipimox receptor (GPR109) was shown in human neutrophils, 5-500 μM acipimox did not affect insulin-induced neutrophil migration. In conclusion, results suggest that acipimox inhibited neutrophil proatherosclerotic functions in the metabolic syndrome through the reduction in circulating levels of insulin.     

Heparin binding to human plasma low-density lipoproteins: dependence on heparin sulfation degree and chain length.

Binding between low-density lipoproteins (LDL) and fluorescein-labeled heparin was studied quantitatively with a modified form of a published procedure [Cardin, A. D., Randall, C. I., Hirose, N., & Jackson, R. L. (1987) Biochemistry 26, 5513-5518], using fluorescence anisotropy titrations. Assumption of binding site equivalence satisfactorily interpreted experimental data. Accordingly, the apparent total capacity, n, and the average dissociation constant, Kd, were estimated as n approximately 24 disaccharides per LDL particle and Kd approximately 4 microM in 0.05 M HEPES/0.1 M NaCl, pH 7.4, 22 degrees C. Competition experiments with unlabeled heparins were exploited for the quantitative study of Kd as a function of heparin chain length and sulfation degree (ns = sulfate groups per disaccharide). The former parameter was investigated with a series of bovine lung heparin fractions with Mw ranging from 1,800 to 21,000 and constant sulfation degree (ns = 2.8 +/- 0.1). A series of physically fractionated or chemically modified heparins having 1.2 less than ns less than 3.5 were used to explore the dependence on sulfation degree. LDL affinity was found to increase with increasing both ns and Mw: an empirical Mw-1.6 dependence represented very well the chain length data set; a linear dependence was observed for log Kd as a function of ns, after appropriate allowance was made for chain length differences among samples. This regularity confirmed that LDL-heparin binding is mainly driven by electrostatic forces.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acute inhibition of hepatic lipase and increase in plasma lipoproteins after alcohol intake

Chronic alcohol intake is associated with an increase in fasting plasma high density lipoproteins (HDL). To study alcohol's acute effects on plasma lipoproteins, we measured plasma lipoprotein concentrations and activities of postheparin plasma lipases in nine normolipemic males after ingestion of 40 g of ethanol (as whiskey). After alcohol there was no change in lipoprotein lipase activity but hepatic lipase was decreased to 67% of baseline at 6 hr. There were associated increases in HDL phospholipids (12 mg/dl) and cholesterol (10 mg/dl) resulting in prominence of larger, lipid-enriched HDL particles. Changes were most pronounced in the HDL3 and HDL2a subclasses. Very low density lipoprotein (VLDL) phospholipids and cholesterol were also increased by 13 and 9 mg/dl, respectively, with no significant change in triglycerides. Changes in lipoproteins and lipase were largely reversed 10 hr after alcohol intake. The transient increases in VLDL and HDL lipids after alcohol may result in part from acute inhibition of hepatic lipase activity. The results suggest a role of hepatic lipase in the catabolism of phospholipids of VLDL and possibly HDL.     

Heparin binding to lipoprotein lipase and low density lipoproteins

Heparin was fractionated on an affinity column of bovine milk lipoprotein lipase (LpL) immobilized to Affi-Gel-15. The bound heparin, designated high-reactive heparin (HRH), enhanced LpL activity, presumably by stabilizing the enzyme against denaturation. The unbound heparin fraction had no observable effect on the initial rate of enzyme activity. However, at longer times of incubation there was inhibition of LpL activity. LpL-specific HRH also showed a high, Ca²⁺-dependent precipitating activity towards human plasma low density lipoproteins (LDL). Since LpL and LDL both bind to heparin-like molecules at the surface of the arterial wall, we suggest that their similar heparin-binding specificity may have physiological consequences as it relates to the development of atherosclerosis.

Heparin binding Lipoprotein lipase LDL Apolipoprotein Lipolysis     

The relationship of circulating plasma progesterone levels and rectal temperatures in gilts

To ascertain if a correlation exists between circulating plasma progesterone levels and rectal temperatures in swine, 10 mixed breed postpubertal ovariectomized gilts were treated with sesame oil containing 500 mg progesterone IM or with saline IM. The average daily rectal temperatures were not correlated with the circulating plasma progesterone levels. Rectal temperature observations do not provide a useful indication of corpus luteum activity in swine.     

IFN-gamma-inducible protein 10 and pentraxin 3 plasma levels are tools for monitoring inflammation and disease activity in Mycobacterium tuberculosis infection

IFN-gamma-inducible protein 10 (IP-10/CXCL10) is a chemokine involved in delayed-type hypersensitivity and attraction of monocytes and activated T lymphocytes at inflammatory foci, whereas pentraxin 3 (PTX3) is part of the innate immune response. In the Republic of Guinea, 220 newly diagnosed, HIV-negative, pulmonary tuberculosis (TB) patients were studied together with 220 healthy household controls and 220 community controls. CXCL10 and PTX3 blood levels were assessed by ELISA at diagnosis, after 2 months and at the end of treatment. In untreated patients, both CXCL10 and PTX3 levels were higher (P < 0.0001) than in controls, although household controls had higher (P < 0.0001) CXCL10 and PTX3 levels than community controls, but lower (P < 0.0001) than those of patients. At the end of treatment, 186 cured patients showed reduction (P < 0.0001) in both CXCL10 and PTX3 levels. In 34 patients with treatment failure, both CXCL10 and PTX3 levels increased further. In five previously healthy households who developed TB during the follow-up and in two patients who relapsed after treatment, a remarkable increase in both CXCL10 and PTX3 plasma levels was observed. Active TB is associated with increased CXCL10 and PTX3 levels in the plasma. Although not specific for TB, measurement of these proteins may help the monitoring of disease activity and efficacy of therapy.     

Enhanced binding of low and high density lipoproteins to human adipocyte plasma membranes: effects of temperature and proteases

The specific binding of 125I-labelled low density lipoprotein ([125I]LDL to human adipocyte plasma membranes was higher at 37 than at 0 degree C. Prior treatment of membranes with pronase had no effect on LDL binding measured at 0 degree C but consistently stimulated binding at 37 degrees C. Plasmin was similar to pronase in enhancing LDL-specific binding, but thrombin was not as effective. 125I-labelled high density lipoprotein ([125I]HDL2) specific binding to human adipocyte plasma membranes was similarly sensitive to temperature and pronase treatment. Addition of the protease inhibitor aprotinin in the adipocyte membrane binding assay significantly reduced [125I]LDL binding at 37 degrees C (p less than 0.05), suggesting the involvement of a protease activity intrinsic to the lipoproteins and (or) membranes. These data demonstrate that both LDL and HDL binding in human adipocyte plasma membranes can be "up-regulated" by specific proteolytic perturbations in a temperature-dependent manner.     

Mutations in Ralstonia solanacearum loci involved in lipopolysaccharide biogenesis,phospholipid trafficking and peptidoglycan recycling render bacteriophage infection

Ralstonia solanacearum causes deadly wilting on many crops worldwide. However, the information on its components important for cell integrity and interactions with phages is limited. By systematically characterizing mutants resistant to a T7-like phage, we showed that the biosynthesis of rough lipopolysaccharides (R-LPS) was crucial for maintaining the membrane integrity, while the production of smooth LPS (S-LPS) was required for the resistance to polymyxin B and phage adsorption. Furthermore, RSc0154/ampG disruption did not affect LPS production and phage adsorption but may have caused aberrant release of peptidoglycan fragments, thus hindering phage DNA injection into or virion release from the cell. Mutations in the RSc2958–RSc2962/mla cluster, although not affecting LPS production, may have caused elevated phospholipid level in the outer leaflet of the outer membrane, consequently sheltering the mutants from phage adsorption on the O-antigen. These results specify important roles of the biogenesis and homeogenesis of envelope components for R. solanacearum-phage interaction.     

Binding of high-density lipoproteins to luteal membranes: the role of prolactin, luteinizing hormone, and circulating lipoproteins

Ovarian and adrenal membranes from immature gonadotropin-primed rats, treated with 4-amino-pyrazolopyrimidine (4APP) to reduce endogenous lipoprotein levels, displayed higher binding of porcine high-density lipoprotein (HDL) when compared to control rats. Immature, hypophysectomized (HYPOX) rats bearing corpora lutea (CL) on Day 5 after ovulation had lower levels of serum progesterone and reduced capacity for HDL and human chorionic gonadotropin (hCG) binding to ovarian membranes when compared with intact animals. Hypophysectomy also reduced the number of HDL binding sites in adrenal membranes. Treatment of HYPOX animals with luteinizing hormone (LH) and prolactin (Prl) alone or in combination increased the HDL binding sites in the ovary relative to HYPOX-untreated rats. Neither hormone affected binding to adrenals, where only adrenocorticotropic hormone (ACTH) enhanced HDL binding. LH treatment reduced the serum progesterone levels and hCG binding to the ovaries, whereas Prl administration increased progesterone levels with no effect on hCG binding. We conclude from this study that HDL binding in the luteinized ovary is regulated by Prl and LH and circulating lipoproteins, whereas in adrenals it is regulated by ACTH and circulating levels of lipoproteins.