Circulating lipoprotein-associated phospholipase A2 in high-grade carotid stenosis: a new biomarker for predicting unstable plaque.

OBJECTIVE: To test plasma levels of lipoprotein-associated phospholipase A2 (Lp-PLA2) in patients with high-grade carotid stenosis according to plaque histology. METHODS: This cross-sectional single-centre study included patients with ≥70% North American Symptomatic Carotid Endarterectomy Trial (NASCET) carotid stenosis, who were treated surgically. Serum Lp-PLA2 and high-sensitivity C-reactive protein (hs-CRP) were determined on the day of surgery. Histopathological analysis classified carotid plaque as stable or unstable, according to AHA classification. RESULTS: Of the 42 patients (mean age 70.4 ± 10.5 years; 67% men), neurological symptoms were present in 16 (38%). Unstable plaques were found in 23 (55%). Median plasma level of Lp-PLA2 was significantly higher in patients with unstable plaque compared to those with stable plaque (222.4 (174.9-437.5) interquartile range (IQR) 63.5 vs. 211.1 (174.9-270.6) IQR 37.2 ng ml(-1); p = 0.02). Moreover, median Lp-PLA2 level were higher in asymptomatic patients with unstable plaque (226.8 ng ml(-1) (174.9-437.5) IQR 76.8) vs. stable plaque (206.9 ng ml(-1) (174.9-270.6) IQR 33.7; p = 0.16). Logistic regression showed that only the neurological symptoms (OR = 30.9 (3.7-244.6); p < 0.001) and the plasma Lp-PLA2 level (OR = 1.7 (1.1-12.3); p = 0.03) were independently associated with unstable carotid plaque as defined by histology. CONCLUSIONS: This study showed that circulating Lp-PLA2 was increased in patients with high-grade carotid stenosis and unstable plaque. Lp-PLA2 may be a relevant biomarker to guide for invasive therapy in asymptomatic patients with carotid artery disease.

[Long-term patency of a popliteal venous aneurysm treated surgically]. / Perméabilité à long terme d'un anévrisme veineux poplité traité chirurgicalement.

Popliteal venous aneurysms are infrequent but should be screened for with venous ultrasound in patients with acute or chronic venous diseases because of the unpredictable high risk of thromboembolism and potential curability. Therapeutic alternatives are discussed: follow-up, anticoagulation, surgery with different techniques. To illustrate this, we report the case of a 51-year-old woman presenting pulmonary embolism and left popliteal venous aneurysm treated surgically. Anticoagulation was stopped 12 months after surgery and primary patency was maintained 40 months after surgery. In patients with thromboembolism disease, clinicians should search for popliteal venous aneurysms in order to prevent recurrent thrombosis and adapt follow-up and treatment.

Band 3 peptides block the adherence of sickle cells to endothelial cells in vitro.

Malaria-parasitized erythrocytes have increased endothelial adherence due to exposure of previously buried intramembranous sites of band 3. Because sickle erythrocytes also show increased adhesiveness and because the membrane portion of band 3 is aggregated in both types of cells, we examined the role of band 3 in sickle cell adhesiveness. Synthetic peptides derived from the second and third exofacial, interhelical regions of band 3 completely inhibited the abnormal adherence of sickle cells to an endothelial monolayer in a static assay. This effect was observed independently of plasma factors, required micromolar levels of peptide, was sequence-specific, and was found with both L- and D-isomers. The active peptides also inhibited the increased adherence induced by low-dose calcium loading of normal red blood cells. Finally, a monoclonal antibody against an active peptide specifically immunostained a fraction of sickle cells. These findings implicate a role for band 3 in at least one type of sickle cell adhesiveness via the exposure of normally cryptic membrane sites.

Distance between Cys-201 in erythrocyte band 3 and the bilayer measured by single-photon radioluminescence.

Single-photon radioluminescence (SPR), the excitation of fluorophores by short-range beta-decay electrons, was developed for the measurement of submicroscopic distances. The cytoplasmic domain of band 3 (cdb3) is the primary, multisite anchorage for the erythrocyte skeleton. To begin to define the membrane arrangement of the highly asymmetrical cdb3 structure, the distance from the bilayer of Cys-201 next to the "hinge" of cdb3 was measured by both SPR and resonance energy transfer (RET). cdb3 was labeled at Cys-201 with fluorescein maleimide. For SPR measurements, the bilayer was labeled with [3H]oleic acid. The corrected cdb3-specific SPR signal was 98 +/- 2 cps microCi-1 [mumol band 3]-1. From this and the signal from a parallel sample in which 3H2O was substituted for [3H]oleic acid to create uniform geometry between 3H and the fluorophores, a Cys-201-to-bilayer separation of 39 +/- 7 A was calculated. Confirmatory distances of 40 and 43 A were obtained by RET between fluorescein on Cys-201 and eosin and rhodamine B lipid probes, respectively. This distance indicates that Cys-201 lies near band 3's vertical axis of symmetry and that the subdomain of cdb3 between the hinge and the membrane is not significantly extended. In addition, these results validate SPR as a measure of molecular distances in biological systems.

Anisotropy decay measurement of segmental dynamics of the anion binding domain in erythrocyte band 3.

Time-resolved anisotropy was utilized to detect nanosecond segmental motions of the band 3 intramembrane domain. Band 3 at lysine 430 was fluorescently labeled in ghost membranes by fluorescein or eosin maleimide treatment of intact human erythrocytes followed by hypotonic lysis. Single lifetimes for fluorescein (3.8-4.1 ns) and eosin (3.2-3.4 ns) were observed. Phase-modulation measurement of anisotropy decay indicated a segmental motion model, r(t) = exp(-t/tau 1c)[r infinity + (ro-r infinity) exp(-t/tau 2c)], defined by rotational correlation times corresponding to band 3 segmental motion (tau 1c, 30-70 ns) and rapid fluorescein motion in its binding pocket (tau 2c, 200-400 ps), and a residual anisotropy (r infinity, 0.23-0.28) describing hindered fluorescein motion. In PBS at pH 7.4, tau 1c, tau 2c, and r infinity were 44 ns, 307 ps, and 0.24, respectively, predicting a steady-state anisotropy of 0.24, in agreement with the measured value of 0.23. Factors that might influence band 3 structure/dynamics were examined. Whereas pH (range 5-10) had little effect on r(t), [NaCl] addition (0-150 mM) remarkably decreased tau 1c from 68 to 44 ns. The decrease in tau 1c correlated with solution ionic strength, and did not depend on osmolality (studied by mannitol addition), or specific anion interactions (comparing Cl, Br, F, SO4, citrate). The ionic strength effect was not observed in fluorescein-labeled carbonic anhydrase and trypsin-cleaved band 3, suggesting a specific effect on intact band 3. Anisotropy decay was relatively insensitive to external lectin or internal 2,3-DPG binding, but was sensitive to temperature, membrane fluidity, urea denaturation, fluid-phase viscosity, and aldehyde fixation.(ABSTRACT TRUNCATED AT 250 WORDS)

Calculation of resonance energy transfer in crowded biological membranes.

Analytical and numerical models were developed to describe fluorescence resonance energy transfer (RET) in crowded biological membranes. It was assumed that fluorescent donors were linked to membrane proteins and that acceptors were linked to membrane lipids. No restrictions were placed on the location of the donor within the protein or the partitioning of acceptors between the two leaflets of the bilayer; however, acceptors were excluded from the area occupied by proteins. Analytical equations were derived that give the average quantum yield of a donor at low protein concentrations. Monte Carlo simulations were used to generate protein and lipid distributions that were linked numerically with RET equations to determine the average quantum yield and the distribution of donor fluorescence lifetimes at high protein concentrations, up to 50% area fraction. The Monte Carlo results show such crowding always reduces the quantum yield, probably because crowding increases acceptor concentrations near donor-bearing proteins; the magnitude of the reduction increases monotonically with protein concentration. The Monte Carlo results also show that the distribution of fluorescence lifetimes can differ markedly, even for systems possessing the same average lifetime. The dependence of energy transfer on acceptor concentration, protein radius, donor position within the protein, and the fraction of acceptors in each leaflet was also examined. The model and results are directly applicable to the analysis of RET data obtained from biological membranes; their application should result in a more complete and accurate determination of the structures of membrane components.

Segmental dynamics of the cytoplasmic domain of erythrocyte band 3 determined by time-resolved fluorescence anisotropy: sensitivity to pH and ligand binding.

Interactions between the erythrocyte membrane and its skeleton are mediated primarily by binding of cytoskeletal components to a conformationally sensitive structure, the cytoplasmic domain of band 3 (cdb3). To examine the nanosecond segmental motions of cdb3, band 3 was labeled selectively by fluorescein maleimide at Cys-201 near the proposed hinge in cdb3 about which pH-dependent conformational changes occur. Time-resolved anisotropy of labeled cdb3 in isolated form and in stripped erythrocyte membranes was measured by parallel-acquisition frequency-domain microfluorimetry. Samples had a single-component fluorescein lifetime of approximately 4 ns. Multifrequency phase and modulation data (5-200 MHz) fitted well to a segmental motion model containing two correlation times (tau 1c and tau 2c) and two limiting anisotropies (r1infinity and r2infinity). Measurements in protease-cleaved and denatured samples indicated that tau 1c (100-150 ps) corresponded to rapid rotation of bound fluorescein and tau 2c (2-5 ns) corresponded to segmental motion of cdb3. Both motions were hindered as quantified by nonzero r1infinity and r2infinity. The strong pH dependence of segmental motion correlated with that of cdb3 conformation measured by intrinsic tryptophan fluorescence. Significant changes in cdb3 segmental motion occurred upon interactions with the small ligands 2,3-bisphosphoglycerate and calcium and several glycolytic enzymes known to bind to the N terminus of band 3. These time-resolved fluorescence measurements of the nanosecond segmental dynamics of a labeled membrane protein provide evidence for the sensitivity of cdb3 conformation to ligand binding and suggest long-range structural communication through cdb3.

A novel photoactivatable cross-linker for the functionally-directed region-specific fluorescent labeling of proteins.

A cleavable cross-linking reagent, sulfosuccinimidyl-2(7-azido-4-methylcoumarin-3-acetamido)-ethyl-1,3'- dithiopropionate (SAED), was synthesized for the selective transfer of a coumarin fluorophore from a 'donor' protein to a position near the binding site of an interacting 'target' protein. SAED contains a terminal N-sulfosuccinimidyl ester for conjugation to the donor, a terminal photoactivatable azido-coumarin species for cross-linking with the interacting target, and a central disulfide spacer for the release of the labeled target after cleavage. To evaluate the effectiveness of this labeling reagent, soybean trypsin inhibitor (STI) was derivatized (approximately 0.5 mol/mol) with SAED and then photolyzed in the presence of trypsin. A single fluorescent cross-linked species (6-7 mol% of total STI) was observed by SDS/PAGE and, after reductive cleavage, was shown to be a 1:1 STI-trypsin complex. This complex was not detected without photolysis or with an inactivated cross-linker. Importantly, complex formation was inhibited by an excess of unmodified STI and prevented by substitution of a non-interacting protein for trypsin. Cleavage of the cross-linked complex revealed that the trypsin, but not the STI, was fluorescent; the uncomplexed trypsin fraction remained unlabeled. These results demonstrated the specificity of the labeling of trypsin by fluorescent-transfer cross-linking with SAED. An efficiency of about 15% for this cross-linking mediated labeling of trypsin was calculated. The short cross-linking span of SAED (less than or equal to 1.8 nm) strictly limited the labeling to the vicinity of the contact region of trypsin with STI. Thus, this novel cross-linker permits the region-specific targeting of a fluorophore near a functionally important binding site.

Conformational changes in the foot protein of the sarcoplasmic reticulum assessed by site-directed fluorescent labeling.

Ca2+ release from sarcoplasmic reticulum during excitation--contraction coupling is likely to be mediated by conformational changes in the foot protein moiety of the triadic vesicles. As a preparative step toward the studies of dynamic conformational changes in the foot protein moiety, we have developed a new method that permits specific labeling of the foot protein moiety of the isolated membranes with a fluorophore. A novel fluorescent cleavable photoaffinity cross-linking reagent, sulfosuccinimidyl 3-((2-(7-azido-4-methylcoumarin-3-acetamido)ethyl)dithio)propionate (SAED), was conjugated with site-directing carriers, polylysine (Ca(2+)-release inducer) and neomycin (Ca(2+)-release blocker). The conjugates were allowed to bind to polylysine- and neomycin-binding sites of the heavy fraction of SR (HSR). After photolysis, the cross-linked reagent was cleaved by reduction and the fluorescently labeled HSR was separated from the carriers by centrifugation. These procedures led to specific incorporation of the methylcoumarin acetate (MCA) into the foot protein. Polylysine and neomycin bound to different sites of the foot protein, since neomycin, at release-blocking concentrations, did not interfere with polylysine binding. The fluorescence intensity of the foot protein labeled with the carrier, neomycin, showed biphasic changes as a function of ryanodine concentration (increasing up to 1 microM ryanodine and decreasing above it), while with the carrier polylysine, ryanodine induced no change in fluorescence intensity. In contrast, the fluorescence intensity of the foot protein labeled with each of the two carriers, neomycin and polylysine, showed almost identical calcium dependence (first increasing from 0.1 microM to about 3.0 microM calcium concentration, and then decreasing at higher calcium concentrations).(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetics and regulation of the ankyrin-band 3 interaction of the human red blood cell membrane.

In an attempt to identify potential regulatory mechanisms for erythrocyte membrane-cytoskeletal interactions, the kinetics and pH dependence of the band 3-ankyrin interaction were investigated. Association of 125I-ankyrin with KI-stripped inside-out erythrocyte membrane vesicles was found to proceed in two kinetic phases. The initial, fast phase (t1/2 approximately 15-30 min) involved predominantly the binding of ankyrin to low affinity sites (KD approximately 130 nM) in a pH-dependent manner. The apparent pKa values describing this reversible pH dependence (7.2 +/- 0.1 and 9.2 +/- 0.1) defined states of band 3 with high, moderate, and no capacity to bind ankyrin (in order of increasing pH). Since the cytoplasmic domain of band 3 also exists in 3 distinct conformational states characterized by apparent pKa values of 7.2 and 9.2, it was hypothesized that the reversible structural equilibrium in band 3 could influence ankyrin binding. The second or slow phase of ankyrin binding to band 3 involved the conversion of low to high affinity sites (KD approximately 13 nM). This phase, which was largely temperature and pH independent, required roughly an order of magnitude longer to reach completion than the fast phase. Unfortunately, even though the slow phase could be cleanly separated from the fast phase at low pH, insufficient data were available to formulate a physical interpretation of its origin. Significantly, however, even after completion of the slow phase under the most quantitative binding conditions identified, a maximum of only 26% of the band 3 was found to bind ankyrin in situ. Although higher ankyrin-band 3 stoichiometries may be achievable with the isolated cytoplasmic fragment of band 3, we interpret the above 1:4 stoichiometry to suggest that the tetramer of band 3 constitutes the predominant ankyrin binding oligomer of band 3 on the membrane.

The redox state of cysteines 201 and 317 of the erythrocyte anion exchanger is critical for ankyrin binding.

Previous studies have demonstrated that modification of erythrocyte membrane cysteine residues via disulfide cross-briding or direct derivatization with thiol reagents promotes massive morphological, rheological, and structural changes in the cell. To determine whether disruption of the band 3-ankyrin interaction, the major membrane-cytoskeletal linkage, might contribute to the above lesions, we quantitatively measured the band 3-ankyrin interaction following modification of Cys-201 and/or Cys-317 of the cytoplasmic domain of band 3. It was observed that irreversible alkylating agents (e.g. N-ethylmaleimide or iodoacetamide and its derivatives), reversible derivatizing compounds (.e.g. p-chloromercuribenzenesulfonate or glutathione), and native disulfide bond formation all blocked the ankyrin interaction. Comparison of the extent of sulfhydryl modification with the degree of inhibition of ankyrin binding further confirmed that cysteine modification was directly responsible for the inhibition. However, analysis of the site of sulfhydryl derivatization revealed that inhibition of ankyrin binding could be initiated in some cases with derivatization of Cys-201, while in other cases obstruction of Cys-317 appeared to be essential. This apparent discrepancy was resolved by demonstrating that Cys-201 of one strand of the cytoplasmic domain of band 3 dimer could disulfide bond with Cys-317 of the opposite strand, thus demonstrating that all four cysteines of the band 3 dimer are clustered at the interface between subunits. We argue that derivatization or disulfide cross-linking of these cysteines can block ankyrin binding by both conformational and steric mechanisms.

Localization of the ankyrin-binding site on erythrocyte membrane protein, band 3.

The predominant attachment site of the spectrin-based cytoskeleton to the erythrocyte membrane occurs via the interaction of ankyrin with the cytoplasmic domain of band 3 (cdb3). In order to further characterize this interaction, we have conducted experiments to localize the ankyrin-binding site on cdb3. Four monoclonal and three antipeptide polyclonal antibodies were raised against cdb3 and used in competition studies to identify regions of close association of cdb3 with ankyrin. Antibodies to regions of cdb3 near the cytoplasmic domain-membrane spanning domain junction had no effect on 125I-ankyrin binding. Likewise, an antibody to a highly conserved region between residues 142 and 154 did not inhibit ankyrin binding. However, antibodies at or near the cysteine 201-317 cluster and the proposed proline-rich hinge in the center of cdb3 were potent inhibitors of ankyrin association, as were antibodies to the acidic NH2 terminus. Additional evidence for interaction of ankyrin with the NH2-terminal region of cdb3 was obtained by demonstrating the ability of ankyrin to inhibit tyrosine phosphorylation of cdb3 at its NH2 terminus by a purified calf thymus tyrosine kinase. These studies reveal two regions of cdb3, distant in primary sequence, which interact with ankyrin. A specific conformation of cdb3 may be required to permit these regions to simultaneously associate with ankyrin and allow binding to occur.