Lipoprotein(a), Lp(a), an athero-thrombotic risk factor, reacts with EO6, a natural monoclonal autoantibody that recognizes the phophorylcholine (PC) group of oxidized phosphatidylcholine (oxPtdPC) either as a lipid or linked by a Schiff base to lysine residues of peptides/proteins. Here we show that EO6 reacts with free apolipoprotein(a) apo(a), its C-terminal domain, F2 (but not the N-terminal F1), kringle V-containing fragments obtained by the enzymatic digestion of apo(a) and also kringle V-containing apo(a) recombinants. The evidence that kringle V is critical for EO6 reactivity is supported by the finding that apo(a) of rhesus monkeys lacking kringle V did not react with EO6. Based on the previously established EO6 specificity requirements, we hypothesized that all or some of the six lysines in human kringle V are involved in Schiff base linkage with oxPtdPC. To test this hypothesis, we made use of a recombinant lysine-containing apo(a) fragment, rIII, containing kringle V but not the protease domain. EO6 reacted with rIII before and after reduction to stabilize the Schiff base and also after extensive ethanol/ ether extraction that yielded no lipids. On the other hand, delipidation of the saponified product yielded an average of two mol of phospholipids/mol of protein consistent with direct analysis of inorganic phosphorous on the nonsaponified rIII. Moreover, only two of the six theoretical free lysine amino groups per mol of rIII were unavailable to chemical modification by 2,4,6-trinitrobenzene sulfonic acid. Finally, rIII, like human apo(a), stimulated the production of interleukin 8 in THP-1 macrophages in culture. Together, our studies provide evidence that in human apo(a), kringle V is the site that reacts with EO6 via lysine-oxPtdPC adducts that may also be involved in the previously reported pro-inflammatory effect of apo(a) in cultured human macrophages.

Objectives-By using proteomics we isolated and identified proteins that were expressed/retained in stable and unstable human carotid artery atherosclerotic plaques.Methods-The criteria for plaque instability were the presence of a thin fibrous cap or fissured cap covering the foamy or necrotic core, and the presence of overt, hemorrhagic, ulcerated or thrombotic plaques. Proteins were extracted from finely minced endarterectomy specimens (19 stable, 29 unstable plaques) and separated by 2-dimensional gel electrophoresis. Coomassie Blue-stained gels were analysed using PD-Quest software.Results-A total of 57 distinct spots corresponding to 33 different proteins were identified by matrix assisted laser desorption/ionization mass spectrometry using the NCBI database. Most of the spots were present in both types of extracts, although significantly (p<0.05) differing in abundance between them. Compared to stable plaque, unstable ones showed reduced abundance of: protective enzymes SOD3 and GST, small heat shock proteins HSP27 and HSP20, annexin A10, and Rho GDI. In unstable plaques the more abundant proteins were: ferritin light subunit, SOD 2 and fibrinogen fragment D. For fibrinogen fragment D, the increased levels in unstable versus stable plaques was confirmed by Western blot analysis.Conclusions-Since many of the differentially expressed proteins are known to have a functional role in inflammation and oxidative stress, we speculate that they may be involved in events relating to plaque stability.

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Clinical and epidemiological studies have shown that HDLs, a class of plasma lipoproteins, heterogeneous in size and density, have an atheroprotective role attributed, for years, to their capacity to promote the efflux of cholesterol from activated cholesterol-loaded arterial macrophages. Recent studies, however, have recognized that the physical heterogeneity of HDLs is associated with multiple functions that involve both the protein and the lipid components of these particles. ApoA-I, quantitatively the major protein constituent, has an amphipathic structure suited for transport of lipids. It readily interacts with the ATP-binding cassette transporter ABCA1, the SR-B1 scavenger receptor; activates the enzyme lecithin-cholesterol acyl transferase (LCAT), which is critical for HDL maturation. It also has antioxidant and antiinflammatory properties, along with the HDL-associated enzymes paraoxonase, platelet activating factor acetylhydrolase (PAF), and glutathione peroxidase. Regarding the lipid moiety, an atheroprotective role has been recognized for lysosphingolipids, particularly sphingosine-1-phosphate (S1P). All of these atheroprotective functions are lost in the post-translational dependent dysfunctional plasma HDLs of subjects with systemic inflammation, coronary heart disease, diabetes, and chronic renal disease. The emerging notion that particle quality has more predictive power than quantity has stimulated further exploration of the HDL proteome, already revealing unsuspected pro- or antiatherogenic proteins/peptides associated with HDL.

Plasma lipoprotein(a) (Lp[a]) level is an independent risk factor of cardiovascular disease that is under strong genetic control. We conducted a genome-wide association study of plasma Lp(a) in 386 members of a founder population that adheres to a communal lifestyle, proscribes cigarette smoking, and prepares and eats meals communally. We identified associations with 77 single nucleotide polymorphisms (SNPs) spanning 12.5 Mb on chromosome 6q26-q27 that met criteria for genome-wide significance (P # 1.3 3 10 27 ) and were within or flanking nine genes, including LPA. We show that variation in at least six genes in addition to LPA are significantly associated with Lp(a) levels independent of each other and of the kringle IV repeat polymorphism in the LPA gene. One novel SNP in intron 37 of the LPA gene was also associated with Lp(a) levels and carotid artery disease number in unrelated Caucasians (P 5 7.3 3 10 212 and 0.024, respectively), also independent of kringle IV number. This study suggests a complex genetic architecture of Lp(a) levels that may involve multiple loci on chromosome 6q26-q27. Lipoprotein (a) [Lp(a)] is recognized as an independent risk factor for atherosclerotic cardiovascular disease (1, 2). The mechanisms underlying this pathogenesis are poorly understood, although proatherogenic, prothrombotic, and inflammatory pathways contribute. Moreover, plasma Lp(a) levels are not responsive to statins and other cholesterol-lowering drugs, except for niacin, for which the long-term efficacy and safety is not yet established (3). Lp(a) is produced in the liver (4) and circulates in the plasma as an LDL particle having as a protein moiety apolipoprotein(a) [apo(a)], encoded by the LPA gene, linked by a disulfide bond to an apolipoprotein B-100 particle, on a 1:1 molecular basis (5). While apolipoprotein B-100 remains relatively constant in size, apo(a) varies in size due to polymorphism in the number of tandemly repeated kringle IV type 2 domains encoded by sequences in exons 1 and 2 of the LPA gene (6).The human LPA gene arose as a duplication of the PLG gene in the primate lineage and retains 80% sequence identity to PLG (7), which has only a single kringle IV structure. The number of kringle IV repeats in Lp(a) is under genetic control and inversely correlates with plasma levels of Lp(a), likely as result of the lower secretion rate in hepatocytes of apo(a) isoforms with larger numbers of kringle IV repeats (8, 9). The LPA locus accounts for 70-90% of the variability in Lp(a) levels in worldwide Abbreviations: apo(a), apolipoprotein(a); BMI, body mass index; LD, linkage disequilibrium; Lp(a), lipoprotein (a); SNP, single nucleotide polymorphism; RSS, residual sum of squares.