RESUMO
Human apolipoprotein(a) (apo(a)), synthesized in the liver, contains oxidized phosphatidylcholine (oxPtdPC) adducts probably generated at the hepatic site. Since plasminogen (Plg), also synthesized in the liver, is genetically related and structurally homologous to apo(a), we wanted to determine whether it contains oxPtdPCs and their location. We used Plg isolated from fresh or frozen normal human plasma and several commercial preparations. Some were freed of non-covalently bound lipids by organic solvent extraction. By immunoblot analyses, all products reacted against T15, a natural IgM monoclonal antibody specific for phosphorylcholine -containing oxidized phospholipids (ox-PLs). This immunoreactivity was retained in urokinase type plasminogen activator -generated plasmin and was abrogated in Plg previously digested with lipoprotein-associated phospholipase A(2) (Lp-PLA(2)), a reaction that generated predominantly C16:0 lysophosphatidylcholine species as determined by mass spectrometry. Lyso derivatives were also generated upon the cleavage by Lp-PLA2 of a model ox-PL chemically linked to a lysine-containing pentapeptide. From inorganic phosphorous analyses, we found 2 mol of oxPtdPC/mole of Plg distributed between the kringles 1-4 and mini-Plg domain. OxPtdPCs were also present in the Plg isolated from the serum-free medium of cultured human HepG2 cells. In conclusion, our results provide strong evidence that naturally occurring Plg contains oxPtdPC probably linked by a Schiff base and also suggest that the linkage occurs at the hepatic site. Given the emerging evidence for the cardiovascular pathogenicity of oxPtdPCs, we speculate that they may impart athero-thrombogenic properties to Plg under inflammatory conditions.
Assuntos
Apoproteína(a)/química , Fosfatidilcolinas/química , Plasminogênio/química , 1-Alquil-2-acetilglicerofosfocolina Esterase/química , Animais , Apoproteína(a)/metabolismo , Células Hep G2 , Humanos , Espectrometria de Massas , Camundongos , Oxirredução , Fosfatidilcolinas/metabolismo , Plasminogênio/metabolismoRESUMO
We investigated whether the presence of endogenous or exogenous lipoprotein-associated phospholipase A2 (Lp-PLA2) can modify the cellular association of oxidized low density lipoprotein (oxLDL) and oxidized lipoprotein(a) (oxLp(a)) by human monocyte-derived macrophages (MDM) and hepatocytes (HepG2). Purified recombinant Lp-PLA2 was used as a source of exogenous enzyme whereas Pefabloc (serine esterase inhibitor) was used to inhibit the endogenous Lp-PLA2 activity associated with isolated lipoproteins. Cellular association studies were performed with DiI-labeled oxLDL or oxLp(a) and human monocyte-derived macrophages and HepG2 cells. Active Lp-PLA2 decreased the cellular association of oxLDL and oxLp(a) in macrophages and HepG2 cells by approximately 30-40%, whereas the inactive enzyme did not significantly change oxidized lipoprotein cellular association by either cell type. OxLDL pretreated by Pefabloc increased oxLDL cellular association by MDM and HepG2 cells compared to untreated oxLDL. Therefore, unlike some lipases, Lp-PLA2 did not appear to have any catalytic independent function in oxLDL cellular association. To assess whether the reduced cellular association mediated by Lp-PLA2 was due to the hydrolysis of oxidized phosphatidylcholine (oxPC), we measured the concentration of lysophosphatidylcholine (lysoPC) in lipoprotein fractions after Lp-PLA2 treatment. LysoPC was increased by 20% (0.4 microM) and 87% (0.7 microM) by active Lp-PLA2 compared to inactive Lp-PLA2 for oxLDL and Lp(a), respectively. LysoPC at higher concentration dose-dependently increased the cellular association of oxLDL and oxLp(a) in MDM and HepG2 cells. We conclude that Lp-PLA2 mediates a decrease in oxidized lipoprotein cellular association in human macrophages and HepG2 cells by reducing the concentration of oxPC within these lipoproteins.
Assuntos
Hepatócitos/metabolismo , Lipoproteína(a)/metabolismo , Lipoproteínas LDL/metabolismo , Macrófagos/metabolismo , Fosfolipases A2/metabolismo , Células Cultivadas , Citometria de Fluxo , Humanos , Monócitos/metabolismo , Fosfolipases A2/genética , Fosfolipases A2/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
Lipoprotein (a) [Lp(a)], a cardiovascular risk factor, is a low-density lipoprotein (LDL) variant shown to bind to oxidized phospholipids (oxPLs); however, its binding mode and origin have not been clearly established. We isolated both LDL and Lp(a) from the plasma of a population of high-Lp(a) subjects and in each Lp(a) particle separated apolipoprotein(a) [apo(a)], from the LDL component, Lp(a(-)). These products were assayed by an ELISA using monoclonal antibody T15 with a known specificity for oxPLs. In each subject, the T15 reactivity was confined to apo(a). Moreover, the amount of oxPL bound to apo(a) was unaffected by plasma Lp(a) levels and apo(a) size polymorphism. We have previously shown that kringle V (KV) is the site of oxPL linkage in human apo(a). In this work, we expressed in human embryonic kidney cells a KV-containing recombinant that, when purified from the medium, contained oxPLs. In summary, in human plasma Lp(a), the oxPLs are located in apo(a) and not in the circulating LDLs, suggesting a cellular origin. This latter concept is supported by the studies in which an expressed KV-containing apo(a) microdomain exhibited oxPL reactivity. Thus, apo(a) can undergo potentially pathogenic posttranslational modifications in a cellular environment able to generate oxPL.
Assuntos
Apoproteína(a)/metabolismo , Lipoproteínas LDL/metabolismo , Fosfolipídeos/metabolismo , Adulto , Idoso , Apoproteína(a)/sangue , Apoproteína(a)/química , Feminino , Humanos , Kringles , Lipoproteínas LDL/química , Masculino , Pessoa de Meia-Idade , Fosfolipídeos/sangue , Fosfolipídeos/química , Processamento de Proteína Pós-TraducionalRESUMO
We previously showed that plasminogen (Plg) isolated from the plasma of normal human subjects contains 1-2 moles of oxidized phosphatidylcholine (oxPtdPC) adducts/mole of protein. Moreover, we suggested that these species are generated at the hepatic site and speculated that they may play a role in the reported cardiovascular pathogenicity of Plg. We aimed to determine whether mouse Plg also harbors linked oxPtdPCs and whether these molecules are metabolized by lipoprotein-associated phospholipase A(2)/PAF acetylhydrolase (Lp-PLA(2)/PAF-AH), an enzyme specific for hydrolysis of oxPtdPCs. We determined the total concentration of Plg in plasma samples from control (WT) and Lp-PLA(2)-deficient (KO) mice, we isolated Plg, and assessed its content of oxPtdPCs by immunoblot analyses. We also evaluated whether human recombinant Lp-PLA(2) metabolized Plg-linked oxPtdPCs in vivo and in vitro. WT and KO mice expressed comparable levels (14.4-15.8 mg/dL) of plasma Plg, as determined by ELISA. We observed no differences in the content of oxPtdPC in Plg isolated from the two mouse strains and in parallel no changes in oxPtdPC content in mouse Plg following incubation with pure recombinant Lp-PLA(2). Plg from mouse plasma contains oxPtdPC adducts that are not affected by the action of Lp-PLA(2), suggesting that linkage to Plg protects oxPtdPCs from metabolism during their transport in the plasma. This modification may have important physio-pathological implications related to the function of Plg, oxPtdPCs, or both.
Assuntos
1-Alquil-2-acetilglicerofosfocolina Esterase/química , Fosfatidilcolinas/química , Plasminogênio/química , 1-Alquil-2-acetilglicerofosfocolina Esterase/sangue , Animais , Humanos , Camundongos , Camundongos Knockout , Oxirredução , Fosfatidilcolinas/sangue , Fosfatidilcolinas/isolamento & purificação , Plasminogênio/isolamento & purificação , Plasminogênio/metabolismoRESUMO
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.
Assuntos
Apolipoproteína A-II/química , Apolipoproteína A-I/química , Lipoproteínas HDL/química , Aterosclerose/prevenção & controle , Glicoesfingolipídeos/metabolismo , Humanos , Lipoproteínas HDL/isolamento & purificação , Lipoproteínas HDL/metabolismo , Fosfolipídeos/metabolismo , Processamento de Proteína Pós-Traducional , Proteômica , Soluções/químicaRESUMO
Over the years, niacin has gained recognition as an atheroprotective agent, in part because of its capacity to lower the plasma levels of cholesterol, triglycerides, and very-low- and low-density lipoproteins and to substantially raise high-density lipoprotein. In high doses, niacin has also been reported to lower the plasma level of lipoprotein(a) (Lp[a]). However, the published research on the subject suffers from a lack of uniformity regarding patient selection, drug dose, length of administration, and methods for plasma Lp(a) quantification. In this report, the authors examine the most relevant niacin-related Lp(a) studies and hypothetical mechanisms of drug action, also considering the emerging notion of Lp(a) as a potential proinflammatory entity.
Assuntos
Anticolesterolemiantes/uso terapêutico , Dislipidemias/tratamento farmacológico , Lipoproteína(a)/efeitos dos fármacos , Niacina/uso terapêutico , Anticolesterolemiantes/farmacologia , HDL-Colesterol/sangue , HDL-Colesterol/efeitos dos fármacos , LDL-Colesterol/sangue , LDL-Colesterol/efeitos dos fármacos , Humanos , Hipertrigliceridemia/tratamento farmacológico , Lipoproteína(a)/sangue , Niacina/farmacologia , Triglicerídeos/sangueRESUMO
OBJECTIVES: Based on previous observations on apolipoprotein(a), apo(a), in human unstable carotid plaques, we explored whether in the inflammatory environment of human atheroma, proteolytic events affect other hepatic and topically generated proteins in relation to the issue of plaque stability. METHODS AND RESULTS: Forty unstable and 24 stable plaques from endarterectomy segments of affected human carotid arteries were extracted with buffered saline (PBS) and then 6 mol/L guanidine-hydrochloride (GdHCl) to identify loosely and tightly bound products, respectively. The extracts were studied before and after ultracentrifugation at d 1.21 g/mL. In the extracts, the concentrations of interleukin (IL)-6, -8, and -18 were significantly higher in the unstable plaques and correlated to those of MMP-2 and MMP-9. By Western blots, both apoB and apo(a) were highly fragmented and mostly present in the d 1.21 bottom that also contained fragments of apoE (10 and 22 kDa), decorin, biglycan, and versican. Fragmentation was higher in the unstable plaques. In baseline plasmas, concentrations of lipids, lipoproteins, and ILs did not differ between patients with unstable and stable plaques. CONCLUSIONS: In unstable and to a lesser extent in stable plaques, there is a proinflammatory and proteolytic microenvironment with the generation of fragments with potential pathobiological significance that requires investigation.
Assuntos
Estenose das Carótidas/metabolismo , Proteínas/metabolismo , Vasculite/metabolismo , Idoso , Apolipoproteínas/análise , Biomarcadores , Estenose das Carótidas/patologia , Estenose das Carótidas/cirurgia , Decorina , Endarterectomia das Carótidas , Proteínas da Matriz Extracelular , Feminino , Guanidina/farmacologia , Humanos , Interleucinas/análise , Masculino , Metaloproteinases da Matriz/análise , Pessoa de Meia-Idade , Fragmentos de Peptídeos/análise , Proteoglicanas/análise , Vasculite/patologiaRESUMO
Most studies on the topic have shown that statin therapy decreases plasma LDL levels but not those of lipoprotein(a) [Lp(a)]. This specificity of action, although previously noted, has not been systematically investigated. In the current study we approached this problem by monitoring LDL- and Lp(a) cholesterol in 80 hypercholesterolemic subjects with high Lp(a) levels, at entry and 8 mon after initiation of statin therapy. We found that commonly used direct and indirect LDL cholesterol assays gave an LDL cholesterol value that comprised both true LDL- and Lp(a) cholesterol. We estimated these two analytes from the values of Lp(a) protein determined by ELISA and from knowledge of the Lp(a) chemical composition, complemented by data from immunochemical and ultracentrifugal analyses. Statin therapy, while not affecting plasma Lp(a) protein levels (21.7+/-10.4, before, and 22.0+/-10.1 mg/dL, after), caused a decrease in the estimated or true LDL cholesterol (P < 0.0001) to values in some cases as low as 10 mg/dL. This drop in true LDL was validated by the decrease in the LDL band in the ultracentrifugation profiles, and its magnitude was proportional to the degree of total cholesterol lowering and to the pretreatment true LDL/Lp(a) cholesterol weight ratio. We conclude that true LDL but not Lp(a) cholesterol is affected by statin therapy and that this specific response cannot be monitored by current LDL cholesterol assays and must, rather, rely on estimates of these two analytes.
Assuntos
Anticolesterolemiantes/uso terapêutico , Hipercolesterolemia/sangue , Hipercolesterolemia/tratamento farmacológico , Lipoproteína(a)/sangue , Lipoproteínas LDL/sangue , Adulto , Idoso , Atorvastatina , Ensaio de Imunoadsorção Enzimática , Feminino , Ácidos Heptanoicos/uso terapêutico , Humanos , Masculino , Pessoa de Meia-Idade , Pravastatina/uso terapêutico , Piridinas/uso terapêutico , Pirróis/uso terapêutico , Sinvastatina/uso terapêuticoRESUMO
Lipoprotein(a) [Lp(a)] represents a class of lipoprotein particles having as a protein moiety apoB-100 linked by a single disulfide bond to apolipoprotein(a) [apo(a)], a multikringle structure with a high degree of homology with plasminogen. A recognized feature of Lp(a) is its instability on storage caused by attendant protein and lipid modifications that affect the structural, functional, and immunological properties of this lipoprotein. Here we present data showing that, under appropriate conditions of cryopreservation, Lp(a) retains the properties of the freshly isolated product, and we provide examples supporting the stability of this cryopreserved product as a primary standard in immunoassay settings and in cell culture systems.
Assuntos
Lipoproteína(a)/normas , Criopreservação/métodos , Estabilidade de Medicamentos , Liofilização , Indicadores e Reagentes , Métodos , Padrões de ReferênciaRESUMO
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 and 29 unstable plaques) and separated by two-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.
Assuntos
Aterosclerose/patologia , Artérias Carótidas/patologia , Estenose das Carótidas/diagnóstico , Estenose das Carótidas/patologia , Proteômica/métodos , Aterosclerose/diagnóstico , Estenose das Carótidas/metabolismo , Eletroforese em Gel Bidimensional , Humanos , Inflamação , Modelos Biológicos , Miócitos de Músculo Liso/metabolismo , Estresse Oxidativo , Processamento de Proteína Pós-Traducional , Corantes de Rosanilina/farmacologia , Transdução de Sinais , Software , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
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 Assuntos
Cromossomos Humanos Par 6/genética
, Estudo de Associação Genômica Ampla
, Lipoproteína(a)
, Isoformas de Proteínas
, Animais
, Mapeamento Cromossômico
, Elementos Facilitadores Genéticos
, Predisposição Genética para Doença
, Genótipo
, Humanos
, Kringles/genética
, Desequilíbrio de Ligação
, Lipoproteína(a)/sangue
, Lipoproteína(a)/genética
, Polimorfismo de Nucleotídeo Único
, Isoformas de Proteínas/sangue
, Isoformas de Proteínas/genética
, Religião
, South Dakota
, População Branca/genética
RESUMO
In previous studies, we showed that the C-terminal domain, F2, but not the N-terminal domain, F1, is responsible for the binding of apolipoprotein [a] (apo[a]) to human fibronectin (Fn). To pursue those observations, we prepared, by both elastase digestion and recombinant technology, subsets of F2 of a different length containing either kringle (K) V or the protease domain (PD). We also studied rhesus monkey apo[a], which is known to contain PD but not KV. In the case of Fn, we used both an intact product and its tenth type III module (10FN-III) expressed in Escherichia coli. The binding studies carried out on microtiter plates showed that the affinity of F2 for immobilized 10FN-III was approximately 6-fold higher than that for Fn (dissociation constants = 1.75 +/- 0.31 nM and 10.25 +/- 1.62 nM, respectively). The binding was also exhibited by rhesus apo[a] and by an F2 subset containing the PD linked to an upstream microdomain comprising KIV-8 to KIV-10 and KV, inactive by itself. Competition experiments on microtiter plates showed that both Fn and 10FN-III, when in solution, are incompetent to bind F2. Together, our results indicate that F2 binds to immobilized 10FN-III more efficiently than whole Fn and that the binding can be sustained by truncated forms of F2 that contain the catalytically inactive PD linked to an upstream four K microdomain.
Assuntos
Apolipoproteínas A/química , Apolipoproteínas A/metabolismo , Fibronectinas/química , Fibronectinas/metabolismo , Sequência de Aminoácidos , Animais , Fibronectinas/classificação , Fibronectinas/genética , Humanos , Macaca mulatta , Dados de Sequência Molecular , Elastase Pancreática/metabolismo , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
A recent genome-scan identified the Leu33Pro polymorphism in the beta3 integrin (ITGB3) gene as a quantitative trait locus for whole blood serotonin level in a large Hutterite pedigree. Because both the Leu33Pro polymorphism and the serotonin system have been implicated in cardiovascular disease (CVD) risk and treatment response, we studied additional variation in ITGB3 and its relationship to intermediate phenotypes associated with CVD in the same population. We examined associations between 15 single nucleotide polymorphisms (SNPs) across ITGB3 and five CVD-related traits in the Hutterites: plasma levels of high density lipoprotein-cholesterol (HDL-c), triglycerides (TG), low density lipoprotein-cholesterol (LDL-c), and lipoprotein(a) [Lp(a)] and blood pressure or hypertension. Seven of these SNPs in ITGB3 were associated with whole blood serotonin. Among the intermediate CVD-related phenotypes, only Lp(a) was associated with multiple ITGB3 SNPs, five of which were also associated with serotonin. A sex-stratified analysis revealed that the association between ITGB3 and Lp(a) is present only in females, whereas the association between ITGB3 and serotonin is concentrated in males. Our results suggest that variation in ITGB3 in addition to Leu33Pro could contribute to susceptibility to CVD and serotonin in a sex-specific manner.
Assuntos
Doenças Cardiovasculares/etiologia , Doenças Cardiovasculares/genética , Integrina beta3/genética , Integrina beta3/fisiologia , Lipoproteínas/sangue , Serotonina/sangue , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Pressão Sanguínea , Criança , Feminino , Efeito Fundador , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Fenótipo , Polimorfismo de Nucleotídeo Único , Precursores de Proteínas , Locos de Características Quantitativas , Fatores Sexuais , South DakotaRESUMO
Although many epidemiologic studies have pointed at an association between plasma levels of lipoprotein(a) (Lp(a)) and cardiovascular risk, the data obtained have been conflicting because of a number of factors, particularly those dealing with plasma storage, lack of assay standardization, population sample size, age, gender, ethnic variations, and variable disease endpoints. Moreover, the attention has been primarily focused on whole Lp(a), with relatively less emphasis on its constituent apolipoprotein(a) and on the apolipoprotein B100-containing lipoprotein, mainly low-density lipoprotein (LDL), to which apolipoprotein(a) is linked. According to recent studies, small-size apolipoprotein(a) isoforms may represent a cardiovascular risk factor either by themselves or synergistically with plasma Lp(a) concentration. Moreover, the density properties of the LDL moiety may have an impact on Lp(a) pathogenicity. It has also become apparent that Lp(a) can be modified by oxidative events and by the action of lipolytic and proteolytic enzymes with the generation of products that exhibit atherothrombogenic potential. The role of the O-glycans linked to the inter-kringle linkers of apolipoprotein(a) is also emerging. This information is raising the awareness of the pleiotropic functions of Lp(a) and is opening new vistas on pathogenetic mechanisms whose knowledge is essential for developing rational therapies against this complex cardiovascular pathogen.
Assuntos
Doença da Artéria Coronariana/sangue , Lipoproteína(a)/sangue , Biomarcadores/sangue , Doença da Artéria Coronariana/genética , Vasos Coronários/metabolismo , Humanos , Lipoproteína(a)/genética , Lipoproteína(a)/metabolismo , Modelos Animais , Oxirredução , Polimorfismo Genético , Túnica Íntima/metabolismoRESUMO
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 non-saponified 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.