Ditta Pfaffinger

Rhesus monkey lipoprotein(a) binds to lysine Sepharose and U937 monocytoid cells less efficiently than human lipoprotein(a). Evidence for the dominant role of kringle 4(37).

Rhesus lipoprotein(a) (Lp[a]) binds less efficiently than human Lp(a) to lysine-Sepharose and to cultured U937 cells. Studies using elastase-derived plasminogen fragments indicated that neither kringle 5 nor the protease domain of Lp(a) are required in these interactions pointing at an involvement of the K4 region. Comparative structural analyses of both the human and simian apo(a) K4 domain, together with molecular modeling studies, supported the conclusion that K4(37) plays a dominant role in the lysine binding function of apo(a) and that the presence of arginine 72 rather than tryptophan in this kringle can account for the functional deficiency observed with rhesus Lp(a). These in vitro results suggest that rhesus Lp(a) may be less thrombogenic than human Lp(a).

Naturally occurring human plasminogen, like genetically related apolipoprotein(a), contains oxidized phosphatidylcholine adducts

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.

Attenuation of immunologic reactivity of lipoprotein(a) by thiols and cysteine-containing compounds. Structural implications.

Samples of human plasma having lipoprotein(a) (Lp[a]) protein levels between 5 and 15 mg/dl and a single apolipoprotein(a) (apo[a]) isoform were incubated in vitro at pH 7.7 with various concentrations (1-20 mM) of N-acetylcysteine, homocysteine, 2-mercaptoethanol (2ME), and dithiothreitol (DTT) for 1 hour at 37 degrees C under a nitrogen atmosphere. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblot analyses using a polyclonal antibody specific for apo(a) showed a progressive decrease in apo(a) immunoreactivity as a function of reductant concentration. This decrease of apo(a) immunoreactivity was corroborated by enzyme-linked immunosorbent assay (ELISA) using anti-apo(a) as the capture antibody and either anti-apo B or anti-apo(a) as the developing antibody. In turn, there was no significant decrease in the immunoreactivity of apo B-100, as assessed by ELISA using anti-apo B as both the capture and the detecting antibody. In the case of high concentrations of DTT the plasma samples had to be diluted to prevent gel formation on addition of the reductant. A progressive drop in immunoreactivity as a function of reagent concentration was also observed in pure preparations of Lp(a) incubated with the reducing agents at pH 7.7. At equivalent stoichiometries the changes were more marked than those observed with whole plasma, suggesting a quenching effect by the plasma proteins on the activity of the reductants. The changes in immunoreactivity were attended by dissociation of apo(a) from Lp(a) as assessed by Western blotting. This dissociation, which we interpret as the result of cleavage of the interchain disulfide bond(s), was complete at 5 mM DTT and 100 mM 2ME.(ABSTRACT TRUNCATED AT 250 WORDS)

The oxidized phospholipids linked to human apolipoprotein(a) do not derive from circulating low-density lipoproteins and are probably of cellular origin

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.— Edelstein, C., Philips, B., Pfaffinger, D., Scanu, A. M. The oxidized phospholipids linked to human apolipoprotein(a) do not derive from circulating low‐density lipoproteins and are probably of cellular origin. FASEB J. 23, 950–956 (2009)

Post-prandial Lp(a): identification of a triglyceride-rich particle containing apo E

A VLDL-like particle containing the apo B100-apo(a) complex was isolated from the post-prandial plasma of subjects fed a fat meal enriched in saturated fatty acids. The abundance of this lipoprotein particle, that we call TG-Lp(a), varied among subjects but not in the same subject. TG-Lp(a), but not the classic Lp(a), contained apo E; this apolipoprotein may cause divergence in cellular uptake and degradation between these two classes of lipoproteins.

Amplification of human APO(a) kringel 4–37 from blood lymphocyte DNA

We have been able to amplify the lysine binding pocket region of human apo(a) kringle type 5 starting from the DNA isolated from peripheral blood lymphocytes. This development now permits the identification of Lp(a) mutants that by lacking their ability to bind to lysine/fibrin would have a lesser thrombogenic potential.

Mouse Plasminogen Has Oxidized Phosphatidylcholine Adducts That Are Not Metabolized by Lipoprotein-Associated Phospholipase A2 under Basal Conditions

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 A2/PAF acetylhydrolase (Lp-PLA2/PAF-AH), an enzyme specific for hydrolysis of oxPtdPCs. We determined the total concentration of Plg in plasma samples from control (WT) and Lp-PLA2-deficient (KO) mice, we isolated Plg, and assessed its content of oxPtdPCs by immunoblot analyses. We also evaluated whether human recombinant Lp-PLA2 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-PLA2. Plg from mouse plasma contains oxPtdPC adducts that are not affected by the action of Lp-PLA2, 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.

Successful utilization of lyophilized lipoprotein(a) as a biological reagent

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.

Molecular Basis for the Lysine Binding Polymorphism of Lipoprotein(a)

Lp(a) and plasminogen share lysine binding property1. For plasminogen this binding function is localized in kringles 1 and 4, whereas for apo(a) the kringle (or kringles) responsible for this function has not been unequivocally identified2. However, there is evidence to suggest that kringle 4-37 contains the necessary structural elements for lysine binding3,4. Experimental support for this prediction has come from studies in rhesus monkeys showing that their apo(a) kringle 4-37 has a mutation associated with a lysine binding deficient Lp(a)5. This mutation is located in the nonpolar trough of the lysine binding pocket that comprises 7 amino acids (two anionic: asp55, asp57; two cationic: arg35, arg71 and three nonpolar: trp62, trp72 and phe64). According to the crystallographic studies on plasminogen kringle 4 by Tulinsky6, trp72 plays a key role in lysine binding. Since lysine binding is related to fibrin binding and to the antifibrinolytic potential of Lp(a), a mutation at this level may render this lipoprotein comparatively less thrombogenic5.

In Vitro Effect of Thiol- and Cysteine-Containing Compounds on the Immunological Reactivity of Human and Rhesus Monkey Lipoprotein(a)

We have examined samples of human and rhesus monkey plasma both with a single and double-band apo(a) isoforms after incubation in vitro at pH 7.7 with various concentrations (1-20 mM) of N-acetylcysteine (NAC), homocysteine (Hcys), 2- mercaptoethanol (2ME) and dithiothreitol (DTT) for 1 hr at 37°C under a nitrogen atmosphere. Incubated samples when fractionated by SDS-polyacrylamide gel electrophoresis followed by immunoblot analyses reacted less intensively than untreated controls as a function of reductant concentration. These data were corroborated by ELISA using as a capture antibody anti apo(a) and as a developing antibody either anti apoB or anti apo(a). Contrary to apo(a) there were no significant changes in the immunoreactivity of apoBl00.