Lipoprotein(a), Lp(a), is an LDL variant, having as a protein moiety apoB100 linked covalently on a 1:1 molar basis to apolipoprotein(a), apo(a), a highly glycosylated multikringle structure. Hallmarks of apo(a) are its heterogeneity due to its size polymorphism dependent on the number of kringle type 2 repeats and susceptibility to proteolytic cleavage. Studies in vitro and in cell culture on cleaved products and recombinants have provided evidence that apo(a) is made of bioactive microdomains (fragments) contained in the C-terminal region that we call F2. Thus far, the bioactivity in F2 has been demonstrated for KIV-containing microdomains. However, we have recently shown that the KV-protease domain, PD, in F2 is able to stimulate the production of interleukin-8 in cultured human macrophages and assigned this effect to lysine residues in KV linked to oxidized phospholipids, ox-PL and also shown that these adducts are recognized by the monoclonal antibody, EO6. On the basis of these observations we will explore the hypothesis that KV-PD contributes to the athero-thrombogenic properties of Lp(a). To this effect, we will pursue the studies on KV-containing recombinants, with no PD, to unequivocally identify the lys residues involved in linkage with ox-PLs and the chemical nature of the latter by mass spectroscopy techniques. We have recently reported discrete fragments of apo(a) in extracts of human carotid plaques in a microenvironment rich in pro-inflammatory cytokines and metalloproteinases. We have also immunological evidence that these fragments contain ox-PL adducts. Thus, we wish to better define the properties of these fragments and compare them with those exhibited by the products generated in vitro by both apo(a) proteolysis and recombinant techniques. For this purpose, we will use immunochemical methods, proteomic and DNA array of laser-microdissected macrophage-rich areas of endarterectomy segments of lesioned human carotid arteries. The results of the proposed studies should shed light on whether ox-PL adducts play a role in the cardiovascular pathogenicity of Lp(a).
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