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

Evidence for a proinflammatory and proteolytic environment in plaques from endarterectomy segments of human carotid arteries

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