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Mouse plasminogen has oxidized phosphatidylcholine adducts that are not metabolized by lipoprotein-associated phospholipase A₂under basal conditions.

Edelstein C, Pfaffinger D, Reichert EC, Stafforini DM, Scanu AM - Int J Mol Sci (2010)

Bottom Line: 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 also evaluated whether human recombinant Lp-PLA(2) metabolized Plg-linked oxPtdPCs in vivo and in vitro.This modification may have important physio-pathological implications related to the function of Plg, oxPtdPCs, or both.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Chicago, Chicago, IL 60637, USA; E-Mails: celina@medicine.bsd.uchicago.edu (C.E.); dpfaffin@medicine.bsd.uchicago.edu (D.P.).

ABSTRACT
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.

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Sandwich ELISA for detection of Plg-bound OxPtdPC. (A) We developed a sandwich ELISA for the detection of OxPtdPC linked to Plg in which T15 was the capture antibody and anti-mouse Plg conjugated to HRP was the detection antibody. (B) Dose response curve obtained with purified mouse Plg. Vertical bars through the data points represent means ± SD.
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f3-ijms-11-05339: Sandwich ELISA for detection of Plg-bound OxPtdPC. (A) We developed a sandwich ELISA for the detection of OxPtdPC linked to Plg in which T15 was the capture antibody and anti-mouse Plg conjugated to HRP was the detection antibody. (B) Dose response curve obtained with purified mouse Plg. Vertical bars through the data points represent means ± SD.

Mentions: To further investigate this issue, we quantified the oxPtdPC molecules bound to Plg isolated from WT and KO mice by ELISA (see “Methods”) (Figure 3A). The assay was highly reproducible over a wide range of concentrations (1.56 to 100 nmol/L, Figure 3B). From the values obtained we calculated an oxPtdPC:Plg ratio of one for both Plg samples, establishing that under basal conditions, deletion of Lp-PLA2 does not affect the extent of Plg conjugation with oxPtdPCs.


Mouse plasminogen has oxidized phosphatidylcholine adducts that are not metabolized by lipoprotein-associated phospholipase A₂under basal conditions.

Edelstein C, Pfaffinger D, Reichert EC, Stafforini DM, Scanu AM - Int J Mol Sci (2010)

Sandwich ELISA for detection of Plg-bound OxPtdPC. (A) We developed a sandwich ELISA for the detection of OxPtdPC linked to Plg in which T15 was the capture antibody and anti-mouse Plg conjugated to HRP was the detection antibody. (B) Dose response curve obtained with purified mouse Plg. Vertical bars through the data points represent means ± SD.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3100823&req=5

f3-ijms-11-05339: Sandwich ELISA for detection of Plg-bound OxPtdPC. (A) We developed a sandwich ELISA for the detection of OxPtdPC linked to Plg in which T15 was the capture antibody and anti-mouse Plg conjugated to HRP was the detection antibody. (B) Dose response curve obtained with purified mouse Plg. Vertical bars through the data points represent means ± SD.
Mentions: To further investigate this issue, we quantified the oxPtdPC molecules bound to Plg isolated from WT and KO mice by ELISA (see “Methods”) (Figure 3A). The assay was highly reproducible over a wide range of concentrations (1.56 to 100 nmol/L, Figure 3B). From the values obtained we calculated an oxPtdPC:Plg ratio of one for both Plg samples, establishing that under basal conditions, deletion of Lp-PLA2 does not affect the extent of Plg conjugation with oxPtdPCs.

Bottom Line: 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 also evaluated whether human recombinant Lp-PLA(2) metabolized Plg-linked oxPtdPCs in vivo and in vitro.This modification may have important physio-pathological implications related to the function of Plg, oxPtdPCs, or both.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Chicago, Chicago, IL 60637, USA; E-Mails: celina@medicine.bsd.uchicago.edu (C.E.); dpfaffin@medicine.bsd.uchicago.edu (D.P.).

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus