Modified Phosphatidylethanolamine as the Active Component of Oxidized Low Density Lipoprotein Promoting Platelet Prothrombinase Activity

Abstract

We analyzed the influence of the atherogenic oxidized low density lipoproteins (LDL) on the activity of the platelet prothrombinase complex, a major contributor to overall thrombin formation in vivo. Platelet dependent thrombin generation was found to be strongly stimulated by in vitro oxidized LDL. The enhancement was additive to that observed with the platelet agonist thrombin. Oxidized LDL increased the platelet binding of annexin-V, suggesting that the augmented surface exposure of aminophospholipids promoted the prothrombinase activity. All of the stimulatory activity of the oxidized LDL could be recovered in the microemulsions prepared from the lipid portion of the modified particles. Phospholipid vesicles were prepared containing the total lipids of the oxidized LDL but lacking specifically in one lipid component. Following the selective removal of the ethanolamine phospholipids (PE) from the LDL lipids, the platelet-dependent thrombin formation was markedly reduced. Vesicles enriched with the isolated PE fraction alone enhanced the thrombin generation. Analyses with autoxidized phospholipids indicated that oxidation products of unsaturated diacyl-PE were mainly responsible for the increased prothrombinase activity. Oxidized LDL and its PE fraction lost their stimulatory activity after treatment with NaCNBH 3 , a chemical reductant of Schiff base adducts. Phospholipid vesicles supplemented with synthetic aldehyde-PE adducts largely reproduced the stimulation of the thrombin generation. We conclude that the oxidized LDL particles elicit a pronounced prothrombotic response by increasing the activity of the platelet prothrombinase complex. Specific oxidative modifications of the LDLassociated ethanolamine phospholipids are mainly responsible for this stimulation.

Import and fate of fluorescent analogs of oxidized phospholipids in vascular smooth muscle cells

“…hand, aldehydes generated by the myeloperoxidase system of phagocytes were shown to covalently modify the amino groups of ethanolamine and serine glycerophospholipids to form biologically active Schiff base adducts (46). Zieseniss et al (38) found that Schiff base adducts between phosphatidylethanolamine and aldehydes in oxidized LDL provoke the stimulation of platelet prothrombinase activity. In the beginning, there was little consideration of the possibility that some of the masking of amino groups during LDL oxidation might be attributable to conjugation with phospholipid aldehydes, as exemplified by POVPC.…”

“…In the beginning, there was little consideration of the possibility that some of the masking of amino groups during LDL oxidation might be attributable to conjugation with phospholipid aldehydes, as exemplified by POVPC. Several studies now exist that demonstrate covalent adduct formation between oxidized phospholipids and apoB (13,38,54). In this work, we found evidence that BODIPY-POVPE reacts with many cellular proteins.…”

“…8A, B). From the literature, it is known that the aldehydolipids might undergo aldol condensation with themselves (39) and form (Schiff base) adducts with endogenous cellular lipids (phosphatidylethanolamine and phosphatidylserine) (38,46).…”

“…After washing with 700 ml of a MgCl 2 solution (0.036% in water) and centrifuging at 80 g for 2 min at room temperature, the lower chloroform phase was collected and evaporated to dryness. In the case of BODIPY-POVPE, extracted lipids were reduced with 10 mM NaCNBH 3 in a biphasic system (a mixture of 1 ml of diethylether and 1 ml of buffer containing 0.75 M NaCl and 1 mM HEPES) (38), leading to the conversion of formed Schiff base adducts to stable amine structures. The reduced lipids were extracted from the aqueous phase with 5 ml of CHCl 3 / MeOH (2:1, v/v).…”

Characterization of Acrolein-Glycerophosphoethanolamine Lipid Adducts Using Electrospray Mass Spectrometry

“…While it has been previously established that GPEtn and GPSer lipids react with aldehydes (13-16), there have not been studies reporting the reaction of either of these aminophospholipids with acrolein. Furthermore, most of these previous aminophospholipid/aldehyde adduct studies have focused primarily on the formation of aldehyde-modified GPEtn and GPSer products in solution, and few studies have shown that these covalently modified GPEtn and GPSer species are present in cell membranes (17,18). …”