Inhibitory effects of in vivo oxidized high-density lipoproteins on platelet aggregation: evidence from patients with abetalipoproteinemia.

There is evidence that high-density lipoproteins (HDLs) may regulate platelet function, but disparate results exist regarding the effects of oxidized HDLs on platelets. The objective of our study was to determine the role of in vivo oxidized HDLs on platelet aggregation. Platelet aggregation and redox status were investigated in 5 patients with abetalipoproteinemia (ABLP) or homozygous hypobetalipoproteinemia, two rare metabolic diseases characterized by the absence of apolipoprotein B-containing lipoproteins, compared to 5 control subjects. Platelets isolated from plasma of patients with ABLP aggregated 4 to 10 times more than control platelets, depending on the agonist. By contrast, no differences in the extent of platelet aggregation were observed between ABLP platelet-rich plasma (PRP) and control PRP, suggesting the presence of a protective factor in ABLP plasma. ABLP HDLs inhibited agonist-induced platelet aggregation by binding to SR-BI, while control HDLs had no effect. On the other hand, lipoprotein-deficient plasma from patients with ABLP did not inhibit platelet aggregation. Severe oxidative stress was evidenced in patients with ABLP. Compared to control HDLs, ABLP HDLs showed a 40% decrease of α-tocopherol and an 11-fold increased malondialdehyde concentration. These results demonstrate that in vivo oxidized HDLs do not lose their antiaggregatory properties despite oxidation.

Alterations in the transfer of phospholipids from very-low density lipoproteins to activated platelets in type 2 diabetes.

Type 2 diabetes is a situation at high cardiovascular risk, characterized by platelet hyperactivation, oxidative stress, elevated very-low density lipoprotein (VLDL) and low high-density lipoprotein concentrations. In the present report, we describe the effects of these alterations on the transfers of phospholipids (PL) from VLDL to platelets in basal conditions or after thrombin (0.1U/mL) or lipoprotein lipase (LPL, 500ng/mL)-mediated platelet activation. In vitro transfer of radiolabelled PL from VLDL (200microM PL) to platelets (2x10(8)/mL) was measured after incubations of 1h at 37 degrees C in a series of recombination experiments using control or diabetic platelets and VLDL, as well as normal or oxidized PL. Basal- and thrombin-stimulated transfers from diabetic VLDL were similar to those from control VLDL. However, LPL-stimulated transfer was decreased when using diabetic VLDL. This was likely due to their lowered ability to be lipolyzed. When we compared the platelets from either diabetic patients or control subjects, we observed that the transfers of PL from control VLDL to diabetic platelets were 20-30% higher than those to control platelets, whether in basal conditions or under LPL or thrombin stimulations. Finally, we observed that, in all conditions tested, the rate of transfers of oxidized PL was two to three times more elevated than that of non oxidized PL. Collective consideration of these data suggests that the transfer of PL from VLDL to platelets might be elevated in type 2 diabetes, favoring oxidative stress-mediated platelet hyperactivation.

The transfer of VLDL-associated phospholipids to activated platelets depends upon cytosolic phospholipase A2 activity.

We previously reported that VLDL could transfer phospholipids (PLs) to activated platelets. To identify the metabolic pathway involved in this process, the transfer of radiolabeled PLs from VLDL (200 microM PL) to platelets (2 x 10(8)/ml) was measured after incubations of 1 h at 37 degrees C, with or without thrombin (0.1 U/ml) or LPL (500 ng/ml), in the presence of various inhibitors, including aspirin, a cyclooxygenase inhibitor (300 microM); esculetin, a 12-lipoxygenase inhibitor (20 microM); methyl-arachidonyl-fluorophosphonate (MAFP), a phospholipase A(2) (PLA(2)) inhibitor (100 microM); 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl) ester (BAPTA-AM), a Ca(2+) chelator (20 microM); bromoenol lactone (BEL), a Ca(2+)- independent phospholipase A(2) (iPLA(2)) inhibitor (100 nM); and 1-[6-[[17beta-3-methoxyestra-1,3,5(10)-trien-17-yl-]amino]hexyl]1H-pyrrole-2,5-dione (U73122), a phospholipase C (PLC) inhibitor (20 microM). Aspirin and esculetin had no effect, showing that PL transfer was not dependent upon cyclooxygenase or lipoxygenase pathways. The transfer of PL was inhibited by MAFP, U73122, and BAPTA-AM. Although MAFP inhibited both cytosolic phospholipase A(2) (cPLA(2)) and iPLA(2), only cPLA(2) is a calcium-dependent enzyme. Because calcium mobilization is favored by PLC and inhibited by BAPTA-AM, the transfer of PL from VLDL to platelets appeared to result from a cPLA(2)-dependent process. The inhibition of iPLA(2) by BEL had no effect on PL transfers.

In vitro glycoxidized low-density lipoproteins and low-density lipoproteins isolated from type 2 diabetic patients activate platelets via p38 mitogen-activated protein kinase.

CONTEXT: Platelet hyperactivation contributes to the increased risk for atherothrombosis in type 2 diabetes and is associated with oxidative stress. Plasma low-density lipoproteins (LDLs) are exposed to both hyperglycemia and oxidative stress, and their role in platelet activation remains to be ascertained. OBJECTIVE: The aim of this study was to investigate the effects of LDLs modified by both glycation and oxidation in vitro or in vivo on platelet arachidonic acid signaling cascade. The activation of platelet p38 MAPK, the stress kinase responsible for the activation of cytosolic phospholipase A(2), and the concentration of thromboxane B(2), the stable catabolite of the proaggregatory arachidonic acid metabolite thromboxane A(2), were assessed. RESULTS: First, in vitro-glycoxidized LDLs increased the phosphorylation of platelet p38 MAPK as well as the concentration of thromboxane B(2). Second, LDLs isolated from plasma of poorly controlled type 2 diabetic patients stimulated both platelet p38 MAPK phosphorylation and thromboxane B(2) production and possessed high levels of malondialdehyde but normal alpha-tocopherol concentrations. By contrast, LDLs from sex- and age-matched healthy volunteers had no activating effects on platelets. CONCLUSIONS: Our results indicate that LDLs modified by glycoxidation may play an important contributing role in platelet hyperactivation observed in type 2 diabetes via activation of p38 MAPK.

Evidence for in situ ethanolamine phospholipid adducts with hydroxy-alkenals.

Hydroxy-alkenals, such as 4-hydroxy-2(E)-nonenal (4-HNE; from n-6 fatty acids), are degradation products of fatty acid hydroperoxides, including those generated by free radical attack of membrane polyunsaturated fatty acyl moieties. The cytotoxic effects of hydroxy-alkenals are well known and are mainly attributable to their interaction with different molecules to form covalent adducts. Indeed, ethanolamine phospholipids (PEs) can be covalently modified in a cellular system by hydroxy-alkenals, such as 4-HNE, 4-hydroxy-2(E)-hexenal (4-HHE; from n-3 fatty acids), and 4-hydroxy-dodecadienal (4-HDDE; from the 12-lipoxygenase product of arachidonic acid), to form mainly Michael adducts. In this study, we describe the formation of PE Michael adducts in human blood platelets in response to oxidative stress and in retinas of streptozotocin-induced diabetic rats. We have successfully characterized and evaluated, for the first time, PEs coupled with 4-HHE, 4-HNE, and 4-HDDE by gas chromatography-mass spectrometry measurement of their ethanolamine moieties. We also report that aggregation of isolated human blood platelets enriched with PE-4-hydroxy-alkenal Michael adducts was altered. These data suggest that these adducts could be used as specific markers of membrane disorders occurring in pathophysiological states with associated oxidative stress and might affect cell function.

Transfer of very low density lipoprotein-associated phospholipids to activated human platelets.

LDL-associated phospholipids (PLs) may be transferred into platelets. In this work, we characterized the role of VLDLs as PL donors. VLDL transferred radiolabeled PLs to platelets in a temperature- and concentration-dependent manner. LPL stimulated this process through its action on VLDL lipolysis, because it was abolished by tetrahydrolipstatin. LPL also stimulated the platelet production of thromboxane B2 (TXB2). Both LPL actions were inhibited in the presence of fatty acid-free albumin, suggesting that they were attributable to fatty acids generated during VLDL lipolysis. To study the relationship between PL transfers and platelet activation, we performed incubations in the presence of HDL, a physiological acceptor of PL released from VLDL. HDL antagonized the transfer of PL from VLDL to platelets but had no effect on the production of TXB2, suggesting that PL transfers were driven by platelet activation. Confirming this idea, thrombin stimulated both the production of TXB2 and the transfers of PL. In conclusion, VLDL can transfer PL to platelets. These transfers are stimulated by LPL and thrombin through their action on platelet activation. They might be enhanced in pathologies characterized by increased VLDL concentrations.

Postprandial increase of plasma apoAV concentrations in Type 2 diabetic patients.

Postprandial hypertriglyceridemia is considered as a risk factor for cardiovascular disease in Type 2 diabetes. However, little is known about the underlying mechanisms. Since the recently discovered apolipoprotein (apo) AV was identified as a modulator of triglyceride (TG) metabolism, the aim of the study was to determine the postprandial apoAV profile of Type 2 diabetic patients. We compared data from 11 patients with Type 2 diabetes mellitus to that of 12 non-diabetic normolipidemic subjects following the ingestion of a lipid-rich cream. Postprandial apoAV was elevated in diabetic patients but no correlation was observed either with plasma TG concentration or with the intensity of lipoprotein lipase-dependent lipolysis. These data obtained in human subjects suggest that plasma apoAV concentration does not play an acute or a direct role in the regulation of plasma TG in the postprandial state.

Alteration in lipoprotein lipase activity bound to triglyceride-rich lipoproteins in the postprandial state in type 2 diabetes.

Postprandial lipid metabolism is largely dependent upon lipoprotein lipase (LPL), which hydrolyses triglycerides (TGs). The time course of LPL activity in the postprandial state following a single meal has never been studied, because its determination required heparin injection. Recently, we have shown that LPL activity could be accurately measured in nonheparinized VLDL using a new assay aiming to determine the LPL-dependent VLDL-TG hydrolysis. Based on the same principle, we used in this study TG-rich lipoprotein (TRL)-bound LPL-dependent TRL-TG hydrolysis (LTTH) to compare the time course of LPL activity of 10 type 2 diabetics to that of 10 controls, following the ingestion of a lipid-rich meal. The peak TG concentration, reached after 4 h, was 67% higher in diabetics than in controls (P < 0.005). Fasting LTTHs were 91.3 +/- 15.6 in controls versus 70.1 +/- 4.8 nmol NEFA/ml/h in diabetics (P < 0.001). LTTH was increased 2 h postprandially by 190% in controls and by only 89% in diabetics, resulting in a 35% lowering of the LTTH area under the curve in diabetics. Postprandial LTTH was inversely correlated with TG or remnant concentrations in controls but not in diabetics, and with insulin resistance in both groups. These data show that TRL-bound LPL activity increases in the postprandial state and is strongly reduced in type 2 diabetes, contributing to postprandial hypertriglyceridemia.

Structural and functional determinants of human plasma phospholipid transfer protein activity as revealed by site-directed mutagenesis of charged amino acids.

Human plasma phospholipid transfer protein (PLTP) exchanges phospholipids between lipoproteins and remodels high-density lipoproteins (HDLs). We determined phospholipid transfer activity and HDL binding ability in wild-type PLTP and in 16 PLTP variants created by replacing 12 charged amino acids by site-directed mutagenesis. The data were analyzed in relation to the structure of a member of the same gene family, bactericidal/permeability-increasing protein, which is a boomerang-shaped molecule containing two symmetrical, hydrophobic pockets that bind phospholipid molecules. When expressed in COS-7 cells, wild-type and all mutant PLTPs accumulated intracellularly to nearly the same extent. Relative to wild-type PLTP, substitution(s) for amino acids with a lateral position totally exposed to the solvent produced reductions in transfer activity proportional to the reductions in the level of HDL binding. Variants containing substitutions for charged amino acids on the concave surface of PLTP did not affect binding to HDL or specific transfer activity. A mutation in the C-terminal pocket (E270R) led to a decrease in both the specific transfer activity and the level of binding to HDLs, whereas mutations in the N-terminal pocket (R25E and D231R) resulted in a large decrease in specific transfer activity without affecting HDL binding. The data support a model of transfer in which N- and C-terminal pockets have different roles in HDL binding and transfer activity. The N-terminal pocket may be critical to PLTP transfer activity but may have no involvement in binding to lipoproteins, whereas amino acid substitutions in the C-terminal pocket might reduce PLTP activity by decreasing PLTP's affinity for HDLs.