Dyslipidemia is associated with a prothrombotic phenotype; however, the mechanisms responsible for enhanced platelet reactivity remain unclear. Proatherosclerotic lipid abnormalities are associated with both enhanced oxidant stress and the generation of biologically active oxidized lipids, including potential ligands for the scavenger receptor CD36, a major platelet glycoprotein. Using multiple mouse in vivo thrombosis models, we now demonstrate that genetic deletion of Cd36 protects mice from hyperlipidemia-associated enhanced platelet reactivity and the accompanying prothrombotic phenotype. Structurally defined oxidized choline glycerophospholipids that serve as high-affinity ligands for CD36 were at markedly increased levels in the plasma of hyperlipidemic mice and in the plasma of humans with low HDL levels, were able to bind platelets via CD36 and, at pathophysiological levels, promoted platelet activation via CD36. Thus, interactions of platelet CD36 with specific endogenous oxidized lipids play a crucial role in the well-known clinical associations between dyslipidemia, oxidant stress and a prothrombotic phenotype.

Rationale A prothrombotic state and increased platelet reactivity are common in pathophysiological conditions associated with oxidative stress and infections. Such conditions are associated with an appearance of altered-self ligands in circulation that can be recognized by Toll-like receptors (TLR). Platelets express a number of TLR, including TLR9, however, the role of TLR in platelet function and thrombosis is poorly understood. Objective To investigate the biological activities of carboxy(alkylpyrrole) protein adducts (CAPs), an altered self-ligand generated in oxidative stress, on platelet function and thrombosis. Methods and Results In this study we show that CAPs represent novel unconventional ligands for TLR9. Furthermore, using human and murine platelets, we demonstrate that CAPs promote platelet activation, granule secretion, and aggregation in vitro and thrombosis in vivo via the TLR9/MyD88 pathway. Platelet activation by TLR9 ligands induces IRAK1 and AKT phosphorylation, and is Src kinase dependent. Physiological platelet agonists act synergistically with TLR9 ligands by inducing TLR9 expression on the platelet surface. Conclusions Our study demonstrates that platelet TLR9 is a functional platelet receptor that links oxidative stress, innate immunity, and thrombosis.

Background-Hepatic stellate cells play a key role in the pathogenesis of hepatic fibrosis. Aims-To examine the inhibitory eVect of oestradiol on stellate cell activation. Methods-In vivo, hepatic fibrosis was induced in rats by dimethylnitrosamine or pig serum. In vitro, rat stellate cells were activated by contact with plastic dishes resulting in their transformation into myofibroblast-like cells. Results-In the dimethylnitrosamine and pig serum models, treatment with oestradiol at gestation related doses resulted in a dose dependent suppression of hepatic fibrosis with restored content of hepatic retinyl palmitate, reduced collagen content, lower areas of stellate cells which express smooth muscle actin ( -SMA) and desmin, and lower procollagen type I and III mRNA levels in the liver. In cultured stellate cells, oestradiol inhibited type I collagen production, -SMA expression, and cell proliferation. These findings suggest that oestradiol is a potent inhibitor of stellate cell transformation. Conclusion-The antifibrogenic role of oestradiol in the liver may contribute to the sex associated diVerences in the progression from hepatic fibrosis to cirrhosis. (Gut 1999;44:127-136)

Hypercholesterolemia is associated with increased platelet sensitivity to agonists and a prothrombotic phenotype. Mechanisms of platelet hypersensitivity are poorly understood; however, increased platelet cholesterol levels associated with hypercholesterolemia were proposed as leading to hypersensitivity. Scavenger receptor class B type I (SR-BI) in the liver controls plasma high-density lipoprotein (HDL) levels, and SR-BI-deficient mice display a profound dyslipoproteinemia. SR-BI is also expressed on platelets, and recent studies have suggested a role for SR-BI in platelet function; however, its role in hemostasis is unknown. Our present studies demonstrated that nonbone marrow-derived SR-BI deficiency and the dyslipidemia associated with it lead to platelet hyperreactivity that was mechanistically linked to increased platelet cholesterol content. Platelet-specific deficiency of SR-BI, on the other hand, was associated with resistance to hyper- IntroductionDyslipidemia is frequently associated with increased platelet reactivity and thrombogenic potential. [1][2][3][4][5] Although subjects with increased measures of platelet reactivity are at increased prospective risk for coronary events and death, 3,6-9 the mechanisms modulating platelet reactivity in vivo during dyslipidemia are still poorly understood. A mechanistic link between oxidative stress associated with dyslipidemia and a prothrombotic phenotype have been recently established by us. 10 Our studies demonstrated that specific oxidized phospholipids accumulate in plasma in dyslipidemia and interact with platelet CD36, leading to enhanced platelet reactivity, activation, and thrombosis. 10 Dyslipidemia is associated with changes in cellular cholesterol balance leading, in some cases, to increases in platelet cholesterol content or cholesterol/phospholipids ratio. A direct role of excessive platelet cholesterol in induction of platelet hyperreactivity has been shown. 1,2,11,12 However, the molecular mechanisms and pathways linking increased platelet reactivity and cholesterol levels in platelets are not well understood. One critical player in cholesterol metabolism is scavenger receptor class B type I (SR-BI), a multiligand receptor of the CD36 superfamily. 13 Its major physiologic function is selective uptake of cholesteryl esters from high-density lipoprotein (HDL) in steroidogenic tissues and liver. 13 SR-BI also stimulates the bidirectional flux of free cholesterol between cells and lipoproteins, modifies membrane cholesterol distribution, and induces signaling events. 14 SR-BI-deficient mice are hypercholesterolemic with abnormally large circulating HDL particles. 15 Platelets of SR-BI-deficient mice exhibit abnormally high unesterified cholesterol, abnormal morphologies, and elevated rates of clearance from the circulation. 16 Surprisingly, despite high platelet cholesterol content, platelets of SR-BIdeficient mice exhibited in vitro either normal or blunted aggregation responses to agonist. 16 Recent studies have shown that SR-BI is exposed on re...

Aging is associated with both the disturbances of circadian rhythms and a prothrombotic phenotype. It remains poorly understood how the circadian system regulates thrombosis, a critical outcome of aging-related cardiovascular disease. Using multiple in vivo models, we now show that mice with genetic ablation of the core clock gene Bmal1, which display pre-mature aging, have a dramatic prothrombotic phenotype. This phenotype is mechanistically linked to changes in the regulation of key risk factors for cardiovascular disease. These include circulating vWF, fibrinogen, and PAI-1, all of which are significantly elevated in Bmal1 −/− mice. We also show that major circadian transcriptional regulators CLOCK and Bmal1 directly regulate the activity of vWF promoter and that lack of Bmal1 results in upregulation of vWF both at mRNA and protein level. Here we report a direct regulation of vWF expression in endothelial cells by biological clock gene Bmal1. This study establishes a mechanistic connection between Bmal1 and cardiovascular phenotype.The circadian clock system regulates a number of physiological functions in humans and provides a mechanism for the adaptation of an organism to daily changes in the environment (Panda et al., 2002;Lowrey and Takahashi, 2004). It is involved in the progression of a number of human pathological processes, and may determine the success or failure of therapeutic interventions. Numerous clinical studies have demonstrated that acute cardiovascular events (myocardial infarction, stroke, and sudden death) follow diurnal pattern (Muller et al., 1985; Willich, 1990) suggesting the involvement of the components of the circadian clock in their regulation. These include two basic helix-loop-helix (bHLH)-PAS domain-containing transcription factors, CLOCK, and Bmal1, which regulate expression of genes through E-box elements in their promoters and their transcriptional