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Platelet hyperreactivity, defined as enhanced sensitivity to activation in response to classical agonists, contributes to the increased risk of arterial thrombosis associated with chronic inflammatory diseases. In this issue of the JCI, Kong and colleagues used an unbiased proteomic approach to identify elevated SEC61B in platelets from patients with diabetes and from hyperglycemic mice. Typically, SEC61B participates in protein transport within the endoplasmic reticulum (ER), but it can also act as an ion channel that allows calcium to leak from ER to cytoplasm. The authors showed that elevated SEC61B expression caused increased calcium leak, elevated basal cytoplasmic calcium concentrations, and platelet hyperreactivity. In vitro and in vivo pharmacological interventions to alter calcium homeostasis through this pathway affected platelet reactivity. The results of this work are consistent with those of previous studies showing that platelets from patients with chronic diseases behaved differently than those from healthy participants. These findings identify potential disease-specific targets to prevent and treat arterial thrombosis.

Platelet hyperreactivity, defined as enhanced sensitivity to activation In response to classical agonists, contributes to the increased risk of arterial thrombosis associated with chronic inflammatory diseases. In this issue of the JCI, Kong and colleagues used an unbiased proteomic approach to identify elevated SEC61B in platelets from patients with diabetes and from hyperglycemic mice. Typically, SEC61B participates in protein transport within the endoplasmic reticulum (ER), but it can also act as an ion channel that allows calcium to leak from ER to cytoplasm. The authors showed that elevated SEC61B expression caused increased calcium leak, elevated basal cytoplasmic calcium concentrations, and platelet hyperreactivity. In vitro and in vivo pharmacological interventions to alter calcium homeostasis through this pathway affected platelet reactivity. The results of this work are consistent with those of previous studies showing that platelets from patients with chronic diseases behaved differently than those from healthy participants. These findings identify potential disease-specific targets to prevent and treat arterial thrombosis.

The trapping of lipid-laden macrophages in the arterial intima is a critical but reversible step in atherogenesis. However, the mechanism by which this occurs is not clearly defined. Here, we tested in mice the hypothesis that CD36, a class B scavenger receptor expressed on macrophages, has a role in this process. Using both in vivo and in vitro migration assays, we found that oxidized LDL (oxLDL), but not native LDL, inhibited migration of WT mouse macrophages but not CD36-deficient cells. We further observed a crucial role for CD36 in modulating the in vitro migratory response of human peripheral blood monocyte-derived macrophages to oxLDL. oxLDL also induced rapid spreading and actin polymerization in CD36-sufficient but not CD36-deficient mouse macrophages in vitro. The underlying mechanism was dependent on oxLDL-mediated CD36 signaling, which resulted in sustained activation of focal adhesion kinase (FAK) and inactivation of Src homology 2-containing phosphotyrosine phosphatase (SHP-2). The latter was due to NADPH oxidase-mediated ROS generation, resulting in oxidative inactivation of critical cysteine residues in the SHP-2-active site. Macrophage migration in the presence of oxLDL was restored by both antioxidants and NADPH oxidase inhibitors, which restored the dynamic activation of FAK. We conclude therefore that CD36 signaling in response to oxLDL alters cytoskeletal dynamics to enhance macrophage spreading, inhibiting migration. This may induce trapping of macrophages in the arterial intima and promote atherosclerosis.

CD36 is a type 2 scavenger receptor with multiple functions. CD36 binding to oxidized LDL triggers signaling cascades that are required for macrophage foam cell formation, but the mechanisms by which CD36 signals remain incompletely understood. Mass spectrometry analysis of anti-CD36 immuno-precipitates from macrophages identified the tetraspanin CD9 as a CD36 interacting protein. Western blot showed that CD9 was precipitated from mouse macrophages by anti-CD36 monoclonal antibody and CD36 was likewise precipitated by anti-CD9, confirming the mass spectrometry results. Macrophages from cd36 null mice were used to demonstrate specificity. Membrane associations of the two proteins on intact cells was analyzed by confocal immunofluorescence microscopy and by a novel cross linking assay that detects proteins in close proximity (<40 nm). Functional significance was determined by assessing lipid accumulation, foam cell formation and JNK activation in wt, cd9 null and cd36 null macrophages exposed to oxLDL. OxLDL uptake, lipid accumulation, foam cell formation, and JNK phosphorylation were partially impaired in cd9 null macrophages. The present study demonstrates that CD9 associates with CD36 on the macrophage surface and may participate in macrophage signaling in response to oxidized LDL.

Oxidative stress increases the risk for clinically significant thrombotic events, yet the mechanisms by which oxidants become prothrombotic are unclear. In this review, we provide an overview of cysteine reactivity and oxidation. We then highlight recent findings on cysteine oxidation events in oxidative stress-related thrombosis. Special emphasis is on the signaling pathway induced by a platelet membrane protein, CD36, in dyslipidemia, and by protein disulfide isomerase (PDI), a member of the thiol oxidoreductase family of proteins. Antioxidative and chemical biology approaches to target cysteine are discussed. Lastly, the knowledge gaps in the field are highlighted as they relate to understanding how oxidative cysteine modification might be targeted to limit thrombosis.

High density lipoproteins (HDL) are considered athero-protective, primarily due to their role in reverse cholesterol transport, where they transport cholesterol from peripheral tissues to the liver for excretion. The current study was designed to determine the impact of HDL modification by acrolein, a highly reactive aldehyde found in high abundance in cigarette smoke, on the cholesterol transport functions of HDL. HDL was chemically-modified with acrolein and immunoblot and mass spectrometry analyses confirmed apolipoprotein crosslinking, as well as acrolein adducts on apolipoproteins A-I and A-II. The ability of acrolein-modified HDL (acro-HDL) to serve as an acceptor of free cholesterol (FC) from COS-7 cells transiently expressing SR-BI was significantly decreased. Further, in contrast to native HDL, acro-HDL promotes higher neutral lipid accumulation in murine macrophages as judged by Oil Red O staining. The ability of acro-HDL to mediate efficient selective uptake of HDL-cholesteryl esters (CE) into SR-BI-expressing cells was reduced compared to native HDL. Together, the findings from our studies suggest that acrolein modification of HDL produces a dysfunctional particle that may ultimately promote atherogenesis by impairing functions that are critical in the reverse cholesterol transport pathway.

CD36 is a membrane glycoprotein expressed on platelets, monocytes, macrophages, and several other cell types that was recently demonstrated to be involved in platelet activation in response to oxidized phospholipids, including oxidized LDL. Although the role of CD36 in other vascular cells has not been well defined, previous studies have demonstrated that cd36-knockout (cd36-/-) mice have prolonged thrombosis times after vascular injury, which can be protective in the state of hyperlipidemia. Here, we found significantly less ROS in the vessel walls of cd36-/- mice compared with WT after chemically induced arterial injury, suggesting that CD36 may contribute to ROS generation in the VSMCs themselves. Gene expression analysis revealed that the antioxidant enzymes peroxiredoxin-2 (Prdx2) and heme oxygenase-1 were upregulated in cd36-/- VSMCs. Molecular dissection of the pathway in isolated mouse VSMCs revealed CD36 ligand-dependent induction of Fyn phosphorylation, with subsequent phosphorylation and degradation of the redox-sensitive transcription factor Nrf2. Chromatin immunoprecipitation experiments further showed that Nrf2 directly occupied the Prdx2 promoter. The importance of this pathway was evidenced by increased ROS generation in prdx2-/- mice and decreased thrombosis times in both prdx2-/- and nrf2-/- mice after vascular injury. These data suggest that CD36-mediated downregulation of antioxidant systems in VSMCs may contribute to its prothrombotic, proinflammatory, and atherogenic effects.

CD36 is a scavenger receptor that binds multiple ligands, including phosphatidyl serine (PS). Although CD36(-) mice do not have a bleeding diathesis, we show here that they do have significantly prolonged thrombotic occlusion times in response to FeCl(3)-induced vascular injury. Because cell-derived microparticles (MPs) are generated in response to vascular injury and circulate in patients with prothrombotic diseases, we hypothesized that PS exposed on their surfaces could be an endogenous CD36 ligand that transmits an activating signal to platelets. We found that MPs prepared from human ECs, monocytes, or platelets or isolated from blood of normal subjects bound to platelets. Binding was not observed with platelets from CD36(-) donors and was inhibited by an anti-CD36 antibody or by blockade of exposed PS by annexin V or anti-PS IgM. Preincubation of platelets with MPs led to CD36-dependent augmentation of platelet activation in response to low doses of ADP, as assessed by measuring alpha(2b)beta(3) activation, P-selectin expression, and aggregation. Immunofluorescence confocal microscopy of murine carotid thrombi from CD36(-) mice showed a significant decrement in endothelial antigen accumulation, which suggests that CD36 plays a role in MP recruitment into thrombi. These results provide what we believe to be a novel role for CD36 in thrombosis.

Epidemiologic studies in diabetic patients as well as research in model organisms have indicated the potential of metformin as a drug candidate for the treatment of various types of cancer, including breast cancer. To date most of the anti-cancer properties of metformin have, in large part, been attributed either to the inhibition of mitochondrial NADH oxidase complex (Complex I in the electron transport chain) or the activation of AMP-activated kinase (AMPK). However, it is becoming increasingly clear that AMPK activation may be critical to alleviate metabolic and energetic stresses associated with tumor progression suggesting that it may, in fact, attenuate the toxicity of metformin instead of promoting it. Here, we demonstrate that AMPK opposes the detrimental effects of mitochondrial complex I inhibition by enhancing glycolysis at the expense of, and in a manner dependent on, pyruvate availability. We also found that metformin forces cells to rewire their metabolic grid in a manner that depends on AMPK, with AMPK-competent cells upregulating glycolysis and AMPK-deficient cell resorting to ketogenesis. In fact, while the killing effects of metformin were largely rescued by pyruvate in AMPKcompetent cells, AMPK-deficient cells required instead acetoacetate, a product of fatty acid catabolism indicating a switch from sugar to fatty acid metabolism as a central resource for ATP production in these cells. In summary, our results indicate that AMPK activation is not responsible for metformin anticancer activity and may instead alleviate energetic stress by activating glycolysis.