Significance determined by two-tailed Students t-test. (e) The cecal contents were harvested from your mice used in Physique 4d, and qPCR was used to quantify the relative expression of microbial as described under Methods. inhibitor selectively accumulated within intestinal microbes to millimolar levels, a concentration over a million-fold higher than needed for a therapeutic effect. These studies uncover that mechanism-based inhibition of gut microbial TMA/TMAO production reduces thrombosis potential, a critical adverse complication in heart disease. They also offer a generalizable approach for the selective non-lethal targeting of gut microbial enzymes linked to host disease, while limiting systemic exposure of the inhibitor in the host. Introduction Recent studies implicate participation of the gut microbiome in numerous facets of human health and disease1C6. For example, less than a decade ago, a link between dietary phosphatidylcholine, a nutrient common in a Western diet, gut microbiota-dependent generation of the metabolite trimethylamine N-oxide (TMAO), and cardiovascular disease (CVD) pathogenesis, was first described7. Since then, multiple human and animal studies supporting both mechanistic and clinical prognostic associations between TMAO formation and cardiometabolic disease risks have been reported8C16. The mechanisms through which TMAO is usually thought to foster enhanced CVD risks are manifold and include alterations in tissue sterol metabolism7,9,17, enhanced endothelial cell activation and vascular inflammation7,18C20, and activation of pro-fibrotic signaling pathways14,15. Historically, gut microbiota are known to impact factors linked to platelet function and hemostasis, including serotonin21, vitamin K22, and von A 286982 Willebrand factor23. In addition, A 286982 recent studies reveal TMAO alters calcium signaling in platelets, enhancing responsiveness and thrombosis potential in animal models15. Parallel clinical studies reveal TMAO levels are associated with thrombotic event risks (heart A 286982 attack and stroke)15, and clinical interventional studies with choline supplementation in healthy vegan or omnivorous volunteers were shown to both increase circulating TMAO levels and heighten platelet responsiveness to agonists24. Finally, several recent meta-analyses confirm a strong clinical association between increased levels of TMAO and incident adverse cardiovascular event and mortality risks in multiple populations25C27. Thus, there is rapidly growing desire for the therapeutic targeting of gut microbiota-dependent TMAO generation for the potential treatment A 286982 of CVD28. TMAO is usually generated via a meta-organismal pathway that begins with gut microbial conversion of dietary nutrients (e.g. phosphatidylcholine, choline, and carnitine) into trimethylamine (TMA), followed by host liver oxidation to TMAO by flavin monooxygenases (FMOs)29,30. Given the Eno2 abundance of the choline moiety in both bile31 and common dietary staples (e.g. eggs, meat/fish, and some fruits/vegetables), microbial conversion of choline into TMA likely accounts for a significant portion of TMAO production in subjects, regardless of diet. A pair of microbial proteins encoded by genes of the choline utilization (and mice on a choline-supplemented diet, plasma TMAO levels were significantly lowered, and concurrently, macrophage cholesterol accumulation, foam cell formation and atherosclerotic lesion development were attenuated35. While atherosclerotic plaque development is usually a defining pathologic feature of coronary artery disease, enhanced platelet reactivity and acute thrombotic occlusion of vessels are the proximate cause of myocardial infarction, stroke and the majority of deaths in patients with CVD36. Use of antiplatelet brokers has become a cornerstone for the treatment of CVD because of substantial reduction in CVD events and mortality37,38. However, more widespread use of antiplatelet brokers has been limited by the increased risk of bleeding, which also leads to nonadherence39C41. Herein we show that a mechanism-based non-lethal inhibitor of the gut microbial TMAO pathway designed to selectively accumulate within the gut microbial compartment, can serve as a new therapeutic approach for attenuating thrombosis while simultaneously limiting systemic exposure in the host. Results DMB, a microbial choline TMA lyase inhibitor, attenuates choline diet-enhanced platelet responsiveness and rate of thrombus formation In initial studies, C57BL/6J mice were maintained on a chemically-defined control Chow diet versus the same diet supplemented with choline (1% w/w). The choline diet elicited no differences in multiple indices of platelet activation, including surface phosphatidylserine content (p=0.84) in ADP-stimulated washed platelets or levels A 286982 of von Willebrand factor (p=0.14), alpha granule release (p=0.31), or prothrombotic microvesicle release (p=0.66) in platelet-rich plasma (PRP) in the absence of agonist.