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. Author manuscript; available in PMC: 2019 Feb 6.
Published in final edited form as: Nat Med. 2018 Aug 6;24(9):1407–1417. doi: 10.1038/s41591-018-0128-1

Figure 1. Proof of concept that microbial choline TMA lyase inhibition can attenuate choline diet-enhanced platelet aggregation and in vivo thrombus formation.

Figure 1

a) Platelet aggregation in PRP of mice fed the indicated diets ± DMB (1.3% v/v) provided in the drinking water for 6 weeks. Platelet aggregation was measured in response to a submaximal concentration of ADP (1 μM). Data points represent aggregation as % of maximum amplitude in PRP recovered from each mouse, and bars represent mean levels for each group. Plasma TMAO levels are also shown and represent mean ± SEM for each group. Significance determined by two-tailed Student’s t-test.

b) Representative vital microscopy images of carotid artery thrombus formation at the indicated time points following FeCl3-induced carotid artery injury in mice fed either a chemically-defined chow or 1% choline diet with or without the addition of DMB (1.3% v/v). The time to complete occlusion is noted in the right-hand panels. Complete study results including replications are shown in Figure 1c.

c) Quantification of in vivo thrombus formation following FeCl3-induced carotid artery injury in mice fed the indicated diets ± DMB (1.3% v/v) provided in the drinking water for 6 weeks. Data points represent the time to cessation of flow for each mouse, and bars represent mean levels for each group. Plasma TMAO levels are also shown and represent mean ± SEM for each group. Significance determined by two-tailed Student’s t-test.

d) Proposed mechanism by which a potential suicide substrate inhibitor of CutC/D, iodomethylcholine (IMC), can form a reactive iodotrimethylamine (I-TMA) product that can promote irreversible CutC/D inhibition via covalent modification of a reactive, nucleophilic active-site residue (Nu).

e) Comparison of the inhibitory potency of IMC (○), DMB (□) and Resveratrol (RESV, △), against (left) wild-type, recombinant P. mirabilis CutC/D lysate, (center) recombinant D. alaskensis CutC/D lysate, and (right) whole-cell (intact live culture) wild-type P. mirabilis. Data points represent the mean ± SEM. Exact numbers used for each data point can be found in the Source Data (n=2–9 technical replicates).