Gut Microbial Metabolite TMAO Enhances Platelet Hyperreactivity and Thrombosis Risk

Abstract

Normal platelet function is critical to blood hemostasis and maintenance of a closed circulatory system. Heightened platelet reactivity, however, is associated with cardiometabolic diseases and enhanced potential for thrombotic events. We now show gut microbes, through generation of trimethylamine N-oxide (TMAO), directly contribute to platelet hyperreactivity and enhanced thrombosis potential. Plasma TMAO levels in subjects (n > 4,000) independently predicted incident (3 years) thrombosis (heart attack, stroke) risk. Direct exposure of platelets to TMAO enhanced sub-maximal stimulus-dependent platelet activation from multiple agonists through augmented Ca(2+) release from intracellular stores. Animal model studies employing dietary choline or TMAO, germ-free mice, and microbial transplantation collectively confirm a role for gut microbiota and TMAO in modulating platelet hyperresponsiveness and thrombosis potential and identify microbial taxa associated with plasma TMAO and thrombosis potential. Collectively, the present results reveal a previously unrecognized mechanistic link between specific dietary nutrients, gut microbes, platelet function, and thrombosis risk.

Figure 1. TMAO is associated with thrombosis potential

(A) Relationship between plasma TMAO level and incident (3 year) thrombotic event (MI or stroke) risk in sequential subjects undergoing elective diagnostic coronary angiography. Kaplan–Meier survival analyses and Cox proportional hazards regression analyses were used for time-to-event analysis to determine adjusted Hazard ratio (HR) and 95% confidence intervals (95%CI). (B) Effect of TMAO on dose response curves for ADP- (left) or thrombin- (right) induced platelet activation monitored using platelet aggregometry. (C and D) Platelet adhesion in whole blood to a microfluidic chip surface (± collagen coating) under physiological sheer conditions ± TMAO, with (C) representative images of platelet adhesion at the indicated times; and (D) adherent platelet area per µm² of chip surface. (E and F) In vivo thrombosis potential using the FeCl₃-induced carotid artery injury model in response to TMAO (vs. normal saline) injected i.p. into mice. Plasma TMAO levels at time of thrombosis model are indicated. (E) Shown are representative vital microscopy images of carotid artery thrombus formation at the indicated time points following arterial injury, and TMAO levels (n≥5 each group); and (F) time to cessation of blood flow in mice from the indicated dietary groups. Bar represents mean time to cessation of blood flow within the indicated group. Significance was measured with either log rank or unpaired Student’s t test. The number of independent biological replicates (n = subjects or animals per group) in all panels are shown. Data are presented as mean ± SEM (B,D,E,F). Linear mixed effect models were used to analyze the repeated measure data in panel D. A Mann-Whitney test was used to compare the time to cessation of flow between the TMAO and control groups. See also Figure S1.

Figure 2. TMAO augments stimulus-dependent release of Ca²⁺ from intracellular stores in human platelets

(A and B) Thrombin evoked changes in intracellular calcium concentration [Ca²⁺]_i in Fura 2-AM loaded washed human platelets incubated with the indicated amounts of TMAO. (A) Representative fluorescent signal from a single subject’s platelet preparation; and (B) mean ± SEM results from n=4 subjects are shown (*, p<0.05 for comparison with vehicle). (C and D) Fluorescence ratio of Fura-2 loaded platelets incubated in medium supplemented with TMAO or vehicle. Cells were first stimulated at the indicated time with a sub-maximal thrombin dose in a Ca²⁺-free medium, and then where indicated, Ca²⁺ was added to the medium. Representative (C) fluorescent signal and (D) mean fluorescence ± SEM (n=4 donors) are shown. (E and F) Washed human platelets pre-incubated (30 min, 22°C) with the indicated concentrations of TMAO wer e stimulated with sub-maximal levels of either (E) thrombin or (F) ADP, and then platelet IP₁ concentrations determined. Data shown represent mean ± SEM (n=4 donors each). Linear mixed effect models were used to analyze the repeated measure data. A Wilcoxon Signed-Rank Test was used to conduct pairwise comparisons. See also Figure S2.

Figure 3. Dietary choline, via gut microbe-generated TMAO, enhances platelet responsiveness and promotes a prothrombotic phenotype

Mice were fed the indicated diets ± oral broad-spectrum antibiotics (ABS). (A, B) Platelet aggregometry was monitored in platelet-rich plasma following addition of sub-maximal (1µM final) ADP; or (D) in vivo thrombosis potential was quantified using the FeCl₃-induced carotid artery injury model. (A) Representative aggregation tracing in response to sub-maximal ADP dose from a mouse within each group, along with the corresponding plasma TMAO level. (B) Aggregation (% of max amplitude of aggregation) in response to ADP, along with plasma TMAO levels, for each group (n>9 each). (C) Aggregation responses to the indicated submaximal concentrations of agonist (ADP, collagen or arachidonic acid, AA) in platelet-rich plasma recovered from mice on the indicated diets (n≥5). (D and E) Time to occlusive thrombus formation in either the (D) FeCl₃-induced carotid artery injury model, or (E) photochemical injury-induced carotid artery injury model. Bar (B,D,E) represents mean value for the indicated group. TMAO levels are reported as mean ± SEM. (F) Aggregation responses to ADP (1µM) within the indicated mixtures of washed platelets and platelet-poor plasma prepared from mice on the indicated diets. Plasma TMAO levels (mean± SEM) of mice on the indicated diets is also shown. Data shown are % of max amplitude of aggregation (± SEM; for n≥=5 each). For significance, a Kruskal Wallis test was used for multiple group comparison and a Mann Whitney test was used for two group comparisons. See also Table S1.

Figure 4. Microbial taxa associated with choline diet-induced TMAO generation and a prothrombotic phenotype

(A) Germ-free mice, conventionally reared mice, and germ-free mice subsequently conventionalized (all groups female, 4 week old, C57BL/6J), were fed the indicated sterile diets for 6 weeks and then both in vivo thrombosis potential (FeCl₃ carotid artery injury model) and plasma TMAO levels were assessed. TMAO data represent mean ± SEM. (B) Correlation (Spearman) between plasma TMAO levels and time to cessation of blood flow among mice in all groups. (C) Microbial DNA encoding 16S rRNA was analyzed from cecum of mice in the indicated diet groups. Principal coordinates analyses demonstrate distinct cecal microbial composition between groups (p < 0.001 for Student’s t test with 1,000 Monte Carlo simulations). Each data point represents a sample from a distinct mouse projected onto the first three principal coordinates (percent variation explained by each PCo is shown in parentheses). (D) Linear Discriminant Analysis (LDA) Effect Size (LEfSe) identified taxa most characteristic (increased abundance) in chow (green) and choline (red) fed groups. (E) Example of choline diet group characteristic taxon (Allobaculum) and chow characteristic taxon (Candidatus Arthromitus) whose proportions are associated with both plasma TMAO levels and time to cessation of blood flow (occlusion time). A Kruskal Wallis test was used for multiple group comparison and a Mann Whitney test was used for two group comparison. See also Figure S3, Figure S4, Figure S5 and Tables S2–S4.

Figure 5. Thrombosis potential is a transmissible trait

(A) Scheme illustrating cecal microbial transplant study design. Germ-free C57BL/6J mice (recipients) had cecal microbes introduced by gastric gavage from conventionally reared C57BL/6J (high TMA/TMAO producer) and NZW/LacJ (low TMA/TMAO producer) donors. Recipients (with and without microbe transplants) were placed on sterile chemically defined chow (0.08% choline) vs. choline supplemented (1.0% total) diets. (B) Cecal choline TMA lyase enzyme activity was quantified in donor mice (left panel), and recipient germ-free mice without (middle) or following (right) microbial transplantation and maintenance on the indicated diets within individual gnotobiotic isolators. Platelet function was assessed following microbial transplantation in the indicated numbers of recipient mice both by (C) ex vivo monitoring of aggregation response to ADP (1 µM), and (D) in vivo measuring of time to occlusive thrombus formation using the FeCl₃-induced carotid artery injury model. (B,C and D) Plasma TMAO levels (mean ±SEM) were also determined at completion of all studies. For significance, a Kruskal Wallis test was used for multiple group comparison and a Mann Whitney test was used for two group comparison. See also Figure S6.

Figure 6. Characterization of donor-characteristic cecal microbiota associated with TMAO and in vivo thrombosis potential in recipients following microbial transplantation

Intestinal microbial community composition was assessed by (A) Principle coordinates analysis for donor and recipient mouse strains on the indicated diets. (B) Linear Discriminant Analysis (LDA) Effect Size (LEfSe) analyses were performed to identify taxa most characteristic (increased abundance) in NZW/LacJ and C57BL/6J donors. (C) Illustration of three taxa identified in recipient cecal microbes whose proportions are significantly associated with both plasma TMAO levels and occlusion time when grouped by dietary treatment (blue squares, recipients of NZW/LacJ donor cecal microbes; red circles, recipients of C57BL/6J microbes). Values in both x and y directions are plotted as mean±SE. Significance was determined using unpaired Student’s t test. See also Figure S7 and Table S5.

Figure 7. Summary schemes illustrating gut-microbial involvement in development of platelet hyperresponsiveness and athero-thrombotic heart disease

(A) Global schema illustrating metaorganismal pathway linking dietary sources of choline abundant in a Western diet, gut microbiota and host hepatic FMOs, resultant TMAO production, and subsequent development of hyperresponsive platelet phenotype and enhanced thrombotic event risk. Also shown are reported pro-atherosclerotic effects of TMAO and the potential involvement of TMAO in development of vulnerable plaque. EC, endothelial cell; FMOs, flavin monooxygenases; M[phage], macrophage; TMA, trimethylamine; TMAO trimethylamine N-oxide. (B) Chronic exposure to a diet rich in choline leads to a shift in the gut microbial composition and function, with consequent enhancement in host TMAO plasma levels. Platelet exposure to high levels of TMAO enhances sub-maximal stimulus (thrombin, ADP, collagen) evoked release of intracellular calcium stores, and platelet hyperresponsiveness. ADP, adenosine diphosphate; GPVI, glycoprotein VI; G, G protein q; IP<3>, inositol 1,4,5-triphosphate; IP<3>R, Inositol 1,4,5-triphosphate receptor; P2Y12, purinergic receptor P2Y12; PAR, protease activated receptor; PLC, phospholipase C; PIP2, phosphatidylinositol 4,5-bisphosphate.