Warfarin untargeted metabolomics study identifies novel procoagulant ethanolamide plasma lipids (2014)

Warfarin is one of the most widely prescribed anticoagulant drugs used to treat and prevent thrombosis in patients with cardiovascular or other thrombotic disorders (Keeling et al, 2011). Warfarin inhibits vitamin K epoxide reductase and decreases vitamin K hydroquinone to attenuate blood coagulation by inhibiting vitamin K-dependent post-translational modifications of plasma coagulation factors in the liver. A recent study showed that R-enantiomers of warfarin interact with the pregnane X receptor (PXR), which is associated with a variety of metabolic clearance functions in vivo (Rulcova et al, 2010), suggesting that warfarin could affect a variety of metabolites in addition to vitamin K-related metabolites. There is a paucity of studies about the effects of warfarin on natural plasma metabolites; thus, we sought to identify metabolites that are regulated by warfarin, which could affect coagulation. Mass spectrometry (MS)-based untargeted metabolomics denotes the profiling of thousands of low molecular weight biochemicals, including lipids, hormones, saccharides, nucleotides, organic acids and amino acids. The method can identify plasma metabolites ‘features’ and detect statistically significant differences between two subject groups without the specific targeting of individual molecules. (Patti et al, 2012a,b). Here, we applied this technology to test the effect of warfarin on plasma metabolites using plasma crossover samples drawn from the 17 subjects while on and off warfarin. Among 9,400 plasma features observed, the metabolic features at 32·2 min with 300·2908 m/z and at 38·6 min with 328·3193 m/z were statistically significantly decreased in patients taking warfarin by 4·0- and 7·2-fold (P = 0·018 and 0·026 by paired t-test, respectively) (Fig 1A and supplemental Table S1). The comparison of their fragments and their relative intensities with their model compounds by tandem MS (MS/MS) defined those metabolic features as palmitoylethanolamide (PEA) and stearoylethanolamide (SEA) from the lipid ethanolamide family (Fig 1A and supplemental Table S1). The change of PEA by warfarin was statistically correlated with the change of SEA (r2 = 0·85, P < 0·0001) (Fig 1B). However, the International Normalized Ratio (INR), which represents the effect of warfarin on vitamin K-dependent coagulation factors, did not correlate with these lipids (data not shown). Palmitoylethanolamide and SEA are naturally occurring saturated N-acylethanolamines (ethanolamides), both of which are structurally related to N-acylethanolamine (AEA) with a longer unsaturated side chain (20:4) which binds to the cannabinoid receptor. PEA and SEA are devoid of affinity for cannabinoid receptors. The role of PEA in inflammation and nociception via a variety of molecular mechanisms has been documented (Petrosino et al, 2010). However, the physiological roles of PEA and SEA remains unclear. Surprisingly, here, we discovered that certain ethanolamide family lipids have procoagulant characteristics. Both the tissue factor (TF)/Ca2+-induced and recalcification-induced thrombin generation were increased by PEA in normal plasma (Fig 2B, A). The precursor molecule of PEA, glycerophospho-N-palmitoyl ethanolamine and analogs of PEA, such as SEA and AEA, also shortened lag time (P values for linear trend were 0·004, 0·0009 and 0·0006, respectively) (Fig 2C) and enhanced thrombin generation in plasma as seen in values for thrombin peak (P values for linear trend were 0·03, 0·16 and 0·00006, respectively) (Fig 2E), whereas certain other analogs of PEA (palmitoyl N-isopropylamide and N-palmitoyl taurine), which are lacking a hydroxyl group in the head group (Figs 1C and 2D, F), showed little influence on thrombin generation. These results suggest that the free hydroxyl group in the head group appears to be a key component for the observed procoagulant activity of certain ethanolamides. Given that the plasma concentrations of PEA, SEA and AEA are approximately 5·7, 1·5 and 0·7 nmol/l respectively (Ozalp & Barroso, 2009; Balvers et al, 2009), the procoagulant effect of ethanolamides is below or within their plasma concentration ranges. These data suggest that the ethanolamide family possibly stimulates the coagulation pathway to increase thrombin generation in plasma. Interestingly, elevated peripheral AEA levels were found in acute stroke patients (Naccarato et al, 2010). The procoagulant activity of AEA and its association with stroke remains to be further evaluated. The physiological and clinical relevance of the procoagulant properties of various ethanolamides is uncertain at this stage and requires further study. The main complication of oral anticoagulant therapy is bleeding, and the bleeding risk is related to the intensity of anticoagulation (Hirsh et al, 2003). Bleeding that occurs at moderate anticoagulation (e.g. INR < 3·0) is frequently associated with trauma or an underlying lesion in the gastrointestinal or urinary tract (Hirsh et al, 2003). However, intracranial haemorrhage frequently occurs without trauma and at moderate or lower levels of anticoagulation (Dowlatshahi et al, 2012). Further, mortality rates of intracranial haemorrhage remained high despite rapid INR correction by the supplementation of coagulation factors (Dowlatshahi et al, 2012). This suggests that certain warfarin-sensitive factors, including plasma lipids (which are not reflected by the INR), might have a role in bleeding complications. Curiously, there was a subset of subjects in whom PEA and SEA were decreased by 70% (dotted circle in Fig 1B). The consequent anticoagulant effect by these reductions might be minor compared to the warfarin anticoagulant effect on vitamin K-dependent coagulation factors. It would be of interest to investigate the association between decreased levels of ethanolamide and warfarin-associated bleeding. Our study has limitations, including small numbers and the lack of a replication study. The cross-over study design lends strength to the small number of patients studied, decreasing intra-individual variability, but it is understood that a study with larger numbers of participants would be required for the confirmation of the findings reported here. However, it is noteworthy that our untargeted metabolomics identified two molecules, together with their procoagulant activity, from the same family. This also suggests future study of the association of ethanolamine family levels with beneficial anticoagulation and unwanted bleeding risk. In summary, the untargeted metabolomics methods identified endogenous plasma lipids whose plasma levels are regulated by warfarin therapy. Further, this led to the discovery of a new family of plasma procoagulant lipid molecules, ethanolamides. These results show the utility of untargeted metabolomics and have implications for future basic and clinic studies related to warfarin's effects and to the procoagulant activities of ethanolamides. This work was supported by the National Institutes of Health grants HL021544, HL101034, and UL1RR025774. The authors declare no competing financial interests. J.H.G, H.D. and G.S. participated in the conception of the study. G.S., S.T., H.M.Z and E.K. were responsible for untargeted metabolomics analysis. H.D. was responsible for performing experiments with ethanolamine activity and statistical analysis. D.J.E. was responsible for organizing the warfarin crossover study, consenting the patients and obtaining blood specimens. H.D., D.J.E. and J.H.G. were responsible for writing the manuscript. Data S1. Materials and experimental methods. Table S1. Plasma metabolites changed by warfarin treatment. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.