Acylcarnitines are anticoagulants that inhibit factor Xa and are reduced in venous thrombosis, based on metabolomics data

Abstract

In many patients with deep vein thrombosis and pulmonary embolism (venous thromboembolism, VTE), biomarkers or genetic risk factors have not been identified. To discover novel plasma metabolites associated with VTE risk, we employed liquid chromatography-mass spectrometry-based untargeted metabolomics, which do not target any specific metabolites. Using the Scripps Venous Thrombosis Registry population for a case-control study, we discovered that 10:1 and 16:1 acylcarnitines were low in plasmas of the VTE patient group compared with matched controls, respectively. Data from targeted metabolomics studies showed that several long-chain acylcarnitines (10:1, 12:0, 12:2, 18:1, and 18:2) were lower in the VTE group. Clotting assays were used to evaluate a causal relationship for low acylcarnitines in patients with VTE. Various acylcarnitines inhibited factor Xa-initiated clotting. Inhibition of factor Xa by acylcarnitines was greater for longer acyl chains. Mechanistic studies showed that 16:0 acylcarnitine had anticoagulant activity in the absence of factor Va or phospholipids. Surface plasmon resonance investigations revealed that 16:0 acylcarnitine was bound to factor Xa and that binding did not require the γ-carboxy glutamic acid domain. In summary, our study identified low plasma levels of acylcarnitines in patients with VTE and showed that acylcarnitines have anticoagulant activity related to an ability to bind and inhibit factor Xa.

Figure 1

Targeted metabolomics data for 5 plasma AC levels show differences between 37 patients with VTE and controls. The distribution of plasma AC levels whose median level was significantly lower in patients with VTE compared with controls are shown in (A) 10:1-AC, (B) 12:0-AC, (C) 12:2-AC, (D) 18-1-AC, and (E) 18:2-AC. The sum of concentrations for all ACs with acyl chain length ≥ 10 carbons is shown in F. The plasma levels of ACs are shown as micromoles, and the bar represents the median of each subgroup. The dotted line indicates the 10th percentile of control for each parameter. The difference of median values between VTE patients and controls was calculated by Mann-Whitney test. To evaluate the association of VTE with low AC level (<10th percentile of control), ORs whose values are seen in C and E were calculated according to the odds of VTE occurring in each of 2 groups; namely, those with AC levels either below the 10th percentile or above the 10th percentile of control.

Figure 2

The anticoagulant effects of long-chain ACs in different clotting assays were determined. Effects of various doses of 16:0-AC are shown for clotting assays in which clotting was induced by endogenously generated factor Xa (RVV-X activated), exogenously added factor Xa, diluted TF, or thrombin. (B) Effect of length of aliphatic side chain of ACs (6:0, 8:0, 10:0, 14:0. 16:0, 18:0, and 18:2) on their anticoagulant activity, measured using RVV-X clotting time.

Figure 3

The anticoagulant effects of 16:0 AC in prothrombinase assay were determined. (A) The effect of 16:0-AC on prothrombin activation by factor Xa, factor Va, and PCPS vesicles. (B) The effect of 16:0-AC on prothrombin activation by factor Xa and factor Va. (C) The effect of 16:0-AC on prothrombin activation by factor Xa and PCPS vesicles. (D) The effect of 16:0-AC on prothrombin activation by factor Xa. (E) The effect of 16:0-AC on desGla-prothrombin activation by factor Xa, factor Va, and PCPS vesicle. (F) The effect of 16:0-AC on prothrombin activation by desGla-factor Xa, factor Va, and PCPS vesicle.

Figure 4

The binding of 16:0 AC to factor Xa was determined. SPR was used to monitor binding of 16:0-AC to BEGR-factor Xa and BEGR-DG-factor Xa. (A) Sensorgram depicting the dose-dependent binding of 16:0-AC (from top to bottom; 50, 37.5, 25, 12.5, 10, 7.5, and 6.25 µM) to BEGR-factor Xa. (B) Sensorgram depicting the dose-dependent binding of 16:0-AC (from top to bottom; 62.5, 25, 20, 10, and 7.5 µM) to BEGR-DG-factor Xa. PCPS vesicles exhibited binding to BEGR-factor Xa, but not to BEGR-DG-factor Xa, indicating the proper coupling of the biotinylated proteins (data not shown).