Abstract

Deep vein thrombosis and pulmonary embolism (VTE) are common causes of morbidity and mortality. No genetic or acquired biomarkers or risk factors can be identified in many VTE patients. Thus, there is a major unmet need to identify new biomarkers and new causal risk factors in VTE patients. Metabolomics is an unexplored frontier for VTE research. Thus, to discover novel plasma metabolite biomarkers for VTE risk, we performed liquid chromatography-mass spectrometry (LC-MS)-based untargeted metabolomics in a pilot study to analyze plasma metabolites without any specific targeting of known metabolites. Untargeted metabolomics data for 40 male idiopathic adult VTE cases and 40 age-matched male controls recorded 9,400 metabolic features for each plasma sample. Data analysis revealed two plasma long-chain acylcarnitines (ACs) (10:1 and 16:1) to be significantly decreased in VTE patients compared to controls, suggesting the association of plasma long-chain ACs levels with the risk of VTE. To validate the association of reduced plasma levels of long-chain ACs with the VTE risk, LC-MS-based targeted metabolomics was used to determine long-chain ACs levels. Data from targeted metabolomics additionally showed that several long-chain ACs (10:1, 12:0, 12:2 and 18:2) were lower in VTE patient plasmas than in control plasmas for subjects in our pilot VTE case-control study.

ACs, which heretofore have no known function in coagulation reactions, consist of a hydrophobic side chain of an acyl moiety which is linked to the carnitine moiety. ACs circulate in plasma and also play key roles in mitochondrial energy metabolism. Studies were initiated to define any procoagulant or anticoagulant properties of ACs. Remarkably, ACs inhibited factor Xa-initiated clotting assays which were triggered by adding either purified factor Xa or RVV-X to plasma. The anticoagulant activities of various ACs were both dose-dependent and acyl chain length-dependent, where ACs with longer acyl chains were more potent procoagulants than ACs with shorter acyl chains (18, 16 > 14, 10 > 6 acyl chain carbons). However, thrombin-induced clotting was not inhibited by ACs, suggesting that ACs were acting on the prothrombinase complex.

For purified prothrombinase reactant systems containing prothrombin, factor Xa, factor Va, and phospholipid vesicles [phosphatidylcholine:phosphatidylserine (PC/PS) 90:10 w/w], thrombin generation was dose-dependently inhibited by 16:0-AC (IC50 = 13 µM). For reactant mixtures lacking either phospholipids or factor Va, prothrombin activation by factor Xa was still dose-dependently inhibited by 16:0-AC (IC50 = 5.6 µM and 5.8 µM, respectively). Prothrombin activation by factor Xa in the absence of both factor Va and phospholipids was still inhibited by 16:0-AC (IC50 = 11 µM). In controls, 16:0-AC did not inhibit the amidolytic activity of either factor Xa or thrombin. These findings indicate that neither factor Va nor phospholipid was required for the anticoagulant property of 16:0-AC and that this lipid did not inhibit the enzyme active sites, suggesting that this lipid disrupts interactions between factor Xa and prothrombin. Although canonical coagulation paradigms emphasize key roles for lipid binding sites that are localized in the amino terminal Gla domain of vitamin K dependent clotting factors, Gla-domainless (DG)-prothrombin activation by factor Xa and prothrombin activation by DG-factor Xa were each similarly inhibited by 16:0-AC with IC50values of 11 and 7.0 μM, respectively. For surface plasmon resonance (SPR) binding studies, biotinylated-Glutamyl-Glycyl-Arginyl-chloromethyl ketone (BEGR) was used to label the active site of factors Xa, IXa, and VIIa. BEGR-factor Xa and BEGR-DG-factor Xa bound with similar affinities to 16:0-AC (10 µM and 23 µM, respectively) whereas no binding of 16:0-AC (60 μM) to BEGR-factor VIIa or BEGR-factor IXa was observed. Thus, these data suggest that a 16:0-AC binding site on factor Xa that is located outside the Gla domain mediates this lipid’s anticoagulant activity.

In summary, first, untargeted and targeted metabolomics data for a pilot VTE case-control study identified ACs as potential biomarkers for VTE, and, second, detailed mechanistic studies show that the AC, 16:0 acyl-carnitine, has anticoagulant activity in the absence of factor Va or phospholipids that is related to its ability to bind factor Xa outside the Gla domain.

Disclosures

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.