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

Abstract

• Untargeted and targeted metabolomics showed association of low plasma acylcarnitines levels with venous thrombosis risk.• Long-chain acylcarnitines are anticoagulants that inhibit factor Xa by binding to factor Xa outside the g-carboxy glutamic acid domain.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 g-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. (Blood. 2015;126(13):1595-1600

Minor plasma lipids modulate clotting factor activities and may affect thrombosis risk

“…A variety of minor abundance soluble plasma lipids manifest either procoagulant or anticoagulant properties by affecting thrombin generation (Figure 2A). Biochemical studies of plasma lipids and thrombin generation show that functionally important lipid binding sites are located on factor Xa outside its N-terminal Gla-domain 32,33 (Factor 2B) which historically has been to only region on factor Xa widely accepted to bind lipids. Thus, the canonical paradigm for clot- Table S2) might act to regulate inflammatory events such as cell proliferation at the same time they act to alter thrombin generation.…”

“…Studies that challenged the widely accepted, simple paradigm for PL‐Gla‐domain lipid effects on prothrombinase structure and activity involved synthetic, soluble dicaproyl‐PS that was reported to promote prothrombinase activity by binding to factor Xa outside the Gla‐domain (Figure B) . Similarly, recent publications both extend and challenge the simple classical paradigm for PL‐Gla‐domain‐factor Xa interactions because it was shown that several plasma lipids carried by lipoproteins or proteins such as albumin and/or lipid binding proteins (e.g, sphingosine, sphinganine, acyl‐carnitines [ACs], and lyso‐sulfatide [LSF]), so called “soluble” lipids, bind factor Xa and alter its procoagulant activity (Figure B), supporting and extending the challenge of the classical, simple paradigm for lipid‐clotting factor interactions. In summary, innovative findings show that certain, minor abundance plasma lipids can bind directly to factor Xa outside the Gla domain and modulate thrombin generation by the prothrombinase complex (Figure B), leading to an expanded framework for the paradigm for lipid‐clotting factor interactions …”

“…In summary, innovative findings show that certain, minor abundance plasma lipids can bind directly to factor Xa outside the Gla domain and modulate thrombin generation by the prothrombinase complex ( Figure 2B), leading to an expanded framework for the paradigm for lipid-clotting factor interactions. 32,33 F I G U R E 1 Blood coagulation and protein C pathways. Thrombin is the major product of the blood coagulation pathways involving sequential enzymatic activations of serine protease zymogens enhanced by nonenzymatic cofactors, factors Va and VIIIa.…”

“…When the association of VTE with GlcCer and PE levels was studied, VTE was associated with plasma GlcCer deficiency but not with any changes in PE plasma levels. 40 Sphingosine 0-70 nmol/L 2.5 [31] Lysosulfatide~5 μmol/L 4 [32] Long-chain acylcarnitine 1-2 μmol/L 5-10 [33] PEA, SEA, AEA 1-6 nmol/L 0.0002 [59] The Table summarizes phosphatidylserine. 56 Their plasma levels can reach 1.5, 35, 1.5, and 1.6 μmol/L in acute coronary syndrome patients.…”

“…Total long-chain ACs (acyl chains >10 carbons) circulate in plasma, with reported concentrations ranging between 1 and 4 μmol/L, 33,[80][81][82] and their levels can reach to 10-30 μmol/L under certain metabolic conditions. [83][84][85][86] Long-chain ACs are lipid metabolites with a hydrophobic side chain and a free amine like sphingosine (Figure 4), but they belong to the long-chain AC family which plays an important role in energy metabolism through mitochondrial β-oxidation of fatty acids.…”