Essential Amino Acids – Essential in Arterial Thrombosis

A proper balanced diet is a mainstay of guidance to improving quality of life and reducing co-morbidities from diet-related obesity and diabetes. Balanced nutritional intake affects the ability of fundamental molecular functions to continue within our cells. Essential amino acids (EAA), such as the branched-chain amino acids (BCAA) leucine, isoleucine and valine, cannot be synthesized by the human body in meaningful amounts and instead must be taken in from external food sources. BCAA have been described to have 3 major roles: 1) serve as a building block for protein synthesis, 2) act as a source of energy, 3) regulate cell growth and autophagy ¹. Despite the term “essential”, as we are increasingly appreciating across multiple disciplines of science, too much of any good thing can turn into a bad thing.

BCAA are popular as an exercise supplement, because it is believed that increased levels of these 3 amino acids promotes building of muscle mass post-exercise ². However, the utility of BCAA supplementation is marked by conflicting evidence of its utility ². Pathologically, cross-sectional studies have shown positive associations of circulating BCAA with cardiovascular risk, hypertension, metabolic dysregulation and insulin resistance. Infusions of healthy individuals with EAAs led to insulin release without driving its clearance from the blood ³. Unsurprisingly, longitudinal studies of BCAA have linked them to metabolic syndrome and type-2 diabetes mellitus (T2DM). Cross-sectional studies have reported positive correlations between increased circulating BCAAs and cardiovascular risk, hypertension, metabolic dysregulation and insulin resistance ¹. However, all of these studies have reported associations between increased levels of BCAAs and disease risk, yet few studies have revealed direct mechanisms by which BCAAs can drive disease progression.

In this issue of Circulation, Xu and Jiang et al describe a novel role for BCAA catabolism in driving platelet activation and promoting cardiovascular disease ⁴. The authors utilize complementary approaches including ex vivo human clinical samples, in vivo mouse models, in vitro cell culture and analytical chemistry techniques to demonstrate that elevated levels of BCAAs and their downstream catabolites lead to hyperactive platelets and subsequent risk of arterial thrombosis. Ingestion of BCAAs by human subjects led to an increase in relative levels of leucine, isoleucine and valine inside platelets. These platelets manifested a corresponding increase in agonist-induced activation, granule release, aggregation and spreading. Thrombin-induced αIIbβ3 activation determined by PAC1 binding to human platelets increased with BCAA ingestion, suggesting that BCAAs increase αIIbβ3-mediated outside-in signaling.

Xu and Jiang et al hypothesized that BCAAs need to be catabolized to exert their effects on platelets. Because BCKD is the rate-limiting enzyme for BCAA catabolism and is controlled by dephosphorylation by PP2Cm, mice deficient in PP2Cm were studied ⁵. PP2Cm^−/− mice lacked the ability to dephosphorylate BCKD, which led to a defect in BCAA catabolism. Compared to WT mouse platelets, platelets from PP2Cm^−/− mice failed to activate in response to agonist, manifested reduced granule release, failed to aggregate, and did not spread in a αIIbβ3-dependent manner as compared to WT. In surgical and laser-induced thrombosis mouse models, BCAA catabolism was demonstrated to be critical for arteriolar, but not venous, thrombus formation in vivo. The authors used intricate molecular techniques to demonstrate that during BCAA catabolism in platelets the cytoskeletal protein, TMOD3, is propionylated and this is necessary for αIIbβ3-mediated cellular spreading. Many groups have reported positive correlations between increased circulating BCAAs and progression of metabolic syndrome, type 2 diabetes, and cardiovascular disease ¹. The authors used their final figure to correlate the increase in BCAAs and their metabolites with increased platelet activation in clinical samples from type 2 diabetes patients. This led the authors to conclude that increased intracellular BCAAs in platelets is a mechanism for hyper-reactive platelets in metabolic syndromes.

These findings have potential clinical implications. As the authors point out, BCAA injections are used clinically for hepatitis, cirrhosis, hepatic encephalopathy and hepatobiliary surgery. This means that we are potentially putting patients at higher risks of thrombotic events by acutely increasing their BCAA levels. These pro-thrombotic effects however may be mitigated in part by the tendency of patients with liver disease to have bleeding complications.

Whereas the clinical data presented here are quite striking, it would be interesting to know the effects of BCAAs in patients on anti-platelet drugs, such as aspirin or clopidogrel, and whether they counteract their effects. The type 2 diabetes patients studied were not given antiplatelet or anticoagulant therapies prior to blood draw which is not likely the case for many of these metabolic syndrome patients. Thus, a limitation of this study is that it remains unknown whether these findings in platelets hold true based on the medications patients are on. These data do suggest that the intake of high levels of BCAA in normal healthy individuals, particularly those taking BCAA supplements seeking to build muscle mass, can be potentially dangerous and counterproductive to a healthy lifestyle they seek through exercise and muscle building.

It would also be interesting to further discern the effects of BCAA metabolism on other circulating cell types, including immune cells that also rely on integrins and shape change for many of their functions. Platelets are not only major regulators of thrombosis and hemostasis, but are also regulators of immune responses ⁶. Whether increased ingestion of BCAAs leads to a change in the function of cells that cross talk with platelets, such as endothelial cells, monocytes, neutrophils and T cells, whereas outside the scope of this study, may also impact thrombotic risk. The use of platelet-specific mouse models could perhaps better elucidate platelet-specific roles in BCAA metabolism ^{7, 8}. BCAA effects on other cell types have been elucidated by other groups. BCAA increased eNOS, E-selectin, and inflammatory response driven by the transcription factor, NF-κB in endothelial cells that may also impact thrombosis ⁹. Previous studies of monocyte-derived dendritic cells suggested that increased extracellular valine can improve dendritic cell function in cirrhotic patients ¹⁰. Too little BCAAs impairs immune function ¹¹, however, the effects of excess BCAA on leukocytes and their regulation of immune responses remains unclear.

This study unveils a novel mechanism whereby platelets become hyper-reactive based on amino acid supplementation and expands on many observational and correlative studies to provide a mechanism by which BCAA metabolism drives TMOD3 propionylation and with it enhanced platelet activation and arterial thrombosis. These findings are clinically relevant and raise new questions that are sure to be explored by many groups in future studies.