ldk378 To date LOX is the only

To date, 12-LOX is the only LOX isoform that has been identified in the platelet [17]. Interestingly, while 12-LOX is highly expressed (∼14000 molecules/platelet) in the platelet, 12(R)-LOX expression is absent in the hematopoietic lineage and has therefore not been reported to be expressed in platelets of any mammal tested to date [17]. The expression of 12-LOX is predominantly restricted to platelets but is also expressed in some hematopoietic and solid tumors [18]. In resting platelets, PUFAs, the most common fatty ldk378 substrates of 12-LOX, are esterified in the membrane glycerolphospholipids to restrict aberrant oxylipin production. Stimulation of platelets triggers the translocation of 12-LOX to the membrane and the hydrolysis of PUFAs from the glycerolphospholipid via cytosolic phospholipase (cPLA2) in a calcium-dependent manner 19, 20, 21, 22, 23, 24 (Figure 2). Unesterified PUFAs are metabolized predominately by 12-LOX and COX, with the most abundant platelet-derived oxylipin being the AA-derived 12-LOX metabolite, 12-HETE [25] (Figure 2). AA-derived oxylipins make up the predominant amount of platelet oxylipins since AA is the most abundant PUFA in the phosphospholids [8]. Both serum and intraplatelet levels of 12-HETE become elevated following thrombus formation in a canine coronary stenosis and endothelial damage model [26]. In vitro, platelets have been shown to produce 12-HETE in response to a myriad of agonists including collagen, thrombin, ADP, and the thromboxane mimetic, U46619 26, 27, 28, 29, 30, 31, 32. Whether an agonist elicits 12-HETE production in platelets is primarily dependent on its ability to increase intracellular calcium levels, since both the release of AA from the membrane and the translocation of 12-LOX to the glycerolphospholipid are calcium dependent 19, 20, 21, 22, 23, 24. Interestingly, in response to PAR stimulation, 12-LOX and COX-1 access different substrate pools and the production of 12-LOX oxylipins is delayed but more sustained than production of COX oxylipins [33]. Despite being chemically identified in platelets 40 years ago the physiological function of 12-LOX and its AA-derived metabolite 12-HETE in platelet function and thrombus formation remain incompletely understood 13, 34. As recently highlighted in a comprehensive review, there is no consensus on the function of 12-HETE in platelets, with studies reporting contrasting evidence of whether 12-HETE potentiates or inhibits platelet activation [14]. A major focus of the field is trying to delineate the mechanism of action of 12-LOX and its metabolites on platelet function to reconcile the differences between these studies, or at the least, to understand under what physiological context each of these published studies adequately explains how 12-LOX functions within the platelet. There are many postulated mechanisms by which 12-LOX and its metabolite 12-HETE may work to regulate platelet function (Figure 3). One such regulatory mechanism focused on an early report showing that 12-HETE activates NADPH oxidase to generate ROS, which are known to potentate platelet activation; however, studies are still required to determine if NADPH oxidase is required for 12-LOX to potentiate platelet activation [35]. While mechanistically incomplete, this study clearly showed that 12-LOX can exhibit a proaggregatory and possibly prothrombotic function in the platelet through formation of its oxylipin 12-HETE. Although most of what we know about 12-HETE comes from studies performed in cancer cells, 12-HETE has been shown to potentiate dense granule secretion in the human platelet [12]. In cancer cells, 12-HETE binds the orphan GPCR, GPR31, which has been renamed the 12-HETE receptor; however, although GPR31 has been reported at conference proceedings to be expressed in the platelet, these reports have not been confirmed in peer-review publications, and continue to be an actively study area of 12-LOX biology in platelets [36]. Due to the lack of selective antibodies to GPR31 or confirmatory publication or reports however, it remains unclear if platelets express GPR31 or another 12-HETE receptor. The reported interaction of 12-LOX with the cytoplasmic tail of β4 integrin in cancer cells has been shown to enhance 12-LOX activity [37]. Interestingly, the cytoplasmic tail of β4 is longer than that of other integrins; therefore, it remains unknown if 12-LOX can also bind platelet integrins. Regulation of platelet reactivity through physical interaction with integrins or other membrane-associated protein complexes in the platelets would establish a unique mechanism for 12-LOX regulation of platelet function independent of its enzymatic activity, and understanding these interactions is essential for delineating the full regulatory potential of 12-LOX on platelet function in both physiological and well as pathophysiological conditions. If, for example, direct 12-LOX binding to the integrin αIIbβ3 is required for normal platelet activation, perturbing this interaction may represent a novel approach for regulation of platelet function and thrombosis. Finally, one of the more recent findings has shown that approximately one-third of the 12-HETE generated by platelets is re-esterified into the phospholipid membrane, supporting the enzymatic activity of 12-LOX as being important to platelet function since esterified 12-HETE has been shown to enhance tissue-factor-dependent thrombin generation, which would be predicted to enhance thrombus formation 38, 39.