UC Santa Cruz

Platelet aggregation and thrombosis are critical processes in hemostasis and wound healing, but their dysregulation can lead to the formation of obstructive blood clots with severe health consequences. To maintain control over these processes, the body relies on specialized pro-resolving mediators (SPMs) derived from polyunsaturated fatty acids (PUFAs) such as arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). Lipoxygenase (LOX) enzymes, which are non-heme iron-containing enzymes, play a vital role in the generation of specific oxylipins, including specialized resolvins, that act as important regulators of platelet function and thrombus resolution.

The interplay between LOX enzymes and platelet aggregation is complex. These enzymes can produce both pro-inflammatory and pro-thrombotic lipid mediators, as well as specialized resolvins that have anti-inflammatory and pro-resolving effects. Targeting LOX activity and promoting the production of specialized resolvins holds promise as potential therapeutic strategies to regulate platelet function and enhance thrombus resolution. Moreover, essential fatty acids like linoleic acid (LA) and α-linolenic acid (ALA) play crucial roles as precursors for the synthesis of bioactive lipid mediators, including omega-6 and omega-3 fatty acids. Understanding the catalytic mechanisms, expression patterns, and allosteric regulation of LOX enzymes is essential for uncovering their physiological functions and developing targeted therapeutic interventions.

In line with these investigations, recent research has focused on the impact of a single nucleotide polymorphism (SNP) in human platelet 12-lipoxygenase (h12-LOX). This SNP results in a tyrosine-to-cysteine mutation at a buried site (Y649C) and has been associated with reduced levels of 12(S)-hydroxyeicosatetraenoic acid (12S-HETE) production in isolated platelets. Detailed characterization of the Y649C mutant revealed that it exhibits reduced catalytic rates, altered membrane affinity, and decreased protein stability compared to the wild-type (WT) h12-LOX enzyme. These subtle changes in activity and protein properties may contribute to the significant physiological alterations observed in platelet biology associated with the Y649C SNP.

Furthermore, lipid metabolism and the involvement of acyl-coenzyme A (acyl-CoA) molecules have gained attention in the context of LOX biochemistry and inflammation. Acyl-CoAs have been found to bind to LOX enzymes, and their inhibitory effects on various LOX isozymes have been investigated. C18 acyl-CoA derivatives were identified as potent inhibitors of h12-LOX, human reticulocyte 15-LOX-1 (h15-LOX-1), and human endothelial 15-LOX-2 (h15-LOX-2), while C16 acyl-CoAs showed higher potency against human 5-LOX. Notably, oleoyl-CoA (18:1) was the most potent inhibitor of h12-LOX and h15-LOX-2, while stearoyl-CoA showed high potency against h15-LOX-1. Additionally, linoleoyl-CoA (18:2) acted as a weak inhibitor but a rapid substrate for h15-LOX-1. These findings highlight the significant role of acyl-CoAs in regulating LOX activity and suggest their involvement in the formation of oxylipin-CoAs, which may serve as novel signaling molecules.

Moreover, omega-3 fatty acids, including docosahexaenoic acid (DHA) and docosapentaenoic acid (DPAn6), have been studied for their health benefits and their oxidation by LOX enzymes to form bioactive oxylipins. The impact of saturation on the kinetic properties and product profiles of h12-LOX, h15-LOX-1, and h15-LOX-2 was investigated using fatty acid substrates with varying degrees of unsaturation. The loss of ∆4 and ∆19 double bonds led to a significant reduction in the kinetic activity of h12-LOX, while h15-LOX-1 and h15-LOX-2 exhibited lower kcat/KM values upon the loss of ∆4 and ∆19. The product profiles varied with the degree of saturation and revealed a preference for 14-oxylipins by h12-LOX, an increase in 14-oxylipin production with loss of saturation by h15-LOX-1, and predominant production of 17-oxylipins by h15-LOX-2.

Additionally, the effects of various 17-oxylipins on platelet activation were investigated, revealing distinct properties of different oxylipins. For instance, 17(S)-hydroxy-4Z,7Z,10Z,13Z,15E,19Z-DHA and 17(S)-hydroxy-4Z,7Z,10Z,13Z,16Z-DPAn6 demonstrated anti-aggregation properties, while 17(S)-hydroxy-7Z,10Z,13Z,15E,19Z-DPAn3 exhibited agonistic effects. The effects of 17(S)-hydroxy-7Z,10Z,13Z,15E-DTA varied depending on concentration, inhibiting aggregation at lower concentrations but promoting it at higher concentrations. In comparison, 17(S)-hydroxy-13Z,15E,19Z-DTrA and 17(S)-hydroxy-13Z,15E-DDiA induced platelet aggregation. Notably, 14-oxylipin counterparts of certain 17-oxylipins, such as 14(S)-hydroxy-13Z,15E,19Z-DTrA, exhibited inhibitory properties when stimulated with collagen or thrombin.

These findings provide valuable insights into the role of oxylipins in platelet aggregation and reveal the influence of fatty acid saturation and specific LOX isozyme activity on their physiological activity. The degree of unsaturation and the location of oxidation appear to influence the physiological properties of these oxylipins, with more unsaturated compounds generally inhibiting aggregation while less unsaturated compounds promoting it. Understanding the mechanisms underlying LOX enzyme regulation, the impact of acyl-CoAs, and the physiological effects of different oxylipins contributes to our knowledge of platelet aggregation and thrombosis, offering potential avenues for the development of targeted therapeutic interventions and improvements in cardiovascular health.