Investigations of human platelet-type 12-lipoxygenase: role of lipoxygenase products in platelet activation

Abstract

Human platelet-type 12-lipoxygenase (12-LOX) has recently been shown to play an important role in regulation of human platelet function by reacting with arachidonic acid (AA). However, a number of other fatty acids are present on the platelet surface that, when cleaved from the phospholipid, can be oxidized by 12-LOX. We sought to characterize the substrate specificity of 12-LOX against six essential fatty acids: AA, dihomo-γ-linolenic acid (DGLA), eicosapentaenoic acid (EPA), α-linolenic acid (ALA), eicosadienoic acid (EDA), and linoleic acid (LA). Three fatty acids were comparable substrates (AA, DGLA, and EPA), one was 5-fold slower (ALA), and two showed no reactivity with 12-LOX (EDA and LA). The bioactive lipid products resulting from 12-LOX oxidation of DGLA, 12-(S)-hydroperoxy-8Z,10E,14Z-eicosatrienoic acid [12(S)-HPETrE], and its reduced product, 12(S)-HETrE, resulted in significant attenuation of agonist-mediated platelet aggregation, granule secretion, αIIbβ3 activation, Rap1 activation, and clot retraction. Treatment with DGLA similarly inhibited PAR1-mediated platelet activation as well as platelet clot retraction. These observations are in surprising contrast to our recent work showing 12(S)-HETE is a prothrombotic bioactive lipid and support our hypothesis that the overall effect of 12-LOX oxidation of fatty acids in the platelet is dependent on the fatty acid substrates available at the platelet membrane.

Fig. 1.

12-LOX product regio-specificity and substrate comparison. (A) The substrates are positioned methyl-end first, relative to the active site iron. The carbon atoms that are labeled with numbers indicate location of oxygenation. (B) Stereochemical structures of 12(S)-HETrE and 13(S)-HOTrE.

Fig. 2.

Fatty acid metabolites regulate platelet aggregation. Washed human platelets were treated with or without increasing concentrations of the exogenously added fatty acid metabolites 12(S)-HETrE, 12(S)-HEPE, 13(S)-HOTrE, or 12(S)-HETE. (A) Representative curves for platelets treated with 20 μM PAR1-AP in the absence (control) or presence of each metabolite. The level of platelet aggregation was measured for 10 min poststimulation (N = 3 for each condition). (B) Composite for all replicates in the presence of increasing concentrations of each metabolite, ranging from 0 to 80 μM (N = 3). Replicates were graphed for the maximal level of platelet aggregation 10 min poststimulation for each condition. *P < 0.05; ***P = 0.002.

Fig. 3.

Fatty acid regulation of platelet dense granule secretion. Washed human platelets were treated with or without increasing concentrations of the exogenously added fatty acid metabolites 12(S)-HETrE, 12(S)-HEPE, 13(S)-HOTrE, or 12(S)-HETE. The maximal level of ATP secretion was measured poststimulation (N = 3 independent donors for each condition). *P < 0.05; **P < 0.01.

Fig. 4.

Agonist-independent regulation of platelet activity by 12-LOX metabolites. (A) Washed human platelets were treated with or without 5 μM DGLA for 10 min followed by stimulation with 20 μM PAR1-AP (PAR1-AP) for 10 min. PAR1-AP induced an immediate and stable platelet aggregation when added to platelets. Maximal and final platelet aggregation following treatment with DGLA was significantly attenuated. Additionally, treatment of DGLA resulted in inhibition of dense granule secretion in the presence of PAR1-AP (N = 3). Treatment with either a COX-1 inhibitor [100 µM aspirin (ASA)] or a 12-LOX inhibitor (100 µM baicalein) partially rescued DGLA-induced inhibition of PAR1-AP-mediated platelet aggregation (N = 3). ***P < 0.001. (B) Platelet aggregation was measured following 10 min stimulation with PAR1-AP, 20 μM ADP, or 5 μg/ml collagen in the presence of 40 μM 12(S)-HPETrE or 40 μM 12(S)-HETrE (N = 4–9). (C) αIIbβ3 activation was measured by flow cytometry following 10 min stimulation with PAR1-AP, ADP, or 100 ng/ml convulxin in the presence of 12(S)-HPETrE or 12(S)-HETrE (N = 4–10). (D) α-Granule secretion was assessed by measuring P-selectin surface expression on the platelet by flow cytometry following 10 min stimulation with PAR1-AP, ADP, or 100 ng/ml convulxin in the presence of 12(S)-HPETrE or 12(S)-HETrE (N = 4–10). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 4.

Agonist-independent regulation of platelet activity by 12-LOX metabolites. (A) Washed human platelets were treated with or without 5 μM DGLA for 10 min followed by stimulation with 20 μM PAR1-AP (PAR1-AP) for 10 min. PAR1-AP induced an immediate and stable platelet aggregation when added to platelets. Maximal and final platelet aggregation following treatment with DGLA was significantly attenuated. Additionally, treatment of DGLA resulted in inhibition of dense granule secretion in the presence of PAR1-AP (N = 3). Treatment with either a COX-1 inhibitor [100 µM aspirin (ASA)] or a 12-LOX inhibitor (100 µM baicalein) partially rescued DGLA-induced inhibition of PAR1-AP-mediated platelet aggregation (N = 3). ***P < 0.001. (B) Platelet aggregation was measured following 10 min stimulation with PAR1-AP, 20 μM ADP, or 5 μg/ml collagen in the presence of 40 μM 12(S)-HPETrE or 40 μM 12(S)-HETrE (N = 4–9). (C) αIIbβ3 activation was measured by flow cytometry following 10 min stimulation with PAR1-AP, ADP, or 100 ng/ml convulxin in the presence of 12(S)-HPETrE or 12(S)-HETrE (N = 4–10). (D) α-Granule secretion was assessed by measuring P-selectin surface expression on the platelet by flow cytometry following 10 min stimulation with PAR1-AP, ADP, or 100 ng/ml convulxin in the presence of 12(S)-HPETrE or 12(S)-HETrE (N = 4–10). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 5.

12-LOX metabolite regulation of Rap1. Washed platelets were treated with 12-LOX metabolites or various fatty acids for 10 min prior to stimulation with 20 μM PAR1-AP to determine their regulatory effects on the level of PAR1-AP-mediated Rap1 activation. (A) Platelets were treated with or without 40 μM 12(S)-HETrE, 12(S)-HETE, 12(S)-HEPE, or 13(S)-HOTrE for 10 min. Following 12-LOX metabolite treatment, Rap1 activation was measured after stimulation with PAR1-AP for 5 min (N = 3 independent experiments). PAR1-AP alone induced a significant increase in Rap1-GTP levels. Treatment with 12(S)-HETrE significantly attenuated PAR1-AP-induced Rap1 activation. Treatment with 12(S)-HETE, 12(S)-HEPE, or 13(S)-HOTrE had no significant effect on PAR1-AP-induced Rap1 activation. (B) Platelets were treated with or without 10 μM AA, DGLA, EPA, LA, or EDA for 10 min. Following fatty acid treatment, Rap1 activation was measured after stimulation with PAR1-AP for 5 min (N = 6). Treatment with DGLA significantly attenuated PAR1-AP-induced Rap1 activation. Treatment with AA, EPA, LA, or EDA had no significant effect on the level of Rap1 activity. *P < 0.05.

Fig. 6.

Fatty acid regulation of platelet-mediated clot retraction. PRP was treated with fatty acids, inhibitors, or eicosanoids, followed by stimulation with thrombin. The rate at which thrombin caused a platelet-dependent clot retraction of the PRP was determined. Pictures were taken at several time points following stimulation (0, 30, 60, and 90 min). The size of the clot was quantified using Image J. PRP formed a complete clot retraction within 60 min without fatty acid treatment. (A) PRP treated with 25 μM fatty acid (AA, DGLA, EPA, LA, or EDA), followed by stimulation with 10 nM thrombin. The rate of clot retraction was not affected by treatment with AA, LA, or EDA, but it was significantly attenuated by DGLA and EPA (N = 4–6). (B) PRP treated with 100 μM aspirin for 40 min, 25 μM DGLA for 10 min, or 200 μM 12(S)-HPETrE for 10 min was stimulated with thrombin (N = 3–5). NS, not significant; ***P < 0.001; ****P < 0.0001.