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.
Highlights
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)
The LOX products are responsible for human inflammatory responses [2], and they are implicated in a variety of human diseases. 5-LOX is involved in asthma [3] and cancer [4, 5]. 12-LOX is involved in psoriasis [6], hypertension [7, 8], hemostasis [9,10,11,12], diabetes [13, 14], and cancer [5, 15, 16], and 15-LOX is involved in atherosclerosis [17] and cancer [5, 18]
The four other fatty acids that were screened [AA, dihomo-␥-linolenic acid (DGLA), eicosapentaenoic acid (EPA), and ␣-linolenic acid (ALA)] all produced over 90% of a single oxygenated product, whose retention time, parent peak mass, and main fragmentation peaks are listed in Tables 1 and 2
Summary
All commercial fatty acids were purchased from Nu-Check prep with purities of 99.0% or greater. Molar extinction coefficients were calculated by first weighing the substrate on an analytical balance, dissolving the substrate with a measured mass of HPLC grade methanol to achieve a stock substrate/methanol solution Small aliquots of this solution were diluted into a quartz cuvette containing a Teflon stir bar and 2 ml of 25 mM HEPES buffer (pH 7.5) at room temperature (22°C). Platelet aggregation Washed platelets (250 l) were pretreated with or without varying concentrations (0–40 M) of fatty acid metabolites for 7 min, followed by stimulation with 20 M protease-activated receptor-activating peptide (PAR1-AP), 20 M ADP, or 100 ng/ml convulxin. Aliquots (40 l) of washed platelets adjusted to a final concentration of 2.5 × 107 platelets/ml were pretreated with metabolites for 15 min followed by addition of 2 l PAC1 and 2 l CD62P. All experiments were repeated at least three times using different subjects
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