Arachidonate 15-Lipoxygenase Enzyme Products Increase Platelet Aggregation and Thrombin Generation

<div><p>Atherosclerotic cardiovascular diseases are the leading causes of morbidity and mortality worldwide. We have previously shown that arachidonate 15-lipoxygenase B (ALOX15B) is highly expressed in atherosclerotic carotid plaques, and elucidation of mechanisms downstream of activated lipoxygenases may be relevant to our understanding of the genesis of atherosclerotic diseases. We examined 120 carotid plaques from patients with symptomatic carotid artery stenosis and showed that the extent of ALOX15B staining was significantly increased in carotid plaques with thrombosis. Impedance aggregometry analyses showed that the ALOX15B enzyme products 15-HETE and 15-HPETE increased platelet aggregation. By using a calibrated automatic thrombin assay, we showed that the ALOX15B products also increased both peak levels of thrombin and the total endogenous thrombin potential. Moreover, platelet aggregation was increased by addition of cell lysates from ischemic human macrophages, whereas platelet aggregation was reduced after knockdown of ALOX15B in human macrophages. Our data show that ALOX15B expression in human carotid plaques is associated with thrombus formation and that enzyme products of ALOX15B increase platelet aggregation and thrombin generation. We therefore propose that activated ALOX15B in macrophages may play a role in the induction of atherothrombotic events by increasing platelet aggregation and thrombin generation.</p></div

ALOX15B is present in atherosclerotic plaques. Serial sections of atherosclerotic plaques from the carotid artery of patients with symptomatic carotid artery stenosis (n = 120) were stained with antibodies against ALOX15B and counterstained with Mayer's hematoxylin.

<p>A representative section is shown in (<b>A</b>). (<b>B</b>) ALOX15B staining in plaques with thrombosis (n = 46) and without thrombosis (n = 74). (<b>C</b>) ALOX15B staining in plaques from patients diagnosed with stroke (n = 62) or TIA (n = 31). (<b>D</b>) ALOX15B staining in plaques classified according to AHA classes: III n = 5; IV n = 38; V n = 18; VI n = 59 (AHA class VI indicates complicated lesions with ruptured fibrous cap and/or surface thrombus). Data are mean ± SEM.</p

Patient and plaque characteristics (n = 120).

<p>AHA class, classification of plaques according to American Heart Association.</p

Platelet aggregation is increased by incubation with lysates from human ischemic macrophages.

<p>(A) ALOX15B mRNA expression in primary human monocyte-derived macrophages incubated in control (21% oxygen) and ischemic (1% oxygen) conditions. (<b>B</b>) 15-HETE production in cell lysates from macrophages incubated in control and ischemic conditions. (<b>C</b>) Area under the curve (AUC) of platelet aggregation measured using cell lysates from control and ischemic macrophages. Data are mean ± SEM from 4 independent experiments (i.e. macrophages from 4 blood donors) analyzed in duplicate.</p

Platelet aggregation and thrombin formation are increased by ALOX15B enzyme products.

<p>(A, B) Platelet aggregation was measured in blood (from healthy volunteers) in the presence or absence of 10 nmol/L 15-HETE or 15-HPETE or ethanol control. (<b>A</b>) A representative experiment showing platelet aggregation over time. (<b>B</b>) Area under the curve (AUC) of platelet aggregation. Data are mean ± SEM from 4 independent experiments (i.e. 4 blood donors) analyzed in duplicate. (<b>C, D</b>) Thrombin generation was measured in pooled normal plasma in the presence or absence of 10 nmol/L 15-HETE or 15-HPETE or 2% DMSO control. (<b>C</b>) A representative experiment showing thrombin generation curves. (<b>D</b>) The total amount of thrombin activity assessed as the area under curve (i.e. the endogenous thrombin potential). Data are mean ± SEM from 4 independent experiments analyzed in duplicate.</p

The Arachidonate 15-Lipoxygenase Enzyme Product 15-HETE Is Present in Heart Tissue from Patients with Ischemic Heart Disease and Enhances Clot Formation

<div><p>Ischemic heart disease is a major cause of death and morbidity and the search for novel therapeutic targets is still required. We have previously shown that the enzyme arachidonate 15 lipoxygenase (ALOX15), which catalyzes the conversion of arachidonic acid to 15-hydroxy eicosatetraenoic acid (15-HETE), is highly expressed in ischemic heart tissue, but its role in the pathogenesis of ischemic heart disease is unclear. Here we showed that expression of ALOX15, but not ALOX12 or ALOX15B, was increased in ischemic versus non-ischemic human heart biopsy samples. A similar ALOX expression pattern was found in hypoxic human cardiomyocytes and cardiac endothelial cells. We also showed that levels of 15-HETE were significantly higher in ischemic versus non-ischemic human heart biopsy samples and showed a tendency to increase in serum from the patients with ischemic heart disease. Moreover, hypoxia increased the production of 15-HETE levels from human cardiomyocytes and cardiac endothelial cells. The hypoxia-induced increase in 15-HETE levels from human cardiomyocytes was inhibited by the ALOX15 inhibitor baicalein. Finally, by using intrinsic rotational thromboelastometry, we showed that human whole blood clotted faster in the presence of 15-HETE. In summary, we propose that increased ALOX15 expression in heart tissue under ischemic conditions may lead to increased production of 15-HETE, potentially contributing to thrombosis.</p></div

Increased expression of ALOX15 and 15-HETE levels in hypoxic human cardiac endothelial cells.

<p>(<b>A</b>) ALOX15, ALOX15B and ALOX12 mRNA expression (relative to the control for their respective gene) in human cardiac endothelial cells incubated under normoxic (21% oxygen; control) or hypoxic conditions (1% oxygen) for 6 h (n = 3) (<b>B</b>) Immunocytochemical staining of human cardiac endothelial cells with antibody against ALOX15 (green) and DAPI (blue) in cells incubated in normoxia (control) or hypoxia. Scale bar 40 μm. <b>(C-D)</b> Representative immunoblot and quantification of ALOX15 protein in human cardiac endothelial cells incubated in normoxia (control) or hypoxia. <b>(E)</b> 15-HETE levels in cell culture media from human cardiac endothelial cells incubated in normoxia (control) or hypoxia. Data are expressed as mean ± SEM (<i>n</i> = 3). Student’s t-test (* <i>p</i> < 0.05; ** <i>p</i> < 0.01).</p

Patient characteristics.

<p>Patient characteristics.</p

Increased expression of ALOX15 and 15-HETE levels in human ischemic heart biopsies.

<p>(<b>A</b>) ALOX15, ALOX15B and ALOX12 mRNA expression n (relative to the control for their respective gene) in right atrium from control adults (n = 3) and from patients undergoing AVR (n = 5) or CABG (n = 5) surgery. (<b>B</b>) Serial sections of biopsies from right atrium of patients undergoing AVR (left panels) or CABG (right panels) surgery. Sections were stained with DAPI (blue) and antibodies against ALOX15 (green, upper panels) or ALOX15 isotype control (green, lower panels). Scale bar = 50 μm. (<b>C)</b> 15-HETE levels in right atrial tissue samples from patients undergoing AVR (n = 5) or CABG (n = 5) surgery. Data are expressed as mean ± SEM. (<b>D)</b> 15-HETE levels in serum from patients undergoing AVR (n = 5) or CABG (n = 5) surgery. Data are expressed as mean ± SEM. (<b>A</b>) One way ANOVA with Tukey’s multiple comparisons test (** <i>p</i> < 0.01). (<b>C</b>) Mann-Whitney test (** <i>p</i> = 0.0079).</p

Increased expression of ALOX15 and 15-HETE levels in hypoxic human cardiomyocytes.

<p>(<b>A</b>) ALOX15, ALOX15B and ALOX12 mRNA expression (relative to the control for their respective gene) in human cardiomyocytes derived from induced pluripotent stem cells incubated under normoxic (21% oxygen; control) or hypoxic conditions (1% oxygen) for 6 h (n = 3). <b>(B)</b> Immunocytochemical staining of human cardiomyocytes with antibody against ALOX15 (green) and DAPI (blue) in cells incubated in normoxia (control) or hypoxia. Scale bar = 40 μm. <b>(C-D)</b> Representative immunoblot and quantification of ALOX15 protein in human cardiomyocytes incubated in normoxia (control) or hypoxia (n = 3), <b>(E)</b> 15-HETE levels in cell culture media from cardiomyocytes incubated in normoxia (control) or hypoxia. <b>(F)</b> 15-HETE levels in lysates from human cardiomyocytes incubated in normoxia (control) or hypoxia with or without baicalein (25 μmol/L) for 6 h (n = 3–6). Data are expressed as mean ± SEM. (<b>A-D</b>) Student’s t-test (* <i>p</i> < 0.05; ** <i>p</i> < 0.01). (<b>F</b>) One-way ANOVA with Tukey's multiple comparisons test (*** <i>p</i> < 0.001).</p