Interindividual variation in the ability of aspirin to inhibit platelet cyclooxygenase-1 (COX-1) could account for some on-treatment cardiovascular events. Here, we sought to determine whether there are clinical phenotypes that are associated with a suboptimal pharmacological effect of aspirin. In a prospective, 2-week study, we evaluated the effect of aspirin 81 mg on platelet COX-1 in 135 patients with stable CAD by measuring serum thromboxane B2 (sTxB2) as an indicator of inhibition of platelet COX-1. A nested randomized study compared enteric-coated with immediate-release formulations of aspirin. We found that sTxB2 was systematically higher among the 83 patients with metabolic syndrome than among the 52 patients without (median 4.0 ng/mL vs. 3.02 ng/mL, P=0.013). Twelve (14%) patients with metabolic syndrome, but none without metabolic syndrome, had sTxB2 levels consistent with inadequate inhibition of COX (sTxB2 ≥13 ng/mL). In linear regression models, metabolic syndrome (but none of its individual components) significantly associated with higher levels of log-transformed sTxB2 (P=0.006). Higher levels of sTxB2 associated with greater residual platelet function measured by aggregometry-based methods. Among the randomized subset, sTxB2 levels were systematically higher among patients receiving enteric-coated aspirin. Last, urinary 11-dehydrothromboxane B2 did not correlate with sTxB2, suggesting that the former should not be used to quantitate aspirin’s pharmacological effect on platelets. In conclusion, metabolic syndrome, which places patients at high risk for thrombotic cardiovascular events, strongly and uniquely associates with less effective inhibition of platelet COX-1 by aspirin.

Meta-analyses have demonstrated that low dose aspirin reduces the risk of developing adenocarcinoma metastasis, and when colon cancer is detected during aspirin treatment, there is a remarkable 83% reduction in risk of metastasis. As platelets participate in the metastatic process, the anti-platelet action of low dose aspirin likely contributes to its anti-metastatic effect. Cycloxooxygenase-2 (COX-2)-derived prostaglandin E2 (PGE2) also contributes to metastasis, and we addressed the hypothesis that low dose aspirin also inhibits PGE2 biosynthesis. We show that low dose aspirin inhibits systemic PGE2 biosynthesis by 45% in healthy volunteers (p <0.0001). Aspirin is found to be more potent in colon adenocarcinoma cells than in the platelet, and in lung adenocarcinoma cells its inhibition is equivalent to that in the platelet. Inhibition of COX by aspirin in colon cancer cells is in the context of the metastasis of colon cancer primarily to the liver, the organ exposed to the same high concentrations of aspirin as the platelet. We find that the interaction of activated platelets with lung adenocarcinoma cells up-regulates COX-2 expression and PGE2 biosynthesis, and inhibition of platelet COX-1 by aspirin inhibits PGE2 production by the platelet-tumor cell aggregates. In conclusion, low dose aspirin has a significant effect on extraplatelet cyclooxygenase, and potently inhibits COX-2 in lung and colon adenocarcinoma cells. This supports a hypothesis that the remarkable prevention of metastasis from adenocarcinomas, and particularly from colon adenocarcinomas, by low dose aspirin results from its effect on platelet COX-1 combined with inhibition of PGE2 biosynthesis in metastasizing tumor cells.

This article is available online at http://www.jlr.org to prostaglandin H 2 (PGH 2 ). In platelets, the thromboxane synthase enzyme catalyzes conversion of PGH 2 to both thromboxane A 2 and malondialdehyde (MDA) in approximately equimolar amounts ( 1 ) (supplementary Fig. 1).MDA is an electrophile that reacts with amino groups, including the -amine of protein lysines. Reaction of MDA with lysine in vitro leads to formation of adducts with several structures ( 2, 3 ), including one that results from the reaction of this dicarbonyl with two lysines to produce intra-and intermolecular cross-links of macromolecules. Such cross-links have been demonstrated when MDA is added to purifi ed apoA-1 in vitro ( 2 ).This evidence that MDA is a major product of the thromboxane synthase and can modify protein structure in vitro suggests a hypothesis that platelet activation could lead to modifi cation of platelet proteins by MDA. However, there previously has been no evidence that this occurs.The investigations reported here demonstrate that activation of platelets ex vivo leads to thromboxane synthasedependent MDA modifi cation of platelet proteins. A stable isotope dilution method for analysis for the dilysyl-MDA cross-link utilizing LC/MS/MS has been developed, making it possible to demonstrate increased levels of MDA adducts of platelet proteins in diseases that are associated with increased platelet activation. Activation of platelets signals cytosolic phospholipase A 2 ␣ activation and an explosive release of arachidonic acid, which is metabolized by cyclooxygenase (COX

Background: Meta-analysis of the trials evaluating the effect of aspirin on cardiovascular outcomes demonstrated that aspirin prevented mortality from adenocarcinomas by 47% and reduced metastasis by 46%. Equivalent benefit was seen with both high and low doses of aspirin. We hypothesize that low dose aspirin blocks metastasis by inhibiting platelet COX-1 and also by inhibiting the biosynthesis of the pro-metastatic PGE2. Methods and Results: The effect of a range of doses of aspirin on PGE2 biosynthesis in three lung adenocarcinoma cell lines was compared with that in washed platelets. PGE2 was measured by GC/MS. In the cancer cell lines, aspirin inhibits COX-2-dependent PGE2 production with IC50s equivalent to or less than that for platelet COX-1. The IC50s were: Platelet = 19.8 ± 1.5 μM; H2122 = 19.5 ± 5 μM; HCC827 = 3.9 ± 2 μM; A549 = 1.6 ± 0.4 μM. To explore the extra-platelet effects of low dose aspirin in vivo, we examined the effect of aspirin 81 mg daily for 2 weeks on the biosynthesis of PGE2 and prostacyclin as reflected by their respective urinary metabolites in 54 healthy humans. The PGE2 metabolite was measured by LC/MS/MS and the prostacyclin metabolite by GC/MS. This dose of aspirin inhibited PGE2 production by 45% (p &lt; 0.0001) and reduced prostacyclin by 37% (p &lt; 0.0001). Adherence of platelets to tumor cells facilitates metastasis. We determined the effect of adherence of ADP-activated washed platelets to lung adenocarcinoma cells (A549) on the PGE2 biosynthetic pathway. COX-2 expression was determined by Western blot, and cytosolic calcium by Fura-2 AM. Activated platelets increased cytosolic calcium in A549 cells by 22% (p &lt; 0.001), increased expression of COX-2 by 8.6 fold (p&lt;0.0001) and increased PGE2 biosynthesis by 8.1 fold (p &lt; 0.0001), indicating that PGE2 production by tumor cells is enhanced by activated platelets. Conclusion: These findings form the basis for a hypothesis that the remarkable effect of low dose aspirin on adenocarcinoma prevention and reduction in metastatic behavior could result from inhibition of platelet activation in concert with inhibition of platelet-induced PGE2 biosynthesis and direct inhibition of the catalytic activity COX-2 in circulating tumor cells. Work funded by the Thoracic Program of the Vanderbilt Ingram Cancer Center Citation Format: John Oates, Pierre Massion, Bjorn Knollmann, James Smith, Elias Haddad, Philip Lammers, Denise Oram, Taneem Amin, Bradford Harris, Megan Hoeksema, Hyun Hwang. Low dose aspirin that reduces mortality from lung adenocarcinoma inhibits both platelet COX-1 and the biosynthesis of PGE2. [abstract]. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr PR05.

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