Niacin and biosynthesis of PGD₂by platelet COX-1 in mice and humans

Abstract

The clinical use of niacin to treat dyslipidemic conditions is limited by noxious side effects, most commonly facial flushing. In mice, niacin-induced flushing results from COX-1-dependent formation of PGD₂ and PGE₂ followed by COX-2-dependent production of PGE₂. Consistent with this, niacin-induced flushing in humans is attenuated when niacin is combined with an antagonist of the PGD₂ receptor DP1. NSAID-mediated suppression of COX-2-derived PGI₂ has negative cardiovascular consequences, yet little is known about the cardiovascular biology of PGD₂. Here, we show that PGD₂ biosynthesis is augmented during platelet activation in humans and, although vascular expression of DP1 is conserved between humans and mice, platelet DP1 is not present in mice. Despite this, DP1 deletion in mice augmented aneurysm formation and the hypertensive response to Ang II and accelerated atherogenesis and thrombogenesis. Furthermore, COX inhibitors in humans, as well as platelet depletion, COX-1 knockdown, and COX-2 deletion in mice, revealed that niacin evoked platelet COX-1-derived PGD₂ biosynthesis. Finally, ADP-induced spreading on fibrinogen was augmented by niacin in washed human platelets, coincident with increased thromboxane (Tx) formation. However, in platelet-rich plasma, where formation of both Tx and PGD₂ was increased, spreading was not as pronounced and was inhibited by DP1 activation. Thus, PGD₂, like PGI₂, may function as a homeostatic response to thrombogenic and hypertensive stimuli and may have particular relevance as a constraint on platelets during niacin therapy.

Figures

Figure 1. Human platelets generate PGD2, and PGD2 inhibits human platelet aggregation.

(A) PGD2 was produced ex vivo by human platelets after aggregation stimulated by 10 μM ADP, 10 μM arachidonic acid (AA), 10 μM collagen (CA), and 10 μM thrombin receptor–activating peptide (TRAP), while pretreatment with 100 μM aspirin (ASA) for 10 minutes prior to addition of the platelet agonist completely suppressed production of PGD2 (n = 4 per group). (B) Urinary PGDM was suppressed by administration of 81 mg/d aspirin orally for 5 days (n = 17). Suppression of urinary PGDM attained after dosing on day 5 was sustained for the entire 24 hours (P < 0.001), consistent with a substantial contribution from anucleated platelets to this metabolite. Cre, creatinine. (C) Urinary dinor-TxM was suppressed by administration of 81 mg/d aspirin orally for 5 days (n = 17). Suppression of urinary dinor-TxM attained after dosing on day 5 was sustained for the entire 24 hours (P < 0.001), consistent with a dominant contribution from anucleate platelets to this metabolite. (D) Urinary PGIM was suppressed by administration of 81 mg/d aspirin orally for 5 days (n = 17). Suppression of urinary PGIM was sustained for only 4 hours after dosing on day 5 (P < 0.05), consistent with a dominant contribution from nucleated cells to this metabolite.

Figure 2. PGD2 biosynthesis is augmented during accelerated platelet-vascular interactions in humans.

(A) Excretion of TxM in successive 6-hour urinary aliquots commencing 6 hours before PTCA. TxM excretion increased significantly in aspirin-allergic patients (n = 3; P < 0.05). Pretreatment with aspirin at either 81 mg/d (n = 3) or 325 mg/d (n = 17) in patients for a minimum of 5 days before the procedure suppressed TxM (P < 0.001) and prevented the procedure-related increase in TxM during PTCA (P < 0.001). (B) Excretion of PGDM in successive 6-hour urinary aliquots commencing 6 hours before PTCA. PGDM excretion increased significantly in aspirin-allergic patients (n = 3; P < 0.05). Pretreatment with aspirin (ASA) at either 81 mg/d (n = 3) or 325 mg/d (n = 17) in control patients for a minimum of 5 days before the procedure suppressed PGDM (P < 0.001) and prevented the increase in PGDM during PTCA (P < 0.001). (C) Excretion of PGIM in successive 6-hour aliquots commencing 6 hours before PTCA. Pretreatment with 325 mg aspirin reduced PGIM significantly in control patients before and during PTCA (P < 0.01); however, 81 mg/d aspirin had no significant effect on urinary PGIM. While urinary PGIM increased significantly during PTCA only in the control group (P < 0.05), there was a significant difference (P < 0.05) among the 3 groups with respect to procedure-related maximal urinary PGIM values.

Figure 3. DP1 deletion augments Ang II–induced aneurysm formation.

(A) Representative images of abdominal aortas after 28 days of Ang II infusion. dKO, double KO. Scale bars: 1 mm. Abdominal aorta wet weights (B) and the outer diameter of abdominal aortas (C) were both significantly increased in DP1-ApoE double KO versus ApoE KO mice (n = 18–21; *P < 0.05). Each data point represents measurement from an individual mouse aorta displaying intergroup variation. The horizontal bars represent mean ± SEM within each group. (D) Distribution of median AAA severity within both groups (n = 18–21), as classified previously (57).

Figure 4. PGD2 restrains thrombogenesis in mice.

(A and B) DP1 deletion shortened the mean time to 50% and 100% vascular occlusion of the carotid artery in female mice (n = 16–18; *P < 0.05). (C and D) No effect on thrombogenesis was evident in male mice (n = 15–18).

Figure 5. Expression of DP1 in atherosclerotic lesions of LDL receptor KO mice.

Staining of atherosclerotic lesions in the (A) aortic root and (B) coronary artery with isotype control, anti-DP1, anti–VCAM-1, and anti-CD11b. Images were composited from approximately 20 images taken with a ×20 objective. Shown are representative composite images (n = 6). Scale bar: 300 μm.

Figure 6. Niacin evokes platelet PGD2 biosynthesis in humans.

Healthy volunteers received placebo or 81 mg aspirin each for 5 days, then 600 mg niacin (NA) was administered either 30 minutes (day 5 NA; n = 9) or 24 hours (day 6 NA; n = 6) after the last dose. Niacin evoked a significant increase in excretion of all prostanoid metabolites under placebo-treated conditions (P < 0.001). Administration of aspirin suppressed excretion of all metabolites when niacin was administered 30 minutes after the last aspirin dose, but only suppressed urinary PGDM and TxM significantly when administered 24 hours after the last dose (P < 0.001).

Figure 7. Stimulation of PGD2 release restrains activation of human platelets by niacin.

While niacin did not alter generation of TxB2 or PGD2 by human platelets in PRP under basal conditions (A and B; n = 4), formation of both eicosanoids was significantly increased in a dose-dependent manner by niacin when it was preincubated with platelets that were then stimulated with 10 μM ADP in PRP (C and D; n = 4). The trivial amount of PGD2 formed in WPs was unaltered by niacin, whereas niacin again significantly increased TxB2 formation (E and F; n = 4). Addition of 100 μM niacin to PRP did not alter platelet spreading, either with or without subsequent induction of spreading by stimulation with 40 μM ADP (G and H; n = 3). However, ADP-induced (40 μM) WP spreading was significantly augmented by preincubation with niacin (I and J; n = 4). Scale bars: 20 μm. *P < 0.05; **P < 0.01; ***P < 0.001.