Human platelets generate PGD 2 , and PGD 2 inhibits human platelet aggregation. Both thromboxane A 2 (TxA 2 ) and PGD 2 are formed by mature human platelets, which exclusively express COX-1 (28, 29). Indeed, activation of human platelets by ADP, arachidonic acid, collagen, and the thrombin receptor–activating peptide all evoked production of PGD 2 , which was suppressed by aspirin (Figure 1A). The capacity of platelets to form agonist-induced PGD 2 was considerably less than TxA 2 (Supplemental Figure 1A; supplemental material available online with this article; doi: 10.1172/JCI59262DS1). DP1 is expressed on human platelets (30), and pretreatment with exogenous PGD 2 increases platelet cAMP production (31); exogenous PGD 2 pretreatment also abolished ADP-induced aggregation in platelet-rich plasma (PRP; Supplemental Figure 1B) at concentrations corresponding to those of the endogenous material formed in response to platelet activation. Low-dose aspirin, which preferentially inhibits platelet COX-1 (32), suppressed both urinary PGDM and 2,3-dinor-TxB 2 (dinor-TxM) in healthy volunteers (Figure 1, B and C), effects that were sustained at steady state for 24 hours after dosing. This delayed recovery pattern is consistent with a dominant contribution from anucleate platelets to systemic biosynthesis of TxA 2 and a substantial contribution to PGD 2 , in contrast to the reversible suppression of 2,3-dinor-6-keto-PGF 1α (PGIM; Figure 1D and refs. 33–35).

Figure 1 Human platelets generate PGD 2 , and PGD 2 inhibits human platelet aggregation. (A) PGD 2 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 PGD 2 (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.

PGD 2 biosynthesis is augmented during accelerated platelet-vascular interactions in humans. Percutaneous transluminal coronary angioplasty (PTCA) is an acute, localized stimulus to platelet and vascular function. Periprocedural cardiovascular complications are reduced by aspirin, presumably due to inhibition of TXA 2 produced by activated platelets. We have previously reported a procedure-related increment in urinary 11-dehydro-TxB 2 (TxM) and PGIM in patients undergoing PTCA, reflecting accelerated platelet vessel wall interactions (36). This is also seen in mice subjected to wire injury, in which deletion of IP augments platelet activation and the proliferative response to injury, consistent with a role for PGI 2 in homeostatic regulation of platelet–vessel wall intractions (37). Here, we recruited aspirin-allergic patients undergoing PTCA and treated with clopidogrel and amciximab to study prostanoid biosynthesis uninhibited by aspirin. Urinary TxM rose significantly in this group, from 1.9 ± 0.5 to 4.0 ± 0.7 ng/mg creatinine during the 6-hour collection corresponding to the procedure, and falling to 2.7 ± 0.9 ng/mg creatinine in the subsequent collection (Figure 2A). As expected, excretion of TxM was markedly and similarly suppressed by pretreatment of PTCA patients with either 81 or 325 mg/d aspirin, to periprocedural values of 0.3 ± 0.2 and 0.5 ± 0.1 ng/mg creatinine, respectively, and remained suppressed thereafter (P < 0.001; Figure 2A). The pattern of urinary PGDM closely resembled that of urinary TxM, consistent with both metabolites deriving predominantly from platelets. Thus, in a setting of platelet activation, urinary PGDM increased in a procedure-related manner in the aspirin-sensitive patients and was maximally suppressed by both aspirin regimens (Figure 2B). In contrast, periprocedural PGIM, reflecting its predominant vascular origin, was dose-dependently suppressed by aspirin, being only partially inhibited by the 81-mg/d regimen (Figure 2C).

Figure 2 PGD 2 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.

DP1 activation restrains the hypertensive and aneurysmal responses to Ang II in male mice. Unlike human platelets, mouse platelets lacked DP1 (Supplemental Figure 2, A and B). Thus, exogenous PGD 2 , unlike the PGI 2 analog cicaprost, failed to inhibit platelet aggregation (data not shown) or disaggregate ADP-induced platelet aggregation (Supplemental Figure 2, C–H). Despite this, the systolic hypertensive response to 4-week infusion of Ang II was significantly augmented in male hyperlipidemic ApoE KO mice by DP1 deletion (Supplemental Figure 3, A and B). By contrast, the elevation in systolic and mean arterial pressure evoked by 14 days on a 4% high-salt diet was unaltered by DP1 deletion (Supplemental Figure 3, C and D). DP1 deletion significantly increased Ang II–induced abdominal aortic aneurysm (AAA) formation in the ApoE KO mice, whether measured by wet weight, external diameter, or blinded allocation of severity (Figure 3).

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).

PGD 2 restrains thrombogenesis in female mice. Despite the absence of DP1 on mouse platelets, its deletion accelerated the partial and complete thrombogenic occlusive response to a photochemical injury to the carotid artery in female mice (Figure 4).

Figure 4 PGD 2 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).

Activation of DP1 restrains atherogenesis in female mice. The impact of DP1 deficiency on atherogenesis was assessed in LDL receptor KO mice. Lesion burden was measured en face at 3, 6, and 9 months on a high-fat diet. Lesion burden increased with time in both genders, and there was no significant impact of DP1 deletion on disease progression in male mice. However, lesion progression was accelerated modestly, but significantly, in female mice lacking DP1 (Supplemental Figure 4). In mice, DP1 immunoreactivity was localized within lesions to activated vascular smooth muscle cells in both the media of the underlying lesion and the neointima, as identified morphologically together with expression of VCAM-1 detected in serial sections as previously described (38), and in areas of inflammatory myeloid infiltrates, as identified by expression of CD11b (Figure 5) and CD45 (data not shown). In humans, DP1 expression was detected in endothelial cells, and in human atheromatous tissue, additional expression was noted in intravascular endothelial cells, macrophages, and vascular smooth muscle cells (Supplemental Figure 5).

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.

Niacin evokes platelet PGD 2 biosynthesis in humans. To address the cellular contribution of platelets to PGD 2 biosynthesis in humans, we randomized healthy volunteers to receive 5 daily doses of aspirin (81 mg/d) or placebo. Niacin (600 mg) was then administered orally either 30 minutes or 24 hours after the final dose of aspirin, on the assumption that a differential, partial recovery of the capacity for prostanoid biosynthesis will have occurred in nucleated cells, but not in platelets, with delayed niacin dosing (34). Niacin evoked a marked increase in urinary PGDM, TxM, PGIM, and PGEM under placebo conditions. Both urinary PGDM and TxM were completely suppressed when niacin was administered either 30 minutes or 24 hours after the last dose of aspirin (Figure 6, A and B). Again, this delayed pattern of recovery is consistent with both prostanoids deriving predominantly from platelets. In contrast, whereas urinary PGIM and PGEM were suppressed at the earlier time point, both had partially recovered when niacin was administered 24 hours after the last dose of aspirin (Figure 6, C and D), consistent with their predominant source being nucleated cells, such as those of vascular origin.

Figure 6 Niacin evokes platelet PGD 2 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).

Niacin evokes platelet COX-1–dependent prostaglandin formation in mice. Niacin evoked an increase in urinary excretion of PGDM, TxM, PGEM, and PGIM in mice as in humans. We have previously demonstrated that COX-1 knockdown (KD) mice exhibit an asymmetric effect on platelet COX-1, reminiscent of the effects of low-dose aspirin in humans (39). In the present study, COX-1 KD ablated the niacin-induced increment in urinary TxM observed in WT littermate controls (average suppression, 97.8% ± 11.9%; P < 0.001) and almost completely suppressed the increment in urinary PGDM (average suppression, 91.0% ± 16.4%; P < 0.001; Supplemental Figure 6, A and B). COX-1 KD had a more modest impact on the niacin-evoked increments in urinary PGIM (average suppression, 70.0% ± 20.5%; P < 0.01) and PGEM (average suppression, 61.3% ± 10.6%; P < 0.01). We used COX-2 KO mice to probe the contribution of COX-2 to the niacin-evoked prostanoid response (40). These experiments in the COX modified mice were not quantitatively comparable, as the mice differ in genetic backgrounds (see Supplemental Methods). For example, the niacin-evoked TxM response in the COX-1 WT littermate controls was quantitatively less than in the COX-2 WT littermate controls (Supplemental Figure 6). In this case, COX-2 deletion failed to alter significantly the niacin-evoked increments in all 4 prostanoid metabolites (Supplemental Figure 6, E–H). Depletion of the platelet count from 8.1 × 108 to 0.1 × 108 platelets/ml was attained with intravenous anti-GPIbα (ref. 41 and Supplemental Methods). However, after antibody administration, there was a transient increase of urinary TxM and PGDM (Supplemental Figure 7), probably reflecting initial platelet activation with subsequent depletion (42). At 24 hours after antibody administration, urine was collected for 12 hours, followed by intraperitoneal injection with niacin (300 mg/kg body weight) or vehicle and urine collection for another 12 hours for prostaglandin analysis. Antibody administration significantly depressed the niacin-evoked increment in urinary TxM (average suppression, 60.3% ± 28.2%; P < 0.05) and PGDM (average suppression, 41.2% ± 15.8%; P < 0.05). No significant effect was observed with either urinary PGEM or PGIM (Supplemental Figure 6, I–L). These data are consistent with a predominant contribution from platelet COX-1 to the niacin-evoked increment in urinary PGDM in mice, as in humans.

The niacin GPR109A receptor is expressed on human platelets. The GPR109A receptor for niacin was identified on human platelets by flow cytometry (Supplemental Figure 8A) and immunofluorescent staining, predominately of the cell membrane (Supplemental Figure 8, B and C). Niacin-induced degradation of its receptor, as observed in other cell types (43), was apparent on Western blotting of platelet cell membranes (Supplemental Figure 8D). Niacin dose-dependently decreased platelet cyclic AMP, as expected, upon activation of this Gi-linked receptor (ref. 44 and Supplemental Figure 8E).

Niacin-induced stimulation of PGD 2 restrains activation of human platelets. Niacin alone failed to alter platelet prostanoid formation or spreading on a fibrinogen-coated plate (ref. 45 and Figure 7, A and B). However, when platelets were preincubated with niacin and then stimulated with ADP, the effects of niacin differed depending on whether the platelets were bathed in plasma, a source of lPGDS (29). Indeed, immunodepletion of lPGDS significantly suppressed PGD 2 formation in PRP (Supplemental Figure 9). Preincubation with niacin in PRP evoked an increased response of both TxB 2 and PGD 2 to ADP (Figure 7, C and D), whereas in washed platelets (WPs), niacin only evoked an increase in TxB 2 (Figure 7, E and F). Despite this differential impact on prostanoid formation, there was no effect of niacin on ADP-induced platelet aggregation in WP or PRP (data not shown). However, ADP-induced platelet spreading on fibrinogen (a more sensitive indicator of platelet activation; ref. 46) was significantly more pronounced in WPs, but not in PRP, after preincubation with niacin (Figure 7, G–J), consistent with a restraining effect on niacin-dependent platelet activation by endogenous PGD 2 . The eicosanoid acts via the DP1 to exert this effect. We conclude that a DP1 antagonist blocks the inhibitory effect of niacin on human platelets activated by a lower dose of ADP (Supplemental Figure 10).