Background— The adenosine diphosphate (ADP) receptor P2Y ₁₂ plays a pivotal role in platelet aggregation, as demonstrated by the benefit conferred by its blockade in patients with cardiovascular disease. Some studies have shown interindividual differences in ADP-induced platelet aggregation responses ex vivo, but the mechanisms underlying this variability are unknown. Methods and Results— We examined ADP-induced platelet aggregation responses in 98 healthy volunteers, and we identified 2 phenotypic groups of subjects with high and low responsiveness to 2 μmol/L ADP. This prompted us to screen the recently identified G _i -coupled ADP receptor gene P2Y ₁₂ for sequence variations. Among the 5 frequent polymorphisms thus identified, 4 were in total linkage disequilibrium, determining haplotypes H1 and H2, with respective allelic frequencies of 0.86 and 0.14. The number of H2 alleles was associated with the maximal aggregation response to ADP in the overall study population ( P =0.007). Downregulation of the platelet cAMP concentration by ADP was more marked in 10 selected H2 carriers than in 10 noncarriers. Conclusions— In healthy subjects, ADP-induced platelet aggregation is associated with a haplotype of the P2Y ₁₂ receptor gene. Given the crucial role of the P2Y ₁₂ receptor in platelet functions, carriers of the H2 haplotype may have an increased risk of atherothrombosis and/or a lesser clinical response to drugs inhibiting platelet function.

Platelet stimulation by thrombin leads to the activation of phosphoinositide 3-kinase (PI 3K) and to the production of the D3 phosphoinositides, phosphatidylinositol 3,4-bisphosphate (PdtIns-3,4P2) and 3,4,5-trisphosphate (PdtIns-3,4,5-P3). Because changes in the levels of these phosphoinositides correlate with the kinetics of actin assembly, they have been proposed to mediate actin assembly, causing cell shape changes. Wortmannin and LY294002, two unrelated inhibitors of PI 3-K, were used to investigate the role of PI 3-K in platelet actin assembly and aggregation. Both PI 3-K inhibitors abrogated the production of PdtIns-3,4-P2 and PdtIns-3,4,5-P3 in thrombin receptor-activating peptide (TRAP)-stimulated cells. However, neither wortmannin nor LY294002 altered the kinetics of actin assembly or the exposure of nucleation sites in TRAP-stimulated cells. In contrast, PI 3-K inhibitors showed a specific inhibitory pattern of cell aggregation, characterized by a primary phase of aggregation followed by progressive disaggregation. Flow cytometry analysis with the PAC1 monoclonal antibody or with FITC-labeled fibrinogen indicated that wortmannin inhibited the maintenance of the platelet integrin GPIIb-IIIa in its active state. Wortmannin also inhibited, in a dose-dependent manner, platelet aggregation induced by the binding of the monoclonal antibodies P256 and LIBS-6 to GPIIb-IIIa. LIBS Fab-induced aggregation also led to the production of PdtIns-3,4-P2. Platelet secretion, as evidenced by the release of preloaded 14C-5-hydroxy-tryptamine secretion or P-selectin up-regulation, was not affected by PI 3-K inhibition. These results demonstrate that the generation of D3 phosphoinositides is not required for actin assembly in TRAP-activated platelets. However, PI 3-K stimulation is necessary for prolonged GPIIb-IIIa activation and irreversible platelet aggregation. PI 3-K stimulation downstream of GPIIb-IIIa engagement may provide positive feedback required to sustain active GPIIb-IIIa.

The P2Y₁₂ receptor is a key player in platelet activation and a major target for antithrombotic drugs. The beneficial effects of P2Y₁₂ receptor antagonists might, however, not be restricted to the primary and secondary prevention of arterial thrombosis. Indeed, it has been established that platelet activation also has an essential role in inflammation. Additionally, nonplatelet P2Y₁₂ receptors present in immune cells and vascular smooth muscle cells might be effective players in the inflammatory response. This review will investigate the biological and clinical impact of P2Y₁₂ receptor inhibition beyond its platelet-driven antithrombotic effects, focusing on its anti-inflammatory role. We will discuss the potential molecular and cellular mechanisms of P2Y₁₂-mediated inflammation, including cytokine release, platelet–leukocyte interactions and neutrophil extracellular trap formation. Then we will summarize the current evidence on the beneficial effects of P2Y₁₂ antagonists during various clinical inflammatory diseases, especially during sepsis, acute lung injury, asthma, atherosclerosis, and cancer.

Protease-activated receptor 1 (PAR-1), the main thrombin receptor on vascular cells, plays a key role in platelet activation. We examined the range of PAR-1 expression on platelets, obtained twice, 1 week apart, from 100 healthy subjects and found a 2-fold interindividual variation in receptor numbers (95% CI = 858-1700). Because PAR-1 density was stable with time (r ² = 76%,P < .001), we sought a genetic explanation for the observed variability. To validate this approach, we also analyzed the α₂β₁ genotype according to receptor density and platelet mRNA expression data. We found that the number of PAR-1 receptors on the platelet surface is associated with the intervening sequence IVSn−14 A/T intronic variation. The number of receptors was also found to govern the platelet response to the SFLLRN agonist, in terms of aggregation and P-selectin expression. The T allele (allelic frequency, 0.14) can be considered as an allele with decreased expression, because it was associated with lower PAR-1 expression on the platelet surface and with a lower response to SFLLRN. The IVSn−14 A/T intronic variation may therefore be clinically relevant.

Summary: Use of oral contraceptives (OC) that combine a progestogen with synthetic ethinyl estradiol (EE) is associated with increased risk of venous thromboembolism. NOMAC/E2 is a new monophasic OC that combines nomegestrol acetate (NOMAC), a highly selective progestogen, with 17β-estradiol (E2). The study objective was to compare the effects on markers of haemostasis of NOMAC/E2 (2.5 mg/1.5 mg) versus the second-generation OC, levonorgestrel (LNG)/EE (100 μg/20 μg). Healthy women (age 18–38 years) received once-daily treatment for three consecutive 28-day cycles in a double-blind, randomised study: either NOMAC/E2 for 24 days with a four-day placebo interval (n=45) or LNG/ EE for 21 days with a seven-day placebo interval (n=45) per cycle. Mean changes from baseline to end-of-treatment in coagulation markers, including prothrombin fragment 1+2 (primary endpoint), fibrinolysis markers and platelet functions were assessed. Mean prothrombin fragment 1+2 levels (primary endpoint) did not increase with NOMAC/E2 compared with LNG/EE ( –0.02 vs. +0.08 nM, p<0.01). Other significant differences between NOMAC/E2 and LNG/EE were mean changes in antithrombin (+0.3% vs. –4.4%, p<0.001), activated protein C resistance – normalised ratio (+0.20 vs. +0.46, p<0.01), D-dimer ( –53 vs. +43 ng/ml, p<0.001), plasminogen (+6% vs. +30%, p<0.0001) and plasminogen activator inhibitor-1 ( –3.1 vs. –8.0 ng/ml, p<0.001). There was no effect of either treatment on platelet aggregation. The NOMAC/E2 pill regimen has fewer adverse effects on blood biological coagulation and fibrinolysis markers than LNG/EE. This suggests that NOMAC/E2 could have a more favourable venous thromboembolism risk profile than LNG/EE; further epidemiological data are required to confirm this.

Rationale:: Sphingosine-1-phosphate (S1P) signaling is essential for vascular development and postnatal vascular homeostasis. The relative importance of S1P sources sustaining these processes remains unclear. Objective:: To address the level of redundancy in bioactive S1P provision to the developing and mature vasculature. Methods and Results:: S1P production was selectively impaired in mouse platelets, erythrocytes, endothelium, or smooth muscle cells by targeted deletion of genes encoding sphingosine kinases -1 and -2. S1P deficiency impaired aggregation and spreading of washed platelets and profoundly reduced their capacity to promote endothelial barrier function ex vivo. However, and in contrast to recent reports, neither platelets nor any other source of S1P was essential for vascular development, vascular integrity, or hemostasis/thrombosis. Yet rapid and profound depletion of plasma S1P during systemic anaphylaxis rendered both platelet- and erythrocyte-derived S1P essential for survival, with a contribution from blood endothelium observed only in the absence of circulating sources. Recovery was sensitive to aspirin in mice with but not without platelet S1P, suggesting that platelet activation and stimulus–response coupling is needed. S1P deficiency aggravated vasoplegia in this model, arguing a vital role for S1P in maintaining vascular resistance during recovery from circulatory shock. Accordingly, the S1P ₂ receptor mediated most of the survival benefit of S1P, whereas the endothelial S1P ₁ receptor was dispensable for survival despite its importance for maintaining vascular integrity. Conclusions:: Although source redundancy normally secures essential S1P signaling in developing and mature blood vessels, profound depletion of plasma S1P renders both erythrocyte and platelet S1P pools necessary for recovery and high basal plasma S1P levels protective during anaphylactic shock.