In the presence of strong P2Y12 receptor blockade, aspirin provides little additional inhibition of platelet aggregation.

BACKGROUND: Aspirin and antagonists of platelet ADP P2Y(12) receptors are often coprescribed for protection against thrombotic events. However, blockade of platelet P2Y(12) receptors can inhibit thromboxane A(2) (TXA(2))-dependent pathways of platelet activation independently of aspirin. OBJECTIVES: To assess in vitro whether aspirin adds additional antiaggregatory effects to strong P2Y(12) receptor blockade. METHODS: With the use of platelet-rich plasma from healthy volunteers, determinations were made in 96-well plates of platelet aggregation, TXA(2) production and ADP/ATP release caused by ADP, arachidonic acid, collagen, epinephrine, TRAP-6 amide and U46619 (six concentrations of each) in the presence of prasugrel active metabolite (PAM; 0.1-10 µmol L(-1)), aspirin (30 µmol L(-1)), PAM + aspirin or vehicle. results: PAM concentration-dependently inhibited aggregation; for example, aggregation in response to all concentrations of ADP and U46619 was inhibited by ≥ 95% by PAM at > 3 µmol L(-1) . In further tests of PAM (3 µmol L(-1)), aspirin (30 µmol L(-1)) and PAM + aspirin, aspirin generally failed to produce more inhibition than PAM or additional inhibition to that caused by PAM. The antiaggregatory effects of PAM were associated with reductions in the platelet release of both TXA(2) and ATP + ADP. Similar effects were found when either citrate or lepirudin were used as anticoagulants, and when traditional light transmission aggregometry was conducted at low stirring speeds. CONCLUSIONS: P2Y(12) receptors are critical to the generation of irreversible aggregation through the TXA(2) -dependent pathway. As a result, strong P2Y(12) receptor blockade alone causes inhibition of platelet aggregation that is little enhanced by aspirin. The clinical relevance of these observations remains to be determined.

Nucleotide oligomerization domain 1 is a dominant pathway for NOS2 induction in vascular smooth muscle cells: comparison with Toll-like receptor 4 responses in macrophages.

BACKGROUND AND PURPOSE: Gram-negative bacteria contain ligands for Toll-like receptor (TLR) 4 and nucleotide oligomerization domain (NOD) 1 receptors. Lipopolysaccharide (LPS) activates TLR4, while peptidoglycan products activate NOD1. Activation of NOD1 by the specific agonist FK565 results in a profound vascular dysfunction and experimental shock in vivo. EXPERIMENTAL APPROACH: Here, we have analysed a number of pharmacological inhibitors to characterize the role of key signalling pathways in the induction of NOS2 following TLR4 or NOD1 activation. KEY RESULTS: Vascular smooth muscle (VSM) cells expressed NOD1 mRNA and protein, and, after challenge with Escherichia coli or FK565, NOS2 protein and activity were induced. Macrophages had negligible levels of NOD1 and were unaffected by FK565, but responded to E. coli and LPS by releasing increased NO and expression of NOS2 protein. Classic pharmacological inhibitors for NF-kappaB (SC-514) and mitogen-activated protein kinase (SB203580, PD98059) signalling pathways inhibited responses in both cell types regardless of agonist. While TLR4-mediated responses in macrophages were specifically inhibited by the pan-caspase inhibitor z-VAD-fmk and the PKC inhibitor Gö6976, NOD1-mediated responses in VSM cells were inhibited by the Rip2 inhibitor PP2. CONCLUSIONS AND IMPLICATIONS: Our findings suggest a selective role for NOD1 in VSM cells, and highlight NOD1 as a potential novel therapeutic target for the treatment of vascular inflammation.

Heparin but not citrate anticoagulation of blood preserves platelet function for prolonged periods.

BACKGROUND: Current guidelines state that platelet aggregation studies should be conducted within 4 h of venepuncture because of the decline in sensitivity to platelet agonists. This constrains studies of platelet activity in clinical situations where samples need to be transported or there are unavoidable delays prior to assessment. OBJECTIVES: The aim of the present study was to compare systematically the responses of platelets stored in the presence of either citrate or heparin, the two most widely used anti-coagulants, using a range of standard techniques. METHODS: Blood was taken from healthy volunteers and either assessed immediately or stored at ambient temperature (18-25 degrees C) for 24 h. Platelet reactivity to a range of agonists was determined by a combination of 96-well plate techniques; light transmission aggregometry, thrombi adhesion, ATP and ADP release, and TxA(2) release; by whole blood aggregometry; and by PFA-100. RESULTS AND CONCLUSIONS: Testing using 96-well plate techniques allowed for the simultaneous measurement of responses to multiple concentrations of multiple agonists. The responses of platelets from blood anti-coagulated with heparin were predominantly preserved in all assays after 24 h storage, whereas, responses of platelets stored in blood anti-coagulated with citrate were greatly diminished. Consequently, anti-coagulation with heparin, but not citrate, preserves platelet responses for up to 24 h as determined by a range of techniques.

Aspirin and the in vitro linear relationship between thromboxane A2-mediated platelet aggregation and platelet production of thromboxane A2.

BACKGROUND: Currently, 'aspirin resistance', the anti-platelet effects of non-steroid anti-inflammatory drugs (NSAIDs) and NSAID-aspirin interactions are hot topics of debate. It is often held in this debate that the relationship between platelet activation and thromboxane (TX) A(2) formation is non-linear and TXA(2) generation must be inhibited by at least 95% to inhibit TXA(2)-dependent aggregation. This relationship, however, has never been rigorously tested. OBJECTIVES: To characterize, in vitro and ex vivo, the concentration-dependent relationships between TXA(2) generation and platelet activity. METHOD: Platelet aggregation, thrombi adhesion and TXA(2) production in response to arachidonic acid (0.03-1 mmol L(-1)), collagen (0.1-30 microg mL(-1)), epinephrine (0.001-100 micromol L(-1)), ADP, TRAP-6 amide and U46619 (all 0.1-30 micromol L(-1)), in the presence of aspirin or vehicle, were determined in 96-well plates using blood taken from naïve individuals or those that had taken aspirin (75 mg, o.d.) for 7 days. RESULTS: Platelet aggregation, adhesion and TXA(2) production induced by either arachidonic acid or collagen were inhibited in concentration-dependent manners by aspirin, with logIC(50) values that did not differ. A linear relationship existed between aggregation and TXA(2) production for all combinations of arachidonic acid or collagen and aspirin (P < 0.01; R(2) 0.92; n = 224). The same relationships were seen in combinations of aspirin-treated and naïve platelets, and in blood from individuals taking an anti-thrombotic dose of aspirin. CONCLUSIONS: These studies demonstrate a linear relationship between inhibition of platelet TXA(2) generation and TXA(2)-mediated aggregation. This finding is important for our understanding of the anti-platelet effects of aspirin and NSAIDs, NSAID-aspirin interactions and 'aspirin resistance'.