Potentiation of clopidogrel active metabolite formation by rifampicin leads to greater P2Y12 receptor blockade and inhibition of platelet aggregation after clopidogrel.

BACKGROUND: The thienopyridine P2Y(12) receptor antagonist clopidogrel reduces the risk of arterial thrombosis and individual pharmacodynamic responses to clopidogrel are believed to reflect the levels of active metabolite (AM) generated. Rifampicin increases the inhibitory effect of clopidogrel on platelet aggregation (PA). We studied the response to clopidogrel before and during administration of rifampicin in order to study the relationship between individual AM levels and P2Y(12) blockade. METHODS: Healthy volunteers received a 600-mg loading dose of clopidogrel followed by 75 mg daily for 7 days and, after a washout period and treatment with rifampicin [300 mg twice a day (b.i.d.)], received the same regimen of clopidogrel. Clopidogrel AM levels were determined over 4 h after the clopidogrel loading dose and unblocked P2Y(12) receptor number was assessed using a (33) P-2MeSADP binding assay. PA was measured by optical aggregometry with ADP and TRAP. RESULTS: Rifampicin enhanced clopidogrel AM production [area-under-the-curve (AUC): clopidogrel 89±22 ng h mL(-1) , clopidogrel+rifampicin 335±86 ng h mL(-1) , P<0.0001], and P2Y(12) blockade (unblocked receptors: clopidogrel 48±24, clopidogrel+rifampicin 4±2, P<0.0001) and reduced PA (5 µmol L(-1) ADP: clopidogrel 20±4, clopidogrel+rifampicin 5±2, P<0.01). Increasing numbers of unblocked receptors were required for an aggregation response with a decreasing concentration of ADP. PA induced by ADP 2 µmol L(-1) was particularly sensitive to low levels of receptor blockade. CONCLUSION: Potentiation of clopidogrel AM production by rifampicin leads to greater P2Y(12) blockade and consequently greater inhibition of PA. PA responses to low concentrations of ADP are more sensitive to P2Y(12) blockade.

Platelet P2Y(12) receptor influences the vessel wall response to arterial injury and thrombosis.

BACKGROUND: Platelets are believed to play an important role in atherogenesis and the vessel response to vascular injury. The P2Y(12) receptor (P2Y(12)) plays a central role in amplifying platelet aggregation, dense granule and alpha-granule secretion, P-selectin expression, microparticle formation, and procoagulant membrane changes, regardless of the activating stimulus. We hypothesized that P2Y(12) deficiency might reduce the vessel wall response to vascular injury as well as thrombosis in murine vascular injury models. METHODS AND RESULTS: P2Y(12)-deficient (-/-) mice and littermate controls (+/+) were bred on a C57 BL/6 background. In vivo murine models of arterial injury were employed alone and in combination with bone marrow transplantation to investigate the role of P2Y(12) in the vessel wall response to arterial injury and thrombosis. At 21 days after ferric chloride injury, neointima formation in P2Y(12)(-/-) arteries was significantly less than that observed in control strain arteries (P<0.025). In agreement with this, the intima-media ratio was significantly greater in femoral wire-injured arteries from P2Y(12)(+/+) compared with P2Y(12)(-/-) animals (P<0.05). Bone marrow transplantation was used to examine the importance of vessel wall P2Y(12) versus platelet P2Y(12). Analysis of arterial sections from chimeric animals at 21 days after injury revealed a smaller intima-media ratio in -/- to +/+ animals than in the positive (+/+ to +/+) control group (P<0.01). CONCLUSIONS: These data demonstrate a role for platelet P2Y(12) in the vessel wall response to arterial injury and thrombosis. This illustrates the manner in which platelets may contribute to atherogenesis and restenosis.

PAR-1 genotype influences platelet aggregation and procoagulant responses in patients with coronary artery disease prior to and during clopidogrel therapy.

Genetic variations of the protease-activated receptor-1 (PAR-1) have been associated with platelet receptor density and linked to thrombin receptor-activating peptide (TRAP)-induced phenotypes of platelet aggregation and P-selectin expression. We investigated whether the PAR-1 intervening sequence-14 A>T dimorphism influences platelet procoagulant activity. We also determined whether the P2Y12 antagonist clopidogrel could offset any observed functional polymorphism of the PAR-1 receptor by inhibiting P2Y12-mediated amplification of TRAP-induced responses. We studied 54 patients listed for elective percutaneous coronary intervention assessing TRAP-induced platelet aggregation and markers of procoagulant activity. Platelet responses were measured at baseline, 4 h post clopidogrel 300 mg, and 10 and 28 days following clopidogrel 75 mg daily. Each patient was genotyped for the PAR-1 intervening sequence-14 A/T dimorphism. Increased platelet aggregation and procoagulant responses were observed with PAR-1 A allele homozygotes. Clopidogrel significantly inhibited these platelet responses regardless of PAR-1 genotype, but did not offset the hyper-reactivity associated with the A/A homozygotes. We conclude that a common sequence variation within the PAR-1 gene influences TRAP-induced platelet procoagulant activity as well as aggregation. Higher platelet reactivity associated with PAR-1 IVSn-14 A allele homozygotes persists despite clopidogrel therapy. These individuals may be at higher risk of thromboembolic events and may require additional anti-platelet medication.

Responses to free lipopolysaccharide and Escherichia coli by normal human intestinal macrophages, following their migration out of the lamina propria.

Intestinal macrophage responses to luminal bacteria and their constituents are important in mucosal inflammatory responses. We investigated the responses of intestinal macrophages to free lipopolysaccharide (LPS) and Escherichia coli. Macrophages were isolated from normal terminal ileum and colon by allowing them to migrate out of the lamina propria of mucosal samples denuded of epithelial cells. Following exposure to free LPS or fluorescein-labelled E. coli, responsiveness was studied by intracellular expression of tumour necrosis factor-alpha (TNF-alpha). CD14, CD33, CD68, TLR2 and TLR4 expression was studied by fluorescence-activated cell sorter (FACS). TLR and NOD2 expression was confirmed by reverse-transcriptase polymerase chain reaction (RT-PCR). CD14 was expressed by 36.5 +/- 4.0% of the macrophages obtained following migration out of the lamina propria. These cells also expressed TLR2, TLR4 and NOD2. Of cells exposed to free LPS or those that had taken up E. coli, a greater proportion of CD14(+) than CD14(-) macrophages expressed intracellular TNF-alpha. Moreover, a greater proportion of macrophages (CD14(+) and CD14(-)) demonstrated responses to E. coli than free LPS. In conclusion, a proportion of macrophages obtained following migration out of the lamina propria of normal terminal ileal and colonic mucosal samples express CD14, TLR2 and TLR4. These cells respond to free LPS and E. coli, as demonstrated by the expression of TNF-alpha.

Interactions of platelets with Synthocytes, a novel platelet substitute.

A platelet substitute, Synthocytes, is being developed for the prevention and treatment of thrombocytopenia. Synthocytes are composed of fibrinogen adsorbed on heat stabilised albumin microcapsules of defined size. The purpose of this study was to perform experiments in vitro to investigate the capacity of Synthocytes to interact with platelets, one of the means through which Synthocytes may contribute to haemostatic plug formation in vivo. Synthocytes were found to interact with platelets as shown by platelet aggregation assays and measurements of [(14)C]5HT release from platelets in whole blood and platelet-rich plasma. Platelet-Synthocytes co-aggregate formation was demonstrated directly using flow cytometry and the presence of activated platelets in these co-aggregates was demonstrated using an antibody to P-selectin. Synthocytes enhanced platelet responsiveness to conventional aggregating agents such as ADP. Indeed, antagonists of the action of ADP on platelets inhibited the direct effects of Synthocytes on platelets in whole blood, as did a GPIIb/IIIa antagonist. Enhancement of annexin V binding was also observed, indicative of increased pro-coagulant activity. Experiments performed with control microcapsules (lacking fibrinogen) confirmed the importance of fibrinogen in the interactions that occurred. The results suggest that fibrinogen on the surface of Synthocytes can interact with GPIIb/IIIa on platelets to induce platelet activation, secretory activity and aggregation, and that ADP contributes to this process. This initial interaction renders platelets more susceptible to the stimulatory effects of other platelet-activating agents. It is considered likely that in the clinical setting of thrombocytopenia any interaction between Synthocytes and residual platelets that are present may contribute to primary haemostasis.

Platelet aggregation and intracellular calcium mobilisation responses are enhanced by cyclosporin A but not by pimecrolimus (SDZ ASM 981).

It has been shown previously that cyclosporin A enhances platelet aggregation responses, particularly to adenosine diphosphate (ADP). In this investigation platelet responses to ADP in the presence of cyclosporin A and pimecrolimus (SDZ ASM 981), a new cell selective inhibitor of inflammatory cytokines, were determined. Measurements were performed in whole blood using a sensitive platelet counting method and in platelet-rich plasma (PRP) using a Biola laser aggregometer. The latter monitors both aggregate formation and aggregate size. In vitro studies were performed using recombinant hirudin as anticoagulant in order that physiological concentrations of divalent cation concentrations were maintained. Studies using both methods confirmed an enhanced aggregation response to ADP in the presence of cyclosporin A. In contrast, aggregation responses were not enhanced in the presence of pimecrolimus, either in PRP or in whole blood where a slight reduction of ADP-induced aggregation was seen at concentrations of pimecrolimus >10(-6) M. The effects of cyclosporin A and pimecrolimus on ADP-induced calcium mobilisation in platelets were determined using a flow cytometric method. A significant increase in intracellular calcium mobilisation was seen in the presence of cyclosporin A but not in the presence of pimecrolimus. Enhanced platelet aggregation responses to ADP observed in the presence of cyclosporin A may be a consequence of enhanced ADP-induced calcium mobilisation.