Population performance of Tetranychus evansi (Acari: Tetranychidae) on African tomato varieties and wild tomato genotypes.

The tomato red spider mite, Tetranychus evansi Baker & Pritchard, is one of the main pests of the tomato crop in several countries, mainly in Africa, where it can reduce tomato yield by up to 90%. The biotic potential of this mite is high and its control is difficult because of low efficiency of chemicals used and the rapid development of resistance to acaricides. We used the two-sex life table to evaluate the effect of two wild tomato genotypes (PI134417 and PI134418) and five tomato varieties widely grown in Benin (Kekefo, Akikon, TLCV15, Tounvi, and TOML4) on demographic characteristics of T. evansi under laboratory conditions. Tetranychus evansi did not develop on the genotypes PI134417 and PI134418, indicating their resistance to this mite. Developmental time of immature stages and female longevity were significantly higher on TLCV15 and Kekefo. Fecundity, net reproductive rate (R0), intrinsic rate of increase (r), and finite rate of increase (λ) of T. evansi on the African varieties were not statistically different among varieties. Generation time (T) was shorter on TOML4 than on TLCV15 and Tounvi. Thus, efforts should be made to prospect varieties with resistance characteristics or to develop other control means, to reduce the use of pesticides to control T. evansi in Africa.

SAR216471, an alternative to the use of currently available P2Y12 receptor inhibitors?

P2Y12 antagonism is a key therapeutic strategy in the management and prevention of arterial thrombosis. The objective of this study was to characterize the pharmacodynamic (PD) and pharmacokinetic (PK) properties of SAR216471, a novel P2Y12 receptor antagonist. SAR216471 blocks the binding of 2MeSADP to P2Y12 receptors in vitro (IC50=17 nM). This inhibition was shown to be reversible. It potently antagonized ADP-induced platelet aggregation in human and rat platelet-rich plasma (IC50=108 and 62 nM, respectively). It also inhibited platelet aggregation when blood was exposed to collagen or thromboxane A2. Its high selectivity was demonstrated against a large panel of receptors, enzymes, and ion channels. Despite its moderate bioavailability in rats, oral administration of SAR216471 resulted in a fast, potent, and sustained inhibition of platelet aggregation where the extent and duration of platelet inhibition were directly proportional to its circulating plasma levels. Pre-clinical study of SAR216471 in a rat shunt thrombosis model demonstrated a dose-dependent antithrombotic activity after oral administration (ED50=6.7 mg/kg). By comparison, ED50 values for clopidogrel, prasugrel and ticagrelor were 6.3, 0.35 and 2.6 mg/kg, respectively. Finally, the anti-hemostatic effect of SAR216471 and its competitors was investigated in a rat tail bleeding model, revealing a favorable safety profile of SAR216471. Together, these findings have established a reliable antiplatelet profile of SAR216471, and support its potential use in clinical practice as an alternative to currently available P2Y12 receptor antagonists.

In vitro maturation of oocytes from Santa Ines ewes subjected to consecutive sessions of follicular aspiration by laparoscopy.

The success of embryo production in vitro depends upon the use of an efficient oocyte retrieval technique, and the best results have been obtained by laparoscopic aspiration. The aim of this study was to evaluate the effect of consecutive sessions of follicular aspiration on the quantity, quality and in vitro maturation competence of oocytes obtained from ewes subjected to hormonal stimulation. Six Santa Ines ewes underwent nine sessions of follicular aspiration by laparoscopy with a 7-day interval between sessions, totalling 56 aspirations. After 24 h of culture, oocytes were stained and classified according to the stage of nuclear and cytoplasmic maturation. Oocyte retrieval rate was 61.4 ± 2%, resulting in a total of 249 oocytes. No significant variation was observed between sessions (p > 0.05). The average number of oocytes retrieved from each ewe was 6.4 ± 2 per session and 42 ± 4 in total. No significant difference was observed between the frequencies of the different stages of nuclear maturation: 32.72% mature, 40.74% immature and 26.54% degenerated/indeterminate oocytes; however, a significant difference was observed between the frequencies of the different stages of cytoplasmic maturation: 10.7% mature, 73.25% immature and 16.05% degenerated/indeterminate oocytes. No significant difference was observed in nuclear or cytoplasmic maturation between the weeks of procedure. We conclude that after nine consecutive sessions of follicular aspiration, the quantity and quality of retrieved oocytes remained unchanged as well as the levels of nuclear and cytoplasmic maturation obtained, demonstrating the viability of this technique for repetitive follicular aspirations on the same donor.

In vitro survival of follicles collected from domestic cats' ovaries at different stages of oestrous cycle and cultured with IGF-1.

Optimal conditions for in vitro culture of feline ovarian follicles have not yet been defined. Follicular development is regulated by intraovarian growth factors, as insulin-like growth factor (IGF-1), and during the different stages of the oestrous cycle, follicles are exposed to specific hormonal environments. The aim of this study was to investigate the effect of IGF-1 on in vitro growth and granulosa cell (GC) viability of preantral follicles collected from domestic cats at follicular and luteal phases of the oestrous cycle. Oestrus and ovulation were induced in 12 cats. A total of 39 and 32 follicles collected at the follicular and luteal phases, respectively, were individually cultured in vitro for 6 days in minimum essential medium media supplemented with or without IGF-1 (100 ng/ml). Follicles collected during the follicular phase and cultured without IGF-1 displayed a significant increase in size and higher GC viability (46.5 ± 22.1 µm, 66.7%, respectively) than that of follicles collected at the luteal phase and cultured without IGF-1 (26.7 ± 14.4 µm, 50%, respectively; p < 0.05). In contrast, when IGF-1 was added to the culture medium, no differences were observed in size or GC viability between follicles collected at the two phases of the cycle. Nonetheless, follicles collected at the luteal phase and cultured with IGF-1 had a significant increase in their diameter and GC viability (31.9 ± 15.9 µm, 63.6%, respectively) than that cultured without IGF-1 (26.7 ± 14.4 µm, 50%, respectively; p < 0.05). These data suggest that in vitro growth and GC survival of feline preantral follicles are affected by the oestrous cycle phase, and the IGF-1 exerts a positive effect on follicles collected at the luteal phase.

TNF-alpha induced PMN apoptosis in whole human blood: protective effect of SSR180575, a potent and selective peripheral benzodiazepine ligand.

The peripheral benzodiazepine receptor (PBR) has been shown to play a key role in the regulation of the mitochondrial process leading to apoptosis. Despite much controversy in the literature on this subject, PBR synthetic ligands (and specifically agonists such as Ro5-4864 and SSR180575) are described as presenting potent anti-apoptotic effect against oxidative stress, TNFalpha- and tamoxifen-induced apoptosis when the PBR ligand is administrated at a low dose, close to the affinity range of the ligand to its receptor. Such anti-apoptotic activity has already been correlated with a protective effect of PBR ligands against ischemia-reperfusion induced tissue dysfunction. Previously, we had shown that SSR180575 is a specific and high affinity PBR ligand of potential interest in pathological cardiovascular, renal and neurodegenerative indications. Beyond its expression in steroid-producing tissues, heart, liver and kidney, the PBR is also known to be highly expressed in blood cells. In this work, we demonstrate by flow cytometry experiments, that SSR180575, at low concentrations, is able to protect polymorphonuclear leukocytes (PMNs) against TNFalpha-induced apoptosis in whole blood. Thus, in a new context, SSR180575 again shows potent anti-apoptotic properties. Moreover, TNFalpha- induced PMN apoptosis appears to be a good surrogate marker for determining SSR180575 blood availability and activity in treated patients.

Apelin/APJ signaling system: a potential link between adipose tissue and endothelial angiogenic processes.

Adipose tissue is an active endocrine organ that produces a variety of secretory factors involved in the initiation of angiogenic processes. The bioactive peptide apelin is the endogenous ligand of the G protein-coupled receptor, APJ. Here we investigated the potential role of apelin and its receptor, APJ, in the angiogenic responses of human endothelial cells and the development of a functional vascular network in a model of adipose tissue development in mice. Treatment of human umbilical vein endothelial cells with apelin dose-dependently increased angiogenic responses, including endothelial cell migration, proliferation, and Matrigel(R) capillary tubelike structure formation. These endothelial effects of apelin were due to activation of APJ, because siRNA directed against APJ, which led to long-lasting down-regulation of APJ mRNA, abolished cell migration induced by apelin in contrast to control nonsilencing siRNA. Hypoxia up-regulated the expression of apelin in 3T3F442A adipocytes, and we therefore determined whether apelin could play a role in adipose tissue angiogenesis in vivo. Epididymal white adipose tissue (EWAT) transplantation was performed as a model of adipose tissue angiogenesis. Transplantation led to increased apelin mRNA levels 2 and 5 days after transplantation associated with tissue hypoxia, as evidenced by hydroxyprobe staining on tissue sections. Graft revascularization evolved in parallel, as the first functional vessels in EWAT grafts were observed 2 days after transplantation and a strong angiogenic response was apparent on day 14. This was confirmed by determination of graft hemoglobin levels, which are indicative of functional vascularization and were strongly increased 5 and 14 days after transplantation. The role of apelin in the graft neovascularization was then assessed by local delivery of stable complex apelin-targeting siRNA leading to dramatically reduced apelin mRNA levels and vascularization (quantified by hemogloblin content) in grafted EWAT on day 5 when compared with control siRNA. Taken together, our data provide the first evidence that apelin/APJ signaling pathways play a critical role in the development of the functional vascular network in adipose tissue. In addition, we have shown that adipocyte-derived apelin can be up-regulated by hypoxia. These findings provide novel insights into the complex relationship between adipose tissue and endothelial vascular function and may lead to new therapeutic strategies to modulate angiogenesis.

Reversible biotinylated oligosaccharides: a new approach for a better management of anticoagulant therapy.

OBJECTIVE: In order to obtain a neutralizable antithrombotic, a chimeric molecule (SSR126517E) containing the sequence of a long-lasting antithrombin (AT)-dependent anti-factor Xa pentasaccharide, idraparinux, linked to a biotin molecule was synthesized and tested for anticoagulant and antithrombotic activity. METHODS: SSR126517E was tested in several models in vitro and in vivo for its pharmacological properties as well as its ability to be neutralized by avidin. RESULTS: SSR126517E displayed exactly the same properties as idraparinux. In vitro, SSR126517E had a very high affinity for AT (K(d) < 1 nm) and showed a potent anti-FXa effect and inhibition of thrombin generation with IC(50) values similar to those of idraparinux. Ex vivo, after intravenous administration to rats, SSR126517E produced a potent and long-lasting anti-FXa effect comparable to that obtained with idraparinux; as with idraparinux, the subcutaneous bioavailability was 100%. In vivo, SSR126517E was a potent antithrombotic in rat and mouse venous and arterial thrombosis models. Direct comparison in rats showed that SSR126517E was as active as idraparinux, when administered at the same molar dose. Furthermore, injection of avidin triggered the immediate elimination of SSR126517E from the bloodstream, resulting in complete neutralization of the antithrombotic activity of SSR126517E. CONCLUSIONS: These results show for the first time that coupling an oligosaccharide with biotin has no effect on the former's pharmacokinetic and pharmacologic properties and renders neutralization easy by injection of avidin.

Gi-dependent and -independent mechanisms downstream of the P2Y12 ADP-receptor.

The P2Y12 ADP receptor is one of the major regulators of platelet activation and the target of antithrombotic thienopyridines (ticlopidine and clopidogrel). It has been recently cloned but the signaling pathways triggered by this receptor are still poorly documented. Here, we show that stimulation of the human P2Y12 receptor stably expressed in Chinese hamster ovary cells activates two major intracellular signaling mechanisms leading either to cell proliferation or to actin cytoskeleton reorganization. Both effects were blocked by the active metabolite of clopidogrel, a specific antagonist of P2Y12. The P2Y12-mediated stimulation of proliferation required the pertussis toxin-sensitive activation of PI3-kinase/Akt upstream of MAP-kinases. A partial contribution of a transactivation mechanism, through the tyrosine kinase receptor platelet-derived growth factor (PDGF)-R-beta, was also observed. Conversely, the P2Y12-mediated reorganization of the actin cytoskeleton was Gi-independent, requiring activation of RhoA and Rho-kinase. Our results provide new insights into the molecular basis of P2Y12-mediated intracellular signaling. These data may prove to be useful for a better understanding of the physiological role of P2Y12, particularly in platelets and glial cells which express this important therapeutic target.

Comparative effects of two synthetic oligosaccharides on platelet activation induced by plasma from HIT patients.

Heparin-induced thrombocytopenia (HIT) is a serious secondary event encountered in the clinical use of heparin. HIT results from the consumption of platelets that are immunologically activated by antibodies directed against complexes formed by platelet factor 4 (PF4) and sulfated polysaccharides that activate platelet aggregation, leading to paradoxical, life-threatening thrombosis. There is strong evidence that the ability of heparin and related compounds to induce HIT is closely linked to the structure of the polysaccharide, and particularly to its negative charge and to the length of the molecule. To test this hypothesis, we synthesized two sulfated oligosaccharides: SanOrg123781, a 16-mer, presenting two terminal charged domains separated by a 7-mer neutral linker, and SR121903, a highly sulfated 17-mer. Both of them displayed strong anti-factor (F) Xa and anti-FIIa activities but their affinities for PF4 were markedly different. SR121903 displaced PF4-bound heparin, whereas SanOrg123781 did not, underlining the importance of the charge of the molecule for the interaction with PF4. Platelet studies, in the presence of HIT serum, showed that SR121903 induced the secretion of platelet-dense granules (measured by the release of serotonin) whereas SanOrg123781 did not, a result in accordance with an absence of affinity of this molecule for PF4. These results were confirmed by measurements of platelet activation by flow cytometry (measured by annexin V binding, CD62 detection and activation of the GpIIb-IIIa complexes). In conclusion, we have demonstrated the importance of the charge of the polysaccharides in the HIT-induced platelet reactions measured by diverse methods, of which some are described for this purpose for the first time.

P2y(12), a new platelet ADP receptor, target of clopidogrel.

The binding characteristics of (33)P-2MeS-ADP, a stable analogue of ADP, were determined on CHO cells transfected with the human P2Y(12) receptor, a novel purinergic receptor. These transfected CHO cells displayed a strong affinity for (33)P-2MeS-ADP, the binding characteristics of which corresponded in all points to those observed on platelets. In particular, this receptor recognised purines with the following order of potency: 2MeS-ADP = 2MeS-ATP > ADP = ATPgammaS = ATP >> UTP, a binding profile which is similar to that obtained in platelets. The binding of (33)P-2MeS-ADP was antagonised by pCMPS but not by MRS2179 and FSBA, antagonists of P2Y(1) and aggregin, respectively. Moreover, the binding of (33)P-2MeS-ADP to these cells was strongly and irreversibly inhibited by the active metabolite of clopidogrel with a potency which was consistent with that observed for this compound on platelets. Like in platelets, 2MeS-ADP induced adenylyl cyclase down-regulation in these P2Y(12) transfected CHO cells, an effect which was absent in the corresponding non-transfected cells. As already shown in platelets, the active metabolite of clopidogrel antagonised 2MeS-ADP-induced inhibition of adenylyl cyclase on transfected cells. Our results confirm that P2Y(12) is the previously called "platelet P2t(AC)" receptor and show that this receptor is antagonised by the active metabolite of clopidogrel.

Studies on the binding of 3H-SR121566, an inhibitor of Gp IIb-IIIa activation.

SR121566 is a new synthetic agent which inhibits the binding of fibrinogen to activated platelets, and platelet aggregation. 3H-SR121566 bound with nanomolar affinity (KD ranging from 45 to 72 nM) to Gp IIb-IIIa expressing cells only. On activated human platelets, this ligand allowed the detection of a maximal number of 100-140,000 binding sites. The binding of SR121566 to platelets, was displaced by several agents including RGD-containing peptides and synthetic RGD mimetics, but not by ReoPro, a humanised monoclonal antibody which inhibits the binding of fibrinogen to the Gp IIb-IIIa complex. Neither the fibrinogen dodecapeptide nor fibrinogen itself were able to compete with SR121566 whether platelets were activated or not. Flow cytometry studies indicated that SR121566 which did not activate Gp IIb-IIIa by itself, dose-dependently prevented the detection of activation-induced binding sites on TRAP-stimulated platelets in the presence or absence of exogenous fibrinogen, indicating a direct effect on the activation state of the Gp IIb-IIIa complex. Moreover, SR121566 was able to reverse the activation of Gp IIb-IIIa and to displace the binding of fibrinogen when added up to 5 min after TRAP stimulation of platelets. When added at later times (15 to 30 min), SR121566 failed to displace fibrinogen binding, even if SR121566 binding sites were still accessible and the Gp IIb-IIIa complex not activated. In conclusion, our study is in accordance with the finding that fibrinogen is recognised by the activated Gp IIb-IIIa complex through the dodecapeptide sequence present on its gamma chain, and that this interaction is inhibited by SR121566 by preventing and reversing the activated conformation of Gp IIb-IIIa and not by direct competition with fibrinogen.

SR123781A, a synthetic heparin mimetic.

SR123781A, a synthetic hexadecasaccharide comprising an antithrombin (AT) binding domain, a thrombin binding domain, and a neutral methylated hexasaccharide sequence, was obtained from glucose through a convergent synthesis. SR123781A showed high affinity for human AT (Kd = 58 +/- 22 nM) and was a potent catalyst of its inhibitory effect with regard to factor Xa (IC50) = 77 +/- 5 ng/ml - 297 +/- 13 U/mg) and thrombin (IC50 = 4.0 +/- 0.5 ng/ml - 150 +/- 30 U/mg). SR123781A which acted exclusively via AT (no effect via heparin cofactor II at a concentration of 6 microg/ml) inhibited thrombin generation occurring via both the extrinsic and intrinsic pathways in vitro in human plasma. SR123781A did not compete for 3H-heparin binding to PF4 and did not activate platelets in the presence of plasma from patients with heparin-induced thrombocytopenia. After intravenous or subcutaneous administration to rats, rabbits or baboons, SR123781A displayed prolonged anti-factor Xa and anti-factor IIa activity ex vivo. After intravenous injection to baboons, decreases of the anti-factor Xa and anti-thrombin activities were parallel and disappeared with the same pharmacodynamics. Intravenous administrations of SR123781A strongly inhibited thrombus formation in an experimental model of thromboplastin-induced venous thrombosis in rats with an ED50 value of 18 +/- 0.1 microg/kg (vs 77 +/- 3 microg/kg for heparin). SR123781A inhibited arterial thrombus formation induced on a silk thread in an arterio-venous shunt and in the vena cava (ED50 values of 225 +/- 10 and 27 +/- 8 microg/kg, respectively). Compared to standard and low molecular weight heparin and to presently used drugs, SR123781A exhibited a highly favourable antithrombotic/bleeding ratio therefore showing that it might be considered as a promising compound in the treatment and prevention of various thrombotic diseases.

Antiplatelet and antithrombotic activity of SL65.0472, a mixed 5-HT1B/5-HT2A receptor antagonist.

The antiplatelet and antithrombotic activity of SL65.0472 (7-fluoro-2-oxo-4-[2-[4-(thieno [3,2-c]pyrin-4-yl) piperazin-1-yl]ethyl]-1,2-di-hydroquinoline-acetamide), a mixed 5-HT1B/5-HT2A receptor antagonist was investigated on 5HT-induced human platelet activation in vitro, and in rat, rabbit and canine platelet dependent thrombosis models. SL65.0472 inhibited 5-HT-induced platelet shape change in the presence of EDTA (IC50 values = 35, 69 and 225 nM in rabbit, rat and human platelet rich plasma (PRP)), and also inhibited aggregation induced in human PRP by 3-5 microM 5-HT + threshold concentrations of ADP (0.5-1 microM) or collagen (0.3 microg/ml) with mean IC50 values of 49 +/- 13 and 48 +/- 6 nM respectively. SL65.0472 inhibited thrombus formation when given both intravenously 5 min and orally 2 h prior to assembly of an arterio-venous (A-V) shunt in rats as from 0.1 and 0.3 mg/kg respectively. It was active in a rabbit A-V shunt model with significant decreases in thrombus weight as from 0.1 mg/kg i. v. and at 10 mg/kg p.o. The delay to occlusion in an electric current-induced rabbit femoral artery thrombosis model was increased by 251% (p <0.05) after 20 mg/kg p.o. SL65.0472 (30 microg/kg i.v.) virtually abolished coronary cyclic flow variations (7.2 +/- 1.0/h to 0.6 +/- 0.6/h, p <0.05) and increased minimum coronary blood flow (1.2 +/- 0.8 ml/min to 31.8 +/- 8.4 ml/min, p <0.05) in a coronary artery thrombosis model in the anaesthetised dog. Finally, SL65.0472 significantly increased the amount of blood lost after rat tail transection at 3 mg/kg p.o. Thus the anti-5-HT2A component of SL65.0472 is reflected by its ability to inhibit 5-HT-induced platelet activation, and platelet-rich thrombus formation.

Cotranscription and intergenic splicing of human P2Y11 and SSF1 genes.

The P2Y(11) receptor is an ATP receptor positively coupled to the cAMP and phosphoinositide pathways. Ssf1 is a Saccharomyces cerevisiae nuclear protein, which plays an important role in mating. The gene encoding the human orthologue of SSF1 is adjacent to the P2Y(11) gene on chromosome 19. During the screening of placenta cDNA libraries, we isolated a chimeric clone resulting from the intergenic splicing between the P2Y(11) and SSF1 genes. The fusion protein was stably expressed in CHO-K1 cells where it generated a cAMP response to ATP qualitatively indistinguishable from that of the P2Y(11) receptor. According to both Western blotting and cAMP response, the expression of the fusion protein in the transfected cells was clearly lower than that of the P2Y(11) receptor. Both P2Y(11) and SSF1 probes detected a 5.6-kb messenger RNA with a similar pattern of intensity in each of 11 human tissues. The ubiquitous presence of chimeric transcripts and their up-regulation during granulocytic differentiation indicate that the transgenic splicing between the P2Y(11) and the SSF1 genes is a common and regulated phenomenon. There are very few examples of intergenic splicing in mammalian cells, and this is the first case involving a G-protein-coupled receptor.

Deficiency or inhibition of Gas6 causes platelet dysfunction and protects mice against thrombosis.

The growth arrest-specific gene 6 product (Gas6) is a secreted protein related to the anticoagulant protein S but its role in hemostasis is unknown. Here we show that inactivation of the Gas6 gene prevented venous and arterial thrombosis in mice, and protected against fatal collagen/epinephrine-induced thrombo embolism. Gas6-/- mice did not, however, suffer spontaneous bleeding and had normal bleeding after tail clipping. In addition, we found that Gas6 antibodies inhibited platelet aggregation in vitro and protected mice against fatal thrombo embolism without causing bleeding in vivo. Gas6 amplified platelet aggregation and secretion in response to known agonists. Platelet dysfunction in Gas6-/- mice resembled that of patients with platelet signaling transduction defects. Thus, Gas6 is a platelet-response amplifier that plays a significant role in thrombosis. These findings warrant further evaluation of the possible therapeutic use of Gas6 inhibition for prevention of thrombosis.

Detection of bradykinin B1 receptors in rat aortic smooth muscle cells.

The tritiated bradykinin B1 receptor agonist [3H]des-Arg(10)-kallidin bound to a single class of high-affinity binding sites (K(d) = 0.5 +/- 0.16 nM; B(max) = 15,000 +/- 8,000 sites/cell) on cultured rat aortic smooth muscle cells. [3H]Des-Arg(10)-kallidin association and dissociation kinetics were monoexponential, making it possible to determine the association and dissociation rate constants (k(+1) = 1.5 10(5) M(-1) sec(-1); k(-1) = 4.2 10(-5) sec(-1)). [3H]Des-Arg(10)-kallidin binding was inhibited by specific ligands of bradykinin B1 and B2 receptors with a rank order of potency consistent with that known for bradykinin B1 receptors in other species (des-Arg(9)-[Leu(8)]bradykinin = des-Arg(10)-kallidin = des-Arg(9)-bradykinin = des-Arg(10)-[Leu(9)]kallidin > des-Arg(10)-HOE-140 >> bradykinin >> HOE-140). Bradykinin B1 receptor mRNA was also detected in these cells. Des-Arg(10)-kallidin increased cytosolic free Ca2+ levels, phosphoinositide turnover, and arachidonic acid release at nanomolar concentrations (respective EC(50) values: 16 +/- 2, 4 +/- 2.7, 6 +/- 2 nM). These functional effects of des-Arg(10)-kallidin could be blocked by the bradykinin B1 receptor antagonist des-Arg(9)-[Leu(8)]bradykinin, but were not sensitive to bradykinin B2 receptor antagonists. These results therefore show that rat aortic smooth muscle cells in culture express functional bradykinin B1 receptors.

Identification and biological activity of the active metabolite of clopidogrel.

Like ticlopidine, the ADP receptor antagonist clopidogrel is inactive in vitro and must be administered i.v. or orally to exhibit antiaggregatory and antithrombotic activities. We have previously shown that hepatic metabolism is necessary for activity. This study demonstrates that an active metabolite can be generated from human liver microsomes incubated with clopidogrel. Using several analytical methodologies (LC/MS, NMR, chiral supercritical fluid chromatography), we have identified its structure. In vitro, this highly unstable compound, different from that formed from ticlopidine, exhibited all the biological activities of clopidogrel observed ex vivo: Irreversible inhibition of the binding of 33P-2MeS-ADP to washed human platelets (IC50) = 0.53 microM), selective inhibition of ADP-induced platelet aggregation (IC)50 = 1.8 microM) and ADP-induced adenylyl cyclase down-regulation. The irreversible modification of the ADP-receptor site which is responsible for the biological activity could be explained by the formation of a disulfide bridge between the reactive thiol group of the active metabolite and a cysteine residue of the platelet ADP receptor.

FcgammaRIIA requires a Gi-dependent pathway for an efficient stimulation of phosphoinositide 3-kinase, calcium mobilization, and platelet aggregation.

FcgammaRIIA, the only Fcgamma receptor present in platelets, is involved in heparin-associated thrombocytopenia (HIT). Recently, adenosine diphosphate (ADP) has been shown to play a major role in platelet activation and aggregation induced by FcgammaRIIA cross-linking or by sera from HIT patients. Herein, we investigated the mechanism of action of ADP as a cofactor in FcgammaRIIA-dependent platelet activation, which is classically known to involve tyrosine kinases. We first got pharmacologic evidence that the ADP receptor coupled to Gi was required for HIT sera or FcgammaRIIA clustering-induced platelet secretion and aggregation. Interestingly, the signaling from this ADP receptor could be replaced by triggering another Gi-coupled receptor, the alpha(2A)-adrenergic receptor. ADP scavengers did not significantly affect the tyrosine phosphorylation cascade initiated by FcgammaRIIA cross-linking. Conversely, the Gi-dependent signaling pathway, initiated either by ADP or epinephrine, was required for FcgammaRIIA-mediated phospholipase C activation and calcium mobilization. Indeed, concomitant signaling from Gi and FcgammaRIIA itself was necessary for an efficient synthesis of phosphatidylinositol 3,4,5-trisphosphate, a second messenger playing a critical role in the process of phospholipase Cgamma2 activation. Altogether, our data demonstrate that converging signaling pathways from Gi and tyrosine kinases are required for platelet secretion and aggregation induced by FcgammaRIIA.

Effect of aspirin on platelet desaggregation induced by SR121566, a potent GP-IIb/IIIa antagonist.

Since results of recent clinical trial, performed in patients suffering from acute coronary syndromes, suggested that aspirin improved the efficacy of an anti-GP-IIb/IIIa therapy, this work was set to study the mechanism underlying such a mechanism of aspirin. SR121566 (3 mg/kg, i.v.), a new selective antagonist of the GP-IIb/IIIa complex inhibited thrombus formation which occurred following electrical stimulation of the carotid artery of rabbits. On rabbits pre-treated with aspirin (10 mg/kg, i.v.), a bolus injection of SR121566 produced a strong reversal of thrombosis compared with the effect obtained on rabbits treated with SR121566 alone. In vitro, the addition of SR121566 to human platelets during the ADP-induced aggregation process resulted in a reversal of the aggregation which was nearly complete when it was added 30 s after the induction of aggregation, but progressively decreased when SR121566 was added at later times. ADP-induced platelet aggregation in the presence of aspirin (1 mM) was comparable to that obtained in control platelet-rich plasma, but the addition of SR121566 at all time points of the aggregation process was followed by a complete reversal of the aggregation even at later time points (> 120 s). Histological examination of the platelet aggregates showed a clear-cut difference in the platelet content between control and aspirin-treated platelets which contained much more dense granules. Inhibition of the release of thrombospondin by aspirin may account for a significant part of its pro-desaggregating activity as demonstrated by the effect of the tetrapeptide VTCG, an inhibitor of the binding of thrombospondin to its receptor, which strongly potentiated the activity of SR121566 as a desaggregating agent. Therefore, our study shows that aspirin pretreatment promotes a 'thrombolytic' activity of GP-IIb/IIIa inhibitors and, in particular, of SR121566, a potent and selective member of this new class of compounds. This effect is mainly due to an inhibition of the release of the platelet granule content and in particular to thrombospondin. This observation may contribute to a greater efficacy of GP-IIb/IIIa inhibitors for the treatment of thrombosis in atherosclerotic patients.