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.

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.

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.

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.

An inherited bleeding disorder linked to a defective interaction between ADP and its receptor on platelets. Its influence on glycoprotein IIb-IIIa complex function.

Much discussion has concerned the central role of ADP in platelet aggregation. We now describe a patient (M.L.) with an inherited bleeding disorder whose specific feature is that ADP induces a limited and rapidly reversible platelet aggregation even at high doses. Platelet shape change and other hemostatic parameters were unmodified. A receptor defect was indicated, for, while epinephrine normally lowered cAMP levels of PGE1-treated (M.L.) platelets, ADP was without effect. The binding of [3H]2-methylthio-ADP decreased from 836 +/- 126 molecules/platelet for normals to 30 +/- 17 molecules/platelet for the patient. Flow cytometry confirmed that ADP induced a much lower fibrinogen binding to (M.L.) platelets. Nonetheless, the binding in whole blood of activation-dependent monoclonal antibodies showed that some activation of GP IIb-IIIa complexes by ADP was occurring. Platelets of a patient with type I Glanzmann's thrombasthenia bound [3H]2-methylthio-ADP and responded normally to ADP in the presence of PGE1. Electron microscopy showed that ADP-induced aggregates of (M. L.) platelets were composed of loosely bound shape-changed platelets with few contact points. Thus this receptor defect has a direct influence on the capacity of platelets to bind to each other in response to ADP.

The mitogenic effect of H2O2 for vascular smooth muscle cells is mediated by an increase of the affinity of basic fibroblast growth factor for its receptor.

Increased generation of active oxygen species such as hydrogen peroxide (H202) may be important in vascular smooth muscle cell growth associated with atherosclerosis and restenosis. In this work, we showed that H202 was a potent mitogen for growth-arrested cultured human aortic smooth muscle cells (SMC), stimulating an increase in cell number at 10 nM to 100 microM concentration. This effect was inhibited in a dose-dependent manner by catalase, deferoxamine, dimethylthiourea or probucol showing that it was dependent on the oxidative activity of H202. H202-induced SMC proliferation was strongly and specifically inhibited by a neutralizing monoclonal antibody directed against basic fibroblast growth factor (bFGF) but was not due to increased expression of bFGF or the bFGF receptor-1 (FGFR-1) by SMC. H202 strongly increased the affinity of bFGF for its receptor-1 at the surface of the SMC, therefore showing that the mitogenic effect of H202 might occur through a direct effect on the bFGF receptor.

IL-4 inhibits LPS-, IL-1 beta- and TNF alpha-induced expression of tissue factor in endothelial cells and monocytes.

Inflammatory mediators such as endotoxin, interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) dose-dependently increased the expression of tissue factor on the surface of cultured bovine aortic endothelial cells (ABAE), human umbilical vein endothelial cells (HUVEC) and human monocytes. In ABAE, endotoxin-, IL-1 beta- and TNF alpha-induced tissue factor expression was suppressed by interleukin-4 (IL-4) which also neutralized the pyrogenic effect of endotoxin in HUVEC and monocytes. IL-4 did not alter TNF-alpha-induced procoagulant changes in HUVEC and monocytes but strongly protected the monocyte surface against IL-1 beta-induced procoagulant changes.

The NK1 receptor is involved in the neurokinin-induced shape change of rabbit platelets.

Substance P and selective neurokinin receptor agonists have been tested for their ability to induce shape change in rabbit platelets. Substance P and the NK1 receptor agonist Ac [Arg6,Sar9,Met(O2)11]-substance P (6-11) induced shape change (EC50 = 3 and 6 nM, respectively), whereas the selective NK2 agonist [Nle10]-Neurokinin A (4-10) and the selective NK3 agonist [MePhe7]-Neurokinin B did not show any effect. Moreover, the specific NK1 receptor antagonist CP-96,345 selectively and dose-dependently counteracted the effect of substance P or of the NK1 receptor agonist (IC50 = 2 and 0.8 nM, respectively), whereas the selective NK2 receptor antagonist, SR 48968, had no effect. Unlike for serotonin or low doses of ADP, epinephrine did not allow substance P or the NK1 receptor agonist to become a proaggregating substance. These data therefore show that the NK1 receptor is solely involved in the neurokinin-induced shape change of rabbit platelets.

Role of P2Y1 purinoceptor in ADP-induced platelet activation.

ADP acts as an agonist of platelet aggregation via specific receptors which are still to be characterised. Amplification by PCR of a human platelet cDNA library confirmed the presence of mRNA of the P2Y1 receptor in platelets. In order to determine if these P2Y1 receptors were involved in ADP-induced platelet activation, we determined the effects of A3P5PS, an antagonist of the P2Y1 receptor, on the binding of [33P]2-MeS-ADP, a potent analogue of ADP. We found that A3P5PS displaced about 27% of [33P]2-MeS-ADP binding, a receptor population which has been shown to be resistant to treatment with clopidogrel, a selective anti-ADP agent. A3P5PS specifically inhibited 2-MeS-ADP-induced shape change and calcium increase but did not affect adenylyl cyclase down-regulation. 2-MeS-ADP-induced platelet aggregation was also inhibited by A3P5PS but was restored when platelets were further activated by serotonin, a non-aggregating compound, therefore suggesting that P2Y1-mediated stimulation is an absolute prerequisite for ADP to induce platelet aggregation and a key event for platelet activation and aggregation to occur. These results therefore show that ADP-induced aggregation cannot be attributed to activation of P2Y1 alone, but must be attributed to the simultaneous activation of the high affinity receptor (P2Y1) and a low affinity receptor of ADP (still to be discovered), each of them essential, but neither able to trigger aggregation alone.

Nerve growth factor (NGF) exerts its pro-apoptotic effect via the P75NTR receptor in a cell cycle-dependent manner.

Nerve growth factor (NGF), the prototypic member of the neurotrophin family of growth factors, exerts its action via two receptors, P75NTR and TrkA, the expression of which varies at the cell surface of neuroblastoma cells (SH-SY5Y cells) in a cycle phase-specific manner. NGF was pro-apoptotic on growing cells expressing preferentially P75NTR and exhibited a potent anti-apoptotic effect on quiescent cells, when TrkA was prevalent at the cell surface, showing that NGF can have a dual action on SH-SY5Y cells depending on the relative cell surface expression of TrkA and P75NTR. The pro-apoptotic activity of NGF but not its anti-apoptotic activity was abrogated by an antibody against the extracellular domain of P75NTR and in cell isolated from P75NTR knock-out mice indicating that NGF exhibits a proapoptotic activity via P75NTR exclusively. On the other hand, we showed that the anti-apoptotic activity of NGF was specifically mediated by an interaction with TrkA with no contribution of P75NTR, as demonstrated on SK-N-BE cells transfected with TrkA in which NGF was a potent anti-apoptotic compound but did not exhibit any pro-apoptotic activity. These results support the hypothesis that the survival response to NGF depends on its binding to TrkA without any involvement of P75NTR which in turn selectively mediates the pro-apoptotic activity of NGF with no contribution of TrkA and show that, depending on the growth state of the cells, NGF exhibits dual pro- or anti-apoptotic properties via P75NTR and TrkA, respectively.

IL-4 and IL-13 exhibit comparable abilities to reduce pyrogen-induced expression of procoagulant activity in endothelial cells and monocytes.

Endotoxin (LPS), interleukin-1 beta (IL-1) and tumor necrosis factor-alpha (TNF) increased the expression of tissue factor, a membrane-anchored glycoprotein that initiates blood coagulation on the surface of cultured bovine aortic endothelial cells (ABAE) and human monocytes. These compounds simultaneously reduced the amount of thrombomodulin on the endothelial cell surface, further contributing to the procoagulant activity of the endothelium or monocytes. On endothelial cells and monocytes, interleukin-4 (IL-4) and interleukin-13 (IL-13), a newly described lymphokine, both strongly inhibited LPS-induced tissue factor expression, a similar activity also being obtained with regard to the pyrogenic effects of IL-1 or TNF. When measured in parallel, IL-4 and IL-13 counteracted thrombomodulin down-regulation induced by LPS, IL-1 or TNF in endothelial cells. These results therefore show that both IL-4 and IL-13 protect the endothelial and the monocyte surface against inflammatory mediator-induced procoagulant changes.

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.

New orally active non-peptide fibrinogen receptor (GpIIb-IIIa) antagonists: identification of ethyl 3-[N-[4-[4-[amino[(ethoxycarbonyl) imino]methyl]phenyl]-1,3-thiazol-2-yl]-N-[1-[(ethoxycarbonyl)methyl]pip erid -4-yl]amino]propionate (SR 121787) as a potent and long-acting antithrombotic agent.

The platelet fibrinogen receptor GpIIb-IIIa is currently considered a target of choice for drugs used in the prevention and treatment of thrombosis. Ethyl 3-[N-[4-[4-[amino[(ethoxycarbonyl)-imino] methyl]phenyl]-1,3-thiazol-2-yl]-N-[1-[(ethoxycarbonyl)methyl] piperid-4-yl] amino]propionate (6, SR 121787) is a new antiaggregating agent which generates in vivo the corresponding diacid 19d (SR 121566), non-peptide GpIIb-IIIa antagonist. In vitro, 19d inhibited ADP-induced aggregation of human and baboon platelets (IC50 = 46 +/- 11 and 54 +/- 6 nM, respectively), and on human platelets, 19d antagonized the binding of 125I-labeled fibrinogen (IC50 = 19.2 +/- 6.2 nM). Ex vivo, 8 h after an i.v. administration of 19d (100 micrograms/kg, i.v.) to baboons, ADP-induced aggregation was strongly inhibited (more than 90%). At 8 h, the ED50 value was 24 +/- 3.3 micrograms/kg), and even 24 h after the administration of a single dose of 100 micrograms/kg of 19d, platelet aggregation was still significantly inhibited (50 +/- 6% inhibition, P < 0.05). In the same species, the oral administration of 500 micrograms/kg of 6 produced a nearly complete inhibition of aggregation for up to 8 h (ED50 at 8 h was 193 +/- 20 micrograms/kg). After an oral dose of 2 mg/kg of 6, an antiaggregating effect was still observed at 24 h (44 +/- 12% inhibition, P < 0.05). 6 was well tolerated in animals, showing that, on the basis of these studies, it is a suitable candidate for development as an orally active antithrombotic agent.

Effect of SR121566A, a potent GP IIb-IIIa antagonist, on the HIT serum/heparin-induced platelet mediated activation of human endothelial cells.

Heparin-induced thrombocytopenia (HIT) is a common adverse effect of heparin therapy that carries a risk of serious thrombotic events. This condition is caused by platelet aggregation, which is mediated by anti-heparin/platelet factor 4 antibodies. Sera from patients with HIT in the presence of platelets, induced the expression of E-selectin, VCAM, ICAM-1 and tissue factor and the release of IL1beta, IL6, TNFalpha and PAI-1 by human umbilical vein endothelial cells (HUVECs) in vitro and initiated platelet adhesion to activated HUVECs. These effects which occurred in a time-dependent manner were significant in the first 1-2 h of incubation and reached a maximum after 6 to 9 h. The GP IIb-1IIa receptor antagonist SR121566A which has been shown to block platelet aggregation induced by a wide variety of agonists including HIT serum/heparin, reduced in a dose-dependent manner the HIT serum/heparin-induced, platelet mediated expression and release of the above mentioned proteins. The IC50 for inhibition of HIT serum/ heparin-induced platelet dependent HUVEC activation by SR121566A was approximately 10-20 nM. ADP, but not serotonin release, also appeared to be involved as apyrase and ATPgammaS blocked platelet-dependent, HIT serum/heparin-induced cell surface protein expression and cytokine release by HUVECs. Increased platelet adherence to HIT serum/heparin-activated HUVECs was inhibited by SR121566A and, to a lesser extent, by apyrase and ATPgammaS, showing that platelet activation and release was at the origin of the HIT serum/heparin-induced expression of these proteins by HUVECs. Thus, sera from patients with HIT induced the expression of adhesive and coagulation proteins and the release of cytokines by HUVECs through the activation of platelets which occurred in a GP IIb-IIIa-dependent manner, a process that could be selectively blocked by SR121566A.

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.

Ticlopidine and clopidogrel (SR 25990C) selectively neutralize ADP inhibition of PGE1-activated platelet adenylate cyclase in rats and rabbits.

Ticlopidine and its potent analogue, clopidogrel, are powerful inhibitors of ADP-induced platelet aggregation. In order to improve the understanding of this ADP-selectivity, we studied the effect of these compounds on PGE1-stimulated adenylate cyclase and on the inhibition of this enzyme by ADP, epinephrine and thrombin. Neither drug changed the basal cAMP levels nor the kinetics of cAMP accumulation upon PGE1-stimulation in rat or rabbit platelets, which excludes any direct effect on adenylate cyclase or on cyclic nucleotide phosphodiesterase. However, the drop in cAMP levels observed after addition of ADP to PGE1-stimulated control platelets was inhibited in platelets from treated animals. In contrast, the drop in cAMP levels produced by epinephrine was not prevented by either drug in rabbit platelets. In rat platelets, thrombin inhibited the PGE1-induced cAMP elevation but this effects seems to be entirely mediated by the released ADP. Under these conditions, it was not surprising to find that clopidogrel also potently inhibited that effect of thrombin on platelet adenylate cyclase. In conclusion, ticlopidine and clopidogrel selectively neutralize the ADP inhibition of PGE1-activated platelet adenylate cyclase in rats and rabbits.

ADP receptor induced activation of guanine nucleotide binding proteins in rat platelet membranes--an effect selectively blocked by the thienopyridine clopidogrel.

The thienopyridine clopidogrel, a potent analog of ticlopidine, is a powerful inhibitor of ADP induced platelet aggregation and ADP induced inhibition of cyclic AMP accumulation in intact platelets but not of ADP induced shape change. We have recently demonstrated that ADP stimulates the binding of GTP gamma S to GTP binding proteins (G proteins) in human platelet membranes. We now studied the effects of clopidogrel, a specific inhibitor of ADP induced platelet aggregation on the stimulation of GTP gamma S binding to rat platelet membranes by ADP. Using the non hydrolyzable stable analog of ADP, 2MeSADP, we demonstrate that 2MeSADP stimulates the binding of [35S]GTP gamma S to rat platelet membranes in a concentration dependent manner, that this effect is inhibited by the specific ADP receptor antagonist Sp-ATP alpha S and that clopidogrel completely and selectively blocks the stimulation by 2MeSADP of [35S]GTP gamma S binding to platelet membranes of treated rats. We conclude that: i) rat platelet membranes possess an ADP receptor coupled to unidentified G protein(s) and ii) the thienopyridine clopidogrel impairs the interaction of the ADP receptor with its G protein by an irreversible modification the ADP receptor itself or its putative G protein.