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Background Although considered as an anti-inflammatory cytokine, interleukin-4 (IL-4) has been shown to be pro-atherogenic in mice models of atherosclerosis. Objectives In order to elucidate this paradox, we have investigated the effects of IL-4 on characteristic atherogenic parameters in human umbilical vein endothelial cells (HUVECs): production of reactive oxygen species, expression of monocyte chemoattractant protein-1 (MCP-1) and nitric oxide (NO) bioavailability. Results Incubation of HUVECs with IL-4 resulted in an increased production of reactive oxygen species and extracellular O 2 − measured using fluorogenic probes and Cytochrome c that was inhibited by superoxide dismutase or gp91ds- tat , a selective NADPH oxidase inhibitor. The latter also inhibited IL-4 induced over-expression of MCP-1 mRNA measured by classical and real time RT-PCR. Incubation of HUVECs with IL-4 reduced thrombin-induced NO release, detected by electrochemistry, an effect which was reversed by incubation with superoxide dismutase. Both production of reactive oxygen species and MCP-1 mRNA over-expression induced by IL-4 were fully inhibited by selective inhibitors of phosphatidyl inositol 3-kinase. Conclusion The data demonstrate that IL-4 up-regulates the expression of MCP-1 and decreases NO bioavailability through activation of NADPH oxidase in endothelial cells. These results are in favor of a pro-inflammatory and pro-atherogenic effect of IL-4 in vascular tissues.

The aim of this review is (i) to classify the different monoclonal antibodies against platelet glycoproteins according to their properties and (ii) to take stock of their many diagnostic and therapeutic uses. Most of these antibodies recognize antigens on resting platelets without inducing activation, sometimes however they inhibit platelet function. Some of these antibodies, especially those against specific antigens (GPIIb/IIIa, CD9, CD36), have the capacity to activate platelets in vitro, either by direct binding of antibodies on the antigen or through the Fc domain binding to its platelet receptor Fc gamma RII. Other antibodies are directed against activation-dependent antigens that are expressed as a result of (i) a modification of a glycoprotein structure during platelet activation (as for GPIIb/IIIa), (ii) platelet release of granular antigens (GMP140, CD63, granulophysin...) or (iii) binding of soluble antigens on the activated platelet surface. Monoclonal antibodies find practical applications for both in vitro and in vivo diagnosis of bleeding or thrombotic pathology with some of them, notably anti-GPIIb/IIIa, having a promising future for antithrombotic therapy.

Thrombin is a serine protease able to evoke biological responses from a variety of cells, including platelets, endothelial cells, fibroblasts and smooth muscle cells. The structure of the thrombin receptor present in the human megakaryoblastic cell line and in hamster fibroblasts has recently been deduced by expression in the Xenopus laevis oocyte. The cloned receptor is a new member of the seven transmembrane domain receptor family that interacts with G proteins. A large amino-terminal extracellular extension has a cleavage site for thrombin (Leu Asp Pro Arg/Ser Phe Leu Leu,/representing the cleavage site). Thrombin cleaves at this site, unmasking a new amino terminus, that functions like a ligand, binding to an as yet undefined site and eliciting receptor activation. Peptides similar to a new amino terminus created after cleavage are able to mimic thrombin cellular effects. These agonist peptides are used to analyse the role of the cloned receptor in the thrombin-specific response.

Synthetic peptides (TRAP or Thrombin Receptor Activating Peptide) corresponding to at least the first five aminoacids of the new N-terminal tail generated after thrombin proteolysis of its receptor are effective to mimic thrombin. We have studied two different TRAPs (SFLLR, and SFLLRN) in their effectiveness to induce the different platelet responses in comparison with thrombin. Using Indo-l/AM-labelled platelets, the maximum rise in cytoplasmic ionized calcium was lower with TRAPs than with thrombin. At threshold concentrations allowing maximal aggregation (50 microM SFLLR, 5 microM SFLLRN and 1 nM thrombin) the TRAPs-induced release reaction was about the same level as with thrombin, except when external calcium was removed by addition of 1 mM EDTA. In these conditions, the dense granule release induced by TRAPs was reduced by over 60%, that of lysosome release by 75%, compared to only 15% of reduction in the presence of thrombin. Thus calcium influx was more important for TRAPs-induced release than for thrombin-induced release. At strong concentrations giving maximal aggregation and release in the absence of secondary mediators (by pretreatment with ADP scavengers plus aspirin), SFLLRN mobilized less calcium, with a fast return towards the basal level and induced smaller lysosome release than did thrombin. The results further demonstrate the essential role of external calcium in triggering sustained and full platelet responses, and emphasize the major difference between TRAP and thrombin in mobilizing [Ca2+]i. Thus, apart from the proteolysis of the seven transmembrane receptor, another thrombin binding site or thrombin receptor interaction is required to obtain full and complete responses.

The aim of this study was to determine if there is a correlation between the activity of a MoAb as an agonist and its ability to bind to the Fc platelet receptor, Fc gamma RIIa. A polymorphism at amino acid 131 [arginine (Arg) or histidine (His)] of Fc gamma RIIa was first shown to be determinant for MoAb-IgG1 binding on monocytes. To clarify the role of this polymorphism in platelet activation by MoAb-IgG1 we (i) established the Fc gamma RIIa polymorphism at the gene level by adapting the denaturating gradient gel electrophoresis method, (ii) analyzed the binding affinity of the MoAbs to Fc gamma RIIa on platelets from homozygous Arg, homozygous His, and heterozygous Arg/His donors, and (iii) characterized the different reactivities of platelets according to the Fc gamma RIIA polymorphism. Among 167 caucasian donors we found 46% heterozygous Arg/His, 36% homozygous His and 18% homozygous Arg. ALB6, and anti CD9, P256 an anti GPIIb-IIIa, and AP3 an anti-GPIIIa were chosen according to their ability (ALB6, P256) or not (AP3) to activate platelets. These 3 MoAbs-IgG1 bind to Fc gamma RIIa with a stronger affinity for the Arg-form of Fc gamma RIIa, a result which was confirmed with the use of diverse MoAbs directed against various antigens. The different abilities of MoAbs to bind to the two Fc gamma RIIa forms were well correlated to the different platelet responses induced by ALB6 and P256. However, low concentrations of ALB6, which allow full activation of platelets from homozygous Arg donors, as did P256, did not induce any activation of platelets from homozygous His donors, whereas P256 is able to induce a low aggregation. The results further define the respective roles of the antigen and the Fc receptor, depending on the MoAb, and the role of the Fc gamma RIIa polymorphism in platelet activation induced by MoAbs. In addition, the results obtained with MoAbs unable to induce platelet activation provided evidence that the binding of a MoAb on Fc gamma RIIa does not predict its ability to activate platelets.

In this study, the clinical history of two patients with the gray platelet syndrome, a rare congenital disorder associating thrombopathia and myelofibrosis is recalled. Complementary studies on platelets and megakaryocytes were performed, mainly with an immunocytochemical approach. In gray platelets, a general decrease of alpha-granule proteins, including PF4, beta tg and PDGF was observed. The decrease in platelet mitogenic activity (PDGF) was confirmed by biological and radio-immunological measurements. An abnormally high level of these compounds was also found in the plasma. In megakaryocytes cultured from the bone marrow of these patients, alpha-granule proteins were normally expressed in early maturation stages, whereas they were found to be absent in the mature megakaryocytes. An alpha-granule membrane glycoprotein, GMP 140 has been studied in resting and thrombin stimulated gray platelets and was found to be normally expressed at the surface of stimulated platelets. GMP140 was studied in resting platelets by immunoelectron microscopy and found to be present in vacuole probably corresponding to empty granules. This observation allows to conclude that alpha-granule membrane is formed in the gray platelet syndrome, but that there is a storage defect of alpha-granule soluble proteins, possibly due to an abnormal targetting of these proteins to the alpha-granule. Synthesis and subsequent release of these proteins, namely of the mitogenic factors, which can induce myelofibrosis and lung fibrosis by abnormal fibroblast stimulation, is discussed.

Platelet prothrombinase activity and aggregation were studied in parallel in the same platelet suspensions with the aim of determining the relationship between these two responses. Stimulation of platelets with thrombin (0.12 U/ml) plus collagen (20 mu g/ml) led to maximum thrombin generation when the aggregation intensity was still only 30%. Since the rate of thrombin formation then remained constant as aggregation intensity was still only 30%. Since the rate of thrombin formation then remained constant as aggregation increased up to its maximum intensity, the aggregation processes did not appear to result in partial masking of any procoagulant surface. This was confirmed by the fact that thrombin generation was the same on aggregated platelets as on activated platelets prevented from aggregating by addition of RGDS. Finally, maximum 5-hydroxy-tryptamine release could be obtained in the absence of thrombin generation. Thus platelet procoagulant activity did not appear to be related to aggregation or secretion and thrombin formation on the surface of activated platelets was in fact two to three times more important in the absence of stirring than under the conditions of continuous stirring required for aggregation. In conclusion, these results suggest procoagulant activity and aggregation to be two independent platelet responses which occur in different physiological situations.

Heparin-induced thrombocytopenia (HIT) involves heparin-dependent antibodies which induce platelet activation. In the present study, we searched for a relationship between the polymorphism of the Fc receptor (FcgammaRIIa) and the development of HIT. In this purpose, all the donors were genotyped for their FcgammaRIIA and HIT patients were selected on the basis of at least one positive answer by 14C-serotonin release assay (SRA). The frequency distribution of the FcgammaRIIa polymorphism in the HIT patient group was similar to that observed in the healthy control group. Moreover, a statistical analysis taking into account our results and those of 3 previously published studies, suggested at most only a weak association between HIT and the FcgammaRIIa-131 polymorphism. Laboratory tests used to diagnose HIT rely on the activation of normal donor platelets but fail to detect every HIT positive patient. We determined the role of FcgammaRIIa-131 polymorphism on the reactivity of control platelets to HIT plasmas. When control platelet FcgammaRIIa-131 was of Arg/Arg form, only 47% of the HIT plasmas were positive by SRA, compared to 81% and 74% for His/His or His/Arg forms, respectively. We also compared the level of anti PF4/heparin antibodies in the HIT plasmas with the response obtained by SRA. The mean anti PF4/heparin antibodies level in HIT plasma was significantly lower in negative SRA than in positive tests when using control platelets from FcgammaRIIa-Arg/Arg 131 and heterozygous donors. Thus, the variability of control platelets to respond to HIT plasmas in the SRA test is related to both the FcgammaRIIa-131 polymorphism, and to the amount of anti PF4/heparin antibodies.