Pro-atherogenic effect of interleukin-4 in endothelial cells: modulation of oxidative stress, nitric oxide and monocyte chemoattractant protein-1 expression.

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 platelet release reaction: granules' constituents, secretion and functions.

Although anucleated, blood platelets are highly organized cells rich in different types of organelles. Three specific granule populations store different types of constituents, some of which are at high concentrations. Platelets thus transport some specific compounds through the whole body. During circulation, platelets are reactive to various stimuli and release the materials stored in the specific granules. This 'release reaction' is an important step of primary haemostasis. Energy and messengers required for platelet reactivity are provided by mitochondria and the dense tubular system. Each granule population has specific properties concerning both the structure and the role played by the released constituents. Dense granules contain small non-protein molecules that are secreted to recruit other platelets. alpha-Granules contain large adhesive and healing proteins. Lysosomes contain hydrolases able to eliminate the circulating platelet aggregate. The extrusion of storage granules' content to the platelet's environment occurs according to regulated secretion events: movements of granules, apposition and fusion of granule and plasma membranes. Typical platelet disorders resulting from a storage granule abnormality are referred to as a storage pool defect and are characterized by a prolonged bleeding time.

A new approach in the study of the molecular and cellular events implicated in heparin-induced thrombocytopenia. Formation of leukocyte-platelet aggregates.

Heparin-induced thrombocytopenia (HIT), a relatively common complication of heparin therapy, results of platelet activation, via the receptor for the Fc domain of IgG (FcgammaRIIa), by heparin-dependent-antibodies, commonly directed against the heparin-platelet factor 4 (H-PF4) antigenic complex. Our strategy was to use whole blood allowing the study of leukocyte-platelet interactions. Experiments were performed with blood from healthy donors incubated with HIT patients' plasma and different concentrations of heparin. We showed that 75% of the HIT patients' plasma induced the formation of leukocyte-platelet-aggregates in a heparin-dependent-manner. The formation of leukocyte-platelet-aggregates induced by HIT plasma in the presence of heparin was (i) independent of the healthy blood donor FcgammaRIIa polymorphism, (ii) correlated with the levels of anti H-PF4 IgG antibodies contained in the patients' plasma, and to a lesser extent to anti H-PF4 IgM antibodies, and (iii) was mediated by P-selectin. This report opens new prospects in the study of the molecular and cellular events implicated in HIT.

Thrombomodulin prolongs thrombin-induced extracellular signal-regulated kinase phosphorylation and nuclear retention in endothelial cells.

On endothelial cells, thrombin binds to thrombomodulin (TM), an integral membrane-bound glycoprotein, and to protease-activated receptors (PARs). Thrombin binding to TM modulates endothelial cell and smooth muscle cell proliferation mediated through PAR1. We studied the phosphorylation and nuclear translocation of extracellular signal-regulated kinases (ERKs) 1 and 2 in human umbilical vein endothelial cells activated by thrombin. Thrombin and thrombin receptor-activating peptide (TRAP)-induced DNA synthesis were significantly inhibited by PD98059, an inhibitor of ERK phosphorylation. Immunoblots of phosphorylated ERKs (pERKs) and immunocytochemical studies of pERK localization revealed differences in the signal generated by thrombin and TRAP. After a short activation (15 minutes), the phosphorylation and the intracellular localization of pERKs were the same with the 2 agonists. After 4 hours, however, pERKs were visualized in the nuclei of thrombin-activated cells but barely detectable in TRAP-activated cells. Moreover, after 4 hours, the pERKs were visualized in the nuclei of cells stimulated by TRAP in the presence of a thrombin mutant that bound to TM, whereas they were around the nuclei in cells stimulated by thrombin in the presence of a monoclonal antibody preventing thrombin binding to TM. The results demonstrate that ERKs are involved in human umbilical vein endothelial cell DNA synthesis mediated by PAR agonists, that the duration of pERK nuclear retention is in inverse ratio to the mitogenic response, and that in addition to its role in the regulation of blood coagulation, TM acts as a thrombin receptor that modulates the duration of pERK nuclear retention and cell proliferation in response to thrombin.

Thiosulfinates inhibit platelet aggregation and microparticle shedding at a calpain-dependent step.

Thiosulfinates (TSs) are sulfur compounds generated through the processing of different Allium species with antiplatelet property. To further define this platelet inhibitory effect we studied diallyl-TS (Al2TS), dipropyl-TS (Pr2TS). and dimethyl-TS (Me2TS) on platelet responses. The three TSs inhibited dose-dependent platelet aggregation, with IC50 values of 15+/-2, 19+/-2, and 9+/-1 microM for Al2TS, Pr2TS and Me2TS, respectively. TSs had no effect on the expression of a platelet procoagulant surface, measured by flow cytometry as the binding of annexin V-FITC. They inhibited the microparticle shedding and clot retraction. Since the microparticle shedding is a calpain-activation dependent step, we assessed calpain activation by analysis of autoproteolysis in shorter active forms and by talin proteolysis in the presence of TSs. Calpain activation was inhibited by TSs independently of fibrinogen binding. Thus, TSs represent a new category of platelet inhibitors, acting on calpain-induced events.

Platelet alpha IIb-beta 3 integrin engagement induces the tyrosine phosphorylation of Cbl and its association with phosphoinositide 3-kinase and Syk.

Agonist-induced platelet activation triggers 'inside-out' signalling which activates alpha IIb-beta 3, the most abundant integrin in platelet membranes. The engagement of activated alpha IIb-beta 3 integrin by linking fibrinogen is necessary for platelet aggregation, and this induces subsequent outside-in signalling, which enhances platelet activation. Here we studied the involvement of Cbl during alpha IIb-beta 3-integrin-mediated signal transduction. During thrombin-induced platelet activation, Cbl was tyrosine phosphorylated, and phosphoinositide 3-kinase (PI 3-kinase) activity measured in Cbl immunoprecipitates was increased. Both Cbl phosphorylation and its association with PI 3-kinase were dependent on alpha IIb-beta 3 engagement by linking fibrinogen. The P256 and anti-LIBS6 (ligand-induced binding site 6) antibodies, which activate platelets directly through alpha IIb-beta 3, induced Cbl phosphorylation and increased the PI 3-kinase activity associated with Cbl. Both thrombin and antibodies to alpha IIb-beta 3 induced association of Cbl with the tyrosine kinase, Syk. Experiments performed with inhibitors of tyrosine kinases indicated that both Src-family kinases and Syk contribute to phosphorylation of Cbl and its consequent association with PI 3-kinase. The results show that, following integrin alpha IIb-beta 3 engagement, Cbl is tyrosine phosphorylated, recruits PI 3-kinase to this integrin signalling pathway and possibly enhances PI 3-kinase activity, downstream of Src-family tyrosine kinases and Syk activation.

Platelets induce human umbilical vein endothelial cell proliferation through P-selectin.

We studied whether platelets could participate in the endothelial cell monolayer regeneration in the case of a vessel damage. Incorporation of [3H]-thymidine into the DNA of human umbilical vein endothelial cells (HUVECs) was measured after 48 h of co-incubation with platelets. The effect of platelets was compared to that of platelet-free supernatants from thrombin-activated platelets that had secreted their active granule constituents. Platelets dose-dependently induced HUVEC proliferation. Platelets preactivated by thrombin induced similar proliferation as did unactivated platelets (proliferation factor = 7 - 8), indicating that preactivation of platelets was not required. Platelets fixed with paraformaldehyde had no effect, suggesting that the platelet mitogenic effect required a mobile, alive membrane. Ketanserine and suramin reduced by at most 30 % the platelet-induced proliferation; supernatants of thrombin-activated platelets caused only minor proliferation (proliferation factor = 2), suggesting that secreted 5-hydroxytryptamine and growth factors poorly contributed to the proliferative effect. When the co-incubation was performed in the presence of an anti P-selectin antibody, the platelet-induced HUVEC proliferation was inhibited. The results suggest that platelet adhesion participate in the control of the endothelial regeneration and that platelet P-selectin is a molecular determinant of the proliferative signal.

Tyrosine phosphorylation and association of FcgammaRII and p72(Syk) are not limited to the FcgammaRII signalling pathway.

The tyrosine kinase p72(Syk) plays a critical role in platelet signal transduction. It associates with the platelet receptor for the Fc domain of IgGs, FcgammaRII, following stimulation by FcgammaRII cross-linking. Here, we show that p72(Syk) and FcgammaRII tyrosine phosphorylation and association occured following platelet stimulation by: (1) two monoclonal antibodies, which form a bridge between a target antigen and FcgammaRII, and (2) the G-protein-coupled receptor agonist thrombin. The kinetics of the p72(Syk)/FcgammaRII association depended on the signalling pathway (i.e., the antigen targeted or the thrombin receptor). We established a direct relationship between the level of FcgammaRII phosphorylation and the detection of its association with p72(Syk). Inhibition of p72(Syk) by piceatannol resulted in partial or total inhibition of FcgammaRII phosphorylation, after immunological activation or addition of thrombin, respectively, suggesting that p72(Syk) participates in FcgammaRII phosphorylation. The results provide evidence that p72(Syk)/FcgammaRII association is not restricted to immunological activation.

Protective effect of a thrombin receptor (protease-activated receptor 1) gene polymorphism toward venous thromboembolism.

The human protease-activated receptor 1 (PAR-1) is activated by thrombin at the surface of platelets and endothelial cells, 2 cells that are implicated in hemostasis and thrombosis. We studied the PAR-1 gene in a large case-control study from the Paris Thrombosis Study (PATHROS), and the possible implication of polymorphisms in venous thromboembolism was evaluated. Two polymorphisms were found in the 5' regulatory region. The first is a C to T transition that is 1426 nucleotides upstream from the translation start site (-1426 C/T), and the second is a 13-bp insertion repeating the preceding -506 5'-CGGCCGCGGGAAG-3' sequence (-506 I/D, where I indicates insertion and D indicates deletion), a putative cis-acting element of the Ets family. The third polymorphism is an A to T transversion in the intervening sequence (IVS) that is 14 nucleotides upstream from the exon 2 start site (IVS-14 A/T). The distribution of the 3 polymorphisms was otherwise similar in the 250 cases and the 1214 controls. A noteworthy sex heterogeneity led us to analyze men and women separately with regard to the -506 I/D polymorphism. We found that allele I was less frequent in male cases than in male controls (0.154 versus 0.247, P<0.01), with an odds ratio at 0.52 (95% CI 0. 32 to 0.82, P<0.01). Furthermore, a reduction of prothrombin fragment 1+2 levels was observed in homozygous carriers of allele -506 I (P=0.04). Altogether, these data suggested a protective effect in men of -506 I/D polymorphism for venous thromboembolism.

Calpain controls the balance between protein tyrosine kinase and tyrosine phosphatase activities during platelet activation.

Protein phosphorylation was studied during platelet stimulation in two ranges of ionized [Ca2+]. At ionized [Ca2+]i< or = 1 microM, proteins were phosphorylated. At ionized [Ca2+]i > or = 4 microM, phosphoproteins disappeared. Protein dephosphorylation was prevented by the combined action of calpeptin and phosphatase inhibitors. Protein tyrosine phosphatase activity was stimulated regardless of the ionized [Ca2+] level. Protein tyrosine kinase activity was stimulated at ionized [Ca2+]i < or =1 microM, whereas at ionized [Ca2+]i > or =4 microM, no protein tyrosine kinase activity was observed except in the presence of calpeptin. Thus, the massive tyrosine phosphoprotein disappearance observed at a high ionized [Ca2+]i resulted not only in protein tyrosine phosphatase activation, but also in calpain-induced protein tyrosine kinase inactivation.

Fc receptor-mediated platelet activation is dependent on phosphatidylinositol 3-kinase activation and involves p120(Cbl).

The platelet receptor for the Fc domain of IgGs (FcgammaRIIa) triggers intracellular signaling through protein tyrosine phosphorylations leading to platelet aggregation. In this study, we focused on the adaptor protein p120(cbl) (Cbl), which became tyrosine-phosphorylated after platelet activation induced by antibodies. Cbl phosphorylation was dependent on Fc receptor engagement. An association of Cbl with the p85 subunit of phosphatidylinositol 3-kinase (PI 3-K) occurred in parallel with Cbl tyrosine phosphorylation. We showed by in vitro experiments that Cbl/p85 association was mediated by the Src homology 3 domain of p85/PI 3-K and the proline-rich region of Cbl. Inhibition of PI 3-K activity by wortmannin led to the blockade of both platelet aggregation and serotonin release mediated by FcgammaRIIa engagement, whereas it only partly inhibited those induced by thrombin. Thus, PI 3-K may play a crucial role in the initiation of platelet responses after FcgammaRIIa engagement. Our results suggest that Cbl is involved in platelet signal transduction by the recruitment of PI 3-K to the FcgammaRIIa pathway, possibly by increasing PI 3-K activity.

Tyrosine unphosphorylated platelet SHP-1 is a substrate for calpain.

The platelet phosphotyrosine phosphatase (PTP) SHP-1 is tyrosine phosphorylated during thrombin-induced activation. Stimulation of platelets by the ionophore A23187 in the presence of CaCl2 induced a calpain dependent cleavage of SHP-1. SHP-1 proteolysis was undetectable during thrombin-induced stimulation. When SHP-1 was tyrosine phosphorylated by thrombin, further addition of A23187 failed to induce its cleavage. In the presence of tyrphostin to inhibit thrombin-induced SHP-1 tyrosine phosphorylation, SHP-1 was cleaved. Thus, only the tyrosine unphosphorylated form of SHP-1 was a substrate for calpain. A23187 induced the disappearance of all platelet phosphotyrosine proteins and a two-fold increase in PTP activity, both inhibited by pervanadate, a PTP inhibitor, but unaffected by calpeptin, a calpain inhibitor. The data show that SHP-1 is either tyrosine phosphorylated or cleaved by calpain, and suggest that SHP-1 cleavage does not contribute to A23187-induced PTP activity.

Thrombomodulin modulates the mitogenic response to thrombin of human umbilical vein endothelial cells.

Thrombin interacts with its receptor and thrombomodulin on endothelial cells. We evaluated the respective roles of these two proteins on human umbilical vein endothelial cell (HUVEC) growth by comparing thrombin, S195A (a mutant thrombin in which the serine of the charge stabilizing system had been replaced by alanine), and the receptor activating peptide (TRAP). Thrombin and TRAP induced DNA synthesis (half maximal cell proliferation with 5 nM and 25 microM, respectively), whereas S195A thrombin was inactive, inferring that growth is mediated through the thrombin receptor. Surprisingly, cells stimulated by TRAP exhibited a maximal proliferation twice greater than that obtained with thrombin. Combination of thrombin and TRAP resulted in a mitogenic response higher than by thrombin alone, but lower than by TRAP alone. The role of thrombomodulin was evaluated by adding an anti-thrombomodulin antibody, which prevents formation of the thrombin-thrombomodulin complex. Antibody did not interfere with cell proliferation induced by TRAP, but enhanced that induced by thrombin. We conclude that formation of the thrombin-thrombomodulin complex restrains HUVEC proliferation mediated through the thrombin receptor.

Importance of the FcgammaRIIa-Arg/His-131 polymorphism in heparin-induced thrombocytopenia diagnosis.

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.

Lonomia obliqua caterpillar spicules trigger human blood coagulation via activation of factor X and prothrombin.

In southern Brazil, envenomation by larvae of the moth Lonomia obliqua (Walker) may result in blood clotting factor depletion, leading to disseminated intravascular coagulation with subsequent haemorrhage and acute renal failure which may prove fatal. We have examined the effect of a crude extract of spicules from these caterpillars on in vitro hemostasis. The extract alone did not aggregate platelets and had no detectable effect on purified fibrinogen, suggesting that extract induces clot formation by triggering activation of the clotting cascade. In agreement with the presence of thrombin-mediated activity, hirudin prevented clot formation. The extract was found to activate both prothrombin and factor X, suggesting that the depletion of blood clotting factors results from the steady activation of factor X and prothrombin. Heating and diisopropylfluorophosphate abolished the procoagulant activity of the extract, indicating that the active component involved is a protein that may belong to the serine protease family of enzymes. The ability of hirudin to inhibit this coagulant activity suggests that this inhibitor could be beneficial in the treatment of patients envenomed by L. obliqua caterpillars.

Platelet and megakaryocyte dense granules contain glycoproteins Ib and IIb-IIIa.

Platelets contain two main types of secretory organelles, the dense granules and the alpha-granules. P-selectin, a specific receptor for leukocytes that is present in the alpha-granule membrane, has also been demonstrated to be associated with the dense granule limiting membrane, showing that a relationship exists between these two types of secretory granules. We have previously shown that the plasma membrane receptors glycoproteins (Gp) IIb-IIIa and Ib are also present in the alpha-granule membrane. To document further the composition of the dense granule membrane, we have used immunoelectron microscopy in the present work to determine if the dense granule membrane also contains these glycoproteins. First, the cytochemical method of Richards and Da Prada (J Histochem Cytochem 25:1322, 1977), which specifically enhances dense body electron density, was combined with immunogold-labeled anti-Gp IIb-IIIa or anti-Gp Ib antibody. A consistent and reproducible labeling for Gp IIb-IIIa, but less for Gp Ib, was found in the membrane of platelet dense granules. Subsequently, double immunogold labeling was performed on frozen thin sections of resting platelets using antibodies directed against the dense body components granulophysin or P-selectin, followed by anti-Gp IIb-IIIa or anti-Gp Ib. Consistent labeling for Gp IIb-IIIa and weaker labeling for Gp Ib were detected in dense bodies. The possibility that the granulophysin-positive structures could be lysosomes was excluded by the presence of P-selectin. Immunogold labeling of isolated dense granule fractions confirmed these results. Identical findings were made on human cultured megakaryocytes using double immunolabeling. In conclusion, this study demonstrates the presence of Gp IIb-IIIa and Gp Ib on the dense granule membrane. This observation provides additional evidence of similarities between the alpha-granule and dense granule membranes and raises the possibility of a dual mechanism responsible for the formation of dense granules similar to that of alpha-granules, ie, endogenous synthesis as well as endocytosis from the plasma membrane.

Interdonor variability of platelet response to thrombin receptor activation: influence of PlA2 polymorphism.

Considering that platelet response to thrombin receptor activation might be critical for the development of arterial thrombosis, we measured the dense granule release under stimulation by the thrombin receptor activating peptide (TRAP) in a series of 102 healthy volunteers. The threshold TRAP concentration which initiated a secretion ranged from 3 to 20 microM. A good concordance (79%, k=0.677) between two tests performed at a 1 month interval indicated that platelet response to thrombin receptor activation was characteristic of each individual donor. Since the threshold concentration required to initiate secretion corresponded to the threshold concentration which induced a biphasic aggregation, all volunteers were genotyped for the PlA2 polymorphism, the Pro33 variant of GPIIIa. Platelets from subjects with the PlA2 polymorphism required higher TRAP concentrations to aggregate than those from subjects with no PlA2 allele (P=0.0012). However, they also required a higher ADP concentration to aggregate. In order to exclude any influence of GPIIIa polymorphism on TRAP-induced secretion, we studied the variability of platelet response to TRAP among the 77 individuals with no PlA2 allele, and found the same interdonor variability with the same distribution of threshold TRAP concentrations as for the 102 individuals. The results suggest that (i) platelet secretion in response to thrombin receptor activation could be a genetically controlled phenotype independent of the GPIIIa polymorphism; (ii) the PlA2 polymorphism is associated with platelet hypoaggregability.