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1.
J Thromb Haemost ; 9(5): 1029-39, 2011 May.
Article in English | MEDLINE | ID: mdl-21320285

ABSTRACT

BACKGROUND: At the site of vascular injury, monocytes (MN) interacting with activated platelets (PLT) synthesize tissue factor (TF) and promote thrombus formation. Intracellular signals necessary for the expression of TF in MN, in the context of a developing thrombus, remain unknown. OBJECTIVE: The study was designed to investigate the role of the glycogen synthase kinase 3 (GSK3, a serine-threonine kinase) downstream insulin receptor pathway, in PLT-induced TF expression in MN. METHODS: To this purpose we used a well-characterized in vitro model of human MN-PLT interactions that allows detailed analysis of TF activity, TF protein and gene expression. RESULTS: The results demonstrated that, in MN interacting with activated PLT: (i) TF activity, antigen and mRNA were low until 8-10 h and dramatically increased thereafter, up to 24 h; (ii) according to the kinetics of TF expression in MN, GSK3ß undergoes phosphorylation on serine 9, a process associated with down-regulation of enzyme activity; (iii) pharmacological blockade of GSK3 further increased TF expression and was accompanied by increased accumulation of NF-kB, in the nucleus; (iv) blockade of phosphoinositide-3 kinase (PI(3)K) by wortmannin inhibited PLT-induced TF expression; and (v) according to the established role of the GSK3 downstream insulin receptor, insulin increased PLT-induced TF expression in a PI(3)K-dependent manner. CONCLUSION: GSK3 acts as a molecular brake on the signaling pathway, leading to TF expression in MN interacting with activated PLT. PI(3)K, through Akt-dependent phosphorylation of GSK3, relieves this brake and allows TF gene expression. This study identifies a novel molecular link between thrombotic risk and metabolic disorders.


Subject(s)
Blood Platelets/cytology , Gene Expression Regulation , Glycogen Synthase Kinase 3/metabolism , Monocytes/cytology , Thromboplastin/biosynthesis , Coagulants/metabolism , Humans , Macrophages/metabolism , Models, Biological , NF-kappa B/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation , Platelet Activation , Serine/chemistry , Signal Transduction , Thromboplastin/metabolism , Thrombosis/metabolism
2.
J Thromb Haemost ; 5(11): 2197-203, 2007 Nov.
Article in English | MEDLINE | ID: mdl-17697141

ABSTRACT

BACKGROUND: The percentage of diabetic patients who do not benefit from the protective effect of aspirin is larger than in other populations at cardiovascular risk. OBJECTIVE: We compared the ability of aspirin to suppress TxA2 and platelet activation in vivo, in type-2 diabetics vs. high-risk non-diabetic patients. METHODS: Urinary 11-dehydro-TXB2, plasma sCD40 L, and sP-selectin were measured, together with indices of low-grade inflammation, glycemic control, and lipid profile, in 82 patients with type-2 diabetes and 39 without diabetes, treated with low doses of aspirin. RESULTS: Urinary 11-dehydro-TxB2, plasma sCD40L and sP-selectin were significantly higher in diabetics than in controls: [38.9 (27.8-63.3) vs. 28.5 (22.5-43.9) ng mmol(-1) of creatinine, P = 0.02], [1.06 (0.42-3.06) vs. 0.35 (0.22-0.95) ng mL(-1); P = 0.0001], [37.0 (16.8-85.6) vs. 20.0 (11.2-35.6) ng mL(-1), P = 0.0001], respectively. The proportion of individuals with diabetes increased across quartiles of 11-dehydro-TxB2, sCD40L, and sP-selectin, with the highest quartiles of 11-dehydro-TxB2, sCD40L and sP-selectin, including 66%, 93.3%, and 93.3% of individuals with diabetes. Markers of platelet activation positively correlated with indices of glycemic control but not with markers of low-grade inflammation. CONCLUSIONS: Platelet dysfunction associated with insufficient glycemic control, may mediate persistent platelet activation under aspirin treatment.


Subject(s)
Aspirin/pharmacology , Diabetes Mellitus, Type 2/blood , Platelet Activation , Aspirin/therapeutic use , Biomarkers/blood , Blood Glucose , Case-Control Studies , Diabetes Mellitus, Type 2/drug therapy , Glycemic Index , Humans , Inflammation , Platelet Activation/drug effects , Thromboxane A2/antagonists & inhibitors
3.
J Thromb Haemost ; 1(2): 314-9, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12871506

ABSTRACT

Epidemiological studies link acute infection of the respiratory tract to a transient increased risk of acute myocardial infarction. The underlying mechanisms remain unknown. We hypothesized that vasoactive mediators produced by inflammatory cells in the lungs and drained in the coronary circulation may trigger acute myocardial ischemia. To test this hypothesis we used an experimental model in the rabbit. Injection of the bacterial-derived peptide N-formyl-Met-Leu-Phe (or N-formyl-Methionyl-Leucyl-Phenylalanine)(fMLP) in the jugular vein induced massive recruitment of both polymorphonuclear leukocytes (PMN) and platelets in the microcirculation of the lungs, accompanied by rapid and marked increase of leukotriene B4, cysteinyl leukotrienes and thromboxane (Tx) A2 in the aortic blood. In all animals, fMLP evoked ischemic electrocardiographic changes: within the first minute of infusion a profound depression of the ST segment and inversion of the T wave were observed. Mean aortic pressure and heart rate fell to 64.0 +/- 6.9 and 83.5 +/- 3.1% of the basal levels at 3 and 10 min, respectively. All these alterations were transient. Aspirin, prevented electrocardiographic ischemic changes, reverted bradycardia and hypotension but did not significantly modify either PMN or platelet recruitment nor leukotriene synthesis. Ridogrel, a Tx-synthase and receptor inhibitor, prevented ECG alterations and bradycardia, but did not prevent and even worsened hypotension; it blocked platelet, but not PMN, sequestration. Pretreatment of animals with intravenous high dose of aspirin prevented ridogrel-dependent hypotension and platelet inhibition, suggesting that PGI2 contributes to the effects of Tx-synthase and receptor inhibitor. In hypercholesterolemic rabbits, ECG alterations persisted longer than in normal controls. In summary, our results indicate that acute activation of PMN and platelets in the lungs provokes transient myocardial ischemia, in normal animals that is exacerbated in hypercholesterolemic rabbits. TxA2 appears to be the major mediator of this phenomenon. Moreover the data suggest that a balance between TxA2 and PGI2 plays a pivotal role in platelet activation and recruitment in our model.


Subject(s)
Myocardial Ischemia/etiology , Myocardial Ischemia/physiopathology , Pneumonia/complications , Pneumonia/physiopathology , Thromboxane A2/physiology , Acute Disease , Animals , Arteriosclerosis/blood , Arteriosclerosis/complications , Arteriosclerosis/physiopathology , Disease Models, Animal , Electrocardiography , Epoprostenol/physiology , Inflammation Mediators/physiology , Male , Myocardial Ischemia/blood , N-Formylmethionine Leucyl-Phenylalanine/toxicity , Platelet Activation/physiology , Pneumonia/blood , Rabbits
4.
J Thromb Haemost ; 1(5): 1048-54, 2003 May.
Article in English | MEDLINE | ID: mdl-12871376

ABSTRACT

BACKGROUND: The initial interaction of human polymorphonuclear leukocytes (PMN) with activated human platelets is mediated by P-selectin and its leukocyte ligand PSGL-1; subsequently the interaction is strengthened by activation of alphaMbeta2 via protein tyrosine phosphorylation mediated by Src kinases and binding of activated alphaMbeta2 to its platelet counterreceptor(s). OBJECTIVES: Because mouse models are being used to define the role of PMN-platelet interactions in thrombosis and the response to vascular injury, we investigated the molecular determinants responsible for the interaction of murine PMNs with activated murine platelets. METHODS: Mouse platelets were labeled with the green fluorescent dye BCECF and then activated with thrombin and fixed with 1% paraformaldehyde. Mouse PMNs were labeled with the red fluorescent dye hydroethidine and then stirred with the fixed platelets. After stopping the reaction with paraformaldehyde, formation of mixed cell conjugates was analyzed by flow cytometry. RESULTS: In time course experiments, 90 +/- 1.9% of PMNs formed mixed conjugates with platelets after 2 min and the mean (+/- SEM) number of platelets per positive PMN was 8.4 +/- 1.5. A monoclonal antibody to P-selectin reduced the percentage of PMNs with attached platelets to 16 +/- 2.4% (P = 0.001), and only 8 +/- 5% of PMNs interacted with platelets from P-selectin-/- mice. In contrast, monoclonal antibodies to PSGL-1, beta2-integrin, and alphaIIbbeta3 had much less or no effect on the production of mixed cell aggregates. To better identify a secondary contribution of beta2-integrins, P-selectin interactions were disrupted by briefly adding 5 mm EGTA to already-formed mixed cell aggregates. Brief EGTA treatment alone reduced the percentage of PMNs with attached platelets to 70 +/- 3.5% (P = 0.004 vs. no treatment), but did not modify the number of platelets per positive PMN (9.5 +/- 1.7). The combination of brief EGTA treatment and a monoclonal antibody to beta2-integrin lowered the percentage of PMN with attached platelets to 50 +/- 7% and reduced the number of platelets attached per positive PMN to 3.6 +/- 0.7 (P = 0.03 vs. brief EGTA treatment only). Brief EGTA treatment did not modify the effect of the other antibodies. When the incubation was stopped with EGTA the Src inhibitors PP1 and PP2 reduced PMN-platelet adhesion, while the inactive analog PP3 was ineffective. CONCLUSIONS: These results confirm that P-selectin plays a prominent role in mediating the initial interactions between mouse PMN and platelets, and provide support for additional contributions from beta2-integrins and Src family kinases.


Subject(s)
CD18 Antigens/physiology , P-Selectin/physiology , Platelet Adhesiveness , src-Family Kinases/physiology , Animals , Antibodies, Monoclonal/pharmacology , Blood Platelets/cytology , Edetic Acid/pharmacology , Flow Cytometry , Membrane Glycoproteins/physiology , Mice , Mice, Knockout , Neutrophils/cytology , Neutrophils/physiology , P-Selectin/genetics , Platelet Glycoprotein GPIIb-IIIa Complex/physiology , Thrombosis/etiology , src-Family Kinases/metabolism
5.
J Med Chem ; 44(21): 3463-8, 2001 Oct 11.
Article in English | MEDLINE | ID: mdl-11585451

ABSTRACT

A new series of nonsteroidal antiinflammatory drugs (NSAIDs) obtained by linking ibuprofen to selected furoxan moieties and to related furazans were synthesized and tested for their antiinflammatory, antiaggregatory, and ulcerogenic properties. All the derivatives are endowed with antiinflammatory activity comparable to that of ibuprofen, but, unlike this drug, they display reduced acute gastrotoxicity. The masking of the ibuprofen-free carboxylic group seems to be principally at the basis of this reduced topical irritant action. The two furoxan derivatives 8 and 9 also trigger potent antiaggregatory effects, principally as a consequence of their NO-donor ability.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/chemical synthesis , Cyclic N-Oxides/chemical synthesis , Ibuprofen/analogs & derivatives , Ibuprofen/chemical synthesis , Nitric Oxide Donors/chemical synthesis , Oxadiazoles/chemical synthesis , Platelet Aggregation Inhibitors/chemical synthesis , Animals , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Anti-Inflammatory Agents, Non-Steroidal/toxicity , Carrageenan , Cyclic N-Oxides/pharmacology , Cyclic N-Oxides/toxicity , Edema/drug therapy , Gastric Mucosa/drug effects , Humans , Ibuprofen/pharmacology , In Vitro Techniques , Male , Nitric Oxide Donors/pharmacology , Nitric Oxide Donors/toxicity , Oxadiazoles/pharmacology , Oxadiazoles/toxicity , Peptic Ulcer/chemically induced , Platelet Aggregation Inhibitors/pharmacology , Platelet Aggregation Inhibitors/toxicity , Rats , Rats, Wistar
6.
Blood ; 98(1): 108-16, 2001 Jul 01.
Article in English | MEDLINE | ID: mdl-11418469

ABSTRACT

Adhesion of polymorphonuclear leukocytes (PMNLs) to activated platelets requires a P-selectin-triggered, tyrosine kinase-dependent adhesiveness of Mac-1 and is accompanied by tyrosine phosphorylation of a 110-kd protein (P-110) in PMNLs. Inhibitors of SRC tyrosine kinases were found to inhibit PMNL adhesion to activated platelets or to P-selectin expressing Chinese hamster ovary (CHO-P) cells and the tyrosine phosphorylation of P-110. Adhesion of PMNLs to activated platelets or to CHO-P cells stimulated activity of LYN and HCK. Monoclonal antibody blockade of P-selectin or beta2-integrins reduced the activation of both kinases. In PMNLs either adherent to platelets or aggregated by P-selectin-IgG chimera, Mac-1 was rapidly redistributed to the Triton X-100-insoluble cytoskeletal fraction, and large clusters of Mac-1 colocalized with patches of F-actin at the sites of cell-cell contact. In PMNLs stimulated by P-selectin-IgG chimera, SRC kinase inhibition impaired Mac-1 clustering, F-actin accumulation, and CD18 redistribution to the cytoskeleton. Disruption of the actin filament network by cytochalasin D prevented PMNL-platelet adhesion and P-selectin-induced PMNL aggregation and impaired the clustering of Mac-1. In agreement with the requirement for the beta2-integrin in the functional up-regulation of LYN and HCK, integrin blockade by monoclonal antibodies resulted in a complete inhibition of P-selectin-induced Mac-1 clustering and F-actin accumulation. Taken together, the results indicate that, after an initial P-selectin-triggered beta2-integrin interaction with the ligand, SRC kinases are activated and allow the remodeling of cytoskeleton-integrin linkages and integrin clustering that finally strengthen cell-cell adhesion. This model highlights a new role for SRC kinases in a regulatory loop by which the Mac-1 promotes its own adhesive function.


Subject(s)
Neutrophils/physiology , Platelet Adhesiveness/drug effects , Actins/metabolism , Animals , CD18 Antigens/pharmacology , CHO Cells , Cell Adhesion , Cricetinae , Cytoskeleton/metabolism , Feedback , Humans , Macrophage-1 Antigen/drug effects , Macrophage-1 Antigen/pharmacology , Macrophage-1 Antigen/physiology , Neutrophils/cytology , P-Selectin/genetics , P-Selectin/pharmacology , Protein-Tyrosine Kinases/drug effects , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins/drug effects , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-hck , Signal Transduction/drug effects , Transfection , src-Family Kinases/drug effects , src-Family Kinases/metabolism , src-Family Kinases/pharmacology , src-Family Kinases/physiology
7.
Blood ; 96(13): 4261-6, 2000 Dec 15.
Article in English | MEDLINE | ID: mdl-11110700

ABSTRACT

Thrombohemorrhagic complications are a major cause of morbidity and mortality in patients with essential thrombocythemia (ET) and polycythemia vera (PV). The pathogenesis of these complications is not completely clarified. Several studies have described abnormalities of red blood cells and platelets in these patients. However, no studies are available on changes in the polymorphonuclear leukocytes (PMNs), which can play an important role in the activation of the hemostatic system. In patients with ET (n = 37) and PV (n = 34), a series of PMN activation parameters (PMN membrane CD11b and leukocyte alkaline phosphatase [LAP] antigen expression, cellular elastase content, plasma elastase, and myeloperoxidase levels) was evaluated simultaneously with the levels of plasma markers of endothelial damage (thrombomodulin and von Willebrand factor antigen) and hypercoagulation (thrombin-antithrombin complex, prothrombin fragment 1 + 2, and D-dimer). The results show the occurrence of PMN activation in both groups of patients compared with a control group of healthy subjects. An increase in CD11b and LAP expression by PMN membrane was observed, together with a significant increase in cellular elastase content, plasma elastase, and myeloperoxidase levels. In addition, patients had high plasma levels of endothelial and hypercoagulation markers compared with controls. For the first time, these data show that in ET and PV, 2 hematologic conditions that place patients at increased risk for thrombosis, an in vivo leukocyte activation occurs and is associated with laboratory signs of endothelium and coagulation system activation. (Blood. 2000;96:4261-4266)


Subject(s)
Hemostasis/physiology , Neutrophils/physiology , Polycythemia Vera/blood , Thrombocythemia, Essential/blood , Adult , Aged , Aged, 80 and over , Alkaline Phosphatase/analysis , Antigens/analysis , Antithrombin III/analysis , Biomarkers , Endothelium, Vascular/pathology , Female , Fibrin Fibrinogen Degradation Products/analysis , Humans , Leukocyte Elastase/analysis , Macrophage-1 Antigen/analysis , Male , Middle Aged , Peptide Fragments/analysis , Peptide Hydrolases/analysis , Peroxidase/analysis , Polycythemia Vera/pathology , Prothrombin/analysis , Thrombocythemia, Essential/pathology , Thrombomodulin/analysis , von Willebrand Factor/immunology
8.
Thromb Haemost ; 84(3): 478-83, 2000 Sep.
Article in English | MEDLINE | ID: mdl-11019975

ABSTRACT

Platelets regulate several polymorphonuclear leukocyte (PMN) functions. We have found that thrombin-stimulated platelets potently inhibited PMN apoptosis. Cell-free supernatant from increasing concentrations of stimulated platelets inhibited PMN apoptosis in a dose-dependent manner, with an effect similar to that of corresponding concentrations of platelets. At the plateau, platelet supernatant inhibited PMN apoptosis by 54.6 +/- 6.8%, the anti-apoptotic activity being higher than that of GM-CSF and comparable to that of LPS. Neither IL-1ra nor a combination of anti-IL1alpha + betamAb affected the activity of platelet supernatant. In contrast a mAb recognizing the active form of TGF-beta1 significantly decreased this activity. Moreover, exogenous TGF-beta1 inhibited PMN apoptosis in a dose-dependent manner. The active form of this cytokine was indeed present in the supernatant of stimulated platelets at a concentration able to elicit an anti-apoptotic effect. The p38 MAPK inhibitor SB203580 prevented the anti-apoptotic effect of TGF-beta1 in a dose-dependent manner. Interestingly, it also prevented the anti-apoptotic effect of IL-1alpha, but not that of GM-CSF, LPS and dexamethasone. In conclusion, we report for the first time that PMN apoptosis is potently inhibited by platelet-released mediators, that TGF-beta1 mediates an important part of this effect, and that p38 MAPK is involved in the TGF-beta1 signaling leading to its anti-apoptotic effect. These results provide novel evidence to support the central role of platelets in inflammation.


Subject(s)
Apoptosis/drug effects , Neutrophils/cytology , Transforming Growth Factor beta/physiology , Blood Platelets/metabolism , Cell Culture Techniques , Dose-Response Relationship, Drug , Enzyme Activation/drug effects , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Humans , Imidazoles/pharmacology , Lipopolysaccharides/pharmacology , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinases/physiology , Pyridines/pharmacology , Signal Transduction , Transforming Growth Factor beta/metabolism , Transforming Growth Factor beta/pharmacology , p38 Mitogen-Activated Protein Kinases
9.
Blood ; 93(8): 2506-14, 1999 Apr 15.
Article in English | MEDLINE | ID: mdl-10194429

ABSTRACT

Granulocyte colony-stimulating factor (G-CSF) enhances neutrophil functions in vitro and in vivo. It is known that neutrophil-derived products can alter the hemostatic balance. To understand whether polymorphonuclear leukocyte (PMN) activation, measured as PMN degranulation and phenotypical change, may be associated to hemostatic alterations in vivo, we have studied the effect of recombinant human G-CSF (rHuG-CSF) administration on leukocyte parameters and hemostatic variables in healthy donors of hematopoietic progenitor cells (HPCs). Twenty-six consecutive healthy donors receiving 10 micrograms/kg/d rHuG-CSF subcutaneously for 5 to 7 days to mobilize HPCs for allogeneic transplants were included in the study. All of them responded to rHuG-CSF with a significant white blood cell count increase. Blood samples were drawn before therapy on days 2 and 5 and 1 week after stopping rHuG-CSF treatment. The following parameters were evaluated: (1) PMN activation parameters, ie, surface CD11b/CD18 antigen expression, plasma elastase antigen levels and cellular elastase activity; (2) plasma markers of endothelium activation, ie, thrombomodulin (TM) and von Willebrand factor (vWF) antigens; (3) plasma markers of blood coagulation activation, ie, F1+2, TAT complex, D-dimer; and (4) mononuclear cell (MNC) procoagulant activity (PCA) expression. The results show that, after starting rHuG-CSF, an in vivo PMN activation occurred, as demonstrated by the significant increment of surface CD11b/CD18 and plasma elastase antigen levels. Moreover, PMN cellular elastase activity, which was significantly increased at 1 day of treatment, returned to baseline at day 5 to 6, in correspondence with the elastase antigen peak in the circulation. This change was accompanied by a parallel significant increase in plasma levels of the two endothelial and the three coagulation markers. The PCA generated in vitro by unstimulated MNC isolated from rHuG-CSF-treated subjects was not different from that of control cells from untreated subjects. However, endotoxin-stimulated MNC isolated from on-treatment individuals produced significantly more PCA compared with both baseline and control samples. All of the parameters were decreased or normal 1 week after stopping treatment. These data show that rHuG-CSF induces PMN activation and transiently affects some hemostatic variables in healthy HPC donor subjects. The clinical significance of these findings remains to be established.


Subject(s)
Blood Donors , Granulocyte Colony-Stimulating Factor/pharmacology , Hematopoietic Stem Cells/physiology , Hemostasis/physiology , Neutrophil Activation/physiology , Adolescent , Adult , Child , Erythrocyte Count , Female , Filgrastim , Hematocrit , Hematopoietic Stem Cells/drug effects , Hemoglobins/metabolism , Hemostasis/drug effects , Humans , Leukocyte Count , Male , Middle Aged , Neutrophil Activation/drug effects , Neutrophils/drug effects , Neutrophils/physiology , Platelet Count , Recombinant Proteins , Reference Values
10.
Thromb Haemost ; 81(3): 442-8, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10102475

ABSTRACT

The contribution of platelets to arachidonic acid transcellular metabolism may represent an important pathway of leukotriene (LT) production. The aim of this study was to investigate the role of platelets on LT production in an acute inflammatory model in the rabbit. Preliminary experiments showed that rabbit whole blood (5 ml) stimulated in vitro with the calcium ionophore A23187 produced LTB4 (52.7+/-13.9 ng) and the mixed 5,12-DiHETE (7.25+/-0.75 ng). In A23187-stimulated thrombocytopenic blood, LTB4 was significantly reduced to 19.5+/-8.6 ng and 5,12-DiHETE was undetectable. Peptido-LTs were undetectable in both conditions. In experiments using washed cells, addition of thrombin-activated platelets to fMLP-activated PMN resulted in the appearance of 5,12-DiHETE and in more than twofold increase of LTB4 synthesis. When 3H-arachidonic acid-labelled platelets were mixed with unlabelled PMN and challenged with fMLP and thrombin, radioactive LTB4 and 5,12-DiHETE were produced, indicating that platelet-derived arachidonic acid was utilized by PMN 5-lipoxygenase. Intravenous infusion of fMLP (2.5 nmol/kg/min) in the rabbit induced marked granulocytopenia, thrombocytopenia and increased TxB2 plasma concentrations within 3 min. Electron microscopy of lungs showed morphologically activated and aggregated platelets occluding the capillary lumen. Activation and recruitment of circulating cells was accompanied by the production of LTB4 (peak levels at 1 min: 30.0+/-9.5 ng/ml) and LTE4 (peak levels at 10 minutes: 77.8+/-11.6 ng/ml). The areas under the blood concentration-time curve (AUC, ng min/ml) corresponded to 812+/-182 and 3692+/-658 for LTB4 and LTE4, respectively. In immunologically thrombocytopenic rabbits, the AUC for LTB4 (86.0+/-23.0) and LTE4 (1165+/-542) were both significantly different from controls while in rabbits treated with an anti-leukocyte antiserum, both LTB4 and LTE4 were similar to controls. This experimental model provides in vivo evidence that platelets, involved in an acute inflammatory event contribute to the transcellular production of LTs.


Subject(s)
Blood Platelets/metabolism , Inflammation/metabolism , Leukotrienes/metabolism , Platelet Activation , Animals , Arachidonic Acid/metabolism , Calcimycin/pharmacology , Inflammation/blood , Ionophores/pharmacology , Leukotriene B4/pharmacology , Male , Platelet Activation/drug effects , Rabbits
11.
Blood ; 93(3): 876-85, 1999 Feb 01.
Article in English | MEDLINE | ID: mdl-9920836

ABSTRACT

Polymorphonuclear leukocyte (PMN) adhesion to activated platelets is important for the recruitment of PMN at sites of vascular damage and thrombus formation. We have recently shown that binding of activated platelets to PMN in mixed cell suspensions under shear involves P-selectin and the activated beta2-integrin CD11b/CD18. Integrin activation required signaling mechanisms that were sensitive to tyrosine kinase inhibitors.1 Here we show that mixing activated, paraformaldehyde (PFA)-fixed platelets with PMNs under shear conditions leads to rapid and fully reversible tyrosine phosphorylation of a prominent protein of 110 kD (P approximately 110). Phosphorylation was both Ca2+ and Mg2+ dependent and was blocked by antibodies against P-selectin or CD11b/CD18, suggesting that both adhesion molecules need to engage with their respective ligands to trigger phosphorylation of P approximately 110. The inhibition of P approximately 110 phosphorylation by tyrosine kinase inhibitors correlates with the inhibition of platelet/PMN aggregation. Similar effects were observed when platelets were substituted by P-selectin-transfected Chinese hamster ovary (CHO-P) cells or when PMN were stimulated with P-selectin-IgG fusion protein. CHO-P/PMN mixed-cell aggregation and P-selectin-IgG-triggered PMN/PMN aggregation as well as P approximately 110 phosphorylation were all blocked by antibodies against P-selectin or CD18. In each case PMN adhesion was sensitive to the tyrosine kinase inhibitor genistein. The antibody PL-1 against P-selectin glycoprotein ligand-1 (PSGL-1) blocked platelet/PMN aggregation, indicating that PSGL-1 was the major tethering ligand for P-selectin in this experimental system. Moreover, engagement of PSGL-1 with a nonadhesion blocking antibody triggered beta2-integrin-dependent genistein-sensitive aggregation as well as tyrosine phosphorylation in PMN. This study shows that binding of P-selectin to PSGL-1 triggers tyrosine kinase-dependent mechanisms that lead to CD11b/CD18 activation in PMN. The availability of the beta2-integrin to engage with its ligands on the neighboring cells is necessary for the tyrosine phosphorylation of P approximately 110.


Subject(s)
Blood Platelets/metabolism , CD18 Antigens/physiology , Macrophage-1 Antigen/physiology , Neutrophils/physiology , P-Selectin/physiology , Adult , Animals , CHO Cells , Calcium/physiology , Cell Adhesion , Cricetinae , Cricetulus , Enzyme Inhibitors/pharmacology , Genistein/pharmacology , Humans , Magnesium/physiology , Membrane Glycoproteins/antagonists & inhibitors , Membrane Glycoproteins/physiology , P-Selectin/genetics , Phosphorylation/drug effects , Platelet Activation , Platelet Adhesiveness/physiology , Protein Processing, Post-Translational/drug effects , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/physiology , Recombinant Fusion Proteins/physiology , Transfection
13.
J Physiol Pharmacol ; 50(4): 661-7, 1999 Dec.
Article in English | MEDLINE | ID: mdl-10639016

ABSTRACT

The occurrence of aspirin resistance has been inferred by the assessment of platelet aggregation ex vivo in patients with ischemic vascular syndromes taking aspirin. Since aspirin is a weak inhibitor of the inducible isoform of prostaglandin H synthase (COX-2), it was suggested that COX-2 may play a role in aspirin resistance. However the cellular source(s) of COX-2 possibly responsible for aspirin resistance remains unknown. Recently, the expression of the inducible isoform of COX-2 in circulating human platelets was reported. To investigate the possible contribution of COX-2 expression in platelet thromboxane (TX) biosynthesis, we have compared the inhibitory effects of NS-398 and aspirin, selective inhibitors of COX-2 and COX-1, respectively, on prostanoid biosynthesis by thrombin-stimulated platelets vs lipopolysaccharide (LPS)stimulated monocytes (expressing high levels of COX-2) isolated from whole blood of healthy subjects. NS-398 was 180-fold more potent in inhibiting monocyte COX-2 activity than platelet TXB2 production. In contrast, aspirin (55 micromol/L) largely suppressed platelet TXB2 production without affecting monocyte COX-2 activity. By using specific Western blot techniques, we failed to detect COX-2 in platelets while COX-1 was readily detectable. Our results argue against the involvement of COX-2 in TX biosynthesis by activated platelets and consequently dispute platelet COX-2 expression as an important mechanism of aspirin resistance.


Subject(s)
Blood Platelets/metabolism , Cyclooxygenase Inhibitors/pharmacology , Isoenzymes/physiology , Monocytes/metabolism , Platelet Activation , Prostaglandin-Endoperoxide Synthases/physiology , Prostaglandins/biosynthesis , Thromboxanes/biosynthesis , Adult , Aspirin/pharmacology , Blotting, Western , Cyclooxygenase 2 , Cyclooxygenase 2 Inhibitors , Dose-Response Relationship, Drug , Female , Humans , In Vitro Techniques , Lipopolysaccharides/pharmacology , Male , Membrane Proteins , Nitrobenzenes/pharmacology , Protein Isoforms , Sulfonamides/pharmacology
14.
Br J Pharmacol ; 123(8): 1691-9, 1998 Apr.
Article in English | MEDLINE | ID: mdl-9605577

ABSTRACT

1. Polymorphonuclear leukocytes (PMN) may contribute to the pathogenesis of acute coronary heart disease (CHD). 2. Epidemiological and laboratory evidence suggests that red wine, by virtue of its polyphenolic constituents, may be more effective than other alcoholic beverages in reducing the risk of CHD mortality. 3 The aim of the present study was to investigate the effects of trans-resveratrol (3,4',5-trihydroxy-trans-stilbene), a polyphenol present in most red wines, on functional and biochemical responses of PMN, upon in vitro activation. 4. trans-Resveratrol exerted a strong inhibitory effect on reactive oxygen species produced by PMN stimulated with 1 microM formyl methionyl leucyl phenylalamine (fMLP) (IC50 1.3+/-0.13 microM, mean+/-s.e.mean), as evaluated by luminol-amplified chemiluminescence. 5. trans-Resveratrol prevented the release of elastase and beta-glucuronidase by PMN stimulated with the receptor agonists fMLP (1 microM, IC50 18.4+/-1.8 and 31+/-1.8 microM), and C5a (0.1 microM, IC50 41.6+/-3.5 and 42+/-8.3 microM), and also inhibited elastase and beta-glucuronidase secretion (IC50 37.7+/-7 and 25.4+/-2.2 microM) and production of 5-lipoxygenase metabolites leukotriene B4 (LTB4), 6-trans-LTB4 and 12-trans-epi-LTB4 (IC50 48+/-7 microM) by PMN stimulated with the calcium ionophore A23187 (5 microM). 6. trans-Resveratrol significantly reduced the expression and activation of the beta2 integrin MAC-1 on PMN surface following stimulation, as revealed by FACS analysis of the binding of an anti-MAC-1 monoclonal antibody (MoAb) and of the CBRM1/5 MoAb, recognizing an activation-dependent epitope on MAC-1. Consistently, PMN homotypic aggregation and formation of mixed cell-conjugates between PMN and thrombin-stimulated fixed platelets in a dynamic system were also prevented by transresveratrol. 7. These results, indicating that trans-resveratrol interferes with the release of inflammatory mediators by activated PMN and down-regulates adhesion-dependent thrombogenic PMN functions, may provide some biological plausibility to the protective effect of red wine consumption against CHD.


Subject(s)
Enzyme Inhibitors/pharmacology , Neutrophils/drug effects , Ribonucleotide Reductases/antagonists & inhibitors , Stilbenes/pharmacology , Blood Platelets/drug effects , Blood Platelets/physiology , Calcium/metabolism , Cell Aggregation/drug effects , Cell Survival/drug effects , Cytoplasmic Granules/drug effects , Cytoplasmic Granules/metabolism , Flow Cytometry , Humans , In Vitro Techniques , Lipoxygenase/metabolism , Neutrophils/enzymology , Phosphorylation , Reactive Oxygen Species/metabolism , Resveratrol , Signal Transduction/drug effects , Tyrosine/metabolism
15.
Thromb Haemost ; 78(2): 919-25, 1997 Aug.
Article in English | MEDLINE | ID: mdl-9268195

ABSTRACT

Previous studies have shown that platelet stimulation with cathepsin G rapidly results in cytoplasmic calcium ([Ca2+]i) increase and activation of protein kinase C (PKC). To elucidate the relationship between these two biochemical events and their relative contribution to the regulation of platelet response to cathepsin G, arachidonic acid (AA) release and serotonin (5HT) secretion were studied. Platelets made Ca2+-depleted and -permeable by treatment with A23187 were compared to intact platelets to better dissociate calcium changes from other receptor-stimulated events. AA release elicited by cathepsin G in intact platelets was prevented by the Ca2+ chelator BAPTA; in Ca2+-depleted, -permeable platelets AA was released in direct response to added Ca2+ and was not increased by simultaneous stimulation with cathepsin G. In intact platelets, PKC inhibition by Ro 31-8220 or PKC induction with PMA either enhanced or reduced, respectively, cathepsin G-induced AA release. Both BAPTA and Ro 31-8220 prevented 5HT secretion from intact platelets; however, in Ca2+-depleted, -permeable platelets, cathepsin G was able to evoke 5HT secretion and p47 phosphorylation independently of [Ca2+]i increase, both effects being hampered by Ro 31-8220. Ca2+ and PKC therefore regulate PLA2 activity and 5HT secretion in cathepsin G-stimulated platelets in a different manner: the former is mainly triggered by [Ca2+]i increase, while PKC represents the major factor in determining dense granule secretion.


Subject(s)
Arachidonic Acid/metabolism , Blood Platelets/metabolism , Calcium/metabolism , Cathepsins/pharmacology , Platelet Activation/drug effects , Protein Kinase C/metabolism , Serotonin/metabolism , Cathepsin G , Cells, Cultured , Humans , Serine Endopeptidases , Signal Transduction
16.
Arch Pharm (Weinheim) ; 330(4): 100-6, 1997 Apr.
Article in English | MEDLINE | ID: mdl-9230527

ABSTRACT

6-[(4-Quinolinyl)oxy]hexanoic acids and the corresponding esters were designed and synthesized as inhibitors of the production of arachidonic acid metabolites. The inhibitory activities were assayed in vitro by evaluation of serum leukotriene B4 and thromboxane B2 production. While all 6-[(4-quinolinyl)oxy]hexanoic acids and their esters proved to be inactive, the N-alkyl-4-quinolones, obtained as by-products in their synthesis, were found to be a new class of leukotriene biosynthesis inhibitors.


Subject(s)
Enzyme Inhibitors/chemical synthesis , Leukotrienes/biosynthesis , Lipoxygenase Inhibitors , Quinolones/pharmacology , Enzyme Inhibitors/pharmacology , Humans , Leukotriene B4/blood , Magnetic Resonance Spectroscopy , Molecular Structure , Quinolones/chemical synthesis , Quinolones/chemistry , Thromboxane B2/blood
17.
Prostaglandins ; 54(4): 699-711, 1997 Oct.
Article in English | MEDLINE | ID: mdl-9440133

ABSTRACT

The kinetic profiles of leukotriene B4 (LTB4) and E4 (LTE4) after intravenous administration (30 nmol/kg) of the inflammatory peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) were evaluated in male rabbits. LTB4 and LTE4 reached the maximal concentration of 84.2 +/- 60.0 and 162.2 +/- 51.4 nmol/L (mean +/- s.d.), at 2 and 5 min, respectively. The first elimination phase for LTB4 and LTE4, after FMLP administration, showed an apparent half-life of 24.6 +/- 6.7 and 36.9 +/- 13.0 min, respectively. The area under the blood concentration-time curve (AUC, nmol min/L) of LTB4 and LTE4 was 2178 +/- 1591 and 7627 +/- 3052, respectively. LTE4 and N-ac-LTE4 were the major components excreted in the urine, mostly in the first time interval (0-12 h) of urinary collection after FMLP treatment; 11-trans-LTE4 was recovered in the second interval (12-24 h). Two other more polar compounds, potential metabolites, were recovered in the first interval of urine collection. Knowledge of the kinetic characteristics of endogenously produced leukotrienes may be useful in understanding the role of these eicosanoids in inflammatory and thrombotic disease, as well as in evaluating the efficacy of drugs designed to modulate their production and effect.


Subject(s)
Leukotriene B4/metabolism , Leukotriene E4/metabolism , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Animals , Disease Models, Animal , Humans , Infant, Newborn , Leukotriene B4/analysis , Leukotriene B4/pharmacology , Leukotriene E4/analysis , Leukotriene E4/pharmacology , Male , Rabbits , Respiratory Distress Syndrome, Newborn/physiopathology
18.
Blood ; 88(11): 4183-94, 1996 Dec 01.
Article in English | MEDLINE | ID: mdl-8943853

ABSTRACT

Adhesion between platelets and polymorphonuclear leukocytes (PMN) is a key event in thrombosis and inflammation. Double color fluorescence-activated cell sorter (FACS) analysis was used to determine the extent and kinetics of adhesion of thrombin-activated platelets to resting or activated PMN when mixed cell populations were incubated in dynamic conditions. Activated platelets bound very rapidly to PMN. Mixed cell conjugates reached a maximum at 1 minute and were reversible within 10 minutes. Platelet/PMN adhesion required both Ca2+ and Mg2+ and was markedly increased by the presence of Mn2+. The latter made mixed cell conjugates stable up to 10 minutes. Adhesion of platelets required metabolic activity of PMN and was abolished by tyrosine kinase inhibitors. Furthermore, adhesion of platelets to PMN resulted in binding of a monoclonal antibody (MoAb 24) known as beta 2 integrins "activation reporter." When PMN were activated by exogenous stimuli, the adhesion of platelets was markedly increased: fMLP induced a rapid and transient effect, while PMA resulted in a slower, but stable, increase in mixed conjugates formation. The hypothesis that activated PMN beta 2 integrins are able to bind a counter-receptor on platelets was directly demonstrated by the increase of mixed cell conjugates following PMN treatment with KIM127 and KIM185, two anti-CD18 antibodies able to induce the active conformation of beta 2 integrins. Consistently, two other anti-CD18, as well as an anti-CD11b inhibitory antibody abolished platelet/PMN adhesion. PMN beta 2 integrin activation was not the only mechanism for activated platelet/PMN adhesion to occur: indeed, this phenomenon could also be inhibited by two anti-P-selectin antibodies. Resting platelets did not adhere to resting PMN, but markedly adhered to fMLP- or PMA-activated PMN. Resting platelet/fMLP-activated PMN adhesion was abolished by anti-CD18 antibodies, but not by anti-P-selectin antibodies. In conclusion, activated platelet/PMN interaction can be modeled as an adhesion cascade involving a P-selectin-dependent recognition step and a functional signal. The latter proceeds through tyrosine kinase activation and enables a beta 2 integrin-dependent adhesion to a not yet identified counter-receptor constitutively expressed on platelet surface.


Subject(s)
Blood Platelets/cytology , CD18 Antigens/physiology , Macrophage-1 Antigen/physiology , Neutrophils/cytology , P-Selectin/physiology , Antibodies, Monoclonal/pharmacology , Blood Platelets/drug effects , Blood Platelets/metabolism , CD18 Antigens/immunology , Calcium/physiology , Cell Adhesion/drug effects , Enzyme Inhibitors/pharmacology , Humans , Macromolecular Substances , Magnesium/physiology , Manganese/pharmacology , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Neutrophils/drug effects , Neutrophils/metabolism , P-Selectin/immunology , Platelet Activation , Protein Conformation/drug effects , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/physiology , Signal Transduction/drug effects , Signal Transduction/physiology , Tetradecanoylphorbol Acetate/pharmacology
19.
J Pharm Pharmacol ; 42(12): 885-7, 1990 Dec.
Article in English | MEDLINE | ID: mdl-1983157

ABSTRACT

The racemic compound indobufen and its (+)- and (-)-enantiomers have been compared for their effects on blood platelet function and rat carrageenan pleurisy. The antiplatelet properties were studied in-vitro in human platelets by measuring the inhibition of platelet aggregation and generation of serum thromboxane (Tx) B2. In-vivo, the antiplatelet and anti-inflammatory properties were studied in rats by measuring the inhibition of serum TxB2, the amount of 6-keto-PGF1 alpha in pleural exudate and pleural exudate volume. In all tests the (+)-enantiomer was slightly more potent than the racemate, while the (-)-enantiomer was far less potent. In the same rats, treatment with the lowest doses of the compounds giving 90% inhibition of serum thromboxane B2 generation was associated with occasional macroscopic lesions of the gastric mucosa.


Subject(s)
Phenylbutyrates/pharmacology , Platelet Aggregation Inhibitors/pharmacology , Platelet Aggregation/drug effects , Thromboxane B2/biosynthesis , Animals , Gastric Mucosa/drug effects , Humans , Isoindoles , Pleurisy/metabolism , Rats , Rats, Inbred Strains , Stereoisomerism , Thromboxane B2/blood
20.
Int Angiol ; 9(4): 256-8, 1990.
Article in English | MEDLINE | ID: mdl-2099958

ABSTRACT

The aim of our study was to evaluate the effects of diet induced hypercholesterolemia and associated atherosclerosis in rabbits on serum thromboxane B2 levels. We have determined thromboxane B2 in serum of hypercholesterolemic rabbits with atherosclerosis and in normocholesterolemic rabbits without atherosclerosis. Our data show only a mildly higher serum thromboxane levels in hypercholesterolemic rabbits and extensive atherosclerosis than in controls without atherosclerosis. In conclusion, these results show that diet induced hypercholesterolemia was not associated with thromboxane B2 generation, in spite of a diffuse experimental atheromatosis.


Subject(s)
Arteriosclerosis/blood , Cholesterol, Dietary/administration & dosage , Cholesterol/blood , Hypercholesterolemia/blood , Thromboxane B2/blood , Animals , Arteriosclerosis/etiology , Hypercholesterolemia/complications , Male , Rabbits
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