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1.
Toxicol In Vitro ; 26(6): 930-8, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22542759

ABSTRACT

OBJECTIVE: Air pollution with fine particulates (PM(10) and PM(2.5)) is associated with an increased incidence of cardiovascular events. The proposed mechanisms include indirect proinflammatory and procoagulant reactions involving activation of pulmonary macrophages, endothelial cells and the TNF/TF pathway, or direct procoagulant effects. Our laboratory has observed a reduction of the platelet responsiveness to collagen after exposure to diesel exhaust particles (DEP). HYPOTHESIS: DEP directly interfere with platelet-collagen interactions by selectively inducing the shedding of platelet signaling receptors via metalloproteinases, which would represent a novel mechanism for DEP action on platelets. METHODS: Citrated blood from healthy volunteers was exposed to highly standardized DEP at concentrations of 0.1, 2.5 and 5.0µg/ml or ultrafine carbon black (ufCB, 0.1µg/ml) and the plasmatic and platelet response was analysed. The closure times with the PFA-100 device and the platelet aggregation in response to a variety of agonists were monitored. Interleukins (IL)-1ß and IL-8 levels were determined by ELISA and soluble P-selectin by the Luminex bead assay. Thrombin activity was measured as the endogenous thrombin potential (ETP) by fluorescence spectrometry. Soluble GPVI and GPIbα (glycocalicin) ectodomain fragments were measured by ELISA. ADAMTS13 activity was determined by a FRETS based assay and plasmin activity with Spectrozyme PL. RESULTS: Aggregation assays where platelets were treated with low dose DEP or ultrafine carbon black (ufCB) revealed a significantly increased response to low doses of collagen (p<0.05, n=5). At higher doses, however, collagen induced aggregation was suppressed by DEP treatment: at 2.5µg/ml, the inhibition was 34±12% (p<0.01, n=10). Aggregations with cross-linked collagen related peptide (CRPxl), convulxin and with the monoclonal antibody 9O12.2 (all known to specifically bind to and activate GPVI) were also diminished. Ristocetin, arachidonic acid and ADP responses were normal at all DEP concentrations. No cleavage of GPVI ectodomain was detected (soluble GPVI 27.8±3 vs. 28±4µg/ml mean±SEM, n=10); however increased plasma glycocalicin (GPIbα ectodomain) was detected upon diesel exposure (2.58±0.11 vs. 2.28±0.03µg/ml p<0.01, n=10). ADAMTS13 and plasmin activity remained unaffected by DEP under the conditions tested. Platelets were not activated by either DEP or ufCB as soluble P-selectin was insensitive to these. CONCLUSIONS: DEP specifically and directly interferes with platelet-collagen interactions. The functional consequences are biphasic and include enhance platelet aggregation at lower DEP concentrations and inhibition at a higher dose. Our data indicate that this interaction does not involve P-selectin or GPVI shedding. It is however associated with an increase in GPIb cleavage.


Subject(s)
Blood Platelets/drug effects , Membrane Glycoproteins/blood , Vehicle Emissions/toxicity , Blood Coagulation/drug effects , Blood Platelets/physiology , Collagen/metabolism , Humans , Platelet Aggregation/drug effects , Platelet Glycoprotein GPIb-IX Complex/metabolism , Platelet Membrane Glycoproteins/metabolism , Soot/toxicity , Thrombin/metabolism
2.
Arterioscler Thromb Vasc Biol ; 31(8): 1772-80, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21571683

ABSTRACT

OBJECTIVE: Plant-derived α-linolenic acid (ALA) may constitute an attractive cardioprotective alternative to fish-derived n-3 fatty acids. However, the effect of dietary ALA on arterial thrombus formation remains unknown. METHODS AND RESULTS: Male C57Bl/6 mice were fed a high-ALA or low-ALA diet for 2 weeks. Arterial thrombus formation was delayed in mice fed a high-ALA diet compared with those on a low-ALA diet (n=7; P<0.005). Dietary ALA impaired platelet aggregation to collagen and thrombin (n=5; P<0.005) and decreased p38 mitogen-activated protein kinase activation in platelets. Dietary ALA impaired arterial tissue factor (TF) expression, TF activity, and nuclear factor-κB activity (n=7; P<0.05); plasma clotting times and plasma thrombin generation did not differ (n=5; P=not significant). In cultured human vascular smooth muscle and endothelial cells, ALA inhibited TF expression and activity (n=4; P<0.01). Inhibition of TF expression occurred at the transcriptional level via the mitogen-activated protein kinase p38 in smooth muscle cells and p38, extracellular signal-regulated kinases 1 and 2, and c-Jun N-terminal kinases 1 and 2 in endothelial cells. CONCLUSIONS: ALA impairs arterial thrombus formation, TF expression, and platelet activation and thereby represents an attractive nutritional intervention with direct dual antithrombotic effects.


Subject(s)
Cardiotonic Agents/administration & dosage , Carotid Artery Thrombosis/prevention & control , Platelet Activation/drug effects , Thromboplastin/metabolism , alpha-Linolenic Acid/administration & dosage , Animals , Carotid Arteries/drug effects , Carotid Arteries/metabolism , Carotid Artery Thrombosis/blood , Carotid Artery Thrombosis/metabolism , Cells, Cultured , Dietary Supplements , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Gene Expression/drug effects , Humans , MAP Kinase Kinase Kinase 5/metabolism , MAP Kinase Signaling System/drug effects , Male , Mice , Mice, Inbred C57BL , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/metabolism , NF-kappa B/metabolism , Platelet Aggregation/drug effects , Thromboplastin/genetics , Tumor Necrosis Factor-alpha/pharmacology , p38 Mitogen-Activated Protein Kinases/metabolism
3.
J Thromb Thrombolysis ; 32(2): 215-22, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21505785

ABSTRACT

The role of platelet receptor gain-of-function single nucleotide polymorphisms (SNP) in cardiovascular disease is controversial. We hypothesised that certain SNPs may accelerate the development of carotid artery stenosis. The intronic PAR-1 receptor intervening sequence-14 A/T (IVSn-14 A/T) polymorphism and three additional platelet receptor polymorphisms, i.e. GPIa (807C/T), GPIbα (5T/C) and HPA-1a/HPA-1b (Pl (A1/A2)) of GPIIIa were studied. The interaction of SNPs with conventional risk factors including male gender, hypertension, high cholesterol, diabetes, advanced age and smoking were investigated. The hypothesis was tested in 114 well-characterised patients with symptomatic carotid or vertebral stenosis from the British CAVATAS population and compared the results with 97 unrelated controls. The allele frequency of the platelet gain-of-function SNP was not significantly different in the CAVATAS population as compared to controls (PAR-1A/T (P = 0.13), GPIa C/T (P = 0.25), GPIIIa HPA-1a/HPA-1b (PlA1/A2) (P = 0.66) and GPIb T/C (P = 0.20)). In the subgroup of smokers, however, the prothrombotic GPIbα C mutated allele was found in a significantly higher frequency in the patient as compared to the control group (P = 0.04). Contrary to the primary hypothesis, the PAR-1A/T SNP as well as the other SNPs tested were not over- or underrepresented in the CAVATAS population. However, a significantly increased prevalence of GPIb-α (5C/T) was found in the subgroup of smokers and may represent an important cofactor in this patient group of our hypothesis-generating study.


Subject(s)
Carotid Stenosis/genetics , Epistasis, Genetic , Platelet Membrane Glycoproteins/genetics , Polymorphism, Single Nucleotide , Vertebrobasilar Insufficiency/genetics , Age Factors , Aged , Carotid Stenosis/therapy , Female , Humans , Male , Middle Aged , Risk Factors , Sex Factors , United Kingdom , Vertebrobasilar Insufficiency/therapy
4.
Blood ; 117(21): 5723-32, 2011 May 26.
Article in English | MEDLINE | ID: mdl-21389318

ABSTRACT

This study investigated the immature platelet fraction (IPF) in assessing treatment effects in immune thrombocytopenia (ITP). IPF was measured on the Sysmex XE2100 autoanalyzer. The mean absolute-IPF (A-IPF) was lower for ITP patients than for healthy controls (3.2 vs 7.8 × 109/L, P < .01), whereas IPF percentage was greater (29.2% vs 3.2%, P < .01). All 5 patients with a platelet response to Eltrombopag, a thrombopoietic agent, but none responding to an anti-FcγRIII antibody, had corresponding A-IPF responses. Seven of 7 patients responding to RhoD immuneglobulin (anti-D) and 6 of 8 responding to intravenous immunoglobulin (IVIG) did not have corresponding increases in A-IPF, but 2 with IVIG and 1 with IVIG anti-D did. This supports inhibition of platelet destruction as the primary mechanism of intravenous anti-D and IVIG, although IVIG may also enhance thrombopoiesis. Plasma glycocalicin, released during platelet destruction, normalized as glycocalicin index, was higher in ITP patients than controls (31.36 vs 1.75, P = .001). There was an inverse correlation between glycocalicin index and A-IPF in ITP patients (r² = -0.578, P = .015), demonstrating the relationship between platelet production and destruction. Nonresponders to thrombopoietic agents had increased megakaryocytes but not increased A-IPF, suggesting that antibodies blocked platelet release. In conclusion, A-IPF measures real-time thrombopoiesis, providing insight into mechanisms of treatment effect.


Subject(s)
Blood Platelets/physiology , Immunoglobulins, Intravenous/therapeutic use , Isoantibodies/therapeutic use , Purpura, Thrombocytopenic, Idiopathic/drug therapy , Thrombopoiesis/drug effects , Adolescent , Adult , Aged , Aged, 80 and over , Benzoates/therapeutic use , Case-Control Studies , Child , Child, Preschool , Female , Humans , Hydrazines/therapeutic use , Infant , Male , Middle Aged , Platelet Count , Prognosis , Purpura, Thrombocytopenic, Idiopathic/immunology , Pyrazoles/therapeutic use , Receptors, IgG/immunology , Retrospective Studies , Rh-Hr Blood-Group System , Rho(D) Immune Globulin , Thrombocytosis , Young Adult
5.
Blood Coagul Fibrinolysis ; 19(8): 801-6, 2008 Dec.
Article in English | MEDLINE | ID: mdl-19002047

ABSTRACT

Our aim was to investigate the role of filamin cleavage and protein tyrosine phosphorylation in shear-stress-induced platelet microparticle formation and of its suppression by the monoclonal antibody (mAb) Ib-23 directed against GPIbalpha. PPACK-anticoagulated or EDTA-anticoagulated platelet-rich plasma or washed platelets were exposed to high shear stress (5000 s-1 for 5 min) in the presence of antagonists of GP Ibalpha (mAb Ib-23), of GP IIb/IIIa (abciximab) and their combination. We assessed the generation of microparticle by flow cytometry, the filamin cleavage and the protein tyrosine phosphorylation by western blotting. Microparticle formation decreased by more than 50% with mAb Ib-23 alone (P < 0.01, n = 23) but filamin cleavage was not inhibited. In contrast, abciximab did not change microparticle numbers nor filamin cleavage under the same conditions. However, when combined, the inhibitors against GP Ibalpha and GP IIb/IIIa decreased microparticle by 70% and the filamin cleavage by 20% (P < 0.05). Focal adhesion kinase and p72Syk phosphorylation was enhanced by mAb Ib-23, whereas treatment with abciximab reduced it. mAb Ib-23 inhibits platelet microparticle induced by high shear stress. The mechanism seems not to depend on filamin cleavage: abciximab allowed for full microparticle formation at similar levels of cleaved filamin, whereas the combined inhibitors reduced it. The effect of mAb Ib-23 on protein tyrosine phosphorylation supports previous data, which correlates microparticle formation with the extent of protein tyrosine dephosphorylation and mirrors the dephosphorylation by protein tyrosine phosphatase (PTP-1B) in platelets of calpain-/- knockout mice.


Subject(s)
Blood Platelets/metabolism , Blood Platelets/ultrastructure , Cell-Derived Microparticles/drug effects , Contractile Proteins/metabolism , Microfilament Proteins/metabolism , Platelet Glycoprotein GPIb-IX Complex/immunology , Proteins/metabolism , Abciximab , Antibodies, Monoclonal/pharmacology , Contractile Proteins/physiology , Filamins , Focal Adhesion Protein-Tyrosine Kinases/metabolism , Humans , Immunoglobulin Fab Fragments/pharmacology , Intracellular Signaling Peptides and Proteins/metabolism , Microfilament Proteins/physiology , Phosphorylation/drug effects , Phosphorylation/physiology , Protein-Tyrosine Kinases/metabolism , Stress, Mechanical , Syk Kinase , Tyrosine/metabolism
6.
Thromb Haemost ; 96(6): 774-80, 2006 Dec.
Article in English | MEDLINE | ID: mdl-17139372

ABSTRACT

We studied the inhibition of platelet microparticle (MP) formation and thrombin generation under high shear forces. We hypothesized that an inhibitor of the GPIb a -von Willebrand factor (vWF) interaction would be more effective in suppressing MP formation and thrombin generation than GPIIb/IIIa inhibitors. Platelet-rich plasma (PRP) anticoagulated with PPACK (D-Phe-Pro-Arg chloromethyl ketone) was exposed in a cone-and-plate viscometer (shear: 5,000 s(-1) for 5 min) in the presence of antagonists to GPIb a (the monoclonal antibody [Mab] Ib-23) or to GPIIb/IIIa (abciximab, tirofiban, eptifibatide) at their IC90 determined in platelet aggregometry with ristocetin or ADP, respectively. We used double labeling (CD41-PE and annexin-V-FITC) for flow cytometric detection of MP and their aminophospholipid exposure. Thrombin generation was measured using PRP prepared from ACD anticoagulated blood. About 40% of the thrombin generation was found to be mediated by the MP fraction of the PRP. Blockade of GPIb a with Mab Ib-23 reduced MP formation and thrombin generation by 50%, and was more effective than any GPIIb/IIIa antagonist. The combination of Mab Ib-23 with one of the GPIIb/IIIa inhibitors further reduced the MP formation to ~ 30%. The antibody also partially inhibited thrombin induced platelet aggregation. Epitope mapping suggested that Mab Ib-23 binds between the amino acids 201 and 268 of GPIb a , explaining the interference with vWF and thrombin interaction. In contrast to the commonly used GPIIb/IIIa antagonists, the blockade of GPIb a with Mab Ib-23 effectively reduces the prothrombotic MP generation and thrombin formation at shear rates typically found in arterial stenoses.


Subject(s)
Antibodies, Monoclonal/immunology , Blood Platelets/metabolism , Platelet Aggregation , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Platelet Glycoprotein GPIb-IX Complex/metabolism , Thrombin/metabolism , Abciximab , Annexin A5/metabolism , Antibodies, Monoclonal/pharmacology , Binding Sites, Antibody , Blood Platelets/drug effects , Dose-Response Relationship, Drug , Epitope Mapping , Eptifibatide , Flow Cytometry , Humans , Immunoglobulin Fab Fragments/pharmacology , In Vitro Techniques , Peptides/pharmacology , Phosphatidylserines/metabolism , Platelet Aggregation/drug effects , Platelet Aggregation Inhibitors/pharmacology , Platelet Function Tests , Platelet Glycoprotein GPIIb-IIIa Complex/antagonists & inhibitors , Platelet Glycoprotein GPIb-IX Complex/immunology , Stress, Mechanical , Tirofiban , Tyrosine/analogs & derivatives , Tyrosine/pharmacology , von Willebrand Factor/metabolism
7.
Eur J Biochem ; 270(13): 2750-8, 2003 Jul.
Article in English | MEDLINE | ID: mdl-12823545

ABSTRACT

We have isolated and characterized a second [Fe]-hydrogenase gene from the green alga, Chlamydomonas reinhardtii. The HydA2 gene encodes a protein of 505 amino acids that is 74% similar and 68% identical to the known HydA1 hydrogenase from C. reinhardtii. HydA2 contains all the conserved residues and motifs found in the catalytic core of the family of [Fe]-hydrogenases. We demonstrate that both the HydA1 and the HydA2 transcripts are expressed upon anaerobic induction, achieved either by neutral gas purging or by sulfur deprivation of the cultures. Furthermore, the expression levels of both transcripts are regulated (in some cases differently) by incubation conditions, such as the length of anaerobiosis, the readdition of O2, the presence of acetate, and/or the absence of nutrients such as sulfate during growth. Antibodies specific for HydA2 recognized a protein of about 49 kDa in extracts from anaerobically induced C. reinhardtii cells, strongly suggesting that HydA2 encodes for an expressed protein. Homology-based 3D modeling of the HydA2 hydrogenase shows that its catalytic site models well to the known structure of Clostridium pasteurianum CpI, including the H2-gas channel. The major differences between HydA1, HydA2 and CpI are the absence of the N-terminal Fe-S centers and the existence of extra sequences in the algal enzymes. To our knowledge, this work represents the first systematic study of expression of two algal [Fe]-hydrogenases in the same organism.


Subject(s)
Chlamydomonas reinhardtii/enzymology , Gene Expression Regulation , Hydrogenase/metabolism , Iron-Sulfur Proteins/metabolism , Amino Acid Sequence , Anaerobiosis , Animals , Chlamydomonas reinhardtii/genetics , Chlamydomonas reinhardtii/metabolism , Hydrogenase/chemistry , Hydrogenase/genetics , Iron-Sulfur Proteins/chemistry , Iron-Sulfur Proteins/genetics , Models, Molecular , Molecular Sequence Data , Protein Structure, Secondary , Sequence Alignment , Sulfur/metabolism
8.
Biochim Biophys Acta ; 1638(1): 29-34, 2003 May 20.
Article in English | MEDLINE | ID: mdl-12757931

ABSTRACT

It has been known for over half a century that homocysteine levels are elevated in liver cirrhosis, but the basis for it is not fully understood. Using differential display, we identified betaine homocysteine methyltransferase (BHMT) as a gene down-regulated in rat liver cirrhosis and most likely involved in this dysregulation. A partial BHMT clone was isolated by screening of a cDNA library with the differential display fragment. The full-length gene was generated by primer extension of cDNA. Expression levels of BHMT in cirrhotic livers of bile duct ligated rats were compared to controls by Northern and Western blotting as well as by enzyme activity measurements. BHMT mRNA levels were reduced to 29+/-23% in established liver cirrhosis induced by bile duct ligation (BDL) as compared to controls. Enzyme assays in crude liver homogenates showed a similar reduction in BHMT activity in bile duct ligated rat livers. By Western blotting, BHMT could be detected in crude liver homogenates of control animals, but was reduced to below the limit of detection in cirrhotic livers. In conclusion, these findings establish a reduced BHMT enzyme activity in cirrhotic rat livers, which may explain the elevated plasma homocysteine levels in cirrhosis.


Subject(s)
Liver Cirrhosis/enzymology , Liver Cirrhosis/genetics , Methyltransferases/genetics , Amino Acid Sequence , Animals , Base Sequence , Betaine-Homocysteine S-Methyltransferase , Cloning, Molecular , DNA, Complementary/genetics , Gene Expression , Gene Expression Profiling , Liver/metabolism , Liver Cirrhosis/metabolism , Male , Methionine/metabolism , Methyltransferases/metabolism , Molecular Sequence Data , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley
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