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1.
J Thromb Haemost ; 14(11): 2253-2260, 2016 11.
Article in English | MEDLINE | ID: mdl-27601054

ABSTRACT

Essentials Relationship of acquired von Willebrand disease (VWD) and platelet dysfunction is explored. Patients with ventricular assist devices and on extracorporeal membrane oxygenation are investigated. Acquired VWD and platelet receptor shedding is demonstrated in the majority of patients. Loss of platelet adhesion receptors glycoprotein (GP) Ibα and GPVI may increase bleeding risk. SUMMARY: Background Ventricular assist devices (VADs) and extracorporeal membrane oxygenation (ECMO) are associated with bleeding that is not fully explained by anticoagulant or antiplatelet use. Exposure of platelets to elevated shear in vitro leads to increased shedding. Objectives To investigate whether loss of platelet receptors occurs in vivo, and the relationship with acquired von Willebrand syndrome (AVWS). Methods Platelet counts, coagulation tests and von Willebrand factor (VWF) analyses were performed on samples from 21 continuous flow VAD (CF-VAD), 20 ECMO, 12 heart failure and seven aortic stenosis patients. Levels of platelet receptors were measured by flow cytometry or ELISA. Results The loss of high molecular weight VWF multimers was observed in 18 of 19 CF-VAD and 14 of 20 ECMO patients, consistent with AVWS. Platelet receptor shedding was demonstrated by elevated soluble glycoprotein (GP) VI levels in plasma and significantly reduced surface GPIbα and GPVI levels in CF-VAD and ECMO patients as compared with healthy donors. Platelet receptor levels were also significantly reduced in heart failure patients. Conclusions These data link AVWS and increased platelet receptor shedding in patients with CF-VADs or ECMO for the first time. Loss of the platelet surface receptors GPIbα and GPVI in heart failure, CF-VAD and ECMO patients may contribute to ablated platelet adhesion/activation, and limit thrombus formation under high/pathologic shear conditions.


Subject(s)
Blood Platelets/cytology , Extracorporeal Membrane Oxygenation , Heart-Assist Devices , Platelet Glycoprotein GPIb-IX Complex/chemistry , Platelet Membrane Glycoproteins/chemistry , Adolescent , Adult , Aged , Aortic Valve Stenosis/physiopathology , Blood Platelets/metabolism , Cohort Studies , Female , Heart Failure/complications , Hemorrhage/complications , Heparin/therapeutic use , Humans , Male , Middle Aged , Platelet Activation , Stress, Mechanical , Thrombosis , Warfarin/therapeutic use , Young Adult , von Willebrand Factor/chemistry
2.
J Thromb Haemost ; 14(8): 1642-54, 2016 08.
Article in English | MEDLINE | ID: mdl-26878340

ABSTRACT

UNLABELLED: Essentials The platelet thrombin receptor, PAR4, is an emerging anti-thrombotic drug target. We examined the anti-platelet & anti-thrombotic effects of PAR4 inhibition in human blood. PAR4 inhibition impaired platelet procoagulant activity in isolated cells and during thrombosis. Our study shows PAR4 is required for platelet procoagulant function & thrombosis in human blood. SUMMARY: Background Thrombin-induced platelet activation is important for arterial thrombosis. Thrombin activates human platelets predominantly via protease-activated receptor (PAR)1 and PAR4. PAR1 has higher affinity for thrombin, and the first PAR1 antagonist, vorapaxar, was recently approved for use as an antiplatelet agent. However, vorapaxar is contraindicated in a significant number of patients, owing to adverse bleeding events. Consequently, there is renewed interest in the role of platelet PAR4 in the setting of thrombus formation. Objectives To determine the specific antiplatelet effects of inhibiting PAR4 function during thrombus formation in human whole blood. Methods and Results We developed a rabbit polyclonal antibody against the thrombin cleavage site of PAR4, and showed it to be a highly specific inhibitor of PAR4-mediated platelet function. This function-blocking anti-PAR4 antibody was used to probe for PAR4-dependent platelet functions in human isolated platelets in the absence and presence of concomitant PAR1 inhibition. The anti-PAR4 antibody alone was sufficient to abolish the sustained elevation of cytosolic calcium level and consequent phosphatidylserine exposure induced by thrombin, but did not significantly inhibit integrin αII b ß3 activation, α-granule secretion, or aggregation. In accord with these in vitro experiments on isolated platelets, selective inhibition of PAR4, but not of PAR1, impaired thrombin activity (fluorescence resonance energy transfer-based thrombin sensor) and fibrin formation (anti-fibrin antibody) in an ex vivo whole blood flow thrombosis assay. Conclusions These findings demonstrate that PAR4 is required for platelet procoagulant function during thrombus formation in human blood, and suggest PAR4 inhibition as a potential target for the prevention of arterial thrombosis.


Subject(s)
Blood Platelets/cytology , Platelet Aggregation , Receptors, Thrombin/antagonists & inhibitors , Thrombosis/metabolism , Adult , Animals , Antibodies/chemistry , Calcium/metabolism , Cytosol/metabolism , Female , Fibrin/chemistry , Fluorescence Resonance Energy Transfer , Healthy Volunteers , Humans , Lactones/therapeutic use , Male , Mice , Mice, Transgenic , Middle Aged , P-Selectin/metabolism , Phosphatidylserines/chemistry , Platelet Activation/drug effects , Platelet Aggregation Inhibitors/chemistry , Platelet Aggregation Inhibitors/therapeutic use , Pyridines/therapeutic use , Receptor, PAR-1/metabolism , Signal Transduction , Thrombin/chemistry , Young Adult
3.
J Diabetes Res ; 2013: 370212, 2013.
Article in English | MEDLINE | ID: mdl-23841102

ABSTRACT

Thrombosis is a life-threatening complication of diabetes. Platelet reactivity is crucial to thrombus formation, particularly in arterial vessels and in thrombotic complications causing myocardial infarction or ischaemic stroke, but diabetic patients often respond poorly to current antiplatelet medication. In this study, we used a nonhuman primate model of Type 1 diabetes to measure early downstream signalling events following engagement of the major platelet collagen receptor, glycoprotein (GP)VI. Diabetic monkeys were given enough insulin to maintain their blood glucose levels either at ~8 mM (well-controlled diabetes) or ~15 mM (poorly controlled diabetes). Flow cytometric analysis was used to measure platelet reactive oxygen species (ROS) generation, calcium mobilisation, receptor surface expression, and immature platelet fraction. We observed exacerbated intracellular ROS and calcium flux associated with engagement of GPVI in monkeys with poorly controlled diabetes. GPVI surface levels did not differ between healthy monkeys or the two diabetic groups. Treatment of platelets with the specific Syk inhibitor BAY61-3606 inhibited GPVI-dependent ROS and, importantly, reduced ROS generation in the poorly controlled diabetes group to that observed in healthy monkeys. These data indicate that glycaemic control is important in reducing GPVI-dependent platelet hyperreactivity and point to a potential antithrombotic therapeutic benefit of Syk inhibition in hyperglycaemic diabetes.


Subject(s)
Blood Platelets/metabolism , Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Type 1/metabolism , Platelet Membrane Glycoproteins/metabolism , Animals , Calcium/metabolism , Female , Macaca mulatta , Male , Niacinamide/analogs & derivatives , Niacinamide/pharmacology , Platelet Aggregation , Pyrimidines/pharmacology , Reactive Oxygen Species/metabolism
4.
J Thromb Haemost ; 10(6): 1133-41, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22489915

ABSTRACT

BACKGROUND: Ligation of the platelet-specific collagen receptor, GPVI/FcRγ, causes rapid, transient disulfide-dependent homodimerization, and the production of intracellular reactive oxygen species (ROS) generated by the NADPH oxidase, linked to GPVI via TRAF4. OBJECTIVES: The aim of this study was to evaluate the role of early signaling events in ROS generation following engagement of either GPVI/FcRγ or a second immunoreceptor tyrosine-based activation motif (ITAM)-containing receptor on platelets, FcγRIIa. METHODS AND RESULTS: Using an H(2) DCF-DA-based flow cytometric assay to measure intracellular ROS, we show that treatment of platelets with either the GPVI agonists, collagen-related peptide (CRP) or convulxin (Cvx), or the FcγRIIa agonist 14A2, increased intraplatelet ROS; other platelet agonists such as ADP and TRAP did not. Basal ROS in platelet-rich plasma from 14 healthy donors displayed little inter-individual variability. CRP, Cvx or 14A2 induced an initial burst of ROS within 2 min followed by additional ROS reaching a plateau after 15-20 min. The Syk inhibitor BAY61-3606, which blocks ITAM-dependent signaling, had no effect on the initial ROS burst, but completely inhibited the second phase. CONCLUSIONS: Together, these results show for the first time that ROS generation downstream of GPVI or FcγRIIa consists of two distinct phases: an initial Syk-independent burst followed by additional Syk-dependent generation.


Subject(s)
Blood Platelets/enzymology , Intracellular Signaling Peptides and Proteins/metabolism , Platelet Membrane Glycoproteins/metabolism , Protein-Tyrosine Kinases/metabolism , Reactive Oxygen Species/metabolism , Receptors, IgG/metabolism , Signal Transduction , Amino Acid Motifs , Blood Platelets/drug effects , Carrier Proteins/pharmacology , Crotalid Venoms/pharmacology , Enzyme Activation , Female , Flow Cytometry , Humans , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Lectins, C-Type , Male , Niacinamide/analogs & derivatives , Niacinamide/pharmacology , Peptides/pharmacology , Platelet Membrane Glycoproteins/agonists , Protein Kinase Inhibitors/pharmacology , Protein Structure, Tertiary , Protein-Tyrosine Kinases/antagonists & inhibitors , Pyrimidines/pharmacology , Receptors, IgG/agonists , Signal Transduction/drug effects , Syk Kinase , Time Factors
5.
Thromb Haemost ; 107(4): 648-55, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22274761

ABSTRACT

New methods for analysing both platelet and plasma forms of the platelet-specific collagen receptor, glycoprotein VI (GPVI) in experimental models or human clinical samples, and the development of the first therapeutic compounds based on dimeric soluble GPVI-Fc or anti-GPVI antibody-based constructs, coincide with increased understanding of the potential pathophysiological role of GPVI ligand binding and shedding. Platelet GPVI not only mediates platelet activation at the site of vascular injury where collagen is exposed, but is also implicated in the pathogenesis of other diseases, such as atherosclerosis and coagulopathy, rheumatoid arthritis and tumour metastasis. Here, we describe some of the critical mechanisms for generating soluble GPVI from platelets, and future avenues for exploiting this unique platelet-specific receptor for diagnosis and/or disease prevention.


Subject(s)
Blood Platelets/metabolism , Platelet Membrane Glycoproteins/metabolism , Thrombocytopenia/blood , Thrombosis/blood , Amino Acid Sequence , Animals , Antibodies, Monoclonal/chemistry , Arthritis, Rheumatoid/metabolism , Atherosclerosis/metabolism , Blood Coagulation , Humans , Immunoglobulin Fc Fragments/chemistry , Ligands , Models, Biological , Molecular Sequence Data , Neoplasm Metastasis , Platelet Membrane Glycoproteins/chemistry , Protein Binding
6.
J Thromb Haemost ; 9(1): 163-72, 2011 Jan.
Article in English | MEDLINE | ID: mdl-20946164

ABSTRACT

BACKGROUND: Reactive oxygen species generation is one consequence of ligand engagement of platelet glycoprotein (GP) receptors GPIb-IX-V and GPVI, which bind VWF/collagen and initiate thrombosis at arterial shear; however, the precise molecular mechanism coupling redox pathway activation to engagement of these receptors is unknown. OBJECTIVE: The objective of this study was to identify novel binding partners for GPIb-IX-V and GPVI that could provide a potential link between redox pathways and early platelet signaling events. METHODS AND RESULTS: Using protein array analysis and affinity-binding assays, we demonstrated that the orphan TNF receptor-associated factor (TRAF) family member, TRAF4, selectively binds cytoplasmic sequences of GPIbß and GPVI. TRAF4, p47(phox) [of the NADPH oxidase (Nox2) enzyme complex] and other redox relevant signaling proteins such as Hic-5, co-immunoprecipitate with GPIb/GPVI from human platelet lysates whilst MBP-TRAF4 or MBP-p47(phox) fusion proteins specifically pull-down GPIb/GPVI. GPIb- or GPVI-selective agonists induce phosphorylation of the TRAF4-associated proteins, Hic-5 and Pyk2, with phosphorylation attenuated by Nox2 inhibition. CONCLUSION: These results describe the first direct association of TRAF4 with a receptor, and identify a novel binding partner for GPIb-IX-V and GPVI, providing a potential link between these platelet receptors and downstream TRAF4/Nox2-dependent redox pathways.


Subject(s)
Blood Platelets/metabolism , Platelet Glycoprotein GPIb-IX Complex/metabolism , Platelet Membrane Glycoproteins/metabolism , TNF Receptor-Associated Factor 4/blood , Chromatography, Affinity , Focal Adhesion Kinase 2/blood , Humans , Immunoprecipitation , Intracellular Signaling Peptides and Proteins/blood , LIM Domain Proteins , NADPH Oxidases/blood , Oxidation-Reduction , Phosphorylation , Protein Array Analysis , Protein Binding , Protein Interaction Domains and Motifs , Protein Interaction Mapping , Recombinant Fusion Proteins/metabolism , Signal Transduction
7.
J Thromb Haemost ; 5(7): 1530-7, 2007 Jul.
Article in English | MEDLINE | ID: mdl-17445093

ABSTRACT

BACKGROUND: Platelet glycoprotein (GP)VI that binds collagen, and GPIb-IX-V that binds von Willebrand factor, initiate thrombus formation. OBJECTIVES: In this study, we investigated the mechanisms of metalloproteinase-mediated ectodomain shedding that regulate the surface expression of GPVI, GPIbalpha (the major ligand-binding subunit) and GPV (that regulates thrombin-dependent activation via GPIbalpha). METHODS AND RESULTS: Immunoblotting human platelet lysates using affinity-purified antibodies against cytoplasmic domains of GPVI, GPIbalpha or GPV allowed simultaneous analysis of intact and cleaved receptor, and revealed (i) that a significant fraction of GPIbalpha, but not GPVI, exists in a cleaved state on platelets, even when isolated in the presence of metalloproteinase inhibitor (GM6001) or EDTA; (ii) the same-sized membrane-associated fragments of GPVI or GPIbalpha are generated by phorbol-ester (PMA), the mitochondrial-targeting reagent CCCP, the calmodulin inhibitor W7, or the thiol-modifying reagent, N-ethylmaleimide, that directly activates ADAM10/ADAM17; and (iii) GPV is shed by both metalloproteinase- and thrombin-dependent mechanisms, depending on the concentration of thrombin. Based on the predicted cleavage area defined by these studies, ADAM10, but not ADAM17, cleaved a GPVI-based synthetic peptide within the extracellular membrane-proximal sequence (PAR;Q(243)YY) as analyzed by MALDI-TOF-MS. In contrast, ADAM17, but not ADAM10, cleaved within the GPIbalpha-based peptide (LRG;V(465)LQ). Both ADAM10 and ADAM17 cleaved within a GPV-based peptide (AQP;V(494)TT). Metalloproteinase-mediated shedding of GPIbalpha from GPIb-IX-transfected or GPVI-transfected cells induced by W7 or N-ethylmaleimide was inhibited by mutagenesis of sequences identified from peptide analysis. CONCLUSIONS: These findings suggest surface levels of GPVI, GPIbalpha and GPV may be controlled by distinct mechanisms involving ADAM10 and/or ADAM17.


Subject(s)
ADAM Proteins/blood , Blood Platelets/metabolism , Platelet Glycoprotein GPIb-IX Complex/metabolism , Platelet Membrane Glycoproteins/metabolism , ADAM Proteins/genetics , ADAM Proteins/metabolism , ADAM10 Protein , ADAM17 Protein , Amino Acid Sequence , Amyloid Precursor Protein Secretases/genetics , Amyloid Precursor Protein Secretases/metabolism , Animals , Binding Sites/genetics , Blood Platelets/drug effects , Cell Line , Dipeptides/pharmacology , Humans , In Vitro Techniques , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mutagenesis, Site-Directed , Platelet Glycoprotein GPIb-IX Complex/genetics , Platelet Membrane Glycoproteins/genetics , Protease Inhibitors/pharmacology , Rats , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Transfection
8.
J Mol Cell Cardiol ; 33(10): 1861-9, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11603927

ABSTRACT

Reperfusion of globally ischemic rat hearts causes rapid generation of inositol(1,4,5) trisphosphate [Ins(1,4,5)P(3)] and the development of arrhythmias, following stimulation of alpha(1)-adrenergic receptors by norepinephrine released from the cardiac sympathetic nerves. The heightened inositol phosphate response in reperfusion depends on the activation of the Na(+)/H(+) exchanger, which might reflect a central role for increased Ca(2+)following reverse mode activation of the Na(+)/Ca(2+) exchanger (NCX). Isolated, perfused rat hearts were subjected to 20 min ischemia followed by 2 min reperfusion and the content of Ins(1,4,5)P(3) measured by mass analysis or by anion-exchange high performance liquid chromatography (HPLC) following [(3)H]inositol labeling. Reperfusion caused generation of Ins(1,4,5)P(3) (1266+/-401 to 3387+/-256 cpm/g tissue, mean+/-s.e.m., n=6, P<0.01) and the development of arrhythmias. Inhibition of NCX either by reperfusion at low Ca(2+) (1133+/-173 cpm/g tissue, mean+/-s.e.m., n=6, P<0.01 relative to reperfusion control) or by adding 10 microm KB-R7943, an inhibitor of reverse mode Na(+)/Ca(2+) exchange, prevented the Ins(1,4,5)P(3) response (1151+/-243 cpm/g tissue, mean+/-s.e.m., n=6, P<0.01 relative to reperfusion control) and the development of ventricular fibrillation. Lower concentrations of KB-R7943 were less effective. Reverse mode activation of NCX is therefore required for the enhanced Ins(1,4,5)P(3) response in early reperfusion, and inhibitors of this transporter may be useful in the prevention of arrhythmias under such conditions.


Subject(s)
Arrhythmias, Cardiac/metabolism , Inositol 1,4,5-Trisphosphate/biosynthesis , Reperfusion , Thiourea/analogs & derivatives , Animals , Calcium/metabolism , Chromatography, High Pressure Liquid , Dose-Response Relationship, Drug , Heart/physiology , Lithium Chloride/pharmacology , Male , Models, Biological , Myocardium/metabolism , Norepinephrine/pharmacology , Perfusion , Rats , Rats, Sprague-Dawley , Sodium-Calcium Exchanger , Thiourea/pharmacology
9.
Circ Res ; 89(4): 343-50, 2001 Aug 17.
Article in English | MEDLINE | ID: mdl-11509451

ABSTRACT

Activation of the alpha(1A)-adrenergic receptor (alpha(1A)-AR)/Gq pathway has been implicated as a critical trigger for the development of cardiac hypertrophy. However, direct evidence from in vivo studies is still lacking. To address this issue, transgenic mice with cardiac-targeted overexpression of the alpha(1A)-AR (4- to 170-fold) were generated, using the rodent alpha-myosin heavy chain promoter. Heterozygous animals displayed marked enhancement of cardiac contractility, evident from increases in dP/dt(max) (80%, P<0.0001), dP/dt(max)/LVP(inst) (76%, P<0.001), dP/dt(max):dP/dt(min) (104%, P<0.0001), and fractional shortening (33%, P<0.05). Moreover, changes in the dP/dt(max)-end-diastolic volume relationship provided load-independent evidence of a primary increase in contractility. Blood pressure and heart rate were largely unchanged, and there was a small increase in (-)norepinephrine-stimulated, but not basal, phospholipase C activity. Increased contractility was directly related to the level of receptor overexpression and could be completely reversed by acute alpha(1A)- but not beta-AR blockade. Despite the robust changes in contractility, transgenic animals displayed no morphological, histological, or echocardiographic evidence of left ventricular hypertrophy. In addition, apart from an increase in atrial natriuretic factor mRNA, expression of other hypertrophy-associated genes was unchanged. To our knowledge, these data provide the first in vivo evidence for an inotropic action of the alpha(1A)-AR.


Subject(s)
Cardiomegaly , Gene Expression/physiology , Gene Targeting , Myocardial Contraction/physiology , Receptors, Adrenergic, alpha-1/biosynthesis , Adenylyl Cyclases/metabolism , Adrenergic alpha-1 Receptor Antagonists , Adrenergic alpha-Agonists/pharmacology , Adrenergic alpha-Antagonists/pharmacology , Animals , Atrial Natriuretic Factor/genetics , Atrial Natriuretic Factor/metabolism , Blood Pressure/genetics , Echocardiography , Electrocardiography/drug effects , Heart Rate/genetics , Heterozygote , Inositol Phosphates/metabolism , Mice , Mice, Transgenic , Myocardial Contraction/drug effects , Myosin Heavy Chains/genetics , Organ Size/genetics , Organ Specificity/genetics , Promoter Regions, Genetic , RNA, Messenger/metabolism , Receptors, Adrenergic, alpha-1/genetics , Transgenes/physiology , Type C Phospholipases/metabolism
10.
J Biol Chem ; 276(40): 37341-6, 2001 Oct 05.
Article in English | MEDLINE | ID: mdl-11489909

ABSTRACT

Activation of phospholipase C (PLC) in neonatal rat cardiomyocytes (NCM) generates primarily inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) in response to rises in intracellular Ca(2+), or inositol 1,4-bisphosphate (Ins(1,4)P(2)) in response to norepinephrine (NE) (Matkovich, S. J. and Woodcock, E. A. (2000) J. Biol. Chem. 275, 10845-10850). To examine the PLC subtype mediating the alpha(1)-adrenergic receptor response, PLC-beta(1) and PLC-beta(3) were overexpressed in NCM using adenoviral infection (Ad-PLC-beta(1) NCM and Ad-PLC-beta(3) NCM, respectively) and PLC responses assessed from [(3)H]inositol phosphate (InsP) generation in the presence of 10 mm LiCl. The [(3)H]InsP response to NE (100 microm) was enhanced in Ad-PLC-beta(1) NCM relative to cells infected with blank virus (Ad-MX NCM), but was reduced in Ad-PLC-beta(3) NCM. In contrast, the [(3)H]InsP response to ATP (100 microm) was not elevated in Ad-PLC-beta(1) NCM, and was enhanced rather than diminished in Ad-PLC-beta(3) NCM, showing that effects of the two PLC-beta isoforms were specific for particular receptor types. PLC-delta(1) overexpression selectively reduced NE-induced [(3)H]InsP responses, without affecting the ATP stimulation. The reduced NE response was associated with a selective loss of PLC-beta(1) expression in Ad-PLC-delta(1) NCM. alpha(1)-Adrenergic receptor activation caused phosphorylation of PLC-beta(1) but not PLC-beta(3), whereas stimulation by ATP induced phosphorylation of PLC-beta(3) but not PLC-beta(1.) Taken together, these studies provide evidence that NE-stimulated InsP generation in NCM is primarily mediated by PLC-beta(1), despite the presence of both PLC-beta(1) and PLC-beta(3) isoforms.


Subject(s)
Isoenzymes/metabolism , Myocardium/metabolism , Receptors, Adrenergic, alpha-1/metabolism , Type C Phospholipases/metabolism , Animals , Animals, Newborn , GTP-Binding Protein alpha Subunits, Gq-G11 , Heterotrimeric GTP-Binding Proteins/metabolism , Inositol Phosphates/metabolism , Phospholipase C beta , Phospholipase C delta , Phosphorylation , Rats , Rats, Sprague-Dawley , Receptors, Purinergic P2/metabolism , Receptors, Purinergic P2Y2
11.
J Mol Cell Cardiol ; 32(10): 1851-8, 2000 Oct.
Article in English | MEDLINE | ID: mdl-11013129

ABSTRACT

Post-ischemic reperfusion causes a change in inositol phosphate responses to norepinephrine from primary generation of inositol(1,4) bis phosphate (Ins(1,4)P(2)) to generation of inositol(1,4,5) tris phosphate (Ins(1,4,5)P(3)) that is required for the initiation of reperfusion arrhythmias. The current study was undertaken to investigate the role of Na(+)/H(+)exchange in facilitating this transient change in inositol phosphate response. Rat hearts were subjected to 20 min ischemia followed by 2 min reperfusion and Ins(1, 4,5)P(3)content was measured by mass analysis or by anion-exchange HPLC following [(3)H]inositol labeling. Reperfusion caused generation of [(3)H]Ins(1,4,5)P(3)(1732+/-398 to 3103+/-214, cpm/g tissue, mean+/-S.E.M., n=5, P<0.01) and the development of arrhythmias. Inhibition of Na(+)/H(+)exchange, by reperfusing at pH 6.3 or by pretreating with HOE-694 (10 n M-3 microM) or HOE-642 (3 microM) prevented the [(3)H]Ins(1,4,5)P(3)generation, without causing any suppression of norepinephrine release. Increases in Ins(1,4,5)P(3)mass were similarly reduced by inhibition of Na(+)/H(+)exchange. Thus, activation of Na(+)/H(+)exchange is required for the enhanced Ins(1,4,5)P(3)response observed under reperfusion conditions, and prevention of Ins(1,4,5)P(3)generation may be an important contributor to the anti-arrhythmic actions of inhibitors of Na(+)/H(+)exchange.


Subject(s)
Inositol 1,4,5-Trisphosphate/metabolism , Myocardial Reperfusion , Sodium-Hydrogen Exchangers/metabolism , Animals , Arrhythmias, Cardiac , Chromatography, High Pressure Liquid , Guanidines/pharmacology , Hydrogen-Ion Concentration , Male , Models, Biological , Norepinephrine/metabolism , Perfusion , Rats , Rats, Sprague-Dawley , Reperfusion Injury , Sodium-Hydrogen Exchangers/antagonists & inhibitors , Sodium-Hydrogen Exchangers/physiology , Sulfones/pharmacology
12.
Clin Exp Pharmacol Physiol ; 27(9): 734-7, 2000 Sep.
Article in English | MEDLINE | ID: mdl-10972542

ABSTRACT

1. The present review focuses on the role of the Ca2+-releasing second messenger inositol 1,4,5-trisphosphate (IP3) in initiating arrhythmias during early reperfusion following a period of myocardial ischaemia. 2. Evidence for an arrhythmogenic action of IP3 was provided by studies showing a correlation between the extent of the increase in IP3 and the incidence of arrhythmias in early reperfusion. In addition, phospholipase C inhibitors selective for thrombin receptor stimulation were anti-arrhythmic only when arrhythmias were thrombin initiated. 3. Mechanisms by which IP3 could initiate arrhythmias are discussed, with particular emphasis on the role of slow and unscheduled Ca2+ release. 4. The reperfusion-induced IP3 and arrhythmogenic responses can be initiated through either alpha1-adrenoceptors or thrombin receptors, but endothelin receptor stimulation was ineffective. Further studies have provided evidence that the noradrenaline-mediated response was mediated by alpha1A-receptors, while the alpha1B-adrenoceptor subtype appeared to be protective. 5. Reperfusion-induced IP3 responses could be inhibited by procedures known to reduce the incidence of arrhythmias under these conditions, including preconditioning, inhibiting Na+/H+ exchange or by dietary supplementation with n-3 polyunsaturated fatty acids. 6. Inositol 1,4,5-trisphosphate generation in cardiomyocytes can be facilitated by raising intracellular Ca2+ and it seems likely that the rise in Ca2+ in ischaemia and reperfusion is responsible for the generation of IP3, which will, in turn, further exacerbate Ca2+ overload.


Subject(s)
Arrhythmias, Cardiac/physiopathology , Inositol 1,4,5-Trisphosphate/physiology , Myocardial Reperfusion Injury/physiopathology , Animals , Arrhythmias, Cardiac/etiology , Arrhythmias, Cardiac/metabolism , Calcium Signaling/physiology , Humans , Myocardial Reperfusion Injury/metabolism
13.
Arterioscler Thromb Vasc Biol ; 17(4): 737-40, 1997 Apr.
Article in English | MEDLINE | ID: mdl-9108788

ABSTRACT

A Silastic collar placed around the common carotid artery of rabbits causes the formation, within 7 days, of an atheroma-like neointima containing cells with the appearance of synthetic-phenotype smooth muscle cells. Using immunohisto-chemistry, we detected the appearance of the cytokine-inducible form of nitric oxide synthase (iNOS, or isoform II) in the neointima of rabbits that had the collar in place for 7 or 14 days. This iNOS immunofluorescence collocalized with anti-smooth muscle myosin in the intima, indicating that it is expressed in smooth muscle cells, and iNOS was also present in a few endothelial cells in collared sections. There was no evidence of iNOS expression in the arterial wall before the neointima was apparent, that is, after only 2 days with the collar. The expression of endothelial NOS (eNOS, or isoform III) immunofluorescence was confined to the endothelial cells in control sections, as it was in collared sections with neointima at 7 and 14 days. Specific immunofluorescence for neuronal NOS (nNOS, or isoform I) was not observed in any sections. Our results suggest that nitric oxide is produced by the inducible isoform of NOS in modified smooth muscle cells of the developing neointima. Activity of iNOS might deprive the endothelium of substrate for nitric oxide production and might explain the compromised endothelium-dependent vasodilatation observed both in this model of atherosclerosis and in human coronary artery disease.


Subject(s)
Arteriosclerosis/enzymology , Muscle, Smooth, Vascular/enzymology , Nitric Oxide Synthase/biosynthesis , Tunica Intima/enzymology , Animals , Carotid Arteries , Endothelium, Vascular/enzymology , Enzyme Induction , Fluorescent Antibody Technique, Indirect , Myosins/metabolism , Rabbits , Regional Blood Flow , Rheology , Time Factors
14.
Cancer Gene Ther ; 4(1): 17-25, 1997.
Article in English | MEDLINE | ID: mdl-9012447

ABSTRACT

Dendritic cells (DCs) are the most potent antigen-presenting cells (APCs) for the initiation of antigen-specific T-cell activation. DCs may be highly enriched from peripheral blood-adherent leukocytes by short-term (7-day) culture in the presence of interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor. Various methods of gene transfer were studied, including DNA/liposome complexes, electroporation, CaPO4 precipitation, and recombinant adenovirus (AdV) vectors. Low levels of expression were obtained with the physical methods tested. In contrast, AdV vectors expressing luciferase, beta-galactosidase, IL-2, and IL-7 all readily transduced human DCs. Increasing levels of gene expression were observed over a range of multiplicity of infection (MOI) of 10:1 to 10,000:1, with transduction efficiencies exceeding 95% at higher MOI. Although levels of maximal gene expression in DCs were significantly lower than those obtained using human tumor cell lines, IL-2 and IL-7 production of up to 5 x 10(2) ng/10(6) DC were achieved. These results suggest that AdV vectors are a promising vehicle for genetically engineering human DCs.


Subject(s)
Dendritic Cells , Gene Transfer Techniques , Dendritic Cells/immunology , Flow Cytometry , Humans , Immunophenotyping , Interleukin-2/biosynthesis , Interleukin-2/genetics , Interleukin-7/biosynthesis , Interleukin-7/genetics , Methods , Recombination, Genetic
15.
Clin Exp Pharmacol Physiol ; 22(6-7): 452-4, 1995.
Article in English | MEDLINE | ID: mdl-8582100

ABSTRACT

1. Nitric oxide (NO) synthase inhibitors and Paf antagonists abrogate hypotension in septic shock. The latter may act by blocking intracellular transduction mechanisms in vascular smooth muscle cells and inflammatory cells. We examined the effect of Paf antagonists on expression of inducible NO synthase. 2. A murine macrophage cell line (J774.2) and rat vascular smooth muscle cells (VSMC) were stimulated with lipopolysaccharide (LPS), either alone or in combination with Paf or Paf antagonists, BN 50739 or E-6123. 3. NO synthase activity in J774.2 was measured by the conversion of [3H]L-arginine to [3H]L-citrulline. Nitrite accumulation was measured in the culture medium of J774.2 and VSM. 4. BN 50739 (10 mumol/L and E-6123 (1 mumol#L) both reduced the expression of calcium-independent NO synthase activity and nitrite accumulation, while Paf alone had no effect. 5. Inhibition of NO synthase induction by Paf antagonists might afford therapeutic benefits in the management of septic shock and possibly other cardiovascular disorders.


Subject(s)
Azepines/pharmacology , Macrophages/drug effects , Muscle, Smooth, Vascular/drug effects , Nitric Oxide Synthase/antagonists & inhibitors , Platelet Activating Factor/antagonists & inhibitors , Triazoles/pharmacology , Animals , Arginine/metabolism , Cell Line , Cells, Cultured , Citrulline/analysis , Citrulline/metabolism , Enzyme Induction/drug effects , Lipopolysaccharides/toxicity , Macrophages/cytology , Macrophages/enzymology , Mice , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/enzymology , Nitric Oxide Synthase/biosynthesis , Nitrites/analysis , Nitrites/metabolism , Platelet Activating Factor/physiology , Rats
16.
J Vasc Res ; 31(4): 187-94, 1994.
Article in English | MEDLINE | ID: mdl-8025180

ABSTRACT

The aim of this study was to use periarterial manipulation to produce an atheroma-like neo-intima in rabbits and study resting blood flow and vascular responsiveness in vivo. One common carotid artery was enclosed in a silastic collar to induce a neo-intima similar to that of human early atherosclerosis, and carotid blood flow was measured periodically over 8 days in 8 conscious rabbits. The vasodilator responses to intravenous infusions of the endothelium-dependent vasodilator, acetylcholine, and glyceryl trinitrate were measured in each artery at 2 and 7 days after surgical placement of the collar, and again following infusion of the nitric oxide synthase inhibitor, N-nitro-L-arginine (NOLA, 15 mg/kg). Histological examination of the arterial segments at completion of the study revealed significant intimal thickening of the regions of artery enclosed in the collar. Resting blood flow was lower in the collared vascular bed as compared with the control, from as early as 2 days after surgery. Acetylcholine- and glyceryl trinitrate-induced decreases in carotid resistance, however, were no different between the arteries after 2 days. At 7 days after surgery, the vasodilator response to acetylcholine was significantly impaired in the collared vascular bed when compared with the control, while the glyceryl trinitrate-induced vasodilatation was similar in the two beds. Following NOLA infusion, mean arterial pressure was significantly increased and blood flow through both arteries was reduced. After NOLA, acetylcholine-induced vasodilatation in the collared vascular bed was no longer different from the vasodilatation in the control bed.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Intracranial Arteriosclerosis/physiopathology , Nitric Oxide/physiology , Tunica Intima/physiopathology , Vasodilation , Acetylcholine/pharmacology , Animals , Carotid Arteries/drug effects , Female , Intracranial Arteriosclerosis/pathology , Male , Nitric Oxide/antagonists & inhibitors , Rabbits , Regional Blood Flow , Rest , Tunica Intima/pathology
17.
N Z Med J ; 105(931): 117-20, 1992 Apr 08.
Article in English | MEDLINE | ID: mdl-1313956

ABSTRACT

Data on all new breast cancer cases in the Auckland area during the nine years September 1976 to September 1985 were used to obtain epidemiological information on breast cancer in the Auckland region. Breast tumours were found in 2706 women (300 per year), yielding a lifetime risk of breast cancer of one in 15. No significant difference in breast cancer incidence was detected between European, Maori and Pacific Island Polynesian women. Confidence limits for incidence were wide in the later groups. Fifty-one percent of women presented with intermediate sized (2-5 cm) tumours, and most (66%) were node negative. Eleven percent had evidence of metastatic disease at presentation. When the relationships between race, tumour size, nodal status and metastases were examined, Pacific Island women more frequently presented with large tumours and metastases, whereas Maori women were more frequently node positive. Eighty-five percent of tumours were invasive ductal carcinomas, 55% grade II, 35% grade III, and 10% grade I. Sixty-seven percent of tumours were oestrogen receptor positive (ER+ve) and ER status was significantly related to age; the proportion of ER+ve tumours was greater in older women. Fifty-seven percent of tumours were progesterone receptor positive (PR+ve), and PR distribution was bimodal with age. These data from the Auckland region are similar to breast cancer figures from other western countries, with some ethnic differences in tumour size and frequency of metastatic disease at presentation.


Subject(s)
Breast Neoplasms/epidemiology , Carcinoma, Intraductal, Noninfiltrating/epidemiology , Adolescent , Adult , Age Factors , Aged , Aged, 80 and over , Breast Neoplasms/ethnology , Breast Neoplasms/pathology , Carcinoma, Intraductal, Noninfiltrating/ethnology , Carcinoma, Intraductal, Noninfiltrating/pathology , Female , Humans , Incidence , Lymphatic Metastasis , Middle Aged , Neoplasm Metastasis , New Zealand/epidemiology , Receptors, Estrogen/analysis , Receptors, Progesterone/analysis
18.
Dig Dis Sci ; 36(4): 532-5, 1991 Apr.
Article in English | MEDLINE | ID: mdl-2007373

ABSTRACT

A case of chronic intestinal pseudoobstruction secondary to systemic amyloidosis in a patient with multiple myeloma is described. Gastrointestinal symptoms and indices of nutrition improved markedly after commencing treatment with cisapride, which may have been responsible for relatively prolonged survival compared with similar reported cases.


Subject(s)
Amyloidosis/complications , Intestinal Diseases/drug therapy , Intestinal Pseudo-Obstruction/drug therapy , Piperidines/therapeutic use , Serotonin Antagonists/therapeutic use , Stomach Diseases/drug therapy , Cisapride , Female , Humans , Intestinal Diseases/etiology , Intestinal Pseudo-Obstruction/etiology , Middle Aged , Stomach Diseases/etiology
19.
Can J Surg ; 26(5): 475-6, 1983 Sep.
Article in English | MEDLINE | ID: mdl-6351989

ABSTRACT

Thirty cases of giant cell tumour of bone are reviewed. The average follow-up for 22 cases was 7.5 years. The recurrence rate in 19 appendicular tumours treated by curettage, with or without bone grafting, was 52%. Treatment of recurrences, however, resulted in cure of an additional 10 tumours, for an overall success rate of primary and secondary treatment of 89%. Pulmonary metastases occurred in two patients (6.7%) and there was one death attributable to the tumour (3.3%). The authors conclude that the conservative primary treatment is justified even with a 52% recurrence rate, since normal function and appearance are preserved and, further, more aggressive treatment of the recurrent tumour can be performed. They prefer limb-saving resection and occasionally amputation for this more aggressive secondary treatment rather than cryosurgery with its complications, allograft replacement with its uncertainties or supervoltage radiation with its risk of later development of malignant tumours.


Subject(s)
Bone Neoplasms/surgery , Giant Cell Tumors/surgery , Adolescent , Adult , Aged , Amputation, Surgical , Bone Neoplasms/radiotherapy , Bone Transplantation , Child , Combined Modality Therapy , Curettage , Female , Follow-Up Studies , Giant Cell Tumors/radiotherapy , Humans , Lung Neoplasms/secondary , Male , Middle Aged , Neoplasm Recurrence, Local/therapy
20.
Cancer ; 51(11): 1992-7, 1983 Jun 01.
Article in English | MEDLINE | ID: mdl-6340820

ABSTRACT

Between September 1976 and May 1980, 135 patients with operable breast cancer and positive axillary nodes received l-phenylalanine mustard, adjunct to surgery, 0.15 mg/kg for five days, six weekly, and were randomised prospectively to levamisole 150 mg for three days, two weekly, or a placebo. Treatment was continued for two years or until evidence of treatment failure, whichever was the sooner. At 4 1/2 years, for all patients, there was no significant difference between the two groups (P = 0.09), but in a subgroup of women less than or equal to 50 with 1-3 positive nodes, levamisole had a negative effect (P = 0.05). Although an analysis of the same age group, independent of the nodal status, did not reach significance, there was a trend in favor of placebo (P = 0.08) which was also apparent in premenopausal women (P = 0.15). In postmenopausal patients, however, and in those with more advanced disease with four or more positive nodes, although the results also failed to reach significance the trend in these subgroups favored levamisole. The results of this study suggest that levamisole has no place in the primary therapy of breast cancer in younger women and those with more favorable disease. The value of this agent in older patients and those with more advanced primary disease, remains unproven, but the favorable trends are in accord with a number of other studies with levamisole in metastatic breast and resectable lung cancer. Retrospective analysis confined to those women who received 75% or more of the total dose of l-phenylalanine mustard showed no evidence for a dose-responsive effect of adjuvant chemotherapy on the described pattern of results.


Subject(s)
Breast Neoplasms/drug therapy , Levamisole/administration & dosage , Melphalan/administration & dosage , Age Factors , Clinical Trials as Topic , Drug Therapy, Combination , Female , Humans , Leukopenia/chemically induced , Lymphatic Metastasis , Menopause , Middle Aged , Placebos , Retrospective Studies , Thrombocytopenia/chemically induced
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