摘要:

Young or reticulated platelets contain some residual mRNA, which is rapidly degraded after platelet release into the circulation. In order to minimize platelet activation and possible loss of large platelets during sample handling a whole blood method has been developed utilising the RNA fluorochrome thiazole orange (TO) in combination with an antibody to anti-glycoprotein Ib (GpIb) directly conjugated to phycoerythrin (PE), to specifically stain reticulated platelets via flow cytometric analysis. In this study whole blood analysis of platelet mRNA was undertaken in healthy normal subjects and a variety of patients with haematological abnormalities. The percentage of Gp Ib positive platelets containing mRNA in normals (n= 22) was 11.61% with a two SD range of 3.19-20.01%. The percentage of reticulated platelets was significantly increased (mean mRNA content±one SD) in sickle cell disorders (n= 22) 38.12%±18.42 (P<0.001); thalassaemia (major, intermedia and trait) (n= 24) 29.76±19.15 (P<0.001); ITP (N= 20) 23.53%±13.04 (P<0.02) and essentialthrombocythemia (N= 32) 37.12±19.84 (P<0.001). Platelets from patients with reactive thrombocytosis (N= 15) were only 12.23% positive (±6.95) and not significantly different from the normal range (P= 0.95). This method offers a rapid and simple procedure for assessment of reticulated platelets in whole blood and suggests that there may be an increased platelet turnover in certain haemoglobinopathies.

ISSN:0953-7104    

DOI:10.3109/09537109609023578

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

The total lipids of the human platelet plasma membrane (HPPM) from 50 ml of blood of healthy subjects were extracted, quantified and related to the mass content of the major intrinsic membrane protein, the glycoprotein IIb/IIIa (GPIIb/IIIa). The HPPM total lipid/GPIIb/IIIa weight ratio determined was 5.40±0.20, independently of the membrane washing procedure used, with the cholesterol/GPIIb/IIIa and phospholipid/GPIIb/IIIa molar ratios of 800±50 and 1200±40, respectively. If the distribution of lipids around each intrinsic protein were proportional to its mass, the lipids around a molecule of GPIIb/IIIa will occupy about 120 nm2of the membrane plane, which is about one and a half times the cross-sectional area of the extracellular head of GPIIb/IIIa, as estimated by electron microscopy. The lipid extracts were further subjected to thin-layer chromatography to separate and quantify the different phospholipid fractions, the free fatty acids and the neutral lipid fraction and the distribution of fatty acids in each fraction was determined by gas chromatography after methanolysis. The phospholipid molar distribution was SPM(22.3±0.9%), PC(36.2±1.0%), PE(24.9±0.9%), PS(12.1±0.6%) and PI(4.5±0.4%) and the free fatty acid fraction represented 2.9±0.4% of the total fatty acids in HPPM. The fatty acid chain length ranged from 14 to 24 carbons, comprising unsaturated fatty acids (47.3% molar per cent of the total) of which 40.7±2.0% were monosaturated and 40.7±0.9% tetraunsaturated. Palmitic, stearic, oleic and arachidonic acids represent 66% of the total fatty acids of HPPM, being: 68.9±5.3% of palmitic acid and 63.3±6.9% of oleic acid in PC; 50.9±3.8% of arachidonic acid in PE; and 30.5±2.4% of stearic acid in PS. We discuss the methodological modifications and the new data in relation with the major differences in HPPM lipid composition found in the literature. The data obtained provides a comprehensive and accurate description of the lipid composition of HPPM on which to rely as a reference for basic and medical studies.

ISSN:0953-7104    

DOI:10.3109/09537109609023579

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

Shedding of microparticles from the platelet surface is usually associated with exposure of platelet procoagulant activity. Platelet-derived microparticles have been detected in blood in various disease states.In vitro, platelet stimulation with a number of different agonists results in formation of microparticles. In the present study, microparticles induced by platelet stimulation by calcium ionophore or by membrane incorporation of the terminal complement complex C5b-9 were studied using electron microscopy, confocal laser microscopy, flow cytometry and radio-immunoelectrophoresis. When studied by electron microscopy, microparticle morphology was found to be dependent upon the induction method. Platelet stimulation with the calcium ionophore resulted in smaller, more homogeneous and electron dense microparticles than those induced by insertion of the terminal complement complex. With flow cytometry and confocal laser immunofluorescence microscopy, microparticle GPIIb-IIIa was demonstrated using a FITC-conjugated antibody to GPIIIa. Surface-bound GPIIb-IIIa was demonstrated on the microparticles by immunoelectron microscopy. Crossed immunoelectrophoresis of detergent-solubilized microparticles visualized a very prominent GPIIb-IIIa immunoprecipitate arc, and binding of [1251]fibrinogen to microparticle GPIIb-IIIa was demonstrated by radio-immunoelectrophoresis. This suggests that the activated GPIIb-IIIa complex is preserved intact during the shedding of microparticles from the platelet surface.

ISSN:0953-7104    

DOI:10.3109/09537109609023580

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

In a family, a moderate bleeding disorder in two patients has been specified as Glanzmann's thrombasthenia because of characteristic defects in platelet function. Analysis of platelet membrane glycoproteins revealed about a 50% decrease in the amount of GP IIb-IIIa complex (α11bβ3integrin), which appeared normal with respect to electrophoretic mobility, apparent M, and isoelectric behaviour of GP IIb and GP IIIa. Content of platelet fibrinogen (Fg) was normal. [125I]Fg binding to ADP-stimulated platelets was strongly reduced but Kdvalues indicated a much higher affinity of the residual receptors for both [125I]Fg and RGD peptide. Fg bound to the isolated complex as detected by crossed immunoelectrophoresis and there was substantial expression of endogenous Fg on the surface of washed thrombin-stimulated platelets. RGD-peptide induced increased binding of conformation-specific monoclonal antibodies (Mabs) LIBS 1 and PMI-1. Flow cytometric analysis revealed defective binding of nine Mabs, among them two out of three tested antibodies specific for GP IIIa (C 17, AP 5). Results indicate a genetic variant of GP IIb-IIIa complex with the structural abnormality possibly related to defective conformational change upon activation.

ISSN:0953-7104    

DOI:10.3109/09537109609023581

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

Human platelets contain an ATP diphosphohydrolase activity (apyrase, EC 3.6.1.5) that is Ca2+dependent, hydrolyses ATP and ADP and also GTP, ITP, CTP, GDP, IDP, CDP. The enzyme does not hydrolyse AMP, p-nitrophenylphosphate, inorganic phosphate or glucose-6-phosphate. Contaminant activities were ruled out because the enzyme was not inhibited by 2μg/d ouabain, 1.0μM levamisole, 10μM ApSA or 1.0 mM azide. The enzyme was sensitive to 100μM orthovanadate, 100μMApSA and 10 mM azide, reagents that have been described as inhibitors of some other apyrases. A strong inhibition by 1.0 mM NEM was observed, indicating that sulphydryl groups are involved in the enzyme activity. The parallel behaviour of ATPase and ADPase activities and the competition plot presented suggest that ATP and ADP hydrolysis occurs at the same active site. ATP diphosphohydrolase from human platelets may be involved in the modulation of nucleotide concentration in the circulation and thus in vascular tonus.

ISSN:0953-7104    

DOI:10.3109/09537109609023582

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

Collagen (5-160μg/ml) induced release of free and sulphate conjugated serotonin (5-HT) from platelets in platelet-rich plasma (PRP) was examined in normal human subjects. Collagen stimulated release of free 5-HT to the plasma (platelet-poor; PPP) increased in a dose dependent manner (P<0.0001) and was mirrored by a decline in platelet free 5-HT content (P<0.0001). The half-maximum response occurred at a collagen concentration of 10.5μg/ml. Non-specific (resting) release of free 5-HT represented 2.5% of the total free 5-HT (sum of PPP and platelet concentrations). On stimulation with 5,10,20,40,80 and 160μg/ml collagen, PPP free 5-HT concentrations were 24.2, 45.5, 63.2, 74.9, 82 and 89.7% of the total respectively. Collagen also elicited dose dependent release of sulphate conjugated 5-HT to PPP (P<0.05), which was reflected by a corresponding decline in platelet sulphate conjugated 5-HT content (P<0.0001). Half maximal release of sulphate conjugated 5-HT occurred with 6μg/ml collagen. Under resting conditions platelet and PPP sulphate conjugated concentrations were respectively 6 and 35.1% of total (sum of free and sulphate conjugated) 5-HT concentrations, whilst under collagen (160μg/ml) stimulated conditions these concentrations represented 12.8 and 7.7% of the total. Mobilisation (i.e. hydrolysis) of platelet sulphate conjugated 5-HT did not occur on collagen stimulation, as evidenced by the absence of alterations in total sulphate conjugated 5-HT concentrations (sum of PPP and platelet sulphate conjugated 5-HT concentrations). PPP concentrations of the 5-HT metabolite 5-hydroxyindoleacetic acid (5-HIAA) were unaltered by collagen stimulation indicating that it did not influence 5-HT metabolism. It is concluded that release of platelet sulphate conjugated 5-HT, in addition to free 5-HT, occurs on platelet activation by collagen, although the sulphate conjugated fraction represents only a minor proportion of the total (free and sulphate conjugated) 5-HT released. Further studies will establish the physiological significance of platelet sulphate conjugated 5-HT.

ISSN:0953-7104    

DOI:10.3109/09537109609023583

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

In 30 patients we investigated the expression of activated GPIIb/IIIa-complex as an indicator ofin vivoplatelet activation before and after coronary angiography/angioplasty. Patients were divided into three groups according to aspirin treatment: group I: patients having a diagnostic coronary angiography without aspirin (n= 6); group II: patients having a diagnostic coronary angiography with low dose aspirin therapy (n= II); group III: patients having a coronary angioplasty with low dose aspirin therapy plus aspirin i.v. (n= 13). Platelets were identified in a flow cytometer by their characteristic light scatter profile and binding of anti-GP Ib, and activated platelets by an antibody to the activated GP IIb/IIIa. Cardiac catheterization lead to an increase of the mean anti-GP IIb/IIIa-fluorescence of the platelets and of the percentage of platelets with an anti-IIb/IIIa-FL exceeding a fixed threshold value (subpopulation). While aspirin substantially inhibited the increase induced by diagnostic angiography, the increase in the angioplasty group was the greatest of all groups despite aspirin.

ISSN:0953-7104    

DOI:10.3109/09537109609023584

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

A textbook of vascular medicine: Tooke, J.E. and Lowe, G.D.O. (eds): Arnold (Hodder Headline Group), 1996. 672 pages. Price:£315.00, ISBN 0 340 55791 5.

ISSN:0953-7104    

DOI:10.3109/09537109609023585

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor