scholarly journals Isolation and characterization of lipid-protein particles containing platelet factor 3 released from human platelets

1982 ◽  
Vol 203 (1) ◽  
pp. 303-311 ◽  
Author(s):  
H Sandberg ◽  
L O Andersson ◽  
S Höglund
Keyword(s):  
Gel Filtration ◽  
Phospholipid Composition ◽  
Chemical Characterization ◽  
Human Platelets ◽  
Electron Microscopic ◽  
Platelet Factor ◽  
Isolation And Characterization ◽  
Microscopic Studies ◽  
Time Test ◽  
Protein Particles

Lipid-protein particles with platelet factor 3 measured by the Stypven clotting-time test [Hardisty & Hutton (1966) Br. J. Haematol. 12, 764-776] have been isolated from platelet-release supernatant. Starting material was washed platelets, which were released by treatment with collagen. Purification of the particles from other components in the release material was accomplished by gel filtration on Sepharose CL-4B followed by affinity chromatography on poly-L-lysine-Sepharose CL-4B gel. Chemical characterization showed that the particles were composed of 40% protein, 42% phospholipids, 13% cholesterol and 5% triacylglycerols. The phospholipid composition was 38% phosphatidylcholine, 25% phosphatidylethanolamine, 9% phosphatidylserine, 2% phosphatidic acid and 26% sphingomyelin. No carbohydrate was detected. Electron-microscopic studies revealed the presence of membranous particles with diameters between 70 and 170 nm.

Estramustine binds MAP-2 to inhibit microtubule assembly in vitro

1988 ◽  
Vol 89 (3) ◽  
pp. 331-342
Author(s):  
M.E. Stearns ◽  
K.D. Tew
Keyword(s):  
Linear Regression Analysis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Microtubule Assembly ◽  
Scatchard Analysis ◽  
Binding Assays ◽  
Electron Microscopic ◽  
Associated Proteins ◽  
Microscopic Studies

We have investigated the ability of estramustine to bind to rat brain microtubule-associated proteins (MAPs) and purified MAP-2 in vitro. [3H]estramustine's relative affinity for tubulin and MAPs was assessed by gel filtration chromatography, immunoprecipitation and binding assays. Scatchard analysis demonstrated a specific affinity of the drug for MAP-2. Calculations from kinetic parameters and non-linear regression analysis gave a Kd of 15 microM, and a Bmax of 3.4 × 10(−7)M ml-1. Extrapolation of this value suggested that each MAP-2 molecule binds approximately 20 molecules of estramustine. Microtubule assembly studies and SDS-polyacrylamide gel electrophoresis revealed that at 20–60 microM levels, estramustine inhibited the association of MAPs with taxol microtubules. Turbidity (A350) studies further demonstrated that 20–60 microM-estramustine inhibited MAP-2-driven tubulin assembly and produced microtubule disassembly. Electron-microscopic studies confirmed the centrifugation and turbidity results. The data demonstrated that estramustine can bind MAPs and MAP-2 specifically, thereby inhibiting microtubule assembly.

Human High Molecular Weight Kininogen - A Secreted Platelet Coagulant Protein

1981 ◽  
Author(s):  
A H Schmaier ◽  
J Kuchibhotla ◽  
R W Colman
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Gel Filtration ◽  
Human Platelets ◽  
Metabolic Inhibitors ◽  
Ionophore A23187 ◽  
High Molecular Weight Kininogen ◽  
Contact Phase ◽  
Platelet Factor ◽  
Coagulant Activity

Platelets have been shown to contain a number of secret- able coagulant proteins, which participate as substrates or cofactors in plasma coagulation reactions. Since we have previously demonstrated that high molecular weight kininogen (HMWK) is immunochemically present in platelet extracts, we posited that HMWK is secreted during activation of platelets. Fresh normal platelets were washed by a combination of albumin-gradient and gel-filtration procedures. In 11 experiments the supernates of freeze-thaw lysates of normal human platelets contained a mean of 5.7 Units (range 3.16 to 8.14) of HMWK coagulant activity/3 × 1011 platelets. This coagulant activity was neutralized by a goat antiki- ninogen antibody. Using a 125I-HMWK tracer in PRP, the supernate of washed activated platelets contained 0.082% radioactivity as the starting PRP, suggesting that 14% of the total HMWK coagulant activity could be accounted for by plasma contamination. In four experiments, ionophore A23187 (15μM) induced a net secretion of 39% of the total platelet HMWK (range 16 to 49%). Platelet HMWK secretion by A23187 was concentration dependent (1 to 15 μM) . At A23187 (15μM) platelets released 75% 14C-5HT (range 61 to 99%) and 81% low affinity platelet Factor 4 (range 60 to 99%). Ninety-five percent of A23187-induced secretion of HMWK could be blocked by platelet pretreatment with metabolic inhibitors. LDH determinations indicated that only 5% (range 0 to 10%) of total secreted platelet HMWK could be attributed to lysis. Collagen and PGH2 also caused secretion of platelet HMWK coagulant activity. This study indicates that human platelets contain functional HMWK which may be secreted locally to modulate the reactions of the contact phase of plasma proteolysis.

Effect of Phospholipase C on Human Platelets

1975 ◽  
Author(s):  
A.-B. Otnœss
Keyword(s):  
Bacillus Cereus ◽  
Phospholipase C ◽  
Electron Micrographs ◽  
Gel Filtration ◽  
Outer Part ◽  
Human Platelets ◽  
Scanning Electron Micrographs ◽  
Platelet Factor ◽  
Asymmetrical Distribution ◽  
Scanning Electron

The effect on human platelets of phospholipase C (Bacillus cereus) has been studied. Platelets prepared by gel filtration lost 20-30% of their phospholipids when incubated with phospholipase C for 20 min. Phosphatidylethanolamine (PE) was reduced by about 50%, whereas phosphatidylcholine and phosphatidylserine were reduced each by about 20%. Sphingomyelin was not reduced.These data suggest an asymmetrical distribution of phospholipids in the platelet membrane, PE being more accessible and therefore probably mainly located in the outer part of the membrane.The loss of phospholipids was not accompanied by aggregation, nor did the platelets lose their ability to aggregate with thrombin or ADP. Data on release of serotonin, platelet factor 3 and 4 and scanning electron micrographs of treated platelets will be given.

scholarly journals Platelet Activation in Heparin-Induced Thrombocytopenia is Followed by Platelet Death via Complex Apoptotic and Non-Apoptotic Pathways

2020 ◽  
Vol 21 (7) ◽  
pp. 2556
Author(s):  
Elmira R. Mordakhanova ◽  
Tatiana A. Nevzorova ◽  
Gulnaz E. Synbulatova ◽  
Lubica Rauova ◽  
John W. Weisel ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Immune Complexes ◽  
Gel Filtration ◽  
Human Platelets ◽  
Platelet Factor 4 ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Mitochondrial Membrane Depolarization ◽  
Low Platelet Count ◽  
Apoptotic Pathways

Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction characterized by thrombocytopenia and a high risk for venous or arterial thrombosis. HIT is caused by antibodies that recognize complexes of platelet factor 4 and heparin. The pathogenic mechanisms of this condition are not fully understood. In this study, we used flow cytometry, fluorimetry, and Western blot analysis to study the direct effects of pathogenic immune complexes containing platelet factor 4 on human platelets isolated by gel-filtration. HIT-like pathogenic immune complexes initially caused pronounced activation of platelets detected by an increased expression of phosphatidylserine and P-selectin. This activation was mediated either directly through the FcγRIIA receptors or indirectly via protease-activated receptor 1 (PAR1) receptors due to thrombin generated on or near the surface of activated platelets. The immune activation was later followed by the biochemical signs of cell death, such as mitochondrial membrane depolarization, up-regulation of Bax, down-regulation of Bcl-XL, and moderate activation of procaspase 3 and increased calpain activity. The results show that platelet activation under the action of HIT-like immune complexes is accompanied by their death through complex apoptotic and calpain-dependent non-apoptotic pathways that may underlie the low platelet count in HIT.

scholarly journals Thrombocytin, A Novel Platelet Activating Enzyme from Bothrops Atrox (MARAJOENSIS) Venom

1977 ◽  
Author(s):  
S. Niewiarowski ◽  
E.P. Kirby ◽  
G.J. Stewart ◽  
R. Turna ◽  
M. Wiedeman ◽  
...  
Keyword(s):  
Platelet Rich Plasma ◽  
Human Platelets ◽  
Activate Factor ◽  
Factor X ◽  
Electron Microscopic ◽  
Platelet Factor ◽  
Small Vessels ◽  
Platelet Aggregates ◽  
Bothrops Atrox ◽  
Induced Aggregation

Thrombocytin (TCN) was purified from Bothrops atrox (BA) venom by precipitation with 1.2% Na-salicylate and chromatography on heparin-agarose column using increasing concentrations of lysine as eluent. It was homogeneous on SDS electrophoresis and had an apparent MW of 36,000. Immunoelectrophoresis with polyvalent anti-BA venom serum gave one cathodic arc indicating an isoelectric point higher than pH 8.6.TCN at a concentration of 1 yg/ml caused aggregation of human platelets, release of low affinity platelet factor 4 and serotonin, and stimulated platelets to retract fibrin.TCN was essentially free of fibrinogen clotting and fibrinolytic activities.TCN action on platelets was not mediated by the formation of thrombin since TCN did not activate Factor X or prothrombin and its action was not inhibited by hirudin.TCN is a serine protease since it was inhibited by DFP and it hydrolyzed a synthetic peptide, chromozyme UK (BZ-Val-Gly-Arg-pNA·HCl).TCN-induced aggregation of human platelets was completely inhibited by soy bean trypsin inhibitor, heparin, prostaglandin E1 and apyrase. Washed human platelets were 2-4 times less sensitive to TCN as compared to platelets in freshly prepared platelet rich plasma (PRP); their sensitivity to TCN gradually deteriorated during incubation of PRP at room temperature for 3 hours. Electron microscopic observations revealed formation of platelet aggregates characterized by pseudopod formation, centralization and partial loss of platelet granules. Infusion of TCN (3 yg) into the main artery of bat wing resulted in the formation of platelet aggregates seen on arterial and venous side which occasionally occluded small vessels.

scholarly journals Purification and Characterization of Human Platelet Factor 3.

1977 ◽  
Author(s):  
H. Sandberg ◽  
L-O. Andersson ◽  
S. Hoglund
Keyword(s):  
Membrane Filtration ◽  
Human Platelet ◽  
Gel Filtration ◽  
Human Platelets ◽  
Purification And Characterization ◽  
Filtration Fraction ◽  
Platelet Factor ◽  
Platelet Suspension ◽  
Main Components

In the purification of platelet factor 3 fresh human platelets prepared by centrifugation were used as starting material. Release material having platelet factor 3 activity, as measured by the Stypven time, was obtained by treating the platelet suspension with collagen or thrombin followed by removal of the platelets by centrifugation. Residual platelets left in supernatant were removed by membrane filtration. Purification of platelet factor 3 from the other components in the release material was accomplished by affinity chromathography followed by gel filtration on Sepharose 2B. The gel filtration fraction having platelet factor 3 activity was studied with respect to chemical composition and appearance in electron mincroscopy. Phospholipids and cholesterol were the main components but small amounts of protein could also be detected. The electron microscopy revealed the presence of numerous membranous particles having diameters between 50-150 Å. The purified fraction was used for immunization of rabbits and an antiserum was obtained which inhibited the platelet factor 3 activity.

Rapid release of peroxidase by peanut cells in suspension culture

1973 ◽  
Vol 51 (6) ◽  
pp. 1169-1175 ◽  
Author(s):  
R. B. van Huystee ◽  
G. Turcon
Keyword(s):  
Molecular Weight ◽  
Metabolic Rate ◽  
Suspension Culture ◽  
Gel Filtration ◽  
Electron Microscopic ◽  
Rapid Release ◽  
Microscopic Studies ◽  
Very High

Peanut cells in a suspension culture were studied with regard to the process of peroxidase release and some ultrastructural features that appeared to relate to this process. By means of gel filtration on Sephadex G-200 it was established that protein released into the medium had the same molecular weight as the peroxidase found in this medium. From information gained through the electron-microscopic studies and from incorporation of labeled leucine, it may be deduced that the metabolic rate of these cells is very high. Within 2 h after incubation with radioactive leucine it was noted that newly synthesized protein was released by the cells into the medium. This protein had also the same characteristics of peroxidase and its synthesis and subsequent appearance in the medium could be reduced by incubation in the presence of cycloheximide. A possible explanation for the presence of these large quantities of peroxidase is discussed.

Purification and Properties of a Low Molecular Weight Protein from Human Platelets and its Relationship to Beta-Thromboglobulin

1979 ◽  
Author(s):  
Daniel A. Walz ◽  
Stefan Niewiarowski
Keyword(s):  
Gel Filtration ◽  
Human Platelets ◽  
Platelet Factor 4 ◽  
Antigenic Determinants ◽  
Platelet Factor ◽  
Amino Terminal ◽  
Molar Activity ◽  
Additional Protein ◽  
Heparin Activity ◽  
C 50

Activated human platelets secrete specific intracellular proteins which have anti-heparin activity. These proteins include platelet factor 4 (PF-4), beta-thromboglobulin (β-TG) low-affinity platelet factor 4 (LA-PF-4), and platelet growth factor. Outdated platelet concentrates were initially heat-treated for 10 minutes at 70°C, centrifuged, and the supernatant fractionated on Sephadex C-50. The fraction eluted with 0.5M NaCl contained the majority of the anti-heparin activity. Subsequent gel filtration resulted in a final product which was homogeneous on SDS electrophoresis (9,000 daltons). Amino terminal analysis of this fraction gave the sequence: Asn-Leu-Ala-Lys-Gly-Lys-Glu-Glu-Ser-. Residues 5-21 were identical to residues 1-16 of β-TG. This protein shared common antigenic determinants with β-TG in a radioimmunoassay. Isoelectric focusing of this material resulted in the majority of protein recovered at an isoelectric point of 8.0 (β-TG = 7.0). This protein fraction possessed four times the specific molar activity of insulin in stimulating the mitogenic expression of Balb 3T3 cells; however, additional protein fractions from the gel filtration procedure were far more potent, though less homogeneous. This protein, therefore, appears identical to LA-PF-4, and is apparently the precursor of β-TG; it is not the most potent of platelet growth factors.(Support: MI Heart; NIH 14217).

The Effect of Phospholipase C on Platelet Factor 3 in Human Blood Platelets

1977 ◽  
Vol 37 (01) ◽  
pp. 029-035 ◽  
Author(s):  
A-B Otnaess ◽  
H Prydz
Keyword(s):  
Phospholipase C ◽  
Human Blood ◽  
Blood Platelets ◽  
Gel Filtration ◽  
Human Platelets ◽  
Small Reduction ◽  
Platelet Factor ◽  
Human Blood Platelets ◽  
External Attack

SummaryIntact human platelets isolated by gel filtration have been treated with purified phospholipase C. The effect of the enzyme on available and total platelet factor 3 has been tested.The available procoagulant platelet factor 3 was very low. A further small reduction was observed after incubation with phospholipase C when the enzyme was washed away before testing.External attack on platelets by phospholipase C led to a marked inactivation of total platelet factor 3.

scholarly journals Effect of platelet-activating factor (PAF) on human platelets

Blood ◽  
1982 ◽  
Vol 59 (3) ◽  
pp. 582-585 ◽  
Author(s):  
CM Chesney ◽  
DD Pifer ◽  
LW Byers ◽  
EE Muirhead
Keyword(s):  
Platelet Rich Plasma ◽  
Gel Filtration ◽  
Platelet Activating Factor ◽  
Creatine Phosphate ◽  
Major Effect ◽  
Human Platelets ◽  
Metabolic Inhibitors ◽  
Platelet Factor ◽  
Platelet Suspension ◽  
Second Wave

Abstract The effect of pure synthetic PAF (1–0-alkyl-2-acetyl-sn-glycero-3- phosphorylcholine) was studied in human platelets. PAF (0.2--2.0 micrograms/ml) produced a dose-dependent aggregation in human platelet- rich plasma (PRP) or platelet suspension obtained by gel-filtration (GFP). In addition, PAF (0.8 microgram/ml) induced secretion of 14C- serotonin (45% +/- 10%; mean +/- SD, n = 9) and platelet factor 4 (PF4) (12.89 +/- 3.81 micrograms/10(9) platelets; n = 9) in PRP. Similar results were obtained in GFP. Aggregation and release of 14C-serotonin and PF4 were inhibited by the metabolic inhibitors 2-deoxyglucose (16.7 mM) and antimycin-A (8.3 micrograms/ml), by the membrane-active drugs mepacrine (10 microM) and chlorpromazine (0.025 mM), by PGI2 (5.34 nM), which elevates intracellular c-AMP, by indomethacin (10 microM) or aspirin (100 microM). The ADP scavengers, creatine phosphate and creatine phosphokinase (CP/CPK), inhibited the second wave of aggregation but not secretion. These data suggest that the major effect of PAF on human platelets is mediated through the cyclo-oxygenase pathway and not through a third pathway.

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