Isolation of a subpopulation of glycoprotein IIb-III from platelet membranes that is bound to membrane actin.

Triton X-100-insoluble residues, or skeletons, of plasma membrane-rich vesicles obtained from unstimulated human platelets were isolated by high speed centrifugation. About 10-15% of the total surface iodinatable glycoproteins IIb and III (GPIIb and GPIII, respectively) co-isolated with the insoluble fraction. After sonication and centrifugation the solubilized material was further purified by affinity chromatography on Lens culinaris lectin-Sepharose. SDS PAGE analysis of this material revealed the presence of at least three major proteins, which were shown to be GPIIb, GPIII, and membrane actin, as judged by their electrophoretic properties and on the basis of immunological criteria. Antibodies directed against platelet surface glycoproteins and antibodies directed against rabbit actin were able to immunoprecipitate all three proteins, which indicates that they were noncovalently associated with one another. Gel filtration of the Lens lectin-purified Triton-insoluble complex on Ultrogel AcA 22 showed that greater than 85% of the total surface GPIIb and III was associated with an actin-rich peak that eluted in the void volume. In contrast, the form of GPIIb-III present in the Triton-soluble membrane fraction behaved as monomeric species when chromatographed under identical conditions. Finally, the GPIIb-III membrane actin complex bound with high efficiency to rabbit f-actin in vitro in a Ca++-independent manner, whereas the monomeric forms found in the Triton-soluble fraction did not bind to actin. These results indicate that two forms of GPIIb and III exist: one that binds directly to endogenous membrane actin and one that does not.

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Studies On Glycocalicin And Glycoprotein Gp Ib

10.1055/s-0038-1652012 ◽

1981 ◽

Author(s):

N O Solum ◽

T Sletbakk ◽

I Hagen ◽

G Gogstad

Keyword(s):

Fast Moving ◽

Gel Filtration ◽

Integral Membrane Protein ◽

Human Platelets ◽

Characteristic Change ◽

Extraction Buffer ◽

Sds Page ◽

Slow Moving ◽

Triton X ◽

Two Peaks

Crossed immunoelectrophoresis (CIE) of extracts of human platelets in 1% Triton X-100 using antiserum to purified glycocalicin shows an immunopre- cipitate consisting of two peaks. Previous experiments have shown the small fast-moving peak to represent free glycocalicin whereas the slow-moving one corresponds to a larger amphiphilic protein, probably the integral membrane protein GP Ib. Glycocalicin is probably derived from the latter secondary to an activation of a calsium-dependent protease during platelet lysis. Further studies on these problems are presented. A gradual reduction of the concentration of Triton X-100 in the extraction buffer (tris-glycine, pH 8.7, 135 mOsM) gradually reduced the area of the slow-moving peak and increased that of the fast-moving one untill all was present as free glycocalicin. Reduction of the concentration of Triton in an extract already prepared with 1% Triton X-100 by adsorption to Bio-Beads SM-2 had no such effect. The presence of the protease inhibitor leupeptin during extraction at a low concentration of Triton (0.2%) reduced the peak corresponding to free glycocalicin. GP Ib was purified from Triton extracts by precipitation with con A, affinity chromatography of the supernatant on WGA-Sepharose and elution with NAGA, and gel filtration of the eluate on Ultrogel AcA 22. Triton X-100, EDTA and sodium azide were present at all steps. The characteristic change in mobility of GP Ib on SDS PAGE comparing unreduced to reduced sampLes deduced from studies on whole platelet proteins, was confirmed with the purified material, as was the correspondence between the GP Ib band on SDS and the slow-moving component of the CIE.

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Platelet activation and microfilament bundling.

The Journal of Cell Biology ◽

10.1083/jcb.89.1.146 ◽

1981 ◽

Vol 89 (1) ◽

pp. 146-151 ◽

Cited By ~ 34

Author(s):

P A Gonnella ◽

V T Nachmias

Keyword(s):

Gel Filtration ◽

Human Platelets ◽

Negative Staining ◽

Low Molecular Weight ◽

Major Band ◽

Activated Platelets ◽

Microfilament Bundles ◽

Activated State ◽

Triton X

Human platelets were obtained in the fully resting state by treating discoid populations with 1.5 mM tetracaine and in the activated state by treatment with 2 microM A-23187. After gel filtration or washing, respectively, platelet suspensions were lysed with 1% Triton X-100 at pH 6.8. The precipitates from resting platelets viewed by negative staining appeared predominantly granular with a few very short microfilaments. They contained polypeptides of 250, 100, 45, 38, 36.5, and 35 Kdaltons, and three small polypeptides including one with the mobility of profilin on SDS gels. Precipitates from activated platelets lacked this low molecular weight band and contained a major band at 200 Kdaltons with the mobility of myosin; these precipitates had significant K+, Ca++ ATPase activity absent from the precipitate of resting platelets. As seen in negative staining, precipitates from activated platelets contained microfilaments arranged as nets or bundles. The granular resting precipitates were transformed in vitro into microfilament bundles by washing the precipitates in buffer at higher pH (7.6) in the presence of 5 X 10(-5) M calcium chloride.

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Loss of 111Indium as indicator of platelet injury

Blood ◽

10.1182/blood.v58.2.350.350 ◽

1981 ◽

Vol 58 (2) ◽

pp. 350-353 ◽

Cited By ~ 18

Author(s):

JH Joist ◽

RK Baker

Keyword(s):

Small Molecules ◽

Adenine Nucleotides ◽

Human Platelets ◽

Metabolic Energy ◽

Platelet Factor ◽

Platelet Protein ◽

Threshold Rate ◽

Immune Injury ◽

Triton X

Abstract We previously demonstrated that platelets can be labeled with 111Inoxine with high labeling efficiency and that 111In is not liberated from labeled platelets during the platelet release reaction or prolonged in vitro storage. In view of these findings, we examined the potential usefulness of loss of 111In from labeled platelets as an indicator or platelet damage by comparing the loss of 111In with that of 51Cr and LDH (in some experiments also with platelet factor 3 availability) under different conditions of platelet injury. When washed human platelets labeled with either 51Cr-chromate or 111In-oxine were exposed to increasing concentrations of detergents (Triton X-100, lysolecithin), threshold, rate, and extent of loss of 111In, 51Cr and, LDH were similar. In contrast, when labeled platelets were depleted of metabolic energy by incubation in glucose-free Tyrode albumin solution or glucose-depleted plasma in the presence of antimycin A and 2-deoxy-D- glucose, loss of 51Cr (and PF3a) occurred earlier and progressed at a faster rate than that of 111In or LDH. Similar results were obtained when platelets were exposed to increasing concentrations of PlA1 antibody, causing complement-mediated immune injury. The findings indicate that with certain agents that cause rapid platelet disruption (lysis), different platelet constituents are lost at similar rates. However, under conditions of more subtle or slowly progressive platelet injury, small molecules such as adenine nucleotides (51Cr) may escape earlier and at faster rates than larger molecules such as LDH or 111In- binding platelet protein. Thus, neither 111In loss nor LDH loss appear to be suitable indicators for sublytic or prelytic platelet injury.

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Impaired Platelet Function in Sept8-Deficient Mice In Vitro

Thrombosis and Haemostasis ◽

10.1055/s-0040-1718733 ◽

2020 ◽

Author(s):

Kerstin Jurk ◽

Katharina Neubauer ◽

Victoria Petermann ◽

Elena Kumm ◽

Barbara Zieger

Keyword(s):

Platelet Function ◽

Thrombin Generation ◽

Human Platelets ◽

Platelet Surface ◽

Integrin Αiibβ3 ◽

Glycoprotein Vi ◽

Fibrinogen Receptor ◽

Static Conditions ◽

The Impact

AbstractSeptins (Septs) are a widely expressed protein family of 13 mammalian members, recognized as a unique component of the cytoskeleton. In human platelets, we previously described that SEPT4 and SEPT8 are localized surrounding α-granules and move to the platelet surface after activation, indicating a possible role in platelet physiology. In this study, we investigated the impact of Sept8 on platelet function in vitro using Sept8-deficient mouse platelets. Deletion of Sept8 in mouse platelets caused a pronounced defect in activation of the fibrinogen receptor integrin αIIbβ3, α-granule exocytosis, and aggregation, especially in response to the glycoprotein VI agonist convulxin. In contrast, δ-granule and lysosome exocytosis of Sept8-deficient platelets was comparable to wild-type platelets. Sept8-deficient platelet binding to immobilized fibrinogen under static conditions was diminished and spreading delayed. The procoagulant activity of Sept8-deficient platelets was reduced in response to convulxin as determined by lactadherin binding. Also thrombin generation was decreased relative to controls. Thus, Sept8 is required for efficient integrin αIIbβ3 activation, α-granule release, platelet aggregation, and contributes to platelet-dependent thrombin generation. These results revealed Sept8 as a modulator of distinct platelet functions involved in primary and secondary hemostatic processes.

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A metalloproteinase extracellularly released byCrithidia deanei

Canadian Journal of Microbiology ◽

10.1139/w03-081 ◽

2003 ◽

Vol 49 (10) ◽

pp. 625-632 ◽

Cited By ~ 15

Author(s):

Claudia Masini d'Avila-Levy ◽

Rodrigo F Souza ◽

Rosana C Gomes ◽

Alane B Vermelho ◽

Marta H Branquinha

Keyword(s):

Molecular Mass ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Residual Activity ◽

Major Protein ◽

Motile Cells ◽

Sds Page ◽

Triton X

Actively motile cells from a cured strain of Crithidia deanei released proteins in phosphate buffer (pH 7.4). The molecular mass of the released polypeptides, which included some proteinases, ranged from 19 to 116 kDa. One of the major protein bands was purified to homogeneity by a combination of anion-exchange and gel filtration chromatographs. The apparent molecular mass of this protein was estimated to be 62 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE). The incorporation of gelatin into SDSPAGE showed that the purified protein presented proteolytic activity in a position corresponding to a molecular mass of 60 kDa. The enzyme was optimally active at 37 °C and pH 6.0 and showed 25% of residual activity at 28 °C for 30 min. The proteinase was inhibited by 1,10-phenanthroline and EDTA, showing that it belonged to the metalloproteinase class. A polyclonal antibody to the leishmanial gp63 reacted strongly with the released C. deanei protease. After Triton X-114 extraction, an enzyme similar to the purified metalloproteinase was detected in aqueous and detergent-rich phases. The detection of an extracellular metalloproteinase produced by C. deanei and some other Crithidia species suggests a potential role of this released enzyme in substrate degradation that may be relevant to the survival of trypanosomatids in the host.Key words: endosymbiont, trypanosomatid, extracellular, proteinase.

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Isolation and Characterization of Two Proteins fromMoraxella catarrhalis That Bear a Common Epitope

Infection and Immunity ◽

10.1128/iai.66.9.4374-4381.1998 ◽

1998 ◽

Vol 66 (9) ◽

pp. 4374-4381 ◽

Cited By ~ 58

Author(s):

John C. McMichael ◽

Michael J. Fiske ◽

Ross A. Fredenburg ◽

Deb N. Chakravarti ◽

Karl R. VanDerMeid ◽

...

Keyword(s):

Molecular Weight ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Bacterial Attachment ◽

Vaccine Candidates ◽

Isolation And Characterization ◽

Sds Page

ABSTRACT The UspA1 and UspA2 proteins of Moraxella catarrhalisare potential vaccine candidates for preventing disease caused by this organism. We have characterized both proteins and evaluated their vaccine potential using both in vitro and in vivo assays. Both proteins were purified from the O35E isolate by Triton X-100 extraction, followed by ion-exchange and hydroxyapatite chromatography. Analysis of the sequences of internal peptides, prepared by enzymatic and chemical cleavage of the proteins, revealed that UspA1 and UspA2 exhibited distinct structural differences but shared a common sequence including an epitope recognized by the monoclonal antibody 17C7. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), purified UspA1 exhibited a molecular weight of approximately 350,000 when unheated and a molecular weight of 100,000 after being heated for 10 min at 100°C. In contrast, purified UspA2 exhibited an apparent molecular weight of 240,000 by SDS-PAGE that did not change with the length of time of heating. Their sizes as determined by gel filtration were 1,150,000 and 830,000 for UspA1 and UspA2, respectively. Preliminary results indicate the proteins have separate functions in bacterial pathogenesis. Purified UspA1 was found to bind HEp-2 cells, and sera against UspA1, but not against UspA2, blocked binding of the O35E isolate to the HEp-2 cells. UspA1 also bound fibronectin and appears to have a role in bacterial attachment. Purified UspA2, however, did not bind fibronectin but had an affinity for vitronectin. Both proteins elicited bactericidal antibodies in mice to homologous and heterologous disease isolates. Finally, mice immunized with each of the proteins, followed by pulmonary challenge with either the homologous or a heterologous isolate, cleared the bacteria more rapidly than mock-immunized mice. These results suggest that UspA1 and UspA2 serve different virulence functions and that both are promising vaccine candidates.

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Production of PEGylated GCSF from Non-classical Inclusion Bodies Expressed in Escherichia coli

Avicenna Journal of Medical Biotechnology ◽

10.18502/ajmb.v13i4.7204 ◽

2021 ◽

Author(s):

Nguyen Thi My Trinh ◽

Tran Linh Thuoc ◽

Dang Thi Phuong Thao

Keyword(s):

Escherichia Coli ◽

Inclusion Bodies ◽

Gel Filtration ◽

Anion Exchange Chromatography ◽

Insoluble Fraction ◽

Exchange Chromatography ◽

Native Page ◽

E Coli ◽

Sds Page ◽

Stimulating Factor

Background: The recombinant human granulocyte colony stimulating factor con-jugated with polyethylene glycol (PEGylated GCSF) has currently been used as an efficient drug for the treatment of neutropenia caused by chemotherapy due to its long circulating half-life. Previous studies showed that Granulocyte Colony Stimula-ting Factor (GCSF) could be expressed as non-classical Inclusion Bodies (ncIBs), which contained likely correctly folded GCSF inside at low temperature. Therefore, in this study, a simple process was developed to produce PEGylated GCSF from ncIBs. Methods: BL21 (DE3)/pET-GCSF cells were cultured in the LiFlus GX 1.5 L bioreactor and the expression of GCSF was induced by adding 0.5 mM IPTG. After 24 hr of fermentation, cells were collected, resuspended, and disrupted. The insoluble fraction was obtained from cell lysates and dissolved in 0.1% N-lauroylsarcosine solution. The presence and structure of dissolved GCSF were verified using SDS-PAGE, Native-PAGE, and RP-HPLC analyses. The dissolved GCSF was directly used for the con-jugation with 5 kDa PEG. The PEGylated GCSF was purified using two purification steps, including anion exchange chromatography and gel filtration chromatography. Results: PEGylated GCSF was obtained with high purity (~97%) and was finally demonstrated as a form containing one GCSF molecule and one 5 kDa PEG molecule (monoPEG-GCSF). Conclusion: These results clearly indicate that the process developed in this study might be a potential and practical approach to produce PEGylated GCSF from ncIBs expressed in Escherichia coli (E. coli).

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Calcium-dependent cysteine protease activity in the sera of patients with thrombotic thrombocytopenic purpura

Blood ◽

10.1182/blood.v70.5.1683.1683 ◽

1987 ◽

Vol 70 (5) ◽

pp. 1683-1687 ◽

Cited By ~ 42

Author(s):

WG Murphy ◽

JC Moore ◽

JG Kelton

Keyword(s):

Thrombotic Thrombocytopenic Purpura ◽

Cysteine Protease ◽

Platelet Activating Factor ◽

Aminocaproic Acid ◽

Human Platelets ◽

Thrombocytopenic Purpura ◽

Platelet Surface ◽

Calcium Dependent

Abstract Plasma and serum from patients with thrombotic thrombocytopenic purpura (TTP) can cause activation and aggregation of normal human platelets in vitro. It is possible that this platelet-activating factor contributes to the disease. In this report we describe studies designed to identify the platelet-activating factor in TTP. Platelet activation by sera from 15 patients with TTP was inhibited by leupeptin, iodoacetamide, and antipain but not by phenylmethylsulphonylfluoride, epsilon-aminocaproic acid, soybean trypsin inhibitor, aprotinin, and D-phenylanyl-1-prolyl-1- arginine chloromethyl ketone. These studies suggested that the platelet- activating factor in TTP serum was a cysteine protease. We confirmed that a calcium-dependent cysteine protease (CDP) was present in the sera of each of the 15 patients when we used an assay based on the ability of CDP to proteolyse platelet membrane glycoprotein 1b (GP1b) and hence to abolish the ability of CDP-treated normal platelets to agglutinate in the presence of ristocetin and von Willebrand factor. This proteolytic activity was inhibited by EDTA, leupeptin, antipain, iodoacetamide, and by N-ethyl-maleamide (NEM) but not by the serine protease inhibitors. Activity was detected in 15 of 15 patients with TTP tested before therapy was begun. In contrast, no activity was detected in the serum of any of five of the TTP patients tested in remission or in any of the sera from 36 patients with thrombocytopenia and 423 nonthrombocytopenic controls. To look for in vivo CDP activity in patients with TTP, we studied platelets from two patients with acute TTP (drawn into acid-citrate-dextrose, NEM, and leupeptin). These platelets showed a loss of GP1b from the platelet surface. Both patients were also studied in remission: GP1b on the platelet surface had returned to normal. These studies provide evidence that CDP is present in the sera of patients with TTP, that it is specific to this disease, and that is is active in vivo as well as in vitro. We postulate that a disorder of CDP homeostasis plays a major role in the pathophysiology of TTP.

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REDISTRIBUTION OF PLATELET GLYCOPROTEINS INDUCED BY ALLO- AND AUTOANTIBODIES

10.1055/s-0038-1644880 ◽

1987 ◽

Author(s):

S Santoso ◽

V Kiefel ◽

C Mueller-Eckhardt

Keyword(s):

Binding Sites ◽

Total Radioactivity ◽

Human Platelets ◽

Membrane Glycoproteins ◽

Platelet Surface ◽

In Vitro Effects ◽

Immunoblot Technique ◽

Antigen Antibody

It is now well established that two of the major membrane glycoproteins (GP) of human platelets, GP lb and Ilb/IIIa, are functionally prominent for adhesion, aggregation and carry the binding sites for allknown types of human platelet specific antibodies (ab). Although a number of in vitro effects of ab on platelet function have been described, the role of the GP specificity of the various ab with regard to membrane mobility and redistribution phenomena is asyet unknown.In this work, we studied the effect on platelet membrane redistribution of allo- ab, auto-aband a quinidine-dependent ab directed against various epitopes on GP lb, lib and Ilia using immunofluorescence and a quantitative radioimmunoassay. The platelet GP's carrying the corresponding epitopes were determined using immunoblot technique or radioimmuno-precipitation. When unfixed platelets were incubated with alio- or auto-ab against epitopes on GP liborGP IlIa cap formation and internalization of antigenantibody complexes were visualized by fluorescence. In contrast, no changes of antigen distribution were seen with auto-ab or quinidine- dependent ab directed against GP lb. To quantitate antigen-antibody complexes internalization a specially designed radioimmunoassay was employed. If unfixed platelets weretreated with allo- or auto-ab against GP lib or GP Ilia precipitous reduction of external radioactivity was found, whereas the total radioactivity remainedessentially unchanged. This indicated that a portionof approximately 50-70% of GP lib or GP Ilia had been removed from the platelet surface and had been internalized. Internalization could not be induced with auto-ab or quinidine dependent ab against GP lb.We conclude that membrane redistribution of human platelets can be induced by various human ab with specificity for GP lib and/or Ilia and is a function of the target GP rather than the source of therespective abSupported by Deutsche Forschungsgemeinschaft (Mu 277/9-6)

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Effect of anti-P1A1 antibody on human platelets. II. Mechanism of the complement-dependent release reaction

Blood ◽

10.1182/blood.v53.4.578.578 ◽

1979 ◽

Vol 53 (4) ◽

pp. 578-587 ◽

Cited By ~ 7

Author(s):

AD Schreiber ◽

DB Cines ◽

C Zmijewski ◽

RW Colman

Keyword(s):

Human Platelets ◽

Metabolic Energy ◽

Igg Antibody ◽

Platelet Surface ◽

Release Reaction ◽

Platelet Release ◽

High Concentrations ◽

Antibody Levels ◽

Pharmacologic Agents

Abstract We studied the mechanism by which complement activated by anti-P1A1 antibody elicits the platelet release reaction. Anti-P1A1 antibody mediates its action through the classic complement pathway, and its effect depends on the concentration of IgG antibody on the platelet surface. At relatively high concentrations of anti-P1A1 antibody the release reaction was mediated by a mechanism in part independent of extracellular ADP and metabolic energy and inhibited by only high concentrations of PGE1. However, at lower concentrations of anti-P1A1 antibody the release reaction was dependent on metabolic energy and ADP, and the concentration of PGE1 required to inhibit platelet release was similar to that required to inhibit ADP-induced release. The cyclooxygenase inhibitor acetylsalicylic acid inhibited the release reaction at all nonlytic antibody levels studied. None of the agents studied inhibited the induction of platelet lysis by very high concentrations of anti-P1A1 antibody, and no effect of antibody on platelet 14C-serotonin uptake was observed at antibody concentrations that did not mediate direct in vitro alteration. These studies suggest the possible use of pharmacologic agents in modifying some complement- mediated platelet alterations.

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