In vivo radiolabeling of platelet proteins: a new method with identification of platelet factor XIII

Abstract A method for radiolabeling platelets in vivo was developed in which 3H- arginine was injected into the bone marrow of normal dogs. On the third day after injection, a maximum of 6%--7% of the radioactivity had been incorporated into the total platelet mass. This method of isotope administration resulted in a 50--60-fold increase in maximum uptake of radiolabel by platelets, as compared to values obtained by others using intravenous injections of various radioactive compounds. Tritium- labeled platelets were harvested from the animals and then were washed to remove unbound 3H-arginine. On polyacrylamide gel electrophoresis 7 labeled protein bands, with molecular weights ranging from 29,000to 132,000, were obtained from the platelet-soluble fraction. One 3H- containing protein with a molecular weight of 81,000 was identified immunologically and enzymatically as platelet factor XIII.

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In vivo radiolabeling of platelet proteins: a new method with identification of platelet factor XIII

Blood ◽

10.1182/blood.v55.4.559.bloodjournal554559 ◽

1980 ◽

Vol 55 (4) ◽

pp. 559-563

Author(s):

DM Rider ◽

RP McDonagh ◽

J McDonagh

Keyword(s):

Gel Electrophoresis ◽

Factor Xiii ◽

Soluble Fraction ◽

Polyacrylamide Gel Electrophoresis ◽

Fold Increase ◽

Platelet Factor ◽

Intravenous Injections ◽

Molecular Weights ◽

The Third

A method for radiolabeling platelets in vivo was developed in which 3H- arginine was injected into the bone marrow of normal dogs. On the third day after injection, a maximum of 6%--7% of the radioactivity had been incorporated into the total platelet mass. This method of isotope administration resulted in a 50--60-fold increase in maximum uptake of radiolabel by platelets, as compared to values obtained by others using intravenous injections of various radioactive compounds. Tritium- labeled platelets were harvested from the animals and then were washed to remove unbound 3H-arginine. On polyacrylamide gel electrophoresis 7 labeled protein bands, with molecular weights ranging from 29,000to 132,000, were obtained from the platelet-soluble fraction. One 3H- containing protein with a molecular weight of 81,000 was identified immunologically and enzymatically as platelet factor XIII.

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Release Of Platelet Factor-4 In Vivo After Heparin Injection

10.1055/s-0038-1652594 ◽

1981 ◽

Author(s):

A Koneti Rao ◽

John C Holt ◽

Pranee James ◽

Stefan Niewiarowski

Keyword(s):

Molecular Weight ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Human Platelets ◽

Platelet Factor 4 ◽

Plasma Samples ◽

Platelet Factor ◽

Immunoreactive Material ◽

Elution Pattern

A single bolus of heparin administered to 8 normal volunteers resulted in a significant increase in levels in platelet poor plasma (PPP) of platelet factor-4 (PF4) but not low-affinity platelet factor-4/β-thromboglobulin (LA-PF4/βTG). However, the presence of heparin interfered with the binding of 125I-PF4 to antibody in radioimmunoassay (RIA). This effect was overcome by increasing the concentration of NaCl from 0.15 to 0.5 M in the buffer used for RIA. In order to establish that the increased amount of immunoreactive material present in PPP was indeed PF4, the protein was isolated from postheparin plasma. A bolus of 5000 units of porcine lung heparin (Upjohn) was administered intravenously to 2 volunteers and plasma samples obtained before and 5 minutes after the injection. The levels of PF4 in PPP rose from 18 and 10 ng/ml before to 185 and 454 ng/ml at 5 minutes after injection in the two volunteers, respectively. The 5 minute samples were adsorbed to heparin agarose columns and PF4 levels decreased to 16 and 10 ng/ml respectively. The immunoreactive material was eluted with 1.2 M NaCl from the heparin agarose columns, showing typical elution pattern for PF4. This material was applied to SDS-polyacrylamide gel electrophoresis in parallel with purified PF4 obtained from human platelets. RIA carried out on eluates from gel slices revealed a species of the same molecular weight as standard PF4. Thus, heparin injection results in appearance in the circulation of a material identical to PF4. LA-PF4/βTG and PF4 are located in same granules and released in parallel during platelet stimulation. Further, LA-PF4 is cleared from plasma 4 times slower than PF4. Therefore, the elevation of PF4alone suggests release from sites other than platelets.

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Gel-electrophoretic identification of hen brain neurotoxic esterase, labelled with tritiated di-isopropyl phosphorofluoridate

Biochemical Journal ◽

10.1042/bj1990323 ◽

1981 ◽

Vol 199 (2) ◽

pp. 323-333 ◽

Cited By ~ 59

Author(s):

D G Williams ◽

M K Johnson

Keyword(s):

Active Site ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

The Other ◽

Particulate Fraction ◽

Molecular Weights ◽

The Brain ◽

Neurotoxic Esterase

The particulate fraction from hen brain was labelled with [3H]di-isopropyl phosphorofluoridate (DiPF) and separated by polyacrylamide-gel electrophoresis. Four radioactive protein bands (1--4) of molecular weights 155000, 92000, 60000, and 30000 were resolved. Most of the labelling of bands 2, 3 and 4 was inhibited by preincubation with Paraoxon. The residue in band 4 was sensitive to pH 5.2. Successive treatments with Paraoxon and pH 5.2 resulted in the abolition of bands 3 and 4. Bands 1 and 2 contained one and two polypeptides respectively, whose labelling was sensitive to Mipafox, but one, in band 2, was sensitive to higher concentrations of Paraoxon. The concentrations of the other two polypeptides were 6.7 and 1.95 pmol of DiPF bound/g of brain in bands 1 and 2 respectively. Both were as sensitive to Mipafox as neurotoxic esterase and were also sensitive to phenyl benzylcarbamate. 4-Nitrophenyl di-n-pentylphosphinate given in vivo inhibited neurotoxic esterase and the labelling of the band-1 polypeptide by 82% and 84% respectively, but inhibited the labelling of the band 2 polypeptide by 51%. The phosphinate in vitro produced 98% inhibition of the labelling of the band-1 polypeptide, with only 26% inhibition of the band-2 polypeptide, under conditions sufficient to inhibit neurotoxic esterase totally. Both neurotoxic esterase and the band-1 polypeptide were found in the forebrain at 1.74-fold their concentration in the rest of the brain, whereas the band-2 polypeptide was uniformly distributed. The evidence indicates that the Mipafox-sensitive polypeptide in band 1 is the [3H]DiPF-labelled active-site subunit of neurotoxic esterase. The catalytic-centre activity of the enzyme for phenyl valerate hydrolysis was found to be 2.6 x 10(5) min-1.

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Soluble High-Molecular-Weight E Fragments in the Plasmin-Induced Degradation Products of Cross-Linked Human Fibrin

Clinical Science ◽

10.1042/cs0490149 ◽

1975 ◽

Vol 49 (2) ◽

pp. 149-156 ◽

Cited By ~ 1

Author(s):

P. J. Gaffney ◽

D. A. Lane ◽

M. Brasher

Keyword(s):

Molecular Weight ◽

High Molecular Weight ◽

Gel Electrophoresis ◽

Factor Xiii ◽

Degradation Products ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Cross Linking ◽

D Dimer

1. The factor XIII-mediated cross-linked α chains in fibrin have no effect on the nature of the fragments released during the solubilization of fibrin by plasmin. 2. Besides the known D dimer and E fragments solubilized during the lysis of cross-linked fibrin, other fragments have been observed on sodium dodecyl sulphate-polyacrylamide gel electrophoresis which have a molecular weight of about 135 000. After prolonged plasmin digestion, these fragments (U fragments) were no longer evident on the gels and the high-molecular-weight E antigen was absent. It is assumed that the E antigen was associated with the U fragments. These fragments also cross-reacted with an anti-D serum. 3. The U fragments have been tentatively presumed to be a factor XIII-mediated cross-linked D–E complex since they degrade only after prolonged degradation with plasmin. Whereas it is known that the fibrin D dimer fragment contains the cross-linked γ chain residues of the originating fibrin, the presumed covalent cross-linking of the D–E fragments has not been proved. 4. The presence of these high-molecular-weight fragments, containing the E antigen, in cross-linked human fibrin digests should be taken into account in the development of D dimer assays to monitor fibrin lysis in vivo.

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On the Plasmin Digestion of Factor XIII

10.1055/s-0039-1689314 ◽

1975 ◽

Author(s):

K. Mikami ◽

T. Mikami ◽

H. Suzuki ◽

M. Fujimaki ◽

K. Fukutake

Keyword(s):

Plasma Protein ◽

Gel Electrophoresis ◽

Factor Xiii ◽

Polyacrylamide Gel Electrophoresis ◽

Polyacrylamide Gel ◽

Platelet Factor ◽

Subunit A ◽

Plasma Factor ◽

Congenital Afibrinogenemia

The plasmin digestion of factor XIII in the plasma with congenital afibrinogenemia has been reported by Suzuki et al. in 1967. The present investigation using polyacrylamide gel electrophoresis and crossimmunoelectrophoresis with anti-factor XIII (A & S) serum demonstrates that the process of plasmin digestion of factor XIII can be divided into three steps ; the subunit A of factor XIII such as placental or platelet factor XIII is ready to get plasmin digestion following the decrease of amount and activity of subunit A, and the subunit S in the A1 S complex like plasma factor XIII is more readily affectable on the plasmin digestion than the subunit A of the complex, and the A2 S complex coexisting with fibrinogen or plasma protein is fairly stable on exposure to plasmin.

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Differentiation between mitochondrial and cytoplasmic protein subunits of blowfly flight muscle mitochondria synthesized in vivo

Journal of Cell Science ◽

10.1242/jcs.50.1.377 ◽

1981 ◽

Vol 50 (1) ◽

pp. 377-390

Author(s):

M.B. Ashour ◽

M. Tribe

Keyword(s):

Gel Electrophoresis ◽

Flight Muscle ◽

Polyacrylamide Gel Electrophoresis ◽

Mitochondrial Protein ◽

Cytoplasmic Protein ◽

Protein Subunits ◽

Molecular Weights ◽

And Function ◽

Made In

Total adult blowfly flight-muscle mitochondrial protein was labelled in vivo with [14C]leucine. The labelled proteins were enumerated and characterized by their electrophoretic mobility using sodium dodcecyl sulphate/polyacrylamide gel electrophoresis and autoradiography. When different gel concentrations were used to resolve the maximum number of bands and their molecular weights, it was possible to observe at least 35 electrophoretic bands after staining and scanning; their molecular weights ranged between 11 000 and 130 000. When mitochondria were labelled in the presence of cycloheximide, only 6–8 bands could be identified on gradient gels after electrophoresis and autoradiography. By comparison, controls (where cycloheximide was absent), which were run alongside the drug-treated mitochondria, revealed 17–20 radioactively labelled bands from densitometric tracings. Whilst the molecular weights of these bands could be estimated, it was difficult to identify the precise nature and function of the proteins made in these mitochondria.

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Low Molecular Weight Trypsin-Plasmin Inhibitors Isolated from Papain Treated Urinary Trypsin Inhibitor

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657115 ◽

1982 ◽

Vol 47 (01) ◽

pp. 014-018 ◽

Cited By ~ 13

Author(s):

H Sumi ◽

N Toki ◽

S Takasugi ◽

S Maehara ◽

M Maruyama ◽

...

Keyword(s):

Trypsin Inhibitor ◽

Gel Electrophoresis ◽

Specific Activity ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Chromatography ◽

Urinary Trypsin Inhibitor ◽

Plasmin Activity ◽

Molecular Weights ◽

Plasmin Inhibitors

SummaryPapain treatment of human urinary trypsin inhibitor (UTI67; mol. wt. 43,000 by SDS-polyacrylamide gel electrophoresis, specific activity 1,897 U/mg protein) produced four new protease inhibitors, which were highly purified by gel chromatography on Sephadex G-100 and isoelectric focusing. The purified inhibitors (UTI26, UTI9-I, UTI9-II, and UTI9-III) were shown to be homogeneous by polyacrylamide disc gel electrophoresis, and had apparent molecular weights of 26,000, 9,000, 9,000, and 9,800, respectively, by sodium dodecyl sulfate gel electrophoresis. During enzymatic degradation of UTI67, the amino acid compositions changed to more basic, and the isoelectric point increased from pH 2.0 (UTI67) to pHs 4.4, 5.2, 6.6, and 8.3 (UTI26, UTI9-I, UTI9-II, and UTI9-III), respectively. Both the parent and degraded inhibitors had anti-plasmin activity as well as antitrypsin and anti-chymotrypsin activities. Much higher anti-plasmin/anti-trypsin and anti-plasmin/anti-chymotrypsin activities were observed in the degraded inhibitors than in the parent UTI67. They competitively inhibited human plasmin with Ki values of 1.13 X 10-7 - 2.12 X 10-6 M (H-D-Val-Leu-Lys-pNA substrate). The reactions were very fast and the active site of the inhibitors to plasmin was thought to be different from that to trypsin or chymotrypsin.

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Platelet Microtubule Subunit Proteins

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657068 ◽

1979 ◽

Vol 42 (05) ◽

pp. 1630-1633 ◽

Cited By ~ 3

Author(s):

A G Castle ◽

N Crawford

Keyword(s):

Epithelial Cells ◽

Gel Electrophoresis ◽

Blood Platelets ◽

Polyacrylamide Gel Electrophoresis ◽

Polyacrylamide Gel ◽

Lens Epithelial Cells ◽

Molecular Weights ◽

Kinetics Of ◽

Microtubular Structures

SummaryBlood platelets contain microtubule proteins (tubulin and HMWs) which can be polymerised “in vitro” to form structures which resemble the microtubules seen in the intact platelet. Platelet tubulin is composed of two non-identical subunits a and p tubulin which have molecular weights around 55,000 but can be resolved in alkaline SDS-polyacrylamide gel electrophoresis. These subunits associate as dimers with sedimentation coefficients of about 5.7 S although it is not known whether the dimer protein is a homo- or hetero-dimer. The dimer tubulin binds the anti-mitotic drug colchicine and the kinetics of this binding are similar to those reported for neurotubulins. Platelet microtubules also contain two HMW proteins which appear to be essential and integral components of the fully assembled microtubule. These proteins have molecular weights greater than 200,000 daltons. Fluorescent labelled antibodies to platelet and brain tubulins stain long filamentous microtubular structures in bovine lens epithelial cells and this pattern of staining is prevented by exposing the cells to conditions known to cause depolymerisation of cell microtubules.

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In-vivo and in-vitro secretion of prolactin by lactating rat adenohypophyses as a function of intracellular age

Journal of Endocrinology ◽

10.1677/joe.0.1010027 ◽

1984 ◽

Vol 101 (1) ◽

pp. 27-32 ◽

Cited By ~ 6

Author(s):

F. Mena ◽

G. Martínez-Escalera ◽

C. Clapp ◽

C. E. Grosvenor

Keyword(s):

Pituitary Gland ◽

Incubation Period ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Polyacrylamide Gel ◽

Pituitary Glands ◽

H 26

ABSTRACT Adenohypophysial prolactin of lactating rats was pulse-labelled by [3H]leucine injected i.v. at the time of removal of the pups. The [3H]prolactin concentration in the pituitary gland, analysed by polyacrylamide-gel electrophoresis, progressively fell as the time from labelling to removal of the pituitary gland increased from 8 to 24 h, which suggests that there was a loss of hormone as it aged within the gland. Suckling effectively provoked the depletion–transformation of total and [3H]prolactin (extracted at pH 7·2) when applied after 8 h but not when applied after either 16 or 24 h after removing the pups. In rats whose pups were removed for 8 h, suckling also depleted–transformed [3H]prolactin labelled 4 h, but not that labelled 1 h before suckling. The pituitary glands of other lactating rats were labelled with [3H]leucine injected i.v. at various times before removing the glands and incubating them in medium 199. The secretion into the medium of [3H]prolactin labelled either 4, 8, 16 or 24 h beforehand was maximal during the first 30 min then declined from 30 to 240 min of incubation. However, secretion of prolactin labelled 1 h and 10 min beforehand reached a maximum after 0·5–1 h and 2 h of incubation respectively, then remained constant during the remainder of the 4-h incubation period. The total 4-h secretion of [3H]prolactin was greatest (65% of preincubation concentration) from those glands labelled 4 h before in contrast to those labelled 10 min (15%) or 1 (38%), 8 (34%), 16 (18%) or 24 h (26%) before incubation. Taken together, these data suggest that prolactin synthesized 4 h earlier is more likely to be released in response to physiological stimuli than is more recently formed prolactin or prolactin which has remained in the pituitary gland for 16 h or longer. J. Endocr. (1984) 101, 27–32

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Effect of detergents and chemicals on purified vaccinia virus: analysis by SDS polyacrylamide gel electrophoresis and electron microscopy

Canadian Journal of Microbiology ◽

10.1139/m77-036 ◽

1977 ◽

Vol 23 (3) ◽

pp. 240-252 ◽

Cited By ~ 4

Author(s):

J. Boisvert ◽

T. Yamamoto

Keyword(s):

Electron Microscopy ◽

Vaccinia Virus ◽

Gel Electrophoresis ◽

Alkali Treatment ◽

Guanidine Hydrochloride ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Ammonium Bromide ◽

Molecular Weights ◽

Viral Particles

Vaccinia virus particles were dissociated into their constituent polypeptides and analysed by sodium dodecyl sulfate (SDS) gel electrophoresis. Thirty-three distinct polypeptide bands were identified and their molecular weights ranged between 11 000 and 150 000 daltons.Specific staining of gels containing polypeptides of dissociated virions revealed the presence of eight glycopeptides. No lipopeptides were detected.Analysis of chemical extracts (urea, guanidine hydrochloride, and alkali treatment) of the virus by SDS gel electrophoresis indicated that a total of 10 to 14 different polypeptides ranging in molecular weights from 11 000 to 70 000 daltons were solubilized.Analysis of detergent extracts and of the remains of extracted viral particles has shown that the detergent Nonidet P-40 (NP-40) solubilized a total of 11 polypeptides of which 6 were glycopeptides. The other detergents sodium deoxycholate (SDC) and cetyl trimethyl ammonium bromide (CTAB) were not as selective, both solubilizing more than 25 of the polypeptides composing the virus. Gel electrophoresis results also indicated that most of the small molecular weight (11 000–70 000 daltons) polypeptides were readily solubilized by NP-40, SDC, and CTAB, while those with molecular weights of 70 000 daltons and higher were not well solubilized.The effects of detergents were also analysed by electron microscopy. Evidence was obtained for subpopulations of viral particles having different susceptibility to detergent extraction.

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