scholarly journals Effects of collagen, ionophore A23187 and prostaglandin E1 on the phosphorylation of specific proteins in blood platelets

1979 ◽  
Vol 178 (2) ◽  
pp. 397-406 ◽  
Author(s):  
Richard J. Haslam ◽  
James A. Lynham ◽  
Joan E. B. Fox
Keyword(s):  
Cyclic Amp ◽  
Prostaglandin E1 ◽  
Blood Platelets ◽  
Short Circuit ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Human Platelets ◽  
Ionophore A23187 ◽  
Release Reaction ◽  
Specific Proteins

Human platelets that had been preincubated with 5-hydroxy[3H]tryptamine and [32P]Pi were stirred with various agents; the secretion of 5-hydroxy[3H]tryptamine from platelet granules and the radioactivity of platelet [32P]phosphopolypeptides separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis were then measured. Exposure of the platelets to collagen fibres or ionophore A23187 selectively increased the phosphorylation of polypeptides with apparent mol.wts. of 47000 (P47) and 20000 (P20) by approx. 3-fold, in association with the release of 5-hydroxy[3H]tryptamine. The 47000-mol.wt. phosphopolypeptide (P47) was clearly separated from platelet actin by the electrophoresis system used. Prostaglandin E1, which inhibits platelet function by increasing platelet cyclic AMP, decreased the phosphorylation of polypeptides caused by collagen as well as the release of 5-hydroxy[3H]tryptamine. Prostaglandin E1 also selectively increased the phosphorylation of distinct polypeptides with apparent mol.wts. of 24000 (P24) and 22000 (P22) by approx. 2-fold. As the phosphorylation reactions caused by collagen are probably mediated by an increase in Ca2+ concentration in the platelet cytosol and may have a role in the release reaction [Haslam & Lynham (1977) Biochem. Biophys. Res. Commun.77, 714–722; (1978) Thromb. Res.12, 619–628], we suggest that a cyclic AMP-dependent phosphorylation of the 24000- and/or 22000-mol.wt. polypeptides caused by prostaglandin E1 may initiate processes that decrease the Ca2+ concentration in the cytosol, so inhibiting both the Ca2+-dependent phosphorylation reactions and the release reaction. Treatment of platelets with prostaglandin E1 did not inhibit the increased phosphorylation of polypeptides with apparent mol.wts. of 47000 and 20000 (P47 and P20) caused by ionophore A23187, which may therefore short-circuit cyclic AMP-dependent mechanisms that decrease the Ca2+ concentration in the platelet cytosol. As prostaglandin E1 did inhibit the release of 5-hydroxy[3H]tryptamine by ionophore A23187, cyclic AMP may also inhibit the release reaction by additional mechanisms.

scholarly journals Human platelets are defective in processing of cholera toxin

1983 ◽  
Vol 212 (3) ◽  
pp. 669-678 ◽  
Author(s):  
R J Hughes ◽  
P A Insel
Keyword(s):  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Cholera Toxin ◽  
Mammalian Cells ◽  
Time Course ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Human Platelets ◽  
Cyclase Activity ◽  
Adenylate Cyclase Activity

Cholera toxin is unable to elevate cyclic AMP levels in intact human platelets despite being very efficacious in this respect in other mammalian cells; in the presence of 0.5 mM-isobutylmethylxanthine, we found that 3-6nM-cholera toxin over 3h at 37 degrees C elevated platelet cyclic AMP from 33 +/- 13 to 39 +/- 12pmol/mg of protein (means +/- S.D.; n = 12). We have investigated the basis for this lack of response. 125I-labelled cholera toxin bound to platelets both saturably and with high affinity (Kd congruent to 60pM; Bmax. congruent to 50fmol/mg of protein). Incubation of platelets with the putative cholera toxin receptor monosialoganglioside GM1 enhanced 125I-labelled cholera toxin binding at least 40-fold but facilitated only a minimal (less than or equal to 3-fold) elevation of platelet cyclic AMP levels. In contrast, dithiothreitol-activated cholera toxin markedly stimulated adenylate cyclase activity in platelet membranes. Platelet cytosol both enhanced stimulation of adenylate cyclase activity by activated cholera toxin (A1 subunit) and supported stimulation by the A1-A2 subunit of cholera toxin. Neither GTP nor NAD+, both necessary for response to cholera toxin, was lacking in intact platelets. However, we found that platelets were unable to cleave cholera toxin to the active A1 subunit (as assessed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis). By contrast, murine S49 lymphoma cells were able to generate the A1 subunit with a time course that closely resembled the kinetics of toxin-mediated cyclic AMP accumulation in these cells. Thus we conclude that human platelets are defective in their ability to process surface-bound cholera toxin. These results indicate that binding of cholera toxin to surface receptors is necessary, but not sufficient, for expression of the toxin effect and the generation of the A1 subunit of the toxin may be rate-limiting for expression of cholera toxin response.

Proteolytic Inhibitors, Contact and Other Variables in the Release Reaction of Human Platelets

1972 ◽  
Vol 28 (03) ◽  
pp. 393-407 ◽  
Author(s):  
Marjorie B. Zucker
Keyword(s):  
Cyclic Amp ◽  
Prostaglandin E1 ◽  
Platelet Rich Plasma ◽  
Human Platelets ◽  
Ph Changes ◽  
Release Reaction ◽  
Platelet Shape

SummaryPlatelet-rich plasma (PRP) containing platelets labelled with 14C-serotonin was used to characterize the release reaction. Heparin promotes release, and the reduced release response to epinephrine observed in heparinized compared to citrated PRP is probably due to the earlier reaction. The release reaction can occur in citrated PRP at pH 7.0, is marked between pH 7.2 and 7.6, and decreases above pH 8.0. It is retained better when PRP is stored below pH 7.7. Adenosine, prostaglandin E1, and theophylline inhibit both aggregation and release, probably because they elevate cyclic AMP. D- and L-tosyl-phenylalanine chloromethylketone (TPCK) also inhibit both aggregation and release at a concentration of 0.1 mM whereas 0.1 mM D- and l-L-chloro-2-tosylamido-7-amino-2-heptanone (TLCK) and 5 mM diisopropylfluoro-phosphate (DFP), like aspirin, inhibit only release. Colchicine (0.1 mM) alters platelet shape but does not affect release or aggregation. Centrifugation of PRP at 37° C causes release which responds to inhibitors and pH changes in a manner similar to release induced by aggregation with ADP or epinephrine. Release occurs in thromb-asthenic platelets centrifuged at 37° C and is increased by epinephrine. Release of ATP and ADP correlates significantly with release of 14C-serotonin. Thrombin plus CaCl2 causes release of β-glucuronidase but not of zinc.

Effects of histamine and histamine receptor antagonists on ion transport in rabbit descending colon

1984 ◽  
Vol 247 (4) ◽  
pp. G411-G418 ◽  
Author(s):  
R. D. McCabe ◽  
P. L. Smith
Keyword(s):  
Ion Transport ◽  
Receptor Antagonist ◽  
Prostaglandin E1 ◽  
Short Circuit ◽  
Bathing Solution ◽  
Ionophore A23187 ◽  
Dependent Manner ◽  
H2 Receptor Antagonist ◽  
Maximal Inhibition ◽  
Descending Colon

The effects of histamine on colonic ion transport were examined in in vitro preparations of rabbit descending colon. Serosal addition of histamine (10(-5) M) produced a transient increase in short-circuit current (Isc) and transepithelial conductance. The Isc response to histamine could be blocked by removing Cl from both bathing solutions, adding furosemide (10(-3) M) to the serosal bathing solution, adding indomethacin to the serosal and mucosal bathing solutions (10(-5) M), or removing Ca from the serosal bathing solution. In addition, the histamine-induced increase in Isc was inhibited in a dose-dependent manner by the H1-receptor antagonist diphenhydramine, with a maximal inhibition at 10(-4) M and a half-maximal inhibition at 3 X 10(-7) M. The H2-receptor antagonist cimetidine (10(-3) M) was without effect on the histamine response. Measurement of unidirectional Na, K, and Cl fluxes revealed that serosal addition of diphenhydramine (10(-3) M) reduced basal Isc due to a decrease in mucosal-to-serosal Na flux. Serosal addition of diphenhydramine (10(-3) M) also inhibited the increase in Isc produced by serosal addition of prostaglandin E1, 8-bromo-cAMP, cholera toxin, or the ionophore A23187. Measurement of unidirectional K and Cl fluxes revealed that prostaglandin E1 alone increased serosal-to-mucosal K and Cl fluxes and reduced the mucosal-to-serosal K flux, thereby increasing net K and Cl secretion. Serosal diphenhydramine (10(-3) M) abolished the changes in Cl fluxes produced by prostaglandin E1 and reduced the magnitude of the changes in K fluxes.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Phosphorylation of synaptic-membrane proteins from ox cerebral cortex in vitro. Preparation of fractions enriched in phosphorylated proteins by using extraction with detergents and urea, and gel filtration

1977 ◽  
Vol 163 (2) ◽  
pp. 369-378 ◽  
Author(s):  
P R Dunkley ◽  
H Holmes ◽  
R Rodnight
Keyword(s):  
Cerebral Cortex ◽  
Cyclic Amp ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Synaptic Membrane ◽  
Hydroxyl Groups ◽  
Phosphorylated Proteins

Synaptic-membrane fragments from ox cerebral cortex contain basal and cyclic AMP-stimulated protein kinase(s) that transfer 32P from [gamma-32P]ATP to hydroxyl groups of serine and threonine residues in membrane-protein substrates. In the present work, labelled membrane fragments were partitioned into soluble and insoluble fractions with Triton X-100, Nonidet P. 40, sodium deoxycholate and urea, and the distribution of 32P-labelled protein in the fractions was determined by polyacrylamide-gel electrophoresis and radioautography. A high percentage of phosphorylated protein sustrates remained insoluble, including those whose phosphorylation was most highly stimulated by cyclic AMP. Whole membrane fragments and samples prepared by detergent extraction were fractionated on Sepharose 6B columns in the presence of low concentrations of sodium dodecyl sulphate and pooled fractions were analysed by polyacrylamide-gel electrophoresis and radioautography. Phosphorylated proteins were fractionated on the basis of their molecular weight, but homogeneous protein was not obtained. The results are discussed in relation to the techniques used and the results obtained in other laboratories.

scholarly journals Glycoprotein V is not the thrombin activation receptor on human blood platelets

Blood ◽  
1986 ◽  
Vol 68 (3) ◽  
pp. 720-725 ◽  
Author(s):  
D Bienz ◽  
W Schnippering ◽  
KJ Clemetson
Keyword(s):  
Platelet Activation ◽  
Polyclonal Antibodies ◽  
Blood Platelets ◽  
Polyacrylamide Gel Electrophoresis ◽  
Human Platelets ◽  
Exchange Chromatography ◽  
Thrombin Activation ◽  
Strong Candidate ◽  
Triton X ◽  
Kinetics Of

Thrombin activation of platelets involves two receptors: glycoprotein Ib (GPIb), which affects the kinetics of the response; and, as a strong candidate for the second, essential receptor, GPV, a hydrophobic, 82-kd glycoprotein with an isoelectric point (pI) of pH 5.85 to 6.55. Whole platelets were treated with endogenous platelets calcium-activated proteases, yielding a major fragment, GPV8, with molecular weight (mol wt) of 79 kilodaltons (kd). The fragment was purified by affinity chromatography on wheat germ agglutinin followed by ion exchange chromatography on DEAE-Sephacel using first a 0 to 0.7-mol/L and then a 0 to 0.3-mol/L NaCl gradient. A rabbit was immunized with the purified GPV8 for preparation of polyclonal antibodies. Crossed immunoelectrophoresis and two-dimensional polyacrylamide gel electrophoresis (PAGE) electrophoretic blotting with the separate phases of a Triton X-114 phase partition of human platelets showed the characteristic pattern of GPV in the hydrophobic phase. During thrombin- induced platelet aggregation GPV is hydrolysed, releasing a fragment, GPVf1, to the supernatant. The fragment GPVf1 still contains a thrombin- binding site. Anti-GPV antibodies blocked GPV proteolysis, but did not inhibit platelet activation induced by thrombin. We conclude that proteolysis of GPV by thrombin is not essential for platelet activation.

Thrombin-Induced Surface Changes of Human Platelets in Plasma: Their Relation to Serotonin Release

1979 ◽  
Author(s):  
K. Subbarao ◽  
V.V. Kakkar
Keyword(s):  
Membrane Proteins ◽  
Blood Platelets ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein Band ◽  
Human Platelets ◽  
Serotonin Release ◽  
Glycoprotein I ◽  
Reversible Aggregation ◽  
Coomassie Blue ◽  
Labeling Pattern

Membrane proteins of both control and thrombin-treated platelets were labeled by NaB3H4, reduction of Schiff bases formed between pyridoxal 5′-phosphate and protein amino groups. Examination of the labeled polypeptides by SDS-polyacrylamide gel electrophoresis and fluorography disclosed a different labeling pattern for thrombin-treated platelets. The distributions of Coomassie blue-stained protein from treated and untreated cells were, by contrast, almost identical. Fluorographs of control platelets showed a single intensely labeled protein band (mol wt 90,000) whereas with cells exposed to thrombin (30-60 milliunits) about 10 protein bands with mol wts ranging from 43,000 to 200,000 were typically present. Among these were: thrombin-sensitive protein (mol wt 188,000), glycoprotein I (mol wt 150,000) and actin (mol wt 43,000). When serotonin release was prevented, either by reversing platelet aggregation with low amounts of ADP (0.1-0.3 μM) or by preincubating with 3',5'-ADP (20 μM), an inhibitor of both ADP- and thrombin-induced platelet function, the labeling patterns on fluorographs were similar to the control. These results indicate that blood platelets can undergo reversible aggregation without major changes in their surface topography, whereas thrombin-induced serotonin release appears related to structural alterations in platelet membrane proteins.

scholarly journals Immunochemical characterization of the platelet-specific alloantigen Leka: a comparative study with the PlA1 alloantigen

Blood ◽  
1984 ◽  
Vol 64 (6) ◽  
pp. 1212-1219 ◽  
Author(s):  
N Kieffer ◽  
B Boizard ◽  
D Didry ◽  
JL Wautier ◽  
AT Nurden
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Human Platelets ◽  
Blue Staining ◽  
Igg Antibody ◽  
Immune Disorder ◽  
Immunochemical Characterization ◽  
Coomassie Blue Staining ◽  
Coomassie Blue

Abstract We report the immunochemical characterization of a new platelet- specific alloantigen detected using an IgG antibody isolated from the serum of a patient with posttransfusion purpura (PTP). In indirect immunoprecipitation experiments, the antibody, termed anti-Leka, predominantly precipitated glycoprotein (GP) IIb from Triton X-100 lysates of normal human platelets. In an immunoblot procedure, which involved the transfer of platelet polypeptides separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to nitrocellulose membrane, anti-Leka bound exclusively to GP IIb. Under identical conditions, four anti-PlA1 antibodies each reacted with GP IIIa. No binding of anti-Leka IgG occurred to Leka (-) platelets or to their separated polypeptides although GP IIb was normally detected by Coomassie blue staining. After electrophoresis of reduced platelet proteins, the Leka determinant was localized to the IIb alpha chain. Thus, unlike the PlA1 antigen, the Leka determinant was not destroyed by disulfide reduction. Analysis of platelets from a patient with Glanzmann's thrombasthenia revealed little or no binding in the GP IIb position. Anti-Leka permitted the identification of 76,000 and 60,000 dalton fragments of GP IIb retained by the platelet following chymotrypsin treatment. Our results further highlight the immunogenicity of the GP IIb-IIIa complex. They also suggest that antibodies against GP IIb can cause the thrombocytopenia observed in PTP and that anti-PlA1 antibodies do not account exclusively for the pathophysiology of this immune disorder.

scholarly journals Survival and recovery of human platelets stored for five days in a non- plasma medium

Blood ◽  
1986 ◽  
Vol 67 (3) ◽  
pp. 672-675 ◽  
Author(s):  
GA Adams ◽  
SD Swenson ◽  
G Rock
Keyword(s):  
Platelet Aggregation ◽  
Human Blood ◽  
Blood Platelets ◽  
Human Platelets ◽  
Plasma Medium ◽  
Release Reaction ◽  
In Vivo Recovery ◽  
Human Blood Platelets

Abstract Human blood platelets were stored for five days as concentrates in 60 mL of: (a) plasma; (b) non-plasma medium with anticoagulant; and (c) non-plasma medium without anticoagulant. All preparations were equally functional when tested for platelet aggregation and release reaction in response to single agonist or synergistic pairs of agonists in vitro. Platelets stored in non-plasma medium with anti-coagulant had lower kallikrein, fibrino(gen)peptide A, lactate, and beta-thromboglobulin than did plasma controls after five days. In vivo recovery and survival of platelets stored in non-plasma medium with anticoagulant were 51.2% +/- 4.3% and 8.7 +/- 0.3 days, respectively, which were not statistically different from plasma controls of 39.2% +/- 4.9% and 7.2 +/- 0.8 days, respectively. It is concluded that platelets can be stored for five days in a non-plasma medium and still have good in vivo recoveries and survivals.

Synthesis and Ultrastructural Localization of Protein C Inhibitor in Human Platelets and Megakaryocytes

Blood ◽  
1999 ◽  
Vol 94 (4) ◽  
pp. 1300-1312
Author(s):  
Maria J. Prendes ◽  
Edith Bielek ◽  
Margareta Zechmeister-Machhart ◽  
Erika Vanyek-Zavadil ◽  
Veronica A. Carroll ◽  
...  
Keyword(s):  
Protein C ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Plasminogen Activator Inhibitor ◽  
Ultrastructural Localization ◽  
Human Platelets ◽  
Plasminogen Activator Inhibitor 1 ◽  
Sds Page ◽  
Protein C Inhibitor

The occurrence of protein C inhibitor (PCI) in human platelets and megakaryocytes was analyzed. As judged from enzyme-linked immunosorbent assays (ELISAs), PCI was present in platelets at a concentration of 160 ng/2 × 109 cells. Its specific activity was 5 times higher than that of plasma PCI. Consistently, mainly the 57-kD form (active PCI) and some high molecular weight (Mr) forms, but no bands corresponding to cleaved PCI, were detected when platelet lysates were immunoprecipitated with monoclonal anti-PCI-IgG and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The localization of PCI in platelets was studied by immunofluorescence histochemistry and immunotransmission electron microscopy: PCI was detected in  granules, in the open canalicular system, and on the plasma membrane. At these sites, colocalization with plasminogen activator inhibitor-1 was seen. Studies were performed to clarify whether platelet PCI is endogenously synthesized or taken up from plasma. Internalization of biotinylated-PCI was analyzed using platelets in suspension and gold-labeled streptavidin for visualization of incorporated biotin. Dose- and time-dependent uptake of PCI was found. PCI mRNA was detected in platelets by reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blotting, as well as in megakaryocytes by in situ hybridization of human bone marrow cryosections. We therefore conclude that platelets contain a functionally active PCI pool that is derived from both endogenous synthesis as well as internalization.

scholarly journals Short communication: possible association of newly absorbed serotonin with nonmetabolic, granule-located adenine nucleotides in human blood platelets

Blood ◽  
1975 ◽  
Vol 45 (3) ◽  
pp. 413-416
Author(s):  
H Holmsen ◽  
CA Setkowsky ◽  
HJ Day
Keyword(s):  
Guinea Pig ◽  
Human Blood ◽  
Short Communication ◽  
Adenine Nucleotides ◽  
Blood Platelets ◽  
Human Platelets ◽  
Serotonin Release ◽  
Release Reaction ◽  
Human Blood Platelets

[3H]-adenine-labeled human platelets in plasma were incubated with or without nonradioactive serotonin. Release reaction was then induced by ADP, epinephrine, collagen, or thrombin. Platelets that had been incubated with serotonin released four times as much serotonin as platelets incubated without serotonin. The specific radioactivities of the ATP and ADP released to plasma during release reaction induced with all four inducers were the same in both systems. This shows that when serotonin is taken up by human platelets, it enters the compartment containing nonmetabolic, granula-stored ATP, and not the compartment with metabolic extragranular ATP. These results suggest that the mechanism of serotonin storage in human platelets is similar to that in other species investigated, i.e., rabbit, guinea pig, and pig.

Sign in / Sign up

Export Citation Format

Share Document