scholarly journals Part of the activating cross-linked immunoglobulin G is internalized by human platelets to sites not accessible for enzymatic digestion

Blood ◽  
1986 ◽  
Vol 67 (1) ◽  
pp. 12-18 ◽  
Author(s):  
MO Spycher ◽  
UE Nydegger
Keyword(s):  
Immunoglobulin G ◽  
Prostaglandin E1 ◽  
Cytochalasin B ◽  
Enzymatic Digestion ◽  
Human Platelets ◽  
Serotonin Release ◽  
Antimycin A ◽  
Energy Dependent ◽  
Increased Susceptibility ◽  
Pronase Treatment

Abstract The differential uptake of tritium-labeled immunoglobulin G (IgG) cross- linked with bisdiazonium-benzidine (BDB) (3H-BDB-IgG) by washed, pooled human platelets to sites inaccessible to pronase digestion was tested. Up to 52% of the 3H-BDB-IgG associated with platelets at 37 degrees C resisted pronase treatment, whereas only 23% of the cross-linked IgG associated with platelets at 4 degrees C, or at 37 degrees C but in the presence of deoxyglucose/antimycin A, remained refractory to pronase. This effect was not due to platelet agglutination. Pronase resistance reached a maximum after a 60-minute incubation period at 37 degrees C. With increasing 3H-BDB-IgG input, both the total cross-linked IgG associated with platelets and the fraction resistant to pronase digestion approached saturation at 4 degrees C, but not at 37 degrees C. The proportion of 3H-BDB-IgG bound to platelets at 4 degrees C that was resistant to pronase treatment increased by 13% within five minutes of warming the platelets to 37 degrees C. Pretreatment of platelets with 10 mmol/L acetylsalicylic acid (or 10 mumol/L prostaglandin E1) prior to the addition of 3H-BDB-IgG led to a 74% (95%) inhibition of the 3H-BDB-IgG-induced 14C-serotonin release, but to only a 44% (49%) inhibition of pronase-digestible bound ligand. In contrast, pretreatment with 10 mumol/L cytochalasin B led to a mere 17% reduction of 14C-serotonin release, whereas acquisition of resistance to pronase digestion by the bound 3H-BDB-IgG was inhibited by 90%. Incubation of platelets at 37 degrees C with 3H-BDB-IgG and removal of unbound material prior to the addition of prostaglandin E1 or deoxyglucose/antimycin A had little effect on the susceptibility of platelet-associated 3H-BDB-IgG to pronase, whereas the addition of cytochalasin B to 3H-BDB-IgG-treated platelets resulted in greatly increased susceptibility of the platelet-associated ligand to pronase. Thus, after binding, 3H-BDB-IgG becomes transferred in an energy- dependent process to pronase-resistant cellular sites, most likely to the open canalicular system.

scholarly journals Part of the activating cross-linked immunoglobulin G is internalized by human platelets to sites not accessible for enzymatic digestion

Blood ◽  
1986 ◽  
Vol 67 (1) ◽  
pp. 12-18
Author(s):  
MO Spycher ◽  
UE Nydegger
Keyword(s):  
Immunoglobulin G ◽  
Prostaglandin E1 ◽  
Cytochalasin B ◽  
Enzymatic Digestion ◽  
Human Platelets ◽  
Serotonin Release ◽  
Antimycin A ◽  
Energy Dependent ◽  
Increased Susceptibility ◽  
Pronase Treatment

The differential uptake of tritium-labeled immunoglobulin G (IgG) cross- linked with bisdiazonium-benzidine (BDB) (3H-BDB-IgG) by washed, pooled human platelets to sites inaccessible to pronase digestion was tested. Up to 52% of the 3H-BDB-IgG associated with platelets at 37 degrees C resisted pronase treatment, whereas only 23% of the cross-linked IgG associated with platelets at 4 degrees C, or at 37 degrees C but in the presence of deoxyglucose/antimycin A, remained refractory to pronase. This effect was not due to platelet agglutination. Pronase resistance reached a maximum after a 60-minute incubation period at 37 degrees C. With increasing 3H-BDB-IgG input, both the total cross-linked IgG associated with platelets and the fraction resistant to pronase digestion approached saturation at 4 degrees C, but not at 37 degrees C. The proportion of 3H-BDB-IgG bound to platelets at 4 degrees C that was resistant to pronase treatment increased by 13% within five minutes of warming the platelets to 37 degrees C. Pretreatment of platelets with 10 mmol/L acetylsalicylic acid (or 10 mumol/L prostaglandin E1) prior to the addition of 3H-BDB-IgG led to a 74% (95%) inhibition of the 3H-BDB-IgG-induced 14C-serotonin release, but to only a 44% (49%) inhibition of pronase-digestible bound ligand. In contrast, pretreatment with 10 mumol/L cytochalasin B led to a mere 17% reduction of 14C-serotonin release, whereas acquisition of resistance to pronase digestion by the bound 3H-BDB-IgG was inhibited by 90%. Incubation of platelets at 37 degrees C with 3H-BDB-IgG and removal of unbound material prior to the addition of prostaglandin E1 or deoxyglucose/antimycin A had little effect on the susceptibility of platelet-associated 3H-BDB-IgG to pronase, whereas the addition of cytochalasin B to 3H-BDB-IgG-treated platelets resulted in greatly increased susceptibility of the platelet-associated ligand to pronase. Thus, after binding, 3H-BDB-IgG becomes transferred in an energy- dependent process to pronase-resistant cellular sites, most likely to the open canalicular system.

scholarly journals Complement-Dependent Action of HLA-Specific Antibodies on Human Platelets

1977 ◽  
Author(s):  
D. Heinrich ◽  
C. Mueller-Eckhardt ◽  
D. Bitter-Suermann
Keyword(s):  
Platelet Function ◽  
Complement Activation ◽  
Prostaglandin E1 ◽  
Platelet Rich Plasma ◽  
Human Platelets ◽  
Antimycin A ◽  
51Cr Release ◽  
Specific Antibodies ◽  
Independent Mechanism ◽  
Hla Antibody

HLA-antibody-induced platelet alterations are mediated by (a) complement-independent (Heinrich et al, Brit. J. Haematol., 35, 443, 1977) and (b) complement-dependent mechanisms. To prove the latter hypothesis platelet-rich plasma (PRP) of HLA-typed donors was incubated with HLA-specific sera and various inhibitors of platelet function (prostaglandin-E1 (PGE1), acetyl-salicylic acid (ASA), N-ethyl maleimide (NEM), adenosin, 2-desoxy-glucose + antimycin A (2-DOG + ANT.A)), furthermore an inhibitor of complement activation (cobra venom factor (CVF)) and EDTA or heparin known to interfer with platelet function and complement activation. Methods: Platelet aggregation (Born), Hc-seroto-nin release and 51Cr release. Results: Contrary to collagen-and thrombin-induced platelet alteration, PGE1, NEM, ASA, adenosin and 2-DOG+ANT. A exhibited only moderate inhibition of antibody-induced platelet alteration. However, EDTA, heparin and CVF+PGE1 strongly inhibited antibody-induced platelet alteration. The results support the concept of two pathways for specific HLA-antibody action on platelets: (a) a complement-independent mechanism resulting in aggregation and release and (b) a complement dependent, lytic mechanism.

scholarly journals Reciprocal transmembranous receptor-cytoskeleton interactions in concanavalin A-activated platelets.

1985 ◽  
Vol 101 (3) ◽  
pp. 993-1000 ◽  
Author(s):  
M E Wheeler ◽  
J M Gerrard ◽  
R C Carroll
Keyword(s):  
Platelet Activation ◽  
Concanavalin A ◽  
Prostaglandin E1 ◽  
Cytochalasin B ◽  
Specific Interaction ◽  
Human Platelets ◽  
Cross Linking ◽  
Con A ◽  
Glycoprotein Complex ◽  
Activated Platelets

Concanavalin A (Con A) has been used to activate platelets, inducing a specific interaction between the glycoprotein IIb-IIIa complex and the cytoskeleton of the activated platelet. In agreement with this, we have shown that Con A activates human platelets, initiating phosphorylation, secretion, and cytoskeletal formation. Con A and cytochalasin B were used to demonstrate a reciprocal interaction of the glycoprotein complex with the platelet cytoskeleton. Additionally, we have shown that a similar reciprocity is provided by the multivalent fibrin-fibrinogen platelet interaction found in the thrombin-induced clot. Con A differs from other activators in precipitating an apparent cytoskeletal core despite a complete inhibition of platelet activation by prostaglandin E1. We suggest, from this result, that Con A may be cross-linking a membrane-associated cytoskeletal complex present in the unactivated platelet.

Role of Internalization in Platelet Activation Induced by Collagen Fibers – Differential Effects of Aspirin, Cytochalasin D, and Prostaglandin E1

1991 ◽  
Vol 66 (06) ◽  
pp. 708-714 ◽  
Author(s):  
Andreas Ruf ◽  
Heinrich Patscheke ◽  
Eberhard Morgenstern
Keyword(s):  
Prostaglandin E1 ◽  
Close Contact ◽  
Shape Change ◽  
Human Platelets ◽  
Serotonin Release ◽  
Collagen Fibers ◽  
Cytochalasin D ◽  
Collagen Membrane ◽  
Computer Assisted ◽  
Initial Contact

SummaryWashed human platelets were stimulated with fibrillar collagen and platelet aggregation was prevented by non-stirring conditions. In these samples, electron microscopy revealed three fractions of platelets: 1) a majority without contacts to the collagen fibers, 2) with focal contacts to collagen, and 3) a small fraction of platelets with internalized collagen. All platelets had undergone shape change, and exhibited an internal contraction and granule release. However, only those with internalized collagen were completely degranulated. The internalized collagen was found to be in close contact to the contractile sphere in the platelet center, as it was demonstrable with computer assisted 3-D reconstruction from serial sections. Aspirin inhibited neither the adhesion to collagen nor its internalization by internal contraction. Also it did not impair the shape change and degranulation in the platelet fractions that internalized collagen. However, aspirin blocked the shape change and internal contraction of the other platelets and inhibited the [3H]serotonin release. Cytochalasin D 0.1 uM also suppressed the internalization of collagen, the shape change, the formation of a contractile gel, the degranulation, and the [3H]serotonin release in all platelets, whereas the number of platelets that adhered to collagen remained unchanged. The same effects were produced by prostaglandin E1. If the platelets were stimulated with the TXA2 mimetic, U46619, cytochalasin D at 0.1 εM had no effect; but at 20 εM it strongly enhanced the degranulation and the [3H]serotonin release, although the platelets remained discoid. It is concluded that collagen triggers a focal activation of an adherent platelet at the site of its initial contact to collagen. This subthreshold activation proceeds to degranulation and TXA2 formation via a feedback cascade which involves internal contraction, internalization of collagen and multiplication of the collagen membrane contacts.

Ticlopidine Facilitates the Deaggregation of Human Platelets Aggregated by Thrombin

1994 ◽  
Vol 71 (01) ◽  
pp. 091-094 ◽  
Author(s):  
M Cattaneo ◽  
B Akkawat ◽  
R L Kinlough-Rathbone ◽  
M A Packham ◽  
C Cimminiello ◽  
...  
Keyword(s):  
Cardiovascular Disease ◽  
Platelet Aggregation ◽  
Prostaglandin E1 ◽  
Human Platelet ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Before And After ◽  
Normal Human ◽  
Platelet Aggregates ◽  
Induced Aggregation

SummaryNormal human platelets aggregated by thrombin undergo the release reaction and are not readily deaggregated by the combination of inhibitors hirudin, prostaglandin E1 (PGE1) and chymotrypsin. Released adenosine diphosphate (ADP) plays an important role in the stabilization of thrombin-induced human platelet aggregates. Since ticlopidine inhibits the platelet responses to ADP, we studied thrombin-induced aggregation and deaggregation of 14C-serotonin-labeled platelets from 12 patients with cardiovascular disease before and 7 days after the oral administration of ticlopidine, 250 mg b.i.d. Before and after ticlopidine, platelets stimulated with 1 U/ml thrombin aggregated, released about 80–90% 14C-serotinin and did not deaggregate spontaneously within 5 min from stimulation. Before ticlopidine, hirudin (5× the activity of thrombin) and PGE1 (10 μmol/1) plus chymotrypsin (10 U/ml) or plasmin (0.06 U/ml), added at the peak of platelet aggregation, caused slight or no platelet deaggregation. After ticlopidine, the extent of platelet deaggregation caused by the same inhibitors was significantly greater than before ticlopidine. The addition of ADP (10 μmol/1) to platelet suspensions 5 s after thrombin did not prevent the deaggregation of ticlopidine-treated platelets. Thus, ticlopidine facilitates the deaggregation of thrombin-induced human platelet aggregates, most probably because it inhibits the effects of ADP on platelets.

Physiological Effects of Nonimmune Platelet Associated Immunoglobulin G

1981 ◽  
Vol 45 (01) ◽  
pp. 027-033 ◽  
Author(s):  
K Sugiura ◽  
M Steiner ◽  
M Baldini
Keyword(s):  
Shape Change ◽  
Fluorescein Isothiocyanate ◽  
Recovery Phase ◽  
Human Platelets ◽  
Serotonin Release ◽  
Platelet Volume ◽  
Igg Antibody ◽  
Heterologous Antiserum ◽  
Igg Binding ◽  
Series Of Experiments

SummaryThe function of nonimmune IgG associated with platelets is unknown. In a series of experiments we have investigated this problem, relating amount of platelet-associated IgG (PAIgG) to platelet volume, serotonin release, adherence of platelets to monocytes and platelet senescence. Most of these studies were performed with human platelets. Platelets freed of preexisting PAIgG by incubation at 22° C were incubated with IgG in a series of concentrations ranging from 0.4 — 27.0 X10-6 M. The IgG preparations used were demonstrably free of aggregated forms of the protein. The amount of PAIgG bound to platelets was determined by the use of fluorescein isothiocyanate-conjugated anti-IgG antibody (F-anti-IgG antibody) which was quantified in a fluorospectrophotometer. Newly bound IgG was assayed similarly by the use of F-IgG. A dose-dependent increase in platelet volume was associated with the binding of nonimmune IgG by platelets. The process which leveled off at an IgG concentration of 1.2 —1.5 X10-5 M was almost fully reversible and was not due to platelet shape change or aggregation. Release of serotonin from IgG-treated platelets was relatively small but to the extent that it occurred was positively related to the IgG concentration to which platelets were exposed. Adherence to autologous monocytes studied quantitatively by the use of formaldehyde-fixed cells was also positively related to the amount of IgG on the platelets. Normal or IgG-defident serum had a potent inhibitory (noncompetitive) action on the binding of F-IgG and F-anti-human IgG antibody to human platelets. Cohorts of platelets prepared in rabbits during the recovery phase of immunological thrombocytopenia induced by injection of heterologous antiserum, showed an age-dependent increase of PAIgG and of IgG binding. These results suggest that PAIgG plays a role in the clearance of senescent platelets.

A Class of Drugs that Inhibits Platelet Release and Aggregation by Apparent Interference with Anion Transport

1979 ◽  
Author(s):  
N.R. Shulman ◽  
H.B. Pollard ◽  
K. Tack-Goldman
Keyword(s):  
Pyridoxal Phosphate ◽  
Human Platelets ◽  
Anion Transport ◽  
Serotonin Release ◽  
Secretory Cells ◽  
Chloride Uptake ◽  
Potential Alternative ◽  
Osmotic Lysis ◽  
Platelet Release ◽  
Ph Range

The platelet release reaction is analogous to the process of exocytosis by which many other secretory cells release hormones or mediators from intracellular granules. Anion transport blocking (ATB) drugs Inhibit release of epinephrine from isolated chromaffin granules (CG) by blocking chloride uptake and preventing osmotic lysis. Studies on platelets analagous to those done on CG showed that increasing osmotic strength in the range 600-1000 m0sm progressively suppressed serotonin release to completion and that ATB drugs (viz, probenecid, SITS, pyridoxal phosphate and suramin) at mM concentrations completely inhibited release and aggregation of human platelets stimulated by thrombin, ADP, A23187, epinephrine or collagen. Sulfinpyrazone has the appropriate structure for anionic blocking, and may suppress platelet function as effectively by this mechanism as by cycloxy-genase inhibition. The ATB drugs acted apparently to prevent movement of OH- from the more alkaline medium into the relatively acidic granule, for platelet release was not inhibited by replacing anions with isethionate or sucrose, but was markedly dependent on OH- in the pH range 6 to 7.5 where inhibition by the ATB drugs was competitive with respect to OH-. Since the ATB compounds include some relatively nontoxic drugs in common use, and since their action on platelets differs markedly from that of aspirin, they should receive attention as potential alternative or auxiliary antithrombotic agents.

scholarly journals Effects of antimycin A and 2-deoxyglucose on secretion in human platelets. Differential inhibition of the secretion of acid hydrolases and adenine nucleotides

1979 ◽  
Vol 182 (2) ◽  
pp. 413-419 ◽  
Author(s):  
Holm Holmsen ◽  
Linda Robkin ◽  
H. James Day
Keyword(s):  
Shape Change ◽  
Adenine Nucleotides ◽  
Human Platelets ◽  
Dense Granule ◽  
Metabolic Inhibitors ◽  
Antimycin A ◽  
Acid Hydrolase ◽  
Acid Hydrolases ◽  
Dense Granule Secretion ◽  
Granule Secretion

1. Shape change, aggregation and secretion of dense-granule constituents in platelets differ in their dependence on cellular energy metabolism. The possibility that such a difference also exists between secretion of dense-granule constituents and acid hydrolases was investigated. 2. Human platelets were incubated with [14C]adenine in plasma, and then washed and resuspended in salt solutions. The effects of incubating the cells with antimycin A and 2-deoxyglucose on the concentrations of [14C]ATP, ADP, AMP, IMP and inosine plus hypoxanthine and on thrombin-induced secretion of ATP plus ADP and acid hydrolases were studied. The metabolic inhibitors only affected 14C-labelled nucleotides, whereas thrombin only liberated unlabelled ATP and ADP. 3. The extent of secretion decreased progressively with time during incubation with the metabolic inhibitors. At any time the secretion of acid hydrolases, β-N-acetylglucosaminidase, β-glucuronidase and β-galactosidase was inhibited to a greater extent than secretion of ATP plus ADP (dense-granule secretion). 4. Incubation with the metabolic inhibitors shifted the log (dose)–response relationship to higher thrombin concentrations, and with a greater shift for acid hydrolase secretion than for dense-granule secretion. 5. Antimycin, when present alone, caused a marked decrease in the rate of acid hydrolase secretion, but had no effect on dense-granule secretion. 6. These results further support the view that acid hydrolase secretion and dense-granule secretion are separate processes with different requirements for ATP energy. Acid hydrolase secretion, but not dense-granule secretion, appears to depend on a simultaneous rapid generation of ATP, which can be accomplished by oxidative, but not by glycolytic, ATP production.

Cyanide-resistant respiration in isolated Asparagus mesophyll cells

1984 ◽  
Vol 62 (6) ◽  
pp. 1122-1126 ◽  
Author(s):  
Nancy M. Shadeed ◽  
Alan W. Bown
Keyword(s):  
Alternative Pathway ◽  
Potassium Cyanide ◽  
Antimycin A ◽  
Mesophyll Cells ◽  
Atp Levels ◽  
Energy Conserving ◽  
Sequential Addition ◽  
Tetraethylthiuram Disulfide ◽  
Energy Dependent ◽  
Cyanide Resistant Respiration

The inhibitors of oxidative phosphorylation antimycin A (0.5 μg/mL), oligomycin (0.01 mg/mL), and sodium azide (3 mM) eliminated energy-dependent H+ efflux from isolated Asparagus mesophyll cells. Antimycin A and oligomycin also reduced ATP levels by 60% or more. In contrast the same concentrations of inhibitors had little or no effect on respiratory O2 consumption. The sequential addition of potassium cyanide to give a final concentration of 5.2 μM resulted in a 60% maximum inhibition of O2 consumption. Subsequent addition of 0.2 mM disulfiram (tetraethylthiuram disulfide), a potent inhibitor of cyanide-resistant respiration, resulted in a further reduction of the oxygen consumption rate. In the absence of cyanide, 0.2 mM disulfiram inhibited O2 consumption by 40 to 80%, depending on the suspension medium. Disulfiram had little or no effect on the ATP levels which varied between 0.7 and 2.2 nmol ATP/106 cells. The results indicate that disulfiram inhibits a non-energy-conserving cyanide-resistant alternative pathway in Asparagus mesophyll cells.

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