scholarly journals Serotonin Release from Filtered and Unfiltered Human Platelets Induced by Antigen/Antibody Complexes of Different Valencies

1977 ◽  
Author(s):  
M. Kazatchkine ◽  
J. Caen ◽  
Anthea H. Johnson ◽  
J.F. Mowbray
Keyword(s):  
Platelet Rich Plasma ◽  
Gel Filtration ◽  
Human Platelets ◽  
Serotonin Release ◽  
Absolute Requirement ◽  
Aggregated Immunoglobulins ◽  
Antigen Antibody ◽  
Free Plasma ◽  
Serum Albumen ◽  
Made In

In order to study the nature of receptors for immune complexes (IC) on the surface of platelets, the release of radiolabelled serotonin produced by exposure of platelets to IC of known composition was studied. Free plasma components might alter this reaction, so platelets were used both in platelet rich plasma (PRP) and after gel filtration. As it has been thought that the aggregation of the immunoglobulin molecules through the antigen was neccessary for the release reaction, we have studied complexes made with antigens of different valencies. The antigen was albumen substituted with different numbers of dinitrophenol (DNP) groups, and the antibody rabbit-anti DNP. Polyvalent antigen complexes were also made from bovine serum albumen (BSA) and rabbit anti-BSA antibody. Soluble and insoluble IC made in a variety of ratios with polyvalent antigen induced release both in PRP and in gel filtered platelets. Soluble complexes in antigen excess were the most effective. Complexes of monovalent antigen prepared, using two methods of monovalent substitution, were also capable of inducing release in PRP and in gel filtered platelets, although the complex could not exist in an aggregated form. Release was less than that produced by polyvalent complex, but could be increased by the addition of CI.These results show that, at least with monovalent IC, the simultaneous involvement of more than one receptor is not an absolute requirement for platelet release. These receptors for monovalent complexes could be different from the Fc receptors for aggregated immunoglobulins or polyvalent complexes.

Fluoride, An Internally Acting Platelet Activator, Causes Phosphorylation Of Platelet Phosphatidic Acid And 20K And 47K Proteins

1981 ◽  
Author(s):  
E H Mürer ◽  
E Siojo ◽  
J L Daniel
Keyword(s):  
Phosphatidic Acid ◽  
Platelet Activation ◽  
Great Increase ◽  
Platelet Rich Plasma ◽  
Gel Filtration ◽  
Human Platelets ◽  
Silica Gels ◽  
Late Step ◽  
Aluminum Plates ◽  
Chloroform Methanol

The effects of fluoride, which is transported into platelets in order to induce secretion, are compared with known effects of thrombin, which acts via external sites. Thus, the changes related to transmission of signal through the platelet membrane will not be common to the two activators, only those changes which are subsequent to the internal triggering of platelet activation. Human platelets were prepared by collection in EDTA and washing in saline-EDTA or by gel filtration of citrated platelet-rich plasma. The two methods gave similar results. Platelets prelabeled in plasma with 32P and them separated were incubated at 37°C with 10 mM fluoride at pH 7.4, and samples removed at intervals. (1) The protein was precipitated with HC104, then solubilized by sonication with SDS buffer and the protein bands separated by acrylamide slab gel electrophoresis. The 20K and 47K bands showed 100 to 200% increase in label, with maximum at 8 min incubation (50% secretion) and a great increase seen already at 3 min incubation, where little secretion is observed. (2) Samples were extracted with chloroform-methanol, evaporated to dryness under N2, redissolved in chloroform and applied on thinlayer silica gels on aluminum plates. Two different systems for separating phosphatidic acid (PA) were used. No significant increase in 32P radioactivity was seen in PA the first 3 min. The label at 20 min was 3x that at 8 min. Thus the labeling related to contractile events, a late step in secretion, precedes the labeling of PA, suggesting that the major part of this labeling is not related to the initial phase of platelet activation.

scholarly journals Ultrastructural and Partial Biochemical Characterization of Platelets from Chediak-Higashi Cattle

1977 ◽  
Author(s):  
K. M. Meyers ◽  
C. I. Seacord ◽  
G. Hopkins ◽  
H. Holmsen
Keyword(s):  
Electron Micrographs ◽  
Biochemical Characterization ◽  
Platelet Rich Plasma ◽  
Gel Filtration ◽  
Human Platelets ◽  
Additional Information ◽  
Storage Pool ◽  
Calcium And Magnesium ◽  
Chediak Higashi Syndrome

To provide additional information on the platelet defect which is associated with the Chediak-Higashi syndrome (CHS), platelet rich plasma from normal and CHS cattle was incubated with 14C-adenine. Platelets were then isolated by gel filtration and treated with thrombin. Both the resting amount and extent of secretion of ATP, ADP, several acid hydrolasis, serotonin, calcium and magnesium was determined. Nucleotide profiles and electron micrographs of resting and thrombin treated platelets were also obtained. The markedly reduced secretion of nucleotides, serotonin, and metals demonstrate that CHS cattle have a storage pool defect. Furthermore, there appears to be significant differences in both the resting amount and extent of secretion of several of these measured substances between normal cattle and human platelets.

Inhibition and potentiation of platelet function by lysolecithin

Blood ◽  
1977 ◽  
Vol 49 (1) ◽  
pp. 101-112 ◽  
Author(s):  
JH Joist ◽  
G Dolezel ◽  
MP Cucuianu ◽  
EE Nishizawa ◽  
JF Mustard
Keyword(s):  
Platelet Function ◽  
Platelet Rich Plasma ◽  
Membrane Damage ◽  
Adenosine Diphosphate ◽  
Lactic Dehydrogenase ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
Serotonin Release ◽  
Prolonged Incubation ◽  
Induced Aggregation

Abstract The effects of lysolecithin (LPC) on aggregation, serotonin release, shape, and lysis of rabbit, pig, or human platelets in platelet-rich plasma (PRP) or Tyrode albumin solution were examined during prolonged incubation. LPC added to citrated or heparinized PRP from humans or rabbits at a final concentration above 100 muM caused instantaneous inhibition of platelet aggregation induced by adenosine diphosphate (ADP), epinephrine (human PRP only), collagen, or thrombin. The inhibitory effect of LPC was found to be partially reversible over a period of 60–90 min. LPC at final concentrations above 30 muM also caused inhibition of ADP-, collagen-, and thrombin-induced aggregation and collagen- and thrombin-induced release of serotonin in suspensions of rabbit, pig, or human platelets. With washed platelets, the inhibitory effect not only rapidly disappeared but was followed by transient potentiation of aggregation and serotonin release. This potentiating effect of LPC was most pronounced when thrombin was used as stimulus. Both inhibition and potentiation were observed at concentrations of LPC that did not cause a significant change in platelet shape or loss from platelets of lactic dehydrogenase. Inhibition and potentiation were also observed when platelets were added to suspending medium containing LPC, although considerably higher concentrations of LPC were required under these conditions. Potentiation was not observed when LPC was added to citrated or heparinized rabbit or human PRP or to washed rabbit platelets suspended in a medium containing 4% bovine serum albumin. It seemed likely that some or all of the observed effects of LPC on platelet function were due to structural modification of the platelet membrane insufficient to result in gross membrane damage or platelet lysis. In addition, the results of experiments using 14C-LPC seemed to indicate that the observed potentiating effect of LPC on platelet function may be related to its rapid uptake and metabolism by the platelets.

scholarly journals The effect of PGI2 and theophylline on the response of platelets subjected to shear stress

Blood ◽  
1981 ◽  
Vol 58 (4) ◽  
pp. 678-681 ◽  
Author(s):  
RA Hardwick ◽  
JD Hellums ◽  
DM Peterson ◽  
JL Moake ◽  
JD Olson
Keyword(s):  
Shear Stress ◽  
Platelet Aggregation ◽  
Platelet Rich Plasma ◽  
Antiplatelet Agent ◽  
Human Platelets ◽  
Clinical Applications ◽  
Serotonin Release ◽  
Large Reduction ◽  
Prior Work ◽  
Rotational Viscometer

A specially designed rotational viscometer was used to investigate the effects of the antiplatelet agent PGI2 in combination with theophylline on the response of human platelets subjected to shear stress. Samples of citrated platelet-rich plasma (PRP) were exposed to shear stress in the viscometer for a period of 5 min at 23 degrees C. The levels of stress studied ranged from 50 to 300 dynes/sq cm. Pretreatment of the platelets with 0.01 microM PGI2 and 500 microM theophylline before exposure to shear stress caused a large reduction in shear-induced platelet aggregation. However, it was also observed that the PGI2-- theophylline pretreatment concomitantly caused a large increase in shear-induced platelet lysis and serotonin release at stress levels equal to or greater than 150 dynes/sq cm. This observed increase in platelet fragility may have important implications for clinical applications of PGI2. The results are discussed and compared to those obtained in prior work in which platelets were pretreated with acetylsalicylic acid or with PGE1.

Effects of Lead Acetate on Platelet Aggregation, Ultrastructure and Serotonin Release

1971 ◽  
Vol 26 (03) ◽  
pp. 455-466 ◽  
Author(s):  
R. B Davis ◽  
G. C Holtz
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Lead Acetate ◽  
Platelet Rich Plasma ◽  
Adenosine Diphosphate ◽  
Blood Platelet ◽  
Human Platelets ◽  
Serotonin Release ◽  
Aggregation Of Platelets

SummaryThe effects of lead on blood platelet function and ultrastructure have been investigated. Lead acetate was injected intravenously in 27 rats and was added to rat and human platelet rich plasma in vitro. In vitro studies showed that concentrations of 2.5 × 10-3 M lead acetate reduced or blocked aggregation of rat and human platelets by adenosine diphosphate, collagen, and thrombin. Radioactive serotonin release from human platelets was inhibited by 10-4 M lead acetate. One hour after the injection of lead, platelet aggregation by thrombin was reduced, but platelet aggregation by adenosine diphosphate and collagen showed little change. Three days after lead, aggregation of platelets by collagen and thrombin was blocked and aggregation by adenosine diphosphate reduced. Thrombocytopenia was present 4 days after intravenous lead acetate. Electron micrographs of platelets showed that the mean number of mitochondria per platelet was increased, whereas alpha granules were reduced. Dense bodies were not significantly changed. Lead acetate affects platelet function in concentrations reported in human bone marrow in lead poisoning, and may relate to the binding of free sulfhydryl groups by lead.

scholarly journals Platelet release reaction and intracellular cGMP

Blood ◽  
1978 ◽  
Vol 52 (3) ◽  
pp. 524-531 ◽  
Author(s):  
A Weiss ◽  
NL Baenziger ◽  
JP Atkinson
Keyword(s):  
Platelet Rich Plasma ◽  
Human Platelets ◽  
Serotonin Release ◽  
Secretory Process ◽  
Release Reaction ◽  
Intracellular Cgmp ◽  
Exogenous Addition ◽  
Human Thrombin ◽  
Platelet Release

Abstract Enchanced cAMP concentrations inhibit the aggregation and release reaction of isolated human platelets and platelet-rich plasma to all known inducing agents. An opposing role for cGMP in this phenomenon has been proposed by some but not by others, and the function of cGMP in this secretory process is unclear. To further elucidate the role of cGMP in the release reaction, the effect of increased concentrations of this cyclic nucleotide on 14C-serotonin release was evaluated utilizing isolated human platelets and highly purified human thrombin or commercially available bovine thrombin. Several recently described stimulators of guanylate cyclase, including sodium nitroprusside, sodium azide, nitrosoquanidines, and ascorbic acid, were found to markedly augment platelet cGMP levels. Enhanced platelet cGMP concentrations produced by these drugs or by the exogenous addition of cGMP and its analogues neither caused these cells to secrete nor modulated the thrombin-induced serotonin release reaction. The inhibition of serotonin release by increased cAMP concentrations was not counteracted by increased cGMP levels. Platelet cGMP concentrations were unaltered by thrombin. These data indicate that cGMP is not an obligatory signal or a modulator of the thrombin-induced platelet release reaction.

scholarly journals Effect of anti-P1A1 antibody on human platelets. I. The role of complement

Blood ◽  
1979 ◽  
Vol 53 (4) ◽  
pp. 567-577 ◽  
Author(s):  
DB Cines ◽  
AD Schreiber
Keyword(s):  
Platelet Aggregation ◽  
Complement Activation ◽  
Platelet Rich Plasma ◽  
Plasma Concentrations ◽  
Human Platelets ◽  
Serotonin Release ◽  
Buffer System ◽  
Induce Platelet Aggregation ◽  
Platelet Surface ◽  
Release Reaction

Abstract We studied the interaction of complement with human platelets. Complement was activated by IgG anti-P1A1 antibody obtained from 3 patients with the post-transfusion purpura syndrome. We used a heparin- plasma buffer system that permits complement activation and also preserves platelet function. With this system complement activation was efficient, and platelet immune alteration was extensive. Anti-P1A1 antibody was effective only in the presence of complement, in which case both platelet lysis and serotonin release (release reaction) in the absence of lysis were observed. Platelet lysis, as assessed by 51Cr loss, required 10-fold more antibody than was necessary to induce platelet aggregation and release of 14C-serotonin. This platelet release reaction required an intact classic complement sequence through C6. The extent of platelet serotonin release parallelled the depletion of C1 and C4 from platelet-rich plasma. Concentrations of antibody insufficient to induce platelet aggregation and serotonin release could still activate C1 and deposit increased C3 on the platelet surface. These studies demonstrated that complement activation by anti-P1A1 antibody can alter human platelets in a nonlytic system. Several phases of complement-mediated human platelet alteration are possible, depending on the concentration of anti-P1A1 antibody.

EFFECTS OF HUMAN ATRIAL NATRIURETIC PEPTIDES ON SECRETION REACTION IN HUMAN PLATELETS

1987 ◽  
Author(s):  
A Tanable ◽  
Y Yatomi ◽  
T Ohashi ◽  
H Oka ◽  
T Kariya ◽  
...  
Keyword(s):  
Platelet Rich Plasma ◽  
Atp Release ◽  
Smooth Muscle Contraction ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
Serotonin Release ◽  
Release Reaction ◽  
Induced Aggregation ◽  
Atrial Natriuretic

Human atrial natriuretic peptide (h-ANP) has vasodilating and natriuretic properties, and inhibits smooth muscle contraction, renal renin secretion and adrenal aldosterone release. Although Schiffrin has reported that human platelets have receptors for ANP, its effects in platelets are not established in vivo. We therefore investigated the influence of h-ANP on secretion reaction in human platelets. Eight healthy subjects, males, aged 20 to 24 years, donated blood for the study. Citrated platelet-rich plasma (PRP) was incubated with or without h-ANP at 37 C for 2.5 minutes. The samples of 0.5 ml PRP then used to measure ADP induced aggregation, ATP release reaction and C-serotonin release reaction. H-ANP, at concentration of 1x10 -6M, decreased ADP induced aggregation (after h-ANP: 77.4±9.7 % of control aggregation), and inhibited ATP release reaction (after h-ANP: 31.8±13.1%). Serotonin releasereaction induced by ADP was also inhibited ( control: 15.3±2.2%, after h-ANP: 8.3±0.5 %). The inhibitory effect of h-ANP on aggregation and secretion reaction was maximal by 3 minutes. These data suggest that h-ANP inhibits secretion reaction in human platelets.

An atypical IgM class platelet cold agglutinin induces GPVI-dependent aggregation of human platelets

2015 ◽  
Vol 114 (08) ◽  
pp. 313-324 ◽  
Author(s):  
Isabel Sánchez Guiu ◽  
Irene Martínez-Martinez ◽  
Constantino Martínez ◽  
José Navarro-Fernandez ◽  
Faustino Garcia-Candel ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Platelet Rich Plasma ◽  
Shape Change ◽  
Human Platelets ◽  
Serotonin Release ◽  
Platelet Glycoprotein ◽  
Causative Role ◽  
Temperature Dependent ◽  
Activation Markers

SummaryPlatelet cold agglutinins (PCA) cause pseudothrombocytopenia, spurious thrombocytopenia due to ex vivo platelet clumping, complicating clinical diagnosis, but mechanisms and consequences of PCA are not well defined. Here, we characterised an atypical immunoglobulin (Ig)M PCA in a 37-year-old woman with lifelong bleeding and chronic moderate thrombocytopenia, that induces activation and aggregation of autologous or allogeneic platelets via interaction with platelet glycoprotein (GP)VI. Patient temperature-dependent pseudothrombocytopenia was EDTA-independent, but was prevented by integrin αIIbβ3 blockade. Unstimulated patient platelets revealed elevated levels of bound IgM, increased expression of activation markers (P-selectin and CD63), low GPVI levels and abnormally high thromboxane (TX)A2 production. Patient serum induced temperature- and αIIbβ3-dependent decrease of platelet count in allogeneic donorcitrated platelet-rich plasma (PRP), but not in PRP from Glanzmann’s thrombasthenia or afibrinogenaemia patients. In allogeneic platelets, patient plasma induced shape change, P-selectin and CD63 expression, 14C-serotonin release, and TXA2 production. Activation was not inhibited by aspirin, cangrelor or blocking anti-Fc receptor (FcγRIIA) antibody, but was abrogated by inhibitors of Src and Syk, and by a soluble GPVI-Fc fusion protein. GPVI-deficient platelets were not activated by patient plasma. These data provide the first evidence for an IgM PCA causing platelet activation/aggregation via GPVI. The PCA activity persisted over a five-year follow-up period, supporting a causative role in patient chronic thrombocytopenia and bleeding.

The Effect of Endotoxins on Generation of Thrombin in the Platelet Atmosphere

1975 ◽  
Author(s):  
J. Vermylen ◽  
D. Fumarola ◽  
N. Semeraro
Keyword(s):  
Human Platelet ◽  
Platelet Rich Plasma ◽  
Gradient Centrifugation ◽  
Human Platelets ◽  
Serotonin Release ◽  
Bacterial Endotoxins ◽  
Normal Human ◽  
Bacterial Lipopolysaccharides ◽  
Vitro Effect

Human platelets, washed by repeated albumin density gradient centrifugation, aggregate strongly, occasionally in 2 waves, 3 to 5 minutes after addition of both calcium ions (2.10−3 M f.c.) and one tenth volume normal human serum, provided the serum contains at least 0.6% residual prothrombin. This aggregation is prevented by heparin or hirudin. Samples removed at the onset of aggregation rapidly clot purified fibrinogen, whereas the serum-CaCl2 mixture alone does not clot purified fibrinogen within 24 hours. It is therefore concluded that thrombin is rapidly generated in the platelet atmosphere.The in vitro effect of endotoxins on human, in contrast to rabbit, platelets is not well established. Using eight different bacterial lipopolysaccharides we failed to demonstrate aggregation in human platelet-rich plasma or of washed platelets or to find significant 14C-serotonin release. On the other hand, all these endotoxins in a final concentration of 100 μg/ml consistently shortened the latent period before aggregation in the washed platelets-serum-CaCl2 system. It is concluded that bacterial endotoxins enhance the generation of thrombin in the atmosphere of human platelets.

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