The Density Distribution Of Intact Platelets Following Their Isolation From All Other Blood Constituents

1981 ◽  
Author(s):  
T Shaw ◽  
J F Martin ◽  
C N Chesterman ◽  
D G Peninqton
Keyword(s):  
Buoyant Density ◽  
Lactic Dehydrogenase ◽  
Blood Platelet ◽  
Dense Granule ◽  
Velocity Sedimentation ◽  
Full Density ◽  
Blood Constituents ◽  
Second Gradient ◽  
Induced Aggregation ◽  
Equilibrium Centrifugation

A method for measuring the buoyant density of platelets which function normally after the procedure, has been developed and critically compared to published methods.Following velocity sedimentation into a gradient of polyvinyl pyrrol 1idone-coated colloidal silica particles (Percoll) at physiological pH and osmolarity, 90% (n=10, SD=10) of the whole blood platelet population was recovered with leukocyte contamination of .002% (n=10, SD=.001); erythrocyte contamination was less.After centrifugation to equilibrium through a second continuous linear Percoll gradient, platelet density showed a Gaussian distribution about a mode of 1.0645 g/ml. (n=13, SD=.0015). Leakage of β thromboglobulin (βTG), lactic dehydrogenase (LDH) and serotonin into the gradients was negligible. Intracellular LDH, serotonin and total protein correlated closely with platelet count, but βTG distribution was skewed towards the denser fractionsPlatelets recovered from the second gradient showed normal aggregation patterns and produced thromboxane B2 and secreted βTG when stimulated by thrombin or arachidonic acid. Addition of plasma was necessary to produce ristocetin-and ADP-induced aggregation, and significant radioactivity was not associated with platelets isolated from blood to which 125I had been addedUnder physiological conditions, arabinogalactan II (Stractan) caused more spontaneous leakage of α-and dense- granule markers than did Percoll. Platelets taken from different interfaces of a discontinuous Stractan gradient were seen to contain platelets of the full density range when they underwent equilibrium centrifugation in linear continuous Stractan or Percoll gradients.True buoyant density of minimally altered platelets has been measured by this new technique.

Platelet Aggregation Caused by Dithiothreitol

1984 ◽  
Vol 51 (01) ◽  
pp. 119-124 ◽  
Author(s):  
M B Zucker ◽  
N C Masiello
Keyword(s):  
Shape Change ◽  
Blood Platelets ◽  
Dense Granule ◽  
Metabolic Inhibitors ◽  
Electrophoretic Patterns ◽  
Discernible Effect ◽  
Variable Extent ◽  
Triton X ◽  
Induced Aggregation ◽  
Platelet Shape

SummaryMacIntyre et al. showed that over 1 mM dithiothreitol (DTT) aggregates blood platelets in the presence of fibrinogen; aggregation is not inhibited by prostaglandin E1. We confirmed their data and found that 70 mM 2-mercaptoethanol was also active. DTT- induced aggregation was not associated with platelet shape change or secretion of dense granule contents, was not inhibited by tetracaine or metabolic inhibitors, was prevented at pH 6.5, and prevented, reversed, or arrested by EDTA, depending on when the EDTA was added. DTT did not cause aggregation of thrombasthenic, EDTA-treated, or cold (0° C) platelets, which also failed to aggregate with ADP. Platelets stimulated with DTT bound 125I-labeled fibrinogen. Thus DTT appears to “expose” the fibrinogen receptors. SDS gel electrophoresis of platelet fractions prepared by use of Triton X-114 showed that aggregating concentrations of DTT reduced proteins of apparent Mr 69,000 and 52,000 (probably platelet albumin) and, to a variable extent, glycoproteins Ib, IIb and III. Exposure of unlabeled or 125I- labeled platelets to ADP had no discernible effect on the electrophoretic patterns.

The Effect of Calcium and Magnesium on Rabbit Blood Platelet Aggregation in Vitro

1964 ◽  
Vol 12 (01) ◽  
pp. 179-200 ◽  
Author(s):  
Torstein Hovig
Keyword(s):  
Platelet Aggregation ◽  
Cation Exchange ◽  
Calcium Concentration ◽  
Blood Platelets ◽  
Blood Platelet ◽  
Rabbit Blood ◽  
Calcium And Magnesium ◽  
Induced Aggregation ◽  
Blood Platelet Aggregation

SummaryThe effect of calcium and magnesium on the aggregation of rabbit blood platelets in vitro was studied, with the following results:1. Platelet aggregation induced by ADP or collagen could be prevented by EGTA or EDTA. The aggregating effect was restored by recalcification. The effect was also restored by addition of magnesium in EDTA-PRP, but not in EGTA-PRP unless a surplus of calcium was present.2. Calcium remained in concentrations of the order of 0.15–0.25 mM after dialysis or cation exchange of plasma. Aggregation of washed platelets resuspended in such plasma could not be produced with ADP or collagen, unless the calcium concentration was increased or that magnesium was added.3. The adhesiveness of blood platelets to collagen was reduced in EGTA-PRP and EDTA-PRP. Release of ADP from platelets influenced by collagen could not be demonstrated either in EGTA-PRP (presence of magnesium) or in EDTA-PRP.4. It is concluded that calcium is a necessary factor both for the reaction leading to release of ADP and for the the aggregation produced by ADP.5. Thrombin induced aggregation of washed platelets suspended in tris-buffered saline in the presence of calcium. No effect of magnesium could be observed unless small quantities of calcium were present.

Megakaryocytes and Platelets as the Main Cause for Vascular Events in Chronic Myeloproliferative Disorders

1996 ◽  
Vol 16 (02) ◽  
pp. 151-163 ◽  
Author(s):  
W. Schneider ◽  
A. Wehmeier
Keyword(s):  
Mononuclear Cells ◽  
Buoyant Density ◽  
Myeloproliferative Disorders ◽  
Term Therapy ◽  
Volume Distribution ◽  
Bleeding Tendency ◽  
Vascular Events ◽  
Chronic Myeloproliferative Disorders ◽  
Clonal Hematopoiesis ◽  
Induced Aggregation

SummaryMegakaryocytes are part of clonal hematopoiesis in chronic myeloproliferative disorders and are responsible for most of the clinical complications in this disease. About 30-40% of patients with polycythemia vera (PV) and essential thrombocythemia (ET) suffer from thrombotic complications, and microcirculatory disorders are common. Spontaneous bleeding mainly from the gastrointestinal tract is another complication that is especially prevalent in myelofibrosis and advanced stages of chronic myeloid leukemia.In vivo, the bone marrow is hypercellular and the concentration of megakaryocytes increased with characteristic morphological abnormalities. Megakaryocytes are enlarged and ploidy is increased in PV and ET but small mononuclear cells with decreased ploidy are a feature of CML. Despite spontaneous growth in cul-ture, megakaryocytes in chronic MPD are hypersensitive to added interleukin-3, interleukin-6 and GM-CSF.Platelets released from these megakaryocytes show abnormal morphology and ultrastructure, reflected in loss of storage granules and organelles, increased volume distribution and low buoyant density. Uptake, storage and secretion of platelet dense granule constituents is abnormal, and the plasma levels of platelet specific proteins which may also include growth factors for fibroblasts are elevated. At high platelet counts, spontaneous aggregation is observed, whereas agonist-induced aggregation in vitro with adrenaline, ADP and collagen is often defective. Platelet thromboxane generation may be stimulated, and production along the lipoxygenase pathway is decreased. Abnormalities of glycoprotein receptors and decreased fibrinogen binding have been reported but their clinical significance is uncertain. Several observations suggest that not only receptor defects but ineffective intracellular signalling may be responsible for platelet function abnormalities.No single underlying defect has been discovered that could explain this variety of pathological findings. Moreover, a combination of intrinsic megakaryocyte abnormalities and increased susceptibility of platelets to activation makes it difficult to differentiate secondary phenomena from effects of clonal hematopoiesis. How-ever, there are some clinical guidelines for therapy.Most elderly patients will be treated with cytoreductive therapy. Alkylating drugs and 32P have been shown to be leukemogenic, but even hydroxyurea may have a 10% incidence of leukemia induction after long-term therapy. Therapy with platelet-inhibitory drugs is often not sufficient to control thrombosis, and may aggravate a bleeding tendency, so that younger patients with PV and ET are increasingly treated with anagrelide or interferon alpha (A-IFN). Anagrelide is a quinazolin derivative that specifically inhibits megakaryocytopoiesis, while A-IFN may suppress clonal hematopoiesis by an unknown mechanism.

ChemInform Abstract: 1,3-Dihydro-2H-imidazo(4,5-b)quinolin-2-ones. Inhibitors of Blood Platelet cAMP Phosphodiesterase and Induced Aggregation.

ChemInform ◽  
2010 ◽  
Vol 23 (10) ◽  
pp. no-no
Author(s):  
N. A. MEANWELL ◽  
H. R. ROTH ◽  
E. C. R. SMITH ◽  
D. L. WEDDING ◽  
J. J. K. WRIGHT ◽  
...  
Keyword(s):  
Blood Platelet ◽  
Camp Phosphodiesterase ◽  
Induced Aggregation

Multiple electrode aggregometry: A new device to measure platelet aggregation in whole blood

2006 ◽  
Vol 96 (12) ◽  
pp. 781-788 ◽  
Author(s):  
Andreas Calatzis ◽  
Sandra Penz ◽  
Hajna Losonczy ◽  
Wolfgang Siess ◽  
Orsolya Tóth
Keyword(s):  
Platelet Aggregation ◽  
Whole Blood ◽  
Blood Platelet ◽  
Platelet Inhibition ◽  
New Device ◽  
Platelet Aggregometry ◽  
Spontaneous Platelet Aggregation ◽  
Induced Aggregation ◽  
Blood Platelet Aggregation ◽  
Multiple Electrode

SummarySeveral methods are used to analyse platelet function in whole blood. A new device to measure whole blood platelet aggregation has been developed, called multiple electrode platelet aggregometry (MEA). Our aim was to evaluate MEA in comparison with the single platelet counting (SPC) method for the measurement of platelet aggregation and platelet inhibition by aspirin or apyrase in diluted whole blood. Platelet aggregation induced by different concentrations of ADP, collagen and TRAP-6 and platelet inhibition by apyrase or aspirin were determined in citrateor hirudin-anticoagulated blood by MEA and SPC. MEA indicated that spontaneous platelet aggregation was lower, and stimulated platelet aggregation was higher in hirudin- than citrate-anticoagulated blood. In hirudin-anticoagulated, but not citrate-anticoagulated blood, spontaneous platelet aggregation measured by MEA was inhibited by apyrase. For MEA compared with SPC the dose response-curves of agonist-induced platelet aggregation in citrate- and hirudin-blood showed similar EC50 values for TRAP, and higher EC50 values for ADP (non-significant) and collagen (p<0.05). MEA and the SPC method gave similar results concerning platelet-inhibition by apyrase and aspirin. MEA was more sensitive than SPC to the inhibitory effect of aspirin in collagen-induced aggregation. In conclusion, MEA is an easy, reproducible and sensitive method for measuring spontaneous and stimulated platelet aggregation, and evaluating antiplatelet drugs in diluted whole blood. The use of hirudin as an anticoagulant is preferable to the use of citrate. MEA is a promising technique for experimental and clinical applications.

Disappearance of the covalently closed circular cryptic plasmid PO-2 in Salmonella pullorum MS35 containing F77

1974 ◽  
Vol 20 (4) ◽  
pp. 551-557
Author(s):  
Paul W. Stiffler ◽  
D. E. Schoenhard
Keyword(s):  
Salmonella Typhimurium ◽  
Sucrose Gradient ◽  
Buoyant Density ◽  
Sedimentation Coefficient ◽  
Apparent Effect ◽  
Donor Property ◽  
Circular Dna ◽  
Salmonella Pullorum ◽  
Cryptic Plasmids ◽  
Equilibrium Centrifugation

The physical basis of the donor property of Salmonella pullorum donor strains MS8300, MS830, and MS831 carrying the F77 factor from Salmonella typhimurium was investigated by dye-buoyant density equilibrium centrifugation and zonal centrifugation in neutral sucrose gradients. Centrifugation of the MS8300 and MS831 closed circular DNA material in a 20 to 31% neutral sucrose gradient resulted in a profile having one sharp peak of radioactivity with a sedimentation coefficient of 17 S and a broad peak extending from 65 to 70 S. The 17- and 65-S species were isolated from the isogenic F− strain MS83. These appeared identical with those isolated previously in S. pullorum MS53 as the cryptic plasmids PO-1 and PO-2 respectively. Cosedimentation of differentially labeled F77 DNA and the lysate containing the 65-S and 70-S species suggested that the 70-S species is the autonomous F77 factor in strains MS8300 and MS831. Lysates of MS830 similarly treated produced a profile containing the 17-S molecule and possibly some 70-S molecules but no 65-S molecules. It was concluded that the F77 factor was integrated in most cells and that the covalently closed circular state of PO-2 plasmid was lost. The mutation in the cysE gene of the F77 factor carried by MS831 had no apparent effect on the covalently closed circular nature of PO-2 plasmid, although F77 no longer seemed to mobilize the chromosome from the cysE locus.

scholarly journals Protein kinase C- and calcium-regulated pathways independently synergize with Gi pathways in agonist-induced fibrinogen receptor activation

2002 ◽  
Vol 368 (2) ◽  
pp. 535-543 ◽  
Author(s):  
Todd M. QUINTON ◽  
Soochong KIM ◽  
Carol DANGELMAIER ◽  
Robert T. DORSAM ◽  
Jianguo JIN ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Intracellular Calcium ◽  
Phospholipase C ◽  
Calcium Release ◽  
Receptor Activation ◽  
Dense Granule ◽  
Fibrinogen Receptor ◽  
Kinase C ◽  
Induced Aggregation

Platelet fibrinogen receptor activation is a critical step in platelet plug formation. The fibrinogen receptor (integrin αIIbβ3) is activated by agonist-mediated Gq stimulation and resultant phospholipase C activation. We investigated the role of downstream signalling events from phospholipase C, namely the activation of protein kinase C (PKC) and rise in intracellular calcium, in agonist-induced fibrinogen receptor activation using Ro 31-8220 (a PKC inhibitor) or dimethyl BAPTA [5,5′-dimethyl-bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid], a high-affinity calcium chelator. All the experiments were performed with human platelets treated with aspirin, to avoid positive feedback from thromboxane A2. In the presence of Ro 31-8220, platelet aggregation caused by U46619 was completely inhibited while no effect or partial inhibition was seen with ADP and the thrombin-receptor-activating peptide SFLLRN, respectively. In the presence of intracellular dimethyl BAPTA, ADP- and U46619-induced aggregation and anti-αIIbβ3 antibody PAC-1 binding were completely abolished. However, similar to the effects of Ro 31-8220, dimethyl BAPTA only partially inhibited SFLLRN-induced aggregation, and was accompanied by diminished dense-granule secretion. When either PKC activation or intracellular calcium release was abrogated, aggregation and fibrinogen receptor activation with U46619 or SFLLRN was partially restored by additional selective activation of the Gi signalling pathway. In contrast, when both PKC activity and intracellular calcium increase were simultaneously inhibited, the complete inhibition of aggregation that occurred in response to either U46619 or SFLLRN could not be restored with concomitant Gi signalling. We conclude that, while the PKC- and calcium-regulated signalling pathways are capable of inducing activating fibrinogen receptor independently and that each can synergize with Gi signalling to cause irreversible fibrinogen receptor activation, both pathways act synergistically to effect irreversible fibrinogen receptor activation.

Protease-activated receptor 1 (PAR1) signalling desensitization is counteracted via PAR4 signalling in human platelets

2011 ◽  
Vol 436 (2) ◽  
pp. 469-480 ◽  
Author(s):  
Knut Fälker ◽  
Linda Haglund ◽  
Peter Gunnarsson ◽  
Martina Nylander ◽  
Tomas L. Lindahl ◽  
...  
Keyword(s):  
Receptor Activation ◽  
Human Platelets ◽  
Dense Granule ◽  
Receptor Internalization ◽  
Functional Responses ◽  
Protease Activated Receptors ◽  
Dense Granules ◽  
Dense Granule Secretion ◽  
Granule Secretion ◽  
Induced Aggregation

PARs (protease-activated receptors) 1 and 4 belong to the family of G-protein-coupled receptors which induce both Gα12/13 and Gαq signalling. By applying the specific PAR1- and PAR4-activating hexapeptides, SFLLRN and AYPGKF respectively, we found that aggregation of isolated human platelets mediated via PAR1, but not via PAR4, is abolished upon homologous receptor activation in a concentration- and time-dependent fashion. This effect was not due to receptor internalization, but to a decrease in Ca2+ mobilization, PKC (protein kinase C) signalling and α-granule secretion, as well as to a complete lack of dense granule secretion. Interestingly, subthreshold PAR4 activation rapidly abrogated PAR1 signalling desensitization by differentially reconstituting these affected signalling events and functional responses, which was sufficient to re-establish aggregation. The lack of ADP release and P2Y12 receptor-induced Gαi signalling accounted for the loss of the aggregation response, as mimicking Gαi/z signalling with 2-MeS-ADP (2-methylthioadenosine-5′-O-diphosphate) or epinephrine (adrenaline) could substitute for intermediate PAR4 activation. Finally, we found that the re-sensitization of PAR1 signalling-induced aggregation via PAR4 relied on PKC-mediated release of both ADP from dense granules and fibrinogen from α-granules. The present study elucidates further differences in human platelet PAR signalling regulation and provides evidence for a cross-talk in which PAR4 signalling counteracts mechanisms involved in PAR1 signalling down-regulation.

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