Presence of P2X1 Purinoceptors in Human Platelets and Megakaryoblastic Cell Lines

SummaryHuman platelets are thought to possess at least two subtypes of purinoceptor, one of which, coupled to G-proteins, could be the P2Y1 receptor (Léon et al. 1997). However, it has been suggested that the unique rapid calcium influx induced by ADP in platelets could involve P2X1 ionotropic receptors (MacKenzie et al. 1996) and the aim of this study was thus to investigate the presence of P2X purinoceptors in platelets and megakaryoblastic cells. Using PCR experiments, we found P2X1 mRNA to be present in human platelets and megakaryoblastic cell lines. In platelets, the selective P2X1 agonist αβMeATP induced a rise in intracellular calcium only in the presence of external calcium and this effect was antagonized by suramin and PPADS. Repeated addition of a�MeATP desensitized the P2X1 purinoceptor but only slightly affected the ADP response, while no calcium response to αβMeATP was observed in megakaryoblastic cells. These results support the existence of functional P2X1 purinoceptors on human platelets and the presence of P2X1 transcripts in megakaryoblastic cell lines.

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Distribution and relative levels of expression of the phosphoinositidase-C-linked G-proteins Gqα and G11α: Absence of G11α in human platelets and haemopoietically derived cell lines

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research ◽

10.1016/0167-4889(93)90143-d ◽

1993 ◽

Vol 1179 (2) ◽

pp. 208-212 ◽

Cited By ~ 35

Author(s):

Graeme Milligan ◽

Ian Mullaney ◽

J.Fraser McCallum

Keyword(s):

G Proteins ◽

Cell Lines ◽

Human Platelets

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Quasi-simultaneous measurement of ionized calcium and alpha-granule release in individual platelets

AJP Cell Physiology ◽

10.1152/ajpcell.1991.260.2.c242 ◽

1991 ◽

Vol 260 (2) ◽

pp. C242-C248 ◽

Cited By ~ 15

Author(s):

A. Oda ◽

J. F. Daley ◽

J. Kang ◽

M. Smith ◽

J. A. Ware ◽

...

Keyword(s):

Monoclonal Antibody ◽

Flow Cytometry ◽

Fluorescein Isothiocyanate ◽

Cytosolic Calcium ◽

Human Platelets ◽

Calcium Mobilization ◽

The Other ◽

Calcium Response ◽

External Calcium ◽

Granule Release

The effect of alpha-thrombin and ADP on calcium mobilization and alpha-granule release in individual platelets was investigated by flow cytometry. alpha-Thrombin (4.5 nM) caused a uniform rise of free cytosolic calcium ([Ca2+]i) among indo-1-loaded human platelets. Despite the uniformity of this effect, approximately 20% of the cells failed to secrete alpha-granule content, as shown by binding of fluorescein isothiocyanate (FITC)-conjugated S12 monoclonal antibody. ADP (10 microM) caused a similar brisk and uniform rise of calcium but did not increase S12 binding to any platelets. On the other hand, with alpha-thrombin (0.5 nM), calcium mobilization was heterogeneous and paralleled granule release. [Ca2+]i increased rapidly in some platelets, while only slowly in others. When an electronic gate was set according to FITC-S12 fluorescence, cells with a greater secretory response proved to be those with a higher calcium level. With both alpha-thrombin and ADP, chelation of external calcium by EGTA (2 mM) reduced calcium response of individual cells. NiCl2 (1 mM) also inhibited calcium rise of individual platelets to the same extent as EGTA (2 mM) in spite of the presence of 1 mM CaCl2 in the extracellular media. The effects of EGTA and NiCl2 were not limited to a particular subpopulation of cells. These data suggest that the putative Ni2(+)-inhibitable divalent cation channel(s) may be responsible for the increased influx of calcium that occurs during platelet activation by alpha-thrombin and ADP. It appears that these calcium channels contribute to the elevation of [Ca2+]i among virtually all the platelets.

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Increased Aggregation and Secretion Responses of Human Platelets when Loaded with the Calcium Fluorescent Probes Quin2 and Fura-2

Thrombosis and Haemostasis ◽

10.1055/s-0038-1645981 ◽

1987 ◽

Vol 58 (02) ◽

pp. 737-743 ◽

Cited By ~ 12

Author(s):

Frarnçois Lanza ◽

Alain Beretz ◽

Martial Kubina ◽

Jean-Pierre Cazenave

Keyword(s):

Intracellular Calcium ◽

Shape Change ◽

Calcium Ionophore ◽

Human Platelets ◽

Arachidonic Acid Metabolism ◽

Free Calcium ◽

Membrane Bilayer ◽

Fluorescent Indicators ◽

Fura 2 ◽

Low Concentrations

SummaryIncorporation into human platelets of the calcium fluorescent indicators quin2 or fura-2 at low concentrations used to measure intracellular free calcium leads to the potentiation of the effects of agonists on platelets. This was shown by increased aggregatory and secretory responses of quin2 or fura-2 loaded platelets after stimulation with ADP, PAP and with low concentrations of thrombin, collagen, the endoperoxide analog U-46619 and the calcium ionophore A 23187. Quin2 and fura-2 mediated platelet sensitisation could be due to altered arachidonic acid metabolism since it was inhibited by prior treatment with the cydooxygenase inhibitor acetylsalicylate. In contrast, platelets loaded with higher concentrations of calcium chelators exhibited diminished aggregation responses to all aggregating agents. This latter effect was accompanied by increased fluidity of the platelet plasma membrane bilayer and by the exposure of a new pool of membranes to the outer surface of platelets, as monitored with trimethylammonium- diphenylhexatriene (TMA-DPH) in platelets loaded with the non-fluorescent calcium probe analog MAPT. In contrast, low concentrations of quin2 did not potentiate shape change of platelets activated with ADP. Thus, shape change and aggregation can be influenced separately by intracellular Ca2+ chelators. We conclude that platelet responses are altered by the incorporation of intracellular calcium chelators at concentrations used to monitor intracellular calcium changes.

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Different Abilities of Thrombin Receptor Activating Peptide and Thrombin to Induce Platelet Calcium Rise and Full Release Reaction

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649934 ◽

1995 ◽

Vol 74 (05) ◽

pp. 1323-1328 ◽

Cited By ~ 15

Author(s):

Dominique Lasne ◽

José Donato ◽

Hervé Falet ◽

Francine Rendu

Keyword(s):

Essential Role ◽

Calcium Influx ◽

Dense Granule ◽

Receptor Interaction ◽

Thrombin Receptor ◽

Release Reaction ◽

External Calcium ◽

Maximum Rise ◽

Granule Release

SummarySynthetic peptides (TRAP or Thrombin Receptor Activating Peptide) corresponding to at least the first five aminoacids of the new N-terminal tail generated after thrombin proteolysis of its receptor are effective to mimic thrombin. We have studied two different TRAPs (SFLLR, and SFLLRN) in their effectiveness to induce the different platelet responses in comparison with thrombin. Using Indo-1/AM- labelled platelets, the maximum rise in cytoplasmic ionized calcium was lower with TRAPs than with thrombin. At threshold concentrations allowing maximal aggregation (50 μM SFLLR, 5 μM SFLLRN and 1 nM thrombin) the TRAPs-induced release reaction was about the same level as with thrombin, except when external calcium was removed by addition of 1 mM EDTA. In these conditions, the dense granule release induced by TRAPs was reduced by over 60%, that of lysosome release by 75%, compared to only 15% of reduction in the presence of thrombin. Thus calcium influx was more important for TRAPs-induced release than for thrombin-induced release. At strong concentrations giving maximal aggregation and release in the absence of secondary mediators (by pretreatment with ADP scavengers plus aspirin), SFLLRN mobilized less calcium, with a fast return towards the basal level and induced smaller lysosome release than did thrombin. The results further demonstrate the essential role of external calcium in triggering sustained and full platelet responses, and emphasize the major difference between TRAP and thrombin in mobilizing [Ca2+]j. Thus, apart from the proteolysis of the seven transmembrane receptor, another thrombin binding site or thrombin receptor interaction is required to obtain full and complete responses.

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Interaction of Human Tumor Cells with Human Platelets and the Coagulation System

Thrombosis and Haemostasis ◽

10.1055/s-0038-1665296 ◽

1983 ◽

Vol 50 (03) ◽

pp. 726-730 ◽

Cited By ~ 11

Author(s):

Hamid Al-Mondhiry ◽

Virginia McGarvey ◽

Kim Leitzel

Keyword(s):

Tumor Cell ◽

Tumor Cells ◽

Cell Lines ◽

Human Tumor ◽

Human Platelets ◽

Factor Vii ◽

Coagulation System ◽

Breast Adenocarcinoma ◽

Activate Factor ◽

Tumor Cell Lines

SummaryThis paper reports studies on the interaction between human platelets, the plasma coagulation system, and two human tumor cell lines grown in tissue culture: Melanoma and breast adenocarcinoma. The interaction was monitored through the use of 125I- labelled fibrinogen, which measures both thrombin activity generated by cell-plasma interaction and fibrin/fibrinogen binding to platelets and tumor cells. Each tumor cell line activates both the platelets and the coagulation system simultaneously resulting in the generation of thrombin or thrombin-like activity. The melanoma cells activate the coagulation system through “the extrinsic pathway” with a tissue factor-like effect on factor VII, but the breast tumor seems to activate factor X directly. Both tumor cell lines activate platelets to “make available” a platelet- derived procoagulant material necessary for the conversion of prothrombin to thrombin. The tumor-derived procoagulant activity and the platelet aggregating potential of cells do not seem to be inter-related, and they are not specific to malignant cells.

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Faculty Opinions recommendation of Cyclic ADP-ribose production by CD38 regulates intracellular calcium release, extracellular calcium influx and chemotaxis in neutrophils and is required for bacterial clearance in vivo.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1001269.37056 ◽

2002 ◽

Author(s):

Marcus Thelen

Keyword(s):

Intracellular Calcium ◽

Calcium Release ◽

Calcium Influx ◽

Extracellular Calcium ◽

Bacterial Clearance ◽

Cyclic Adp Ribose ◽

Intracellular Calcium Release

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Chimeric Measles Virus (MV/RSV), Having Ectodomains of Respiratory Syncytial Virus (RSV) F and G Proteins Instead of Measles Envelope Proteins, Induced Protective Antibodies against RSV

Vaccines ◽

10.3390/vaccines9020156 ◽

2021 ◽

Vol 9 (2) ◽

pp. 156

Author(s):

Akihito Sawada ◽

Takashi Ito ◽

Yoshiaki Yamaji ◽

Tetsuo Nakayama

Keyword(s):

Respiratory Syncytial Virus ◽

G Proteins ◽

Cell Lines ◽

Measles Virus ◽

Pathological Finding ◽

Control Group ◽

Protein Coding ◽

Virus Growth ◽

Protective Antibodies ◽

Syncytial Virus

In our previous study, fusion (F) or glyco (G) protein coding sequence of respiratory syncytial virus (RSV) was inserted at the P/M junction of the measles AIK-C vector (MVAIK), and the recombinant measles virus induced protective immune responses. In the present study, the ectodomains of measles fusion (F) and hemagglutinin (HA) proteins were replaced with those of RSV F and G proteins, and a chimeric MV/RSV vaccine was developed. It expressed F and G proteins of RSV and induced cytopathic effect (CPE) in epithelial cell lines (Vero, A549, and HEp-2 cells), but not in lymphoid cell lines (B95a, Jurkat, and U937 cells). A chimeric MV/RSV grew similarly to AIK-C with no virus growth at 39 °C. It induced NT antibodies against RSV in cotton rats three weeks after immunization through intramuscular route and enhanced response was observed after the second dose at eight weeks. After the RSV challenge with 106 PFU, significantly lower virus (101.4±0.1 PFU of RSV) was recovered from lung tissue in the chimeric MV/RSV vaccine group than in the MVAIK control group with 104.6±0.2 PFU (p < 0.001) and no obvious inflammatory pathological finding was noted. The strategy of ectodomain replacement in the measles virus vector is expected to lead to the development of safe and effective vaccines for other enveloped viruses.

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Caulerpenyne Affects Bradykinin-Induced Intracellular Calcium Kinetics in LoVo Cells

Applied Sciences ◽

10.3390/app11062697 ◽

2021 ◽

Vol 11 (6) ◽

pp. 2697

Author(s):

Giovanna Traina

Keyword(s):

Intracellular Calcium ◽

Cell Lines ◽

Calcium Homeostasis ◽

Human Colon ◽

Caulerpa Taxifolia ◽

Green Macroalgae ◽

Calcium Kinetics ◽

Lovo Cells ◽

Pharmacological Interest ◽

Antiproliferative Activities

Sesquiterpene caulerpenyne (CYN) is the major metabolite present in green macroalgae Caulerpa taxifolia. This metabolite has been shown to be cytotoxic in some cell lines and was found to be active in various assays of pharmacological interest. In addition, it exerts antibiotic, antiviral, phytotoxic, antidyslipidemic, and antiproliferative activities. In the present study, we report that pretreatment with CYN decreases the bradykinin-induced calcium peak in human colon LoVo cells. We hypothesize that CYN pretreatment may adversely affect bradykinin-induced intracellular calcium increases. The data suggest that CYN, by reducing the increase in intracellular calcium, exerts an inhibitory role on calcium homeostasis and, likely, intercellular transmission.

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