Effect of the calcium antagonist TMB-6 on intracellular calcium redistribution associated with platelet shape change

SummaryCalcium is considered to have an essential role in various platelet reactions. Using platelets preincubated with Chlortetracycline, a fluorescent divalent cation indicator, and suspended in a calcium free medium, it was shown that collagen-induced intracellular calcium redistribution occurred before the platelet shape change, the release reaction and thromboxane B2 formation. Verapamil, at concentrations which affect intracellular calcium movements, inhibited intracellular calcium redistribution in platelets and the subsequent collagen-induced platelet reactions. Low concentrations of the ionophore A23187 overcame the inhibitory effect of verapamil. These experiments provide evidence that intracellular calcium mobilization is involved in the activation of platelets by collagen. Furthermore, calcium may be released from different cellular pools since platelet secretion, aggregation and thromboxane B2 formation were inhibited at lower concentrations of verapamil than was the platelet shape change.

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INCREASED AGGREGATION AND SECRETION RESPONSES OF HUMAN PLATELETS WHEN LOADED WITH THE CALCIUM FLUORESCENT PROBES QUIN2 AND FURA-2

10.1055/s-0038-1643760 ◽

1987 ◽

Author(s):

F Lanza ◽

A Beretz ◽

M Kubina ◽

J-P Cazenave

Keyword(s):

Intracellular Calcium ◽

Shape Change ◽

Calcium Ionophore ◽

Human Platelets ◽

Arachidonic Acid Metabolism ◽

Free Calcium ◽

Membrane Bilayer ◽

Fura 2 ◽

Low Concentrations ◽

Platelet Shape

Incubation of human platelets with the fluorescent dye esters quin2-AM (10 μM) or fura-2-AM (1 μM) makes possible the direct measurement of intracellular free calcium ([Ca2+1).Underthese conditions, basal levels of [Ca2+]i of 120 ± 16 nM (n=23) using quin2 and 137 ± 15 nM (n=5) using fura-2 can be measured. Both probes record comparable increases of [Ca2 ]i after stimulation with ADP, thrombin, PAF, or U-46619. Incorporation into human platelets of quin2 or fura-2 at the concentrations used to monitor [Ca2+]i leads to the activation of platelets. This was shown by increased aggregation and secretion responses of quin2or fura-2 loaded platelets after stimulationwith ADP (5 μM), PAF (1 μM) and with low concentrations of thrombin (0.015U/ml), collagen (0.5 μg/ml), the endoperoxide analog U-46619 (0.5 μM) or the calcium ionophore A 23187 (1 μM). Quin2 and fura-2 mediated platelet activation could be due to altered arachidonic acid metabolism, since it was partly inhibited by prior treatment with the cyclooxygenase inhibitor acetylsalicylate (1 μM). In contrast, platelets loaded with higher concentrations of calcium chelators (20 to 100 μM quin2-AM)exhibited diminished aggregation responses to all aggregating agents. Thislatter effectwas accompanied by increased fluidity of theplatelet plasma membrane bilayer and by the exposure of a new pool of membranes at the outer surface of platelets, as monitored withtrimethylammonium-diphenylhexatriene (TMA-DPH) in platelets loaded with thenon-fluorescent calcium probe analog MAPT. Platelet shape change, as measured in the aggregometer, was dose-dependently inhibited after loading of quin2 (10-50 μM quin2-AM), even at concentrations which potentiated aggregation. We conclude that incorporation of intracellular calcium chelators alters platelet responses, including at concentrations used to monitor intracellular calcium changes.

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Trypsin causes platelet activation independently of known protease-activated receptors

Thrombosis and Haemostasis ◽

10.1160/th13-03-0189 ◽

2013 ◽

Vol 110 (12) ◽

pp. 1241-1249 ◽

Cited By ~ 4

Author(s):

Yingying Mao ◽

Satya P. Kunapuli

Keyword(s):

Intracellular Calcium ◽

Platelet Activation ◽

Light Chain ◽

Myosin Light Chain ◽

Shape Change ◽

Signalling Pathway ◽

Protease Activated Receptors ◽

Similar Shape ◽

High Concentrations ◽

Platelet Shape

SummaryTo identify a physiological agonist of PAR3, we used PAR4 null murine platelets, which were known to express only PAR3. In this study, we tested several proteases and found that trypsin, but not heat-inactivated trypsin, activated PAR4 null murine platelets. Even at high concentrations, trypsin caused shape change without increasing intracellular calcium levels in PAR4 null murine platelets. Consistent with this result, the Gq inhibitor YM-254890 had no effect on trypsin-induced shape change. However, trypsin-induced platelet shape change was abolished by either p160ROCK inhibitor, Y27632 or H1152. Furthermore, trypsin caused phosphorylation of myosin light chain (Thr18), but not Akt or Erk. Surprisingly, trypsin caused a similar shape change in PAR4-desensitised PAR3 null murine platelets as in PAR4 null murine platelets, indicating that trypsin did not activate PAR3 to cause shape change. More interestingly, the Src family kinase (SFK) inhibitor PP2 abolished trypsin-induced, but not AYPGKF-induced, shape change. Hence, trypsin activated a novel signalling pathway through RhoA/p160ROCK and was regulated by SFKs. In conclusion, our study demonstrates a novel protease signalling pathway in platelets that is independent of PARs. This protease-induced novel signalling pathway regulates platelet shape change through SFKs and p160ROCK.

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Mechanism of shape change in chilled human platelets

Blood ◽

10.1182/blood.v85.7.1796.bloodjournal8571796 ◽

1995 ◽

Vol 85 (7) ◽

pp. 1796-1804 ◽

Cited By ~ 116

Author(s):

R Winokur ◽

JH Hartwig

Keyword(s):

Intracellular Calcium ◽

Actin Filament ◽

Actin Filaments ◽

Cytochalasin B ◽

Shape Change ◽

Receptor Activation ◽

Actin Assembly ◽

Nucleation Sites ◽

Shape Changes ◽

Platelet Shape

The so-called cold activation of platelets that precludes refrigeration of platelets for storage has long been recognized, but its mechanism has remained a mystery. Cooling of discoid resting platelets to temperatures below 15 degrees C causes shape distortions, and the chilled cells rewarmed to above 25 degrees C are spheres rather than discs. As platelet shape change responsive to receptor activation at normal temperatures requires the remodeling of an actin scaffolding (Hartwig JH, 1992, J Cell Biol 118:1421–1442), we examined the role of actin in the morphologic changes induced by cooling. The addition of actin monomers onto the fast-exchanging (barbed) ends of actin filaments accompanies the initial physiologic platelet shape changes, and a key control point in this growth is the removal of proteins (caps) from the filament ends. This uncapping of actin filament ends is mediated by polyphosphoinositide aggregates in vitro, suggesting that cold-induced phase changes in membrane lipids might uncap actin filaments and thereby account for actin assembly-mediated shape alterations during cooling. Consistent with this hypothesis, reversible inhibition of actin assembly with cytochalasin B prevented the distortions in shape, although cooled platelets had increased actin nucleation sites and became spherical. Another step in normal platelet shape changes requires the severing of actin filaments that maintain the resting platelet. The proteins that sever initially bind to the broken filament ends, and uncapping of these fragmented filaments provides numerous nucleation sites for growth of actin filaments to fill in spreading filopodia and lamellae. Actin filament fragmentation requires a rise in intracellular calcium, and we showed that chilling platelets from 37 degrees C to 4 degrees C increases free cytosolic calcium levels from 80 nmol/L to approximately 200 nmol/L in minutes, thus providing an explanation for the spherical shape of cooled, rewarmed platelets. Blocking the calcium transient with nanomolar concentrations of the permeant calcium chelators Quin-2 and Fura-2 prevented the increase in nucleation sites and the sphering, but not the other shape changes of chilled and rewarmed platelets. However, a combination of micromolar cytochalasin B and millimolar intracellular calcium chelators preserved the discoid shapes of chilled and rewarmed platelets. After removal of cytochalasin B and addition of sufficient extracellular calcium, these platelets responded with normal morphologic alterations to glass and thrombin activation.

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Specific features of intracellular calcium signalling, distinctive for Wiskott-Aldrich syndrome patients

Pediatric Hematology/Oncology and Immunopathology ◽

10.24287/1726-1708-2020-19-1-100-107 ◽

2020 ◽

Vol 19 (1) ◽

pp. 100-107

Author(s):

A. A. Martyanov ◽

D. S. Morozova ◽

A. L. Khoreva ◽

M. A. Panteleev ◽

A. Yu. Shcherbina ◽

...

Keyword(s):

Intracellular Calcium ◽

Intracellular Signaling ◽

Shape Change ◽

Calcium Signalling ◽

Functional Responses ◽

Membrane Area ◽

Wiskott Aldrich Syndrome ◽

Pediatric Hematology ◽

Healthy Donors ◽

Platelet Shape

Wiskott–Aldrich syndrome (WAS) is a rare X-linked immunodeficient disease accompanied by microthrombocytopenia, which leads to spontaneous/post-traumatic haemorrhages. It has been demonstrated that WAS is caused by gene mutation of WASP protein, which is participating in the processes of actin polarization and actin cytoskeleton re-organisation. It is yet unknown how this mutation affects intracellular signalling and functional responses of platelets of patients with WAS. Assessment of the intracellular calcium signalling, shape change and fibrinogen binging by the platelets of WAS patients. The study was approved by the Independent Ethics Committee of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology, and Immunology. Three patients with WAS and three healthy volunteers were included in the study. Intracellular signaling and platelet functional responses were observed on a BD Facs Canto II flow cytometer. To measure the calcium concentration in the platelet cytosol, the Fura-Red fluorophore was used, platelet shape change upon activation was evaluated by side scattering of cells at a wavelength of 488 nm, platelet integrin activation was evaluated by binding of fluorescently-labeled fibrinogen. During activation, the platelet concentration was 1000 cells per ul to avoid the effects of secondary activation. In quescent state of platelets, an increased concentration of calcium in the cytosol of platelets of patients was observed compared with platelets of healthy donors. In response to stimulation, the highest achievable calcium concentrations were comparable in both cases. The binding of fibrinogen to platelets in patients was not significantly changed compared to healthy donors. On the other hand, the change in the shape of the cells in response to activation, expressed as a percentage, was more significant in patients than the change in the shape of the platelets of healthy donors. With similar maximum responses to stimulation by all agonists, the concentration of calcium in resting platelets, as well as the change in the platelet shape of patients with WAS is significantly higher than that of healthy platelet donors. These results can be explained by the increased ratio of the platelet membrane area to their volume.

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ADP Initiates Phosphorylation Of Platelet Myosin Light Chain (MLC), A 40,000 Dalton Protein (40P) And Actin Binding Protein (ADP) Independent Of Secretion

10.1055/s-0038-1652234 ◽

1981 ◽

Author(s):

J M Gerrard ◽

R C Carroll

Keyword(s):

Intracellular Calcium ◽

Protein Interactions ◽

Platelet Rich Plasma ◽

Shape Change ◽

Actin Binding ◽

Phosphorylated Proteins ◽

Reversible Aggregation ◽

Granule Secretion ◽

Platelet Shape ◽

Platelet Proteins

The mechanism by which ADP initiates platelet shape change and granule centralization is unknown. Recent studies of platelet proteins have provided evidence that raised intracellular calcium produces its effect by promoting protein phosphorylation. We have studied the influence of ADP on the phosphorylation of platelet proteins in both platelet rich plasma (PRP) and washed platelets, using the incorporation of 32p to identify phosphorylated proteins. 10u M ADP was found to cause a 2-5 fold increased phosphorylation of MLC, 40P and ABP. Pretreatment of the platelets with 100uM ASA prevented secretion of serotonin but decreased the ADP induced phosphorylation only by about 1/3. 2u M ADP stirred with platelets in PRP caused reversible aggregation without secretion and was associated with increased phosphorylation of MLC, 40P and ABP. The results show that ADP can cause phosphorylation of platelet proteins independent of secretion. We suggest ADP raises intracellular calcium levels to cause the phosphorylations which initiate contractile protein interactions to produce shape change and granule centralization. The results also show that platelet granule secretion must be dependent on other factors in addition to phosphorylation of these proteins.

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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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Platelet Aggregation Caused by Dithiothreitol

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661036 ◽

1984 ◽

Vol 51 (01) ◽

pp. 119-124 ◽

Cited By ~ 59

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.

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Adenosine Diphosphate (ADP)-Induced ⍺-Granules Release from Platelets of Native Whole Blood Is Reduced by Ticlopidine but Not by Aspirin or Dipyridamole

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661629 ◽

1986 ◽

Vol 56 (02) ◽

pp. 147-150 ◽

Cited By ~ 18

Author(s):

V Pengo ◽

M Boschello ◽

A Marzari ◽

M Baca ◽

L Schivazappa ◽

...

Keyword(s):

Whole Blood ◽

Light Transmission ◽

Ex Vivo ◽

Early Stage ◽

Shape Change ◽

Adenosine Diphosphate ◽

Dose Dependent ◽

Plateletderived Growth Factor ◽

Platelet Shape

SummaryA brief contact between native whole blood and ADP promotes a dose-dependent release of platelet a-granules without a fall in the platelet number. We assessed the “ex vivo” effect of three widely used antiplatelet drugs, aspirin dipyridamole and ticlopidine, on this system. Aspirin (a single 800 mg dose) and dipyridamole (300 mg/die for four days) had no effect, while ticlopidine (500 mg/die for four days) significantly reduced the a-granules release for an ADP stimulation of 0.4 (p <0.02), 1.2 (p <0.01) and 2 pM (p <0.01). No drug, however, completeley inhibits this early stage of platelet activation. The platelet release of α-granules may be related to platelet shape change of the light transmission aggregometer and may be important “in vivo” by enhancing platelet adhesiveness and by liberating the plateletderived growth factor.

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