The Role of Platelet Membrane Potential in the Initiation of Platelet Aggregation

1982 ◽  
Vol 47 (01) ◽  
pp. 022-026 ◽  
Author(s):  
D E MacIntyre ◽  
T J Rink
Keyword(s):  
Membrane Potential ◽  
Platelet Aggregation ◽  
Human Platelets ◽  
Null Point ◽  
Platelet Membrane ◽  
Resting Potential ◽  
Permselective Membrane ◽  
High K ◽  
Transduction Mechanisms

SummaryThe membrane potential of human platelets, and the role of this potential in platelet aggregation, was assessed using the non-covalent, fluorescent probe DiS-C3-5. High K+ and Gramicidin depolarised the cells, whereas valinomycin in standard (4 mMK+) solution produced a hyperpolarisation. Very small changes in potential were observed when choline Cl replaced NaCl. These findings indicate that platelets possess a relatively K+-permselective membrane. The resting potential calculated from the “valinomycin null point” (the K+ concentration gradient at which valinomycin did not change the potential) was approximately – 60 mV. Other factors that contribute to the platelet membrane potential include a significant Cl− permeability, demonstrated by replacing Cl− with methylsulphate, and an electrogenic Na+ pump, demonstrated using strophanthidin. Little or no change in potential was observed upon addition of ADP, collagen, U44069 or thrombin. Neither strong depolarisation with high K+ or gramicidin nor hyperpolarisation with valinomycin induced platelet aggregation or altered platelet responses to agonists. It is concluded that the information transduction mechanisms involved in platelet activation do not include changes in platelet membrane potential.

Further Observations On The Possible Association Of Human Platelet Membrane Glycoproteins IIb And IIIa

1981 ◽  
Author(s):  
J-P Rosa ◽  
D Pidard ◽  
T Kunicki ◽  
A T Nurden
Keyword(s):  
Platelet Aggregation ◽  
Membrane Proteins ◽  
Divalent Cation ◽  
Human Platelet ◽  
Human Platelets ◽  
Platelet Membrane ◽  
Membrane Glycoproteins ◽  
Normal Human ◽  
Triton X

Studies are described which represent a continuation of our investigation into the role of membrane glycoproteins (GP’s) IIb and IIIa during human platelet aggregation. The surface proteins of washed platelets were labelled with 125I by the lactoperoxidase-catalysed method prior to membrane isolation by the glycerol lysis procedure. Solubilisation of the membrane proteins by triton X-100 was followed by their analysis by crossed immunoelectrophoresis (CIE) using a rabbit antibody prepared against normal human platelets. In the absence of divalent cation chelation GP IIb and Ilia were contained within a single 125I-labelled immunoprecipitate. When the isolated membranes were solubilised by triton X-100 in the presence of 5mM EDTA, GP IIb and IIIa formed distinctand separate immunoprecipitates during CIE. In order to further investigate this finding 125I-labelled membrane proteins solubilised by triton X-100 in the presence or absence of EDTA were subjected to centrifugation for 18 h at 100,000 g over a 10-40% sucrose gradient containing the nonionic detergent. The results confirmed that in the presence of divalent cations lib and Ilia were associated in a complex, and that this complex is dissociated by EDTA. The IgG..L is an alloantibody isolated from a polytransfused thrombasthenic patient that has been shown in previous studies to inhibit ADP-induced platelet aggregation and the binding of 125I-fibrinogen to normal human platelets in the presence of ADP. When the IgG..L was incorporated in an intermediate gel during CIE it was shown to precipitate the complex containing IIb/IIIa but under identical conditions it did not precipitate the individual glycoproteins dissociated by EDTA. Divalent cation-mediated changes in the orientation of lib and Ilia in the platelet membrane should be considered in assessing the role of these GP’s in platelet function.

Platelet Membrane Potential: Simultaneous Measurement of diSC3(5) Fluorescence and Optical Density

1985 ◽  
Vol 54 (03) ◽  
pp. 645-649 ◽  
Author(s):  
Eva Pipili
Keyword(s):  
Membrane Potential ◽  
Time Course ◽  
Shape Change ◽  
Human Platelets ◽  
Platelet Membrane ◽  
Receptor Interaction ◽  
Na Channel ◽  
Similar Time ◽  
High K ◽  
Contrast Exposure

SummaryThe role of membrane potential in the activation of human platelets by thrombin, ADP and PAF was assessed, using the fluorescent probe diSC3(5). Thrombin, ADP and PAF transiently depolarised the platelet membrane by 6-8 mV from its resting level (—70 mV). This depolarisation had a similar time course to that of shape change. The ionophores valinomycin and gramicidin hyperpolarised and depolarised the platelets respectively but did not activate them. In contrast, exposure of platelets to high K+ media both depolarised and caused them to change shape. Removal of Na+ from the suspension media abolished the depolarisation induced by thrombin, ADP and PAF but the platelets under these conditions were still capable of changing shape and aggregating. This result indicates that the observed depolarisation depends on Na+ fluxes. Amiloride or tetrodotoxin did not mimic the effect of Na+ removal suggesting that any Na+ movement involved does not go through the classic “Na+ channel”. Thrombin, ADP and PAF still depolarised the platelet membrane in the absence of added Ca+ +. Under these conditions, however, the membrane did not repolarise. It is evident that all three agents, thrombin, ADP and PAF, change the membrane potential of human washed platelets through a similar mechanism and this change seems to be a consequence of stimulus-receptor interaction (and platelet activation?). A causal relationship however between these events cannot be clearly shown.

Collagen-Induced Platelet Aggregation: -Evidence Against the Essential Role of Platelet Adenosine Diphosphate

1979 ◽  
Vol 42 (04) ◽  
pp. 1193-1206 ◽  
Author(s):  
Barbara Nunn
Keyword(s):  
Platelet Aggregation ◽  
Time Course ◽  
Essential Role ◽  
Atp Release ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
High Doses ◽  
Induced Aggregation

SummaryThe hypothesis that platelet ADP is responsible for collagen-induced aggregation has been re-examined. It was found that the concentration of ADP obtaining in human PRP at the onset of aggregation was not sufficient to account for that aggregation. Furthermore, the time-course of collagen-induced release in human PRP was the same as that in sheep PRP where ADP does not cause release. These findings are not consistent with claims that ADP alone perpetuates a collagen-initiated release-aggregation-release sequence. The effects of high doses of collagen, which released 4-5 μM ADP, were not inhibited by 500 pM adenosine, a concentration that greatly reduced the effect of 300 μM ADP. Collagen caused aggregation in ADP-refractory PRP and in platelet suspensions unresponsive to 1 mM ADP. Thus human platelets can aggregate in response to collagen under circumstances in which they cannot respond to ADP. Apyrase inhibited aggregation and ATP release in platelet suspensions but not in human PRP. Evidence is presented that the means currently used to examine the role of ADP in aggregation require investigation.

Modulation of cell membrane potential in cultured vascular endothelium

1996 ◽  
Vol 270 (3) ◽  
pp. C819-C824 ◽  
Author(s):  
L. Vaca ◽  
A. Licea ◽  
L. D. Possani
Keyword(s):  
Membrane Potential ◽  
Cell Membrane ◽  
Cell Membrane Potential ◽  
Resting Potential ◽  
Similar Response ◽  
Aortic Endothelial Cells ◽  
Ionic Conductances ◽  
Selective Channel ◽  
Inwardly Rectifying

The present study explores the role of different ionic conductances in the regulation of membrane potential under resting conditions and after bradykinin (BK) or thapsigargin (TG) stimulation of cultured bovine aortic endothelial cells. Under resting conditions, the cell membrane potential observed was -62+/- 5 mV. The main conductance under these conditions is an inwardly rectifying potassium (IRK) channel. Application of 50 nM BK induced a transient hyperpolarization to -87 +/- 4 mV followed by sustained depolarization to -35 +/- 5 mV. The transient hyperpolarization was eliminated by 1 microM noxiustoxin, a blocker of calcium-activated postassium channels (K(Ca)). the sustained depolarization induced by BK was prevented by incubating the cells with the calcium channel blocker lanthanum. TG evoked a similar response in membrane potential, with the exception that the onset of the hyperpolarization was slower compared with BK. The results presented here indicate that the cell resting potential is maintained at -62 +/- 2 mV by the IRK channel. BK or TG stimulation induces a transient hyperpolarization of approximately -20 mV produced by activation of a KCa. This hyperpolarization is followed by a sustained depolarization produced by activation of a calcium-selective channel sensitive to lanthanum.

Role of phosphoinositide 3-kinase β in platelet aggregation and thromboxane A2 generation mediated by Gi signalling pathways

2010 ◽  
Vol 429 (2) ◽  
pp. 369-377 ◽  
Author(s):  
Analia Garcia ◽  
Soochong Kim ◽  
Kamala Bhavaraju ◽  
Simone M. Schoenwaelder ◽  
Satya P. Kunapuli
Keyword(s):  
Platelet Aggregation ◽  
Critical Role ◽  
Thromboxane A2 ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
P2y12 Receptor ◽  
Functional Responses ◽  
Induce Platelet Aggregation ◽  
Erk Phosphorylation

PI3Ks (phosphoinositide 3-kinases) play a critical role in platelet functional responses. PI3Ks are activated upon P2Y12 receptor stimulation and generate pro-aggregatory signals. P2Y12 receptor has been shown to play a key role in the platelet aggregation and thromboxane A2 generation caused by co-stimulation with Gq or Gz, or super-stimulation of Gi pathways. In the present study, we evaluated the role of specific PI3K isoforms α, β, γ and δ in platelet aggregation, thromboxane A2 generation and ERK (extracellular-signal-regulated kinase) activation. Our results show that loss of the PI3K signal impaired the ability of ADP to induce platelet aggregation, ERK phosphorylation and thromboxane A2 generation. We also show that Gq plus Gi- or Gi plus Gz-mediated platelet aggregation, ERK phosphorylation and thromboxane A2 generation in human platelets was inhibited by TGX-221, a PI3Kβ-selective inhibitor, but not by PIK75 (a PI3Kα inhibitor), AS252424 (a PI3Kγ inhibitor) or IC87114 (a PI3Kδ inhibitor). TGX-221 also showed a similar inhibitory effect on the Gi plus Gz-mediated platelet responses in platelets from P2Y1−/− mice. Finally, 2MeSADP (2-methyl-thio-ADP)-induced Akt phosphorylation was significantly inhibited in the presence of TGX-221, suggesting a critical role for PI3Kβ in Gi-mediated signalling. Taken together, our results demonstrate that PI3Kβ plays an important role in ADP-induced platelet aggregation. Moreover, PI3Kβ mediates ADP-induced thromboxane A2 generation by regulating ERK phosphorylation.

ADRENALINE (ADR) INTERACTIONS WITH THROMBIN AND COLLAGEN -EFFECTS ON PLATELET ARACHIDONATE RELEASE AND 5HT SECRETION AND ROLE OF ENDOGENOUSLY FORMED THROMBOXANE A2 (TxA2)

1987 ◽  
Author(s):  
Y Patel ◽  
S Krishnamurthi ◽  
V V Kakkar
Keyword(s):  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Adenylate Cyclase ◽  
Synergistic Effects ◽  
Thromboxane A2 ◽  
Human Platelets ◽  
Presence And Absence ◽  
Pre Treatment ◽  
Arachidonate Release

We have examined the effect of combinations of ADR + thrombin (T) and ADR + collagen (C) on platelet arachidonate release and 5HT secretion, and assessed the role of endogenously formed TxA2 on these responses using indomethacin (I). Washed, human platelets prelabelled with [3H]-arachidonic acid (AA) or [14C]-5HT were used, ADR was added 10 sec before T or C and the reaction was terminated 3 min later. In the range 1-100μM, ADR induced no detectable aggregation or 5HT secretion but potentiated platelet aggregation when added with sub-threshold concentrations of T or C, which on their own induced no aggregation. At 2-4 fold higher concentrations of T and C (threshold for 5HT secretion), 5HT secretion and AA/TXB2 release were also potentiated by ADR (1-10μM) by 30-50%. Pre-treatment of platelets with I (10μM) abolished threshold T and C-induced 5HT secretion, as well as its potentiation by ADR. However, approximately 2-fold and 5-fold higher concentrations of T and C respectively were able to induce 'I-insensitive'secretion, which was further potentiated by ADR. In I-treated platelets, C-induced AA release and its potentiation by ADR were also abolished suggesting a role for endogenously formed TxA2 This was confirmed by addition of the TxA2 mimetic, U46619 (0.3μM), which potentiated C-induced AA release in the presence and absence of ADR, even though it induced no AA release on its own or, in combination with ADR alone in the absence of collagen. The latter suggests agonist specificity regarding the ability of TxA2 to synergistically stimulate AA release. Finally, unstirred platelets in PRP pre-incubated with ADR (10μM) for 120 min lost their responsiveness to ADR, when eventually stirred; however, these 'ADR-desensitised' platelets when washed and resuspended, were able to demonstrate synergistic effects on secretion when stimulated with ADR+T or ADR+C. This is analogous to the previously demonstrated ability of ADR to inhibit adenylate cyclase even in 'ADR-desensitised' platelets and re-inforces the separation regarding the mechanisms underlying the various effects of ADR on platelets.

The Endocannabinoid 2-Arachidonoylglycerol Regulates Platelet Function.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3904-3904
Author(s):  
Samantha Baldassarri ◽  
Alessandra Bertoni ◽  
Paolo Lova ◽  
Stefania Reineri ◽  
Chiara Sarasso ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Platelet Function ◽  
Cannabinoid Receptors ◽  
Thromboxane A2 ◽  
Human Platelets ◽  
Dependent Manner ◽  
Cb2 Receptor ◽  
Thromboxane A2 Receptor

Abstract 2-Arachidonoylglycerol (2-AG) is a naturally occurring monoglyceride that activates cannabinoid receptors and meets several key requisites of an endogenous cannabinoid substance. It is present in the brain and hematopoietic cells, including macrophages, lymphocytes and platelets. 2-AG is released from cells in a stimulus-dependent manner and is rapidly eliminated by uptake into cells and enzymatic hydrolysis in arachidonic acid and glycerol. 2-AG might exert a very fine control on platelet function either through mechanisms intertwining with the signal transduction pathways used by platelet agonists or through mechanisms modulating specific receptors. The aim of this study was to define the role of 2-AG in human platelets and characterize the mechanisms by which it performs its action. Platelets from healthy donors were isolated from plasma by differential centrifugations and gel-filtration on Sepharose 2B. The samples were incubated with 2-AG (10–100 μM) under constant stirring in the presence or absence of various inhibitors. Platelet aggregation was measured by Born technique. We have found that stimulation of human platelets with 2-AG induced irreversible aggregation, which was significantly enhanced by co-stimulation with ADP (1–10 μM). Furthermore, 2-AG-dependent platelet aggregation was completely inhibited by ADP scavengers, aspirin, and Rho kinase inhibitor, as well as by antagonists of the 2-AG receptor (CB2), of the ADP P2Y12 receptor, and of the thromboxane A2 receptor. We further investigated the role of endocannabinoids on calcium mobilization. Intracellular [Ca2+] was measured using FURA-2-loaded platelets prewarmed at 37°C under gentle stirring in a spectrofluorimeter. 2-AG induced rapid increase of cytosolic [Ca2+] in a dose-dependent manner. This effect was partially blocked by ADP scavengers and CB2 receptor antagonists. Furthermore, 2-AG-induced [Ca2+] mobilization was totally suppressed by aspirin or the thromboxane A2 receptor antagonist. These results suggest that 2-AG is able to trigger platelet activation, and that this action is partially mediated by CB2 receptor and ADP. Furthmore, 2-AG-dependent platelet activation is totally dependent on thromboxane A2 generation.

An Important Role for Rac1 and RhoA In Regulating Platelet Microparticle Formation and Phosphatidylserine Exposure

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2023-2023
Author(s):  
Michael Keegan Delaney ◽  
Junling Liu ◽  
Yi Zheng ◽  
Xiaoping Du
Keyword(s):  
Platelet Aggregation ◽  
Conflicts Of Interest ◽  
Human Platelets ◽  
Small Gtpases ◽  
Procoagulant Activity ◽  
Null Mouse ◽  
Signaling Mechanisms ◽  
Granule Secretion ◽  
Microparticle Formation

Abstract Abstract 2023 Platelets activated by physiological agonists such as thrombin and collagen shed procoagulant microparticles (MPs) and externalize the procoagulant phospholipid phosphatidylserine (PS), both of which are critical to hemostasis and play an important role in inflammation. To date, the signaling mechanisms that regulate agonist-induced MP formation and PS exposure in platelets remain unclear. In this study, we demonstrate that the small GTPases Rac1 and RhoA play important roles in regulating the procoagulant activity of platelets. Rac1 null (-/-) mouse platelets or human platelets treated with the Rac1 inhibitor NSC23766 (NSC) displayed a significant defect in MP formation and PS exposure induced by various agonists. Furthermore, Rac1-/- platelets and NSC-treated human platelets displayed a defect in procoagulant activity as demonstrated by a prolonged coagulation time following recalcification of citrated PRP. The stimulatory role of Rac1 in platelet MP formation and PS exposure is distinct from the known function of Rac1 in facilitating platelet granule secretion and secretion-dependent amplification of platelet aggregation, because supplementation of the granule content ADP rescued the defect in platelet aggregation caused by Rac1 inhibition, but failed to rescue the defect in MP formation caused by Rac1 inhibition. In contrast to Rac1, RhoA plays an inhibitory role in regulating platelet procoagulant activity, because treatment of platelets with the Rho inhibitor C3-toxin (C3) significantly enhanced agonist-induced MP formation, PS exposure, and procoagulant activity. The enhancing effect of C3 on platelet procoagulant activity is not caused by an overall enhancement of platelet activation because C3 significantly inhibited platelet secretion and aggregation. Thus, our data demonstrates that while Rac1 and RhoA both play important stimulatory roles in platelet granule secretion and aggregation, they play opposing roles in MP formation and PS exposure in platelets. Rac1 is important for stimulating platelet MP formation, PS exposure, and procoagulant activity, which is antagonized by RhoA. Disclosures: No relevant conflicts of interest to declare.

scholarly journals ZP3-dependent activation of sperm cation channels regulates acrosomal secretion during mammalian fertilization.

1996 ◽  
Vol 134 (3) ◽  
pp. 637-645 ◽  
Author(s):  
C Arnoult ◽  
Y Zeng ◽  
H M Florman
Keyword(s):  
Membrane Potential ◽  
Pertussis Toxin ◽  
Zona Pellucida ◽  
Mammalian Sperm ◽  
Transient Rise ◽  
Transduction Mechanisms ◽  
Dependent Signal ◽  
Sperm Membrane ◽  
Mammalian Fertilization

The sperm acrosome reaction is a Ca(2+)-dependent secretory event required for fertilization. Adhesion to the egg's zona pellucida promotes Ca2+ influx through voltage-sensitive channels, thereby initiating secretion. We used potentiometric fluorescent probes to determine the role of sperm membrane potential in regulating Ca2+ entry. ZP3, the glycoprotein agonist of the zona pellucida, depolarizes sperm membranes by activating a pertussis toxin-insensitive mechanism with the characteristics of a poorly selective cation channel. ZP3 also activates a pertussis toxin-sensitive pathway that produces a transient rise in internal pH. The concerted effects of depolarization and alkalinization open voltage-sensitive Ca2+ channels. These observations suggest that mammalian sperm utilize membrane potential-dependent signal transduction mechanisms and that a depolarization pathway is an upstream transducing element coupling adhesion to secretion during fertilization.

Functional role of a polymorphism in the Pannexin1 gene in collageninduced platelet aggregation

2015 ◽  
Vol 114 (08) ◽  
pp. 325-336 ◽  
Author(s):  
Filippo Molica ◽  
Jean-François Denis ◽  
Paul Bradfield ◽  
Silvia Penuela ◽  
Anne Zufferey ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Reactivity ◽  
Critical Role ◽  
Atp Release ◽  
Human Platelets ◽  
Immune Synapse ◽  
Cardiovascular Patients ◽  
Exogenous Expression ◽  
First Time

SummaryPannexin1 (Panx1) forms ATP channels that play a critical role in the immune response by reinforcing purinergic signal amplification in the immune synapse. Platelets express Panx1 and given the importance of ATP release in platelets, we investigated Panx1 function in platelet aggregation and the potential impact of genetic polymorphisms on Panx1 channels. We show here that Panx1 forms ATP release channels in human platelets and that inhibiting Panx1 channel function with probenecid, mefloquine or specific 10Panx1 peptides reduces collagen-induced platelet aggregation but not the response induced by arachidonic acid or ADP. These results were confirmed using Panx1-/- platelets. Natural variations have been described in the human Panx1 gene, which are predicted to induce non-conservative amino acid substitutions in its coding sequence. Healthy subjects homozygous for Panx1–400C, display enhanced platelet reactivity in response to collagen compared with those bearing the Panx1–400A allele. Conversely, the frequency of Panx1–400C homozygotes was increased among cardiovascular patients with hyper-reactive platelets compared with patients with hypo-reactive platelets. Exogenous expression of polymorphic Panx1 channels in a Panx-deficient cell line revealed increased basal and stimulated ATP release from cells transfected with Panx1–400C channels compared with Panx1–400A expressing transfectants. In conclusion, we demonstrate a specific role for Panx1 channels in the signalling pathway leading to collagen-induced platelet aggregation. Our study further identifies for the first time an association between a Panx1–400A>C genetic polymorphism and collagen-induced platelet reactivity. The Panx1–400C variant encodes for a gain-of-function channel that may adversely affect atherothrombosis by specifically enhancing collagen-induced ATP release and platelet aggregation.

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