Ca2+ AND AMINE STORAGE GRANULE SECRETION FROM HUMAN PLATELETS

Fura 2 is one of a recently-introduced family of Ca++ indicators with improved fluorescent properties compared to quin 2 (Grynkiewicz et al 1985). This study has examined the role of [Ca++]i in thrombin-induced dense granule release using prostacyclin-washed human platelets loaded with either thedense granule marker 14C-5HT (5HT) alone or with 5HT together with quin 2 ([quin2]i = 0.8mM) or fura 2 ([fura 2]i 20-30µM). In the presence of ImM extracellular calcium concentration ([Ca++]i) the [Ca++]e in quin 2 and fura 2 loaded platelets was 93±2 (n=10 experiments) and 133±0.3nM (n=12 experiments) respectively. In either quin 2 or fura 2 loaded platelets suspended in the presence of ImM [Ca++]e, thrombin (0.23-23.InM) promoted a rapid (in secs)concentration-dependent elevation of [Ca++]i from basal values to levels l-2µM, together with a parallel release of dense granules almost identical to that obtained with thrombin in non dye loaded platelets. In fura 2 loaded cells, removal of [Ca++]e inhibited the elevation of [Ca++]i induced by a sub-maximal concentration of thrombin (0.77nM) by 43+5% (n=4) but interestingly had no significant effect (p<0.05) on the rise in [Ca++]i elicited by low thrombin doses (0.231nM). Neither did lowering [Ca++]e inhibit the release of 5HT evoked by thrombin ( 0.231-23.InM) from either fura 2 loaded or non dye loaded platelets. In contrast, in quin 2 loaded platelets, removal of [Ca++]e inhibited the thrombin (0.231-23.InM) stimulated rise in [Ca++]i-by 90% and the 5HT release response to either low (0.231nM), sub-maximal (0.77nM) or maximal (23.InM) thrombin by 100% (n=4), 87+2°/o (n=6)and 2+l°/o (n=4) respectively. Fura 2 but not quin 2 loaded cells suspended in ImM [Ca++]e exhibited a Ca++ response to thrombin concentrations >2.31nM which could be separated into a rapid phasic component and a more sustained 'tonic' like component inhibitable by removal of [Ca++]e or by addition of ImM Ni++ . These data suggest the use of fura 2 rather than quin 2 for investigating stimulus response coupling in platelets, particularly when [Ca++]e is less than physiological. We thank the British Heart Foundation and Ciba-Geigy USA for financial support.

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Effects of antimycin A and 2-deoxyglucose on secretion in human platelets. Differential inhibition of the secretion of acid hydrolases and adenine nucleotides

Biochemical Journal ◽

10.1042/bj1820413 ◽

1979 ◽

Vol 182 (2) ◽

pp. 413-419 ◽

Cited By ~ 14

Author(s):

Holm Holmsen ◽

Linda Robkin ◽

H. James Day

Keyword(s):

Shape Change ◽

Adenine Nucleotides ◽

Human Platelets ◽

Dense Granule ◽

Metabolic Inhibitors ◽

Antimycin A ◽

Acid Hydrolase ◽

Acid Hydrolases ◽

Dense Granule Secretion ◽

Granule Secretion

1. Shape change, aggregation and secretion of dense-granule constituents in platelets differ in their dependence on cellular energy metabolism. The possibility that such a difference also exists between secretion of dense-granule constituents and acid hydrolases was investigated. 2. Human platelets were incubated with [14C]adenine in plasma, and then washed and resuspended in salt solutions. The effects of incubating the cells with antimycin A and 2-deoxyglucose on the concentrations of [14C]ATP, ADP, AMP, IMP and inosine plus hypoxanthine and on thrombin-induced secretion of ATP plus ADP and acid hydrolases were studied. The metabolic inhibitors only affected 14C-labelled nucleotides, whereas thrombin only liberated unlabelled ATP and ADP. 3. The extent of secretion decreased progressively with time during incubation with the metabolic inhibitors. At any time the secretion of acid hydrolases, β-N-acetylglucosaminidase, β-glucuronidase and β-galactosidase was inhibited to a greater extent than secretion of ATP plus ADP (dense-granule secretion). 4. Incubation with the metabolic inhibitors shifted the log (dose)–response relationship to higher thrombin concentrations, and with a greater shift for acid hydrolase secretion than for dense-granule secretion. 5. Antimycin, when present alone, caused a marked decrease in the rate of acid hydrolase secretion, but had no effect on dense-granule secretion. 6. These results further support the view that acid hydrolase secretion and dense-granule secretion are separate processes with different requirements for ATP energy. Acid hydrolase secretion, but not dense-granule secretion, appears to depend on a simultaneous rapid generation of ATP, which can be accomplished by oxidative, but not by glycolytic, ATP production.

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The secretory pathway of bovine platelets

Blood ◽

10.1182/blood.v69.3.878.878 ◽

1987 ◽

Vol 69 (3) ◽

pp. 878-885 ◽

Cited By ~ 5

Author(s):

JG White

Keyword(s):

Plasma Membrane ◽

Secretory Pathway ◽

Human Platelet ◽

Surface Membrane ◽

Surrounding Medium ◽

Human Platelets ◽

Resting Cells ◽

Granule Secretion ◽

New Perspective ◽

Platelet Secretion

Abstract Human platelets contain tortuous channels in their cytoplasm, the surface-connected or open canalicular system (OCS), that communicate directly with the surrounding medium through openings on the surface membrane. Some workers have suggested that the OCS serves as the egress route for products secreted during the release reaction. Others have proposed alternate secretory pathways. Since bovine platelets lack the OCS found in human cells, the present study has examined the secretory mechanism of these cells to see whether it can shed light on the mystery of human platelet secretion. Bovine platelet granules, in contrast to human granules, are located more peripherally in resting cells (often in contact with the plasma membrane), most do not move centrally following thrombin stimulation as do human platelet granules, and many fuse directly with the external plasma membrane without any intermediate channel. The lack of peripheral location of human granules, their central rather than peripheral movement during secretion, and the presence of extensive channels are all consistent with the larger importance of the secretory channel to human platelets. Thus, though studies of bovine secretion do show that platelets can secrete their granules by direct fusion of granule and surface membranes, other differences from human platelets emphasize that this pathway, although important to bovine platelet secretion, is less important in human platelets. Studies of bovine platelets also show that the OCS is more dynamic than might have been considered from human studies and can form rapidly in response to stimulation. Such newly formed channels are used as a conduit for secretion of granule contents. The finding emphasizes the importance of channels for granule secretion in platelets generally and puts a new perspective on the ability of these cells to form channels rapidly in response to stimulation.

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

Biochemical Journal ◽

10.1042/bj20101360 ◽

2011 ◽

Vol 436 (2) ◽

pp. 469-480 ◽

Cited By ~ 21

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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Human platelets express and are activated by galectin-8

Biochemical Journal ◽

10.1042/bj20100538 ◽

2010 ◽

Vol 432 (3) ◽

pp. 535-547 ◽

Cited By ~ 45

Author(s):

Maria Albertina Romaniuk ◽

Maria Virginia Tribulatti ◽

Valentina Cattaneo ◽

Maria Jose Lapponi ◽

Felisa Concepcion Molinas ◽

...

Keyword(s):

Thrombus Formation ◽

Endothelial Activation ◽

Human Platelets ◽

Target Cells ◽

Fibrinogen Binding ◽

Extracellular Signal ◽

Carbohydrate Recognition Domains ◽

Thrombin Activation ◽

Granule Secretion ◽

Phosphoinositide 3 Kinase

Gals (galectins) are proteins with glycan affinity that are emerging as mediators of atherosclerosis. Despite the similarities in structure and sequence, different Gals exert distinct effects on their target cells. We have shown that Gal-1 triggers platelet activation, suggesting a role for Gals in thrombus formation. Since Gal-8 is expressed upon endothelial activation and also contributes to inflammation, to understand further the role of these lectins in haemostasis, we evaluated the effect of Gal-8 on human platelets. Gal-8 bound specific glycans in the platelet membrane and triggered spreading, calcium mobilization and fibrinogen binding. It also promoted aggregation, thromboxane generation, P-selectin expression and granule secretion. GP (glycoprotein) αIIb and Ib-V were identified as putative Gal-8 counter-receptors by MS. Studies performed using platelets from Glanzmann's thromboasthenia and Bernard–Soulier syndrome patients confirmed that GPIb is essential for transducing Gal-8 signalling. Accordingly, Src, PLC2γ (phospholipase C2γ), ERK (extracellular-signal-regulated kinase) and PI3K (phosphoinositide 3-kinase)/Akt downstream molecules were involved in the Gal-8 signalling pathway. Gal-8 fragments containing either the N- or C-terminal carbohydrate-recognition domains showed that activation is exerted through the N-terminus. Western blotting and cytometry showed that platelets not only contain Gal-8, but also expose Gal-8 after thrombin activation. These findings reveal Gal-8 as a potent platelet activator, supporting a role for this lectin in thrombosis and inflammation.

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Comparative study of platelet dense granule constituents

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.1982.243.3.r454 ◽

1982 ◽

Vol 243 (3) ◽

pp. R454-R461 ◽

Cited By ~ 11

Author(s):

K. M. Meyers ◽

H. Holmsen ◽

C. L. Seachord

Keyword(s):

Comparative Study ◽

Gel Filtration ◽

Human Platelets ◽

Dense Granule ◽

Releasable Pool ◽

Rabbit Platelets ◽

Granule Secretion

Cat, cattle, dog, horse, human, mink, pig, and rabbit platelets were separated from plasma by gel filtration. The gel-filtered platelets (GFP) were treated with thrombin to induce maximal granule secretion and the potential dense granule constituents ATP, ADP, serotonin (5-HT), Ca2+, and Mg2+ were measured in GFP and in the control and thrombin-treated platelets and in the respective supernatants. The amount of Ca2+, Mg2+, 5-HT, ATP, and ADP within the nonreleasable pool for all species varied between 3.1 and 10.0 mumol/10(11) platelets for Ca2+ and Mg2+ was less than 1.5 mumol/10(11) platelets for ADP and 5-HT and was between 2.0 and 5.0 mumol/10(11) platelets for ATP. Marked differences were observed in the releasable fraction. Human platelets were characterized by the largest releasable Ca2+ pool (greater than 10 mumol/10(11) platelets), the smallest secretable 5-HT and Mg2+ pool (less than 0.5 mumol/10(11) platelets), and the lowest ATP-to-ADP ratio (greater than 1.0). Pig platelets had the highest amount of releasable Mg2+ (approximately 8.0 mumol/10(11) platelets). Rabbits platelets released the most 5-HT (greater than 3.0 mumol/10(11)) and had the highest ATP/ADP (greater than 5.0). The releasable pool of Ca2+, Mg2+, ATP, and ADP in the remaining species varied in mumol/10(11) platelets from approximately 1.5-4.0, approximately 1.0-3.0, 0.5-3.5, and approximately 0.5-1.5, respectively.

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Synergistic potentiation of 5-hydroxytryptamine secretion by platelet agonists and phorbol myristate acetate despite inhibition of agonist-induced arachidonate/thromboxane and β-thromboglobulin release and Ca2+ mobilization by phorbol myristate acetate

Biochemical Journal ◽

10.1042/bj2380193 ◽

1986 ◽

Vol 238 (1) ◽

pp. 193-199 ◽

Cited By ~ 27

Author(s):

S Krishnamurthi ◽

S Joseph ◽

V V Kakkar

Keyword(s):

Phorbol Myristate Acetate ◽

Phorbol Esters ◽

Synergistic Effects ◽

Human Platelets ◽

Significant Rise ◽

Inhibitory Effects ◽

Myristate Acetate ◽

Inositol Phospholipid ◽

Granule Secretion ◽

Inositol Phospholipid Breakdown

Previous studies have demonstrated an inhibition of agonist-induced inositol phospholipid breakdown and intracellular Ca2+ ([Ca2+]i) mobilization by phorbol esters in platelets. In this study, we have examined the effect of phorbol 12-myristate 13-acetate (PMA) on agonist-induced granule secretion and correlated it with agonist-induced [Ca2+]i mobilization, arachidonate and thromboxane (Tx) release in human platelets. With increasing times of incubation with PMA (10 s-5 min), the rise in [Ca2+]i induced by thrombin and the TxA2 mimetic, U46619, was increasingly inhibited (90-100% with 5 min incubation) and, correlating with this, thrombin-induced [3H]arachidonate, TxB2 and beta-thromboglobulin (beta TG) release were also inhibited. In addition, the conversion of exogenously added arachidonate to TxB2 was inhibited (50-80%) by a 10 s-5 min pretreatment with PMA. However, secretion of 5-hydroxy[14C]tryptamine (5HT) induced by thrombin or U46619 was not inhibited by 10 s-2 min incubations with PMA and, on the contrary, with low agonist concentrations, was potentiated by PMA in the absence of a significant rise in [Ca2+]i or endogenous Tx formation, to levels significantly greater than or equal to the sum of that obtained when agonist and PMA were added separately. With longer times of incubation with PMA (5 min), these synergistic effects became less pronounced as inhibitory effects of PMA on agonist-induced [14C]5HT secretion became apparent. The results indicate that, while PMA may cause an inhibition of agonist-induced [Ca2+]i mobilization resulting in an inhibition of agonist-induced arachidonate, TxB2 and beta TG release, its effects on agonist-induced 5HT secretion may be complicated by [Ca2+]i-independent synergistic effects of agonist and PMA.

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

Blood ◽

10.1182/blood.v116.21.2023.2023 ◽

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.

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