scholarly journals Role of Munc13-4 as a Ca2+-dependent tether during platelet secretion

2016 ◽  
Vol 473 (5) ◽  
pp. 627-639 ◽  
Author(s):  
Michael C. Chicka ◽  
Qiansheng Ren ◽  
David Richards ◽  
Lance M. Hellman ◽  
Jinchao Zhang ◽  
...  
Keyword(s):  
Dense Granule ◽  
Rapid Response ◽  
Vascular Damage ◽  
Dependent Manner ◽  
Dense Granule Secretion ◽  
Granule Secretion ◽  
Platelet Exocytosis ◽  
Platelet Secretion

Platelet exocytosis, mediated by SNAREs and Ca2+-dependent regulators, is critical for haemostasis. Munc13-4 binds membranes in a Ca2+- and phosphatidylserine (PS)-dependent manner and acts as a tethering factor for pre-docked platelet dense granule secretion to mediate rapid response to vascular damage.

nPKC Epsilon Negatively Regulates Platelet Functional Responses

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2855-2855
Author(s):  
Yamini Saraswathy Bynagari ◽  
Bela Nagy ◽  
Kamala Bhavaraju ◽  
Donna Woulfe ◽  
Soochong Kim ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Ex Vivo ◽  
Dense Granule ◽  
Dependent Manner ◽  
P2y1 Receptor ◽  
Wild Type ◽  
Functional Responses ◽  
Dense Granule Secretion ◽  
Granule Secretion

Abstract Protein Kinase C (PKC) are family of serine threonine kinases, known to regulate various platelet functional responses. Among them novel class of PKC isoforms (nPKC) including delta(δ), theta(𝛉), eta(η), and epsilon(ε) are expressed in platelets. Although, the role of nPKC ε and η in platelets is fairly understood, not much is known about nPKC ε and η in platelets. In this study, we investigated the role of nPKC ε in platelet functional responses using ADP-induced signaling as our stereotype. ADP causes platelet activation via Gq-coupled P2Y1 receptor and Gi-coupled P2Y12 receptor. Thus, we primarily studied the role of P2Y1 receptor in nPKC ε activation. ADP activated nPKC ε in time- and concentration- dependent manner. In the presence of P2Y1 receptor antagonist MRS-2179 and in P2Y1 knockout (KO) murine platelets ADP failed to activate nPKC ε, suggesting that ADP activates nPKC ε via P2Y1 receptor. We further investigated the functional role of nPKC ε using specific nPKC ε inhibitory RACK peptide (ε V1-2). ε V1-2 is a peptide designed to compete with native nPKC ε to bind ε-Receptors for activated C Kinase (ε-RACK) and thereby inhibits nPKC ε catalytic activity due to decreased substrate accessibility. ADP-induced thromboxane generation in human platelets pretreated with ε V1-2 peptide was more compared to the platelets pretreated with control peptide. Similarly, ADP-induced thromboxane generation in platelets derived from nPKC ε KO mouse was more compared to the wild type (WT) littermates. However, ADP- induced alpha granule secretion and aggregation in aspirin treated platelets derived from PKC ε KO mice was not significantly different from platelets derived from wild type littermates. These data suggest that nPKC e regulates an unknown pathway, which primarily regulates thromboxane generation with minimal effects on aggregation and alpha granule secretion. Furthermore, we also investigated the role of nPKC ε in PAR- and GPVI- mediated platelet aggregation and dense granule secretion. Interestingly, in both aspirin-treated and non-aspirin-treated platelets PAR- and GPVI- mediated platelet aggregation and dense granule secretion were potentiated. Consistent with ex vivo studies, FeCl3-induced arterial thrombosis was enhanced in nPKC ε KO mice compared to WT littermates.

THE ROLE OF IONISED INTRACELLULAR FREE CALCIUM ([Ca++]i) IN THROMBIN-INDUCED DENSE GRANULE SECRETION

1987 ◽  
Author(s):  
C T Poll ◽  
J Westwick
Keyword(s):  
Human Platelets ◽  
Dense Granule ◽  
Free Calcium ◽  
Fluorescent Properties ◽  
Fura 2 ◽  
Stimulus Response ◽  
Dense Granules ◽  
Dense Granule Secretion ◽  
Granule Secretion

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.

REQUIREMENT FOR THROMBIN RECEPTOR OCCUPANY DURING PLATELET SECRETION UNDER AGGREGATING CONDITIONS

1987 ◽  
Author(s):  
Kazuo Koike ◽  
Holm Holmsen
Keyword(s):  
Receptor Occupancy ◽  
Dense Granule ◽  
Secretory Process ◽  
Thrombin Receptor ◽  
Cell Contact ◽  
Acid Hydrolase ◽  
High Concentration ◽  
Dense Granule Secretion ◽  
Granule Secretion ◽  
Platelet Secretion

We have previously showed that hirudin abruptly arrests thrombin-induced secretion of acid hydrolase at any stage of its progress, whereas it only affects dense granule secretion only at its initial stages; these results have been interpreted to show that acid hydrolase secretion requires sustained while dense granule secreion ony requires a brief receptor occupancy (Holmsen et al. JBC 256(1981)9393). The requirement for receptor occupancy in thrombin-induced α-granule secretion and secretion during aggregation have been studied. Increasing concentrations of thrombin were added to gel-fitered platelets containing a constant, high concentration of hirudin. Dense granule secretion was initiated at lower thrombin concentration than those required for α-granule secretion and aggregation; acid hydrolase secretion required higher concentrations. A 14-fold exess of hirudin produced abrupt stop of dense granule secretion and α-granule secretion when added to platelets shortly after thrombin; it had no affect after these secretory process had reached 30% of their maximal values. Acid hydrolase secretion was, however, abruptly stopped by hirudin at any stage. When the platelets were allowed to aggregate, all three secretory processes increased their rates and could now be abruptly stopped by hirudin at any stage. Aggregation (optical) occurred slower than dense granule andoαgranule secretion, and was reversed by hirudin when added before it had reached 30% of its maximum. It is concluded thatαgranule secretion, like dense granule secretion, only requires a short receptor occupancy to be completed, in contrast to the requirement for sustained occupancy for hydrolase secretion.α-granule secretion might, however, require longer occupancy than dense granule secretion. It is possible that aggregation potentiates all secretory responses through close cell contact and that the abrupt inhibition by hirudin of all secretions may have been caused by its effect on the slower aggregation.

scholarly journals A dual role for integrin-linked kinase in platelets: regulating integrin function and α-granule secretion

Blood ◽  
2008 ◽  
Vol 112 (12) ◽  
pp. 4523-4531 ◽  
Author(s):  
Katherine L. Tucker ◽  
Tanya Sage ◽  
Joanne M. Stevens ◽  
Peter A. Jordan ◽  
Sarah Jones ◽  
...  
Keyword(s):  
Thrombus Formation ◽  
Dense Granule ◽  
Conditional Knockout ◽  
Knockout Mouse Model ◽  
Conditional Knockout Mouse ◽  
Fibrinogen Binding ◽  
Integrin Linked Kinase ◽  
Dense Granule Secretion ◽  
Granule Secretion

Abstract Integrin-linked kinase (ILK) has been implicated in the regulation of a range of fundamental biological processes such as cell survival, growth, differentiation, and adhesion. In platelets ILK associates with β1- and β3-containing integrins, which are of paramount importance for the function of platelets. Upon stimulation of platelets this association with the integrins is increased and ILK kinase activity is up-regulated, suggesting that ILK may be important for the coordination of platelet responses. In this study a conditional knockout mouse model was developed to examine the role of ILK in platelets. The ILK-deficient mice showed an increased bleeding time and volume, and despite normal ultrastructure the function of ILK-deficient platelets was decreased significantly. This included reduced aggregation, fibrinogen binding, and thrombus formation under arterial flow conditions. Furthermore, although early collagen stimulated signaling such as PLCγ2 phosphorylation and calcium mobilization were unaffected in ILK-deficient platelets, a selective defect in α-granule, but not dense-granule, secretion was observed. These results indicate that as well as involvement in the control of integrin affinity, ILK is required for α-granule secretion and therefore may play a central role in the regulation of platelet function.

The actin cytoskeleton differentially regulates platelet α-granule and dense-granule secretion

Blood ◽  
2005 ◽  
Vol 105 (10) ◽  
pp. 3879-3887 ◽  
Author(s):  
Robert Flaumenhaft ◽  
James R. Dilks ◽  
Nataliya Rozenvayn ◽  
Rita A. Monahan-Earley ◽  
Dian Feng ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Membrane Fusion ◽  
Dense Granule ◽  
Protein Receptor ◽  
Low Concentrations ◽  
Latrunculin A ◽  
High Concentrations ◽  
Dense Granule Secretion ◽  
Granule Secretion ◽  
Platelet Exocytosis

AbstractStimulation of platelets with strong agonists results in centralization of cytoplasmic organelles and secretion of granules. These observations have led to the supposition that cytoskeletal contraction facilitates granule release by promoting the interaction of granules with one another and with membranes of the open canalicular system. Yet, the influence of the actin cytoskeleton in controlling the membrane fusion events that mediate granule secretion remains largely unknown. To evaluate the role of the actin cytoskeleton in platelet granule secretion, we have assessed the effects of latrunculin A and cytochalasin E on granule secretion. Exposure of platelets to low concentrations of these reagents resulted in acceleration and augmentation of agonist-induced α-granule secretion with comparatively modest effects on dense granule secretion. In contrast, exposure of platelets to high concentrations of latrunculin A inhibited agonist-induced α-granule secretion but stimulated dense granule secretion. Incubation of permeabilized platelets with low concentrations of latrunculin A primed platelets for Ca2+- or guanosine triphosphate (GTP)-γ-S-induced α-granule secretion. Latrunculin A-dependent α-granule secretion was inhibited by antibodies directed at vesicle-associated membrane protein (VAMP), demonstrating that latrunculin A supports soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein-dependent membrane fusion. These results indicate that the actin cytoskeleton interferes with platelet exocytosis and differentially regulates α-granule and dense granule secretion.

Platelet 12-Lipoxygenase Is Required for Dense Granule Secretion and Platelet Aggregation: Role of 12-hLO In Platelet Hemostasis and Thrombosis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3203-3203
Author(s):  
Patrick Apopa ◽  
Megha Patel ◽  
Olivier Boutaud ◽  
Michael Holinstat
Keyword(s):  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Platelet Activation ◽  
Platelet Reactivity ◽  
Dense Granule ◽  
Clot Formation ◽  
Dense Granule Secretion ◽  
Granule Secretion ◽  
12 Lipoxygenase

Abstract Abstract 3203 Platelet activation plays a central role in regulating hemostasis. Uncontrolled activation of circulating platelets can result in the formation of occlusive thrombi and stroke. Following activation, metabolism of arachidonic acid by 12-lipoxygenase (12-hLO) may play a significant role in regulating the degree and stability of platelet reactivity. Using specific inhibitors for 12-hLO which do not interact with other lipoxygenases or enzymes in the COX-1 pathway, we were able for the first time to asses the involvement of 12-hLO in platelet reactivity. In order to assess the role of 12-hLO in platelet activation and thrombosis, dense granule secretion, platelet aggregation, alpha granule secretion, and platelet adhesion and clot formation under flow were measured. Inhibiting 12-hLO results in a complete inhibition of dense granule secretion with only a partial attenuation of alpha granule secretion indicating a novel regulatory scheme for modulating positive autocrine reinforcement of platelet reactivity and clot formation. Addition of the 12-hLO metabolite, 12-HETE (as low as 250 nM), resulted in a significant (25%) increase in PAR1-mediated dense granule secretion compare to agonist alone indicating that 12-HETE may be the crucial metabolite formed by 12-hLO metabolism of arachidonic acid. Importantly, platelet aggregation and adhesion are also significantly attenuated in the absence of 12-hLO. In fact, collagen-mediated platelet aggregation was shifted over 25 fold to the right in the absence of 12-hLO. These studies support the role of 12-hLO in hemostasis and may be a good target for anti-platelet therapy. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Peroxynitrite can affect platelet responses by inhibiting energy production.

2006 ◽  
Vol 53 (4) ◽  
pp. 769-776 ◽  
Author(s):  
Tomasz Rusak ◽  
Marian Tomasiak ◽  
Michal Ciborowski
Keyword(s):  
Oxygen Consumption ◽  
Energy Production ◽  
Dense Granule ◽  
Resting Cells ◽  
Antimycin A ◽  
Dependent Manner ◽  
Atp Content ◽  
Dense Granule Secretion ◽  
Granule Secretion ◽  
Induced Aggregation

Peroxynitrite (ONOO-) strongly inhibits agonist-induced platelet responses. However, the mechanisms involved are not completely defined. Using porcine platelets, we tested the hypothesis that ONOO- reduces platelet aggregation and dense granule secretion by inhibiting energy production. It was found that ONOO- (25-300 microM) inhibited collagen-induced dense granule secretion (IC50 = 55 +/- 7 microM) more strongly than aggregation (IC(50) = 124 +/- 16 microM). The antiaggregatory and antisecretory effects of ONOO- were only slightly (5-10%) reduced by 1H-[1,2,4]-oxadiazolo-[4,3-alpha]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase. In resting platelets ONOO- (50-300 microM) enhanced glycolysis rate and reduced oxygen consumption, in a dose dependent manner. The ONOO- effects on glycolysis rate and oxygen consumption were not abolished by ODQ. The extent of glycolysis stimulation exerted by ONOO- was similar to that produced by respiratory chain inhibitors (cyanide and antimycin A) or an uncoupler (2,4-dinitrophenol). Stimulation of platelets by collagen was associated with a rise in mitochondrial oxygen consumption, accelerated lactate production, and unchanged intracellular ATP content. In contrast to resting cells, in collagen-stimulated platelets, ONOO- (200 microM) distinctly decreased the cellular ATP content. The glycolytic activity and oxygen consumption of resting platelets were not affected by 8-bromoguanosine 3',5'-cyclic monophosphate. Blocking of the mitochondrial ATP production by antimycin A slightly reduced collagen-induced aggregation and strongly inhibited dense granule secretion. Treatment of platelets with ONOO- (50-300 microM) resulted in decreased activities of NADH : ubiquinone oxidoreductase, succinate dehydrogenase and cytochrome oxidase. It is concluded that the inhibitory effect of ONOO- on platelet secretion and to a lesser extent on aggregation may be mediated, at least in part, by the reduction of mitochondrial energy production.

scholarly journals The Predominant Role of Arrestin3 in General GPCR Desensitization in Platelets

2021 ◽  
Vol 10 (20) ◽  
pp. 4743
Author(s):  
Preeti Kumari Chaudhary ◽  
Sanggu Kim ◽  
Soochong Kim
Keyword(s):  
Platelet Aggregation ◽  
Thrombus Formation ◽  
Dense Granule ◽  
Functional Responses ◽  
Erk Phosphorylation ◽  
Dense Granule Secretion ◽  
Granule Secretion ◽  
Gpcr Desensitization

Arrestins in concert with GPCR kinases (GRKs) function in G protein-coupled receptor (GPCR) desensitization in various cells. Therefore, we characterized the functional differences of arrestin3 versus arrestin2 in the regulation of GPCR signaling and its desensitization in platelets using mice lacking arrestin3 and arrestin2. In contrast to arrestin2, platelet aggregation and dense granule secretion induced by 2-MeSADP, U46619, thrombin, and AYPGKF were significantly potentiated in arrestin3-deficient platelets compared to wild-type (WT) platelets, while non-GPCR agonist CRP-induced platelet aggregation and secretion were not affected. Surprisingly, in contrast to GRK6, platelet aggregation induced by the co-stimulation of serotonin and epinephrine was significantly potentiated in arrestin3-deficient platelets, suggesting the central role of arrestin3 in general GPCR desensitization in platelets. In addition, the second challenge of ADP and AYPGKF restored platelet aggregation in arrestin3-deficient platelets but failed to do so in WT and arrestin2-deficient platelets, confirming that arrestin3 contributes to GPCR desensitization. Furthermore, ADP- and AYPGKF-induced Akt and ERK phosphorylation were significantly increased in arrestin3-deficient platelets. Finally, we found that arrestin3 is critical for thrombus formation in vivo. In conclusion, arrestin3, not arrestin2, plays a central role in the regulation of platelet functional responses and thrombus formation through general GPCR desensitization in platelets.

scholarly journals Cyclin Y Is Expressed in Platelets and Modulates Integrin Outside-in Signaling

2020 ◽  
Vol 21 (21) ◽  
pp. 8239
Author(s):  
Anastasia Kyselova ◽  
Mauro Siragusa ◽  
Julian Anthes ◽  
Fiorella Andrea Solari ◽  
Stefan Loroch ◽  
...  
Keyword(s):  
Dense Granule ◽  
Diabetic Patients ◽  
Clot Retraction ◽  
Healthy Donors ◽  
Β3 Integrin ◽  
Dense Granule Secretion ◽  
Granule Secretion ◽  
Thromboxane A2 Analogue ◽  
Increased Activity

Diabetes is associated with platelet hyper-reactivity and enhanced risk of thrombosis development. Here we compared protein expression in platelets from healthy donors and diabetic patients to identify differentially expressed proteins and their possible function in platelet activation. Mass spectrometry analyses identified cyclin Y (CCNY) in platelets and its reduced expression in platelets from diabetic patients, a phenomenon that could be attributed to the increased activity of calpains. To determine the role of CCNY in platelets, mice globally lacking the protein were studied. CCNY-/- mice demonstrated lower numbers of circulating platelets but platelet responsiveness to thrombin and a thromboxane A2 analogue were comparable with that of wild-type mice, as was agonist-induced α and dense granule secretion. CCNY-deficient platelets demonstrated enhanced adhesion to fibronectin and collagen as well as an attenuated spreading and clot retraction, indicating an alteration in “outside in” integrin signalling. This phenotype was accompanied by a significant reduction in the agonist-induced tyrosine phosphorylation of β3 integrin. Taken together we have shown that CCNY is present in anucleated platelets where it is involved in the regulation of integrin-mediated outside in signalling associated with thrombin stimulation.

scholarly journals Differential Role of Protein Kinase Cδ Isoform in Agonist-induced Dense Granule Secretion in Human Platelets

2003 ◽  
Vol 279 (4) ◽  
pp. 2360-2367 ◽  
Author(s):  
Swaminathan Murugappan ◽  
Florin Tuluc ◽  
Robert T. Dorsam ◽  
Haripriya Shankar ◽  
Satya P. Kunapuli
Keyword(s):  
Protein Kinase ◽  
Human Platelets ◽  
Dense Granule ◽  
Dense Granule Secretion ◽  
Granule Secretion ◽  
Protein Kinase Cδ
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