scholarly journals Hemostasis vs. homeostasis: Platelets are essential for preserving vascular barrier function in the absence of injury or inflammation

2020 ◽  
Vol 117 (39) ◽  
pp. 24316-24325 ◽  
Author(s):  
Shuchi Gupta ◽  
Christoph Konradt ◽  
Adam Corken ◽  
Jerry Ware ◽  
Bernhard Nieswandt ◽  
...  
Keyword(s):  
Barrier Function ◽  
Dense Granule ◽  
Severe Thrombocytopenia ◽  
Barrier Dysfunction ◽  
Vascular Integrity ◽  
Lectin Receptor ◽  
Dense Granules ◽  
Dense Granule Secretion ◽  
Granule Secretion ◽  
Time Required

Platelets are best known for their vasoprotective responses to injury and inflammation. Here, we have asked whether they also support vascular integrity when neither injury nor inflammation is present. Changes in vascular barrier function in dermal and meningeal vessels were measured in real time in mouse models using the differential extravasation of fluorescent tracers as a biomarker. Severe thrombocytopenia produced by two distinct methods caused increased extravasation of 40-kDa dextran from capillaries and postcapillary venules but had no effect on extravasation of 70-kDa dextran or albumin. This reduction in barrier function required more than 4 h to emerge after thrombocytopenia was established, reverting to normal as the platelet count recovered. Barrier dysfunction was also observed in mice that lacked platelet-dense granules, dense granule secretion machinery, glycoprotein (GP) VI, or the GPVI signaling effector phospholipase C (PLC) γ2. It did not occur in mice lacking α-granules, C type lectin receptor-2 (CLEC-2), or protease activated receptor 4 (PAR4). Notably, although both meningeal and dermal vessels were affected, intracerebral vessels, which are known for their tighter junctions between endothelial cells, were not. Collectively, these observations 1) highlight a role for platelets in maintaining vascular homeostasis in the absence of injury or inflammation, 2) provide a sensitive biomarker for detecting changes in platelet-dependent barrier function, 3) identify which platelet processes are required, and 4) suggest that the absence of competent platelets causes changes in the vessel wall itself, accounting for the time required for dysfunction to emerge.

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.

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

2011 ◽  
Vol 436 (2) ◽  
pp. 469-480 ◽  
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.

Assessment Of Energy Costs Of Secretory Responses In Platelets By Abrupt Arrest Of ATP Regeneration

1981 ◽  
Author(s):  
J W N Akkerman ◽  
G Gorter ◽  
H Holmsen
Keyword(s):  
Energy Consumption ◽  
Dense Granule ◽  
Metabolic Energy ◽  
Metabolic Inhibitors ◽  
Energy Costs ◽  
Dense Granules ◽  
Metabolic Conditions ◽  
Energy Consuming ◽  
Dense Granule Secretion ◽  
Granule Secretion

A new method has been developed for the quantitative assessment of energy consuming processes in platelets. Under carefully controlled metabolic conditions ATP resynthesis is abruptly blocked by a cocktail of metabolic inhibitors. This leads to a fall in metabolic ATP, which is linear with time between 0 and 30 sec after addition of the inhibitors. Evidence is presented that this fall reflects the velocity by which the platelets consume metabolic energy prior to addition of the inhibitors. Resting platelets consume 4 μmol ATP equivalents/min/1011 cells at 37° and 0.5 μmol (same units) at 15°C. When thrombin (5 U/ml) is included in the inhibitor-mixture, aggregation and secretion of dense granules (3H-serotonin), α-granules (β-thromboglobulin) and lysosomal granules (N acetyl β glucosaminidase) follow despite the arrest in ATP resynthesis. The fall in metabolic ATP is now much steeper, reflecting an increase in energy consumption during these functions. Using changes in temperature as a means to affect secretion and energy metabolism, secretion velocity (measured between 0 and 10 sec after thrombin addition) can be compared with simultaneous energy consumption (measured between 0 and 30 sec after thrombin addition). At a consumption of 12 ymol ATP/min/1011 cells secretion velocity of dense-, α- and lysosomal granules is 100, 95 and 50% of uninhibited suspensions, respectively. At 6 μmol (same units) these percentages are 70, 35 and 25%.If thrombin is added after addition of the inhibitors thereby initiating secretion at lowered metabolic ATP levels, secretion is slower as metabolic ATP is lower. Again lysosomal granule secretion is more inhibited than α-granule secretioiy. which is slower than dense granule secretion. These data reflect an increasing need for metabolic energy in the order: dense-, α- and lysosomal granule secretion.

scholarly journals Analysis of microvascular thrombus mechanobiology with a novel particle-based model

2021 ◽  
Author(s):  
Anastasia A Masalceva ◽  
Valeriia N Kaneva ◽  
Mikhail A Panteleev ◽  
Fazoil Ataullahanov ◽  
Vitaly Volpert ◽  
...  
Keyword(s):  
Thrombus Formation ◽  
Dense Granule ◽  
Aggregate Formation ◽  
Mechanical Stabilization ◽  
Dense Granules ◽  
Virtual Particles ◽  
Platelet Accumulation ◽  
Dense Granule Secretion ◽  
Granule Secretion

Platelet accumulation at the site of vascular injury is regulated by soluble platelet agonists, which induce various types of platelet responses, including integrin activation and granule secretion. The interplay between local biochemical cues, mechanical interactions between platelets and macroscopic thrombus dynamics is poorly understood. Here we describe a novel computational model of microvascular thrombus formation for detailed analysis of thrombus mechanics. Adopting a previously developed two-dimensional particle-based model focused on the thrombus shell formation, we revise it to introduce platelet agonists. Blood flow is simulated via computational fluid dynamics approach. In order to model soluble platelet activators, we apply Langevin dynamics to a large number of non-dimensional virtual particles. Taking advantage of the available data on platelet dense granule secretion kinetics, we model platelet degranulation as a stochastic agonist-dependent process. The new model qualitatively reproduces enhanced thrombus formation due to granule secretion in line with in vivo findings and provides a mechanism for thrombin confinement at the early stages of aggregate formation. Our calculations also predict that release of dense granules results in additional mechanical stabilization of the inner layers of the thrombus. Distribution of the inter-platelet forces throughout the aggregate reveals multiple weak spots in the outer regions of thrombus, which are expected to result in mechanical disruptions at the later stages of thrombus formation.

Prostaglandin Endoperoxide – Thromboxane Synthesis and Dense Granule Secretion as Positive Feedback Loops in the Propagation of Platelet Responses during “The Basic Platelet Reaction”

1977 ◽  
Vol 38 (04) ◽  
pp. 1030-1041 ◽  
Author(s):  
Holm Holmsen
Keyword(s):  
Positive Feedback ◽  
Shape Change ◽  
Second Messenger ◽  
Dense Granule ◽  
Feedback Loops ◽  
Dense Granules ◽  
Platelet Reaction ◽  
Dense Granule Secretion ◽  
Granule Secretion ◽  
External Stimuli

SummaryPlatelets respond to a great variety of stimuli by a sequential display of shape change, aggregation, prostaglandin/thromboxane synthesis – dense granule secretion and α-granule secretion. It is suggested that these responses are independent of each other, and caused by an increase in the concentration of a second messenger, liberated to the cytoplasm through the interaction between an extracellular agonist and the platelet membrane. The extent of the propagation of responses is determined by the strength of the stimulus. Stimuli can be subdivided into 1) original, applied stimuli and 2) platelet-produced stimuli (substances secreted from dense granules, prostaglandins and thromboxanes); these stimuli may act synergistically. In this way the platelet has two apparently independent means of potentiating their response to external stimuli which act as two separate positive feedback loops.

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

1979 ◽  
Vol 182 (2) ◽  
pp. 413-419 ◽  
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.

scholarly journals Syntaxin 8 Regulates Platelet Dense Granule Secretion, Aggregation, and Thrombus Stability

2014 ◽  
Vol 290 (3) ◽  
pp. 1536-1545 ◽  
Author(s):  
Ewelina M. Golebiewska ◽  
Matthew T. Harper ◽  
Christopher M. Williams ◽  
Joshua S. Savage ◽  
Robert Goggs ◽  
...  
Keyword(s):  
Dense Granule ◽  
Dense Granule Secretion ◽  
Granule Secretion

scholarly journals Loss of the exocyst complex component EXOC3 promotes hemostasis and accelerates arterial thrombosis

2021 ◽  
Vol 5 (3) ◽  
pp. 674-686
Author(s):  
Tony G. Walsh ◽  
Yong Li ◽  
Christopher M. Williams ◽  
Elizabeth W. Aitken ◽  
Robert K. Andrews ◽  
...  
Keyword(s):  
Arterial Thrombosis ◽  
Surface Expression ◽  
Dense Granule ◽  
Conditional Knockout ◽  
Integrin Activation ◽  
Platelet Factor ◽  
Glycoprotein Vi ◽  
Exocyst Complex ◽  
Dense Granule Secretion ◽  
Granule Secretion

Abstract The exocyst is an octameric complex comprising 8 distinct protein subunits, exocyst complex components (EXOC) 1 to 8. It has an established role in tethering secretory vesicles to the plasma membrane, but its relevance to platelet granule secretion and function remains to be determined. Here, EXOC3 conditional knockout (KO) mice in the megakaryocyte/platelet lineage were generated to assess exocyst function in platelets. Significant defects in platelet aggregation, integrin activation, α-granule (P-selectin and platelet factor 4), dense granule, and lysosomal granule secretion were detected in EXOC3 KO platelets after treatment with a glycoprotein VI (GPVI)-selective agonist, collagen-related peptide (CRP). Except for P-selectin exposure, these defects were completely recovered by maximal CRP concentrations. GPVI surface levels were also significantly decreased by 14.5% in KO platelets, whereas defects in proximal GPVI signaling responses, Syk and LAT phosphorylation, and calcium mobilization were also detected, implying an indirect mechanism for these recoverable defects due to decreased surface GPVI. Paradoxically, dense granule secretion, integrin activation, and changes in surface expression of integrin αIIb (CD41) were significantly increased in KO platelets after protease-activated receptor 4 activation, but calcium responses were unaltered. Elevated integrin activation responses were completely suppressed with a P2Y12 receptor antagonist, suggesting enhanced dense granule secretion of adenosine 5′-diphosphate as a critical mediator of these responses. Finally, arterial thrombosis was significantly accelerated in KO mice, which also displayed improved hemostasis determined by reduced tail bleeding times. These findings reveal a regulatory role for the exocyst in controlling critical aspects of platelet function pertinent to thrombosis and hemostasis.

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.

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