Platelet Aggregation By Membrane Glycoprotein I

1981 ◽  
Author(s):  
K Watanabe ◽  
M Yamamoto ◽  
Y Ando ◽  
H Iri ◽  
K Furihata ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Rich Plasma ◽  
Lag Phase ◽  
Affinity Column ◽  
Membrane Glycoprotein ◽  
Dependent Manner ◽  
Membrane Glycoproteins ◽  
Aggregation Mechanism ◽  
Membrane Components ◽  
Aggregation Of Platelets

It has been recently shown that platelet membrane components, particularly glycoproteins, have a lectin activity, thus mediating an aggregation of platelets. To obtain further evidences for a crucial role of glycoproteins in an aggregation mechanism,we have investigated the possibility that membrane glycoprotein can directly induce an aggregation of platelets. The membrane glycoproteins ( GP I, GP II and GP III ) were isolated from 3-4 mg of human platelet membranes using preparative electrophoresis on 5 % polyacrylamide gels with 0.1% SDS. Platelet aggregation by isolated GP I, GP II or GP III was examined under phase_contrast microscopy after the incubation of these peptides with platelet rich plasma at 37°C for 15 min.. Among glycoproteins tested, only GP I( 20 μg/ml < ) exerted an apparent platelet aggregation. No such aggregation was induced by either GP II or GP III even at concentration of 80 μg/ml. GP I isolated separately using the wheat germ agglutinin affinity column also produced a platelet aggregation. Aggregation curve recorded with an aggregometer showed a long lag phase ( 10 min. < ) followed by an irreversible aggregation. The GP I-induced platelet aggregation occured in a dose dependent manner. This aggregation was completely inhibited by the addition of aggregating inhibitors such as indomethacin ( 25 μM ), PGE1 ( 1 μM ), EDTA ( 0.5 mM ) and TMB-8 ( lmg/ml ). A significant amount of serotonin ( 27% ) and β-thromboglobulin ( 14.6% ) was released from platelets by GP I ( 100 μg/ml ). Treatment of GP I with either trypsin ( 50 μg/ml ) or chymotrypsin ( 40 μg/ml ) reduced the aggregating activity of this glycopeptides. The platelet aggregation by GP I was inhibited in the presense of 30 mM N-acetylneuraminic acid, arginin or L-lysine, but N-acetyl- ated amino sugars and neutral sugars were without effect. This GP I-induced platelet aggregation may be an important findings in elucidating platelet aggregation mechanism.

scholarly journals Both Complement- and Fibrinogen-Dependent Mechanisms Contribute to Platelet Aggregation Mediated by Staphylococcus aureus Clumping Factor B

2007 ◽  
Vol 75 (7) ◽  
pp. 3335-3343 ◽  
Author(s):  
Helen Miajlovic ◽  
Anthony Loughman ◽  
Marian Brennan ◽  
Dermot Cox ◽  
Timothy J. Foster
Keyword(s):  
Staphylococcus Aureus ◽  
Platelet Aggregation ◽  
Platelet Rich Plasma ◽  
Lag Time ◽  
Dependent Manner ◽  
Wild Type ◽  
Factor B ◽  
Fibrinogen Binding ◽  
Aggregation Of Platelets ◽  
Dependent Mechanism

ABSTRACT Staphylococcus aureus can stimulate activation and aggregation of platelets, which are thought to be factors in the development of infective endocarditis. Previous studies have identified clumping factor A (ClfA) and fibronectin binding proteins A and B (FnBPA and FnBPB) as potent platelet aggregators. These proteins are able to stimulate rapid platelet aggregation by either a fibrinogen- or a fibronectin-dependent process which also requires antibodies specific to each protein. Slower aggregation has been seen in other systems where specific fibrinogen binding ligands are absent and platelet aggregation is mediated by complement and specific antibodies. Bacteria expressing ClfB aggregate platelets with a longer lag time than ClfA or FnBPA and FnBPB. In order to investigate whether ClfB causes platelet aggregation in a complement- or fibrinogen-dependent manner, a non-fibrinogen-binding mutant of ClfB (ClfB Q235A) was constructed. Lactococcus lactis expressing ClfB Q235A was able to stimulate platelet aggregation in platelet-rich plasma without a significant increase in lag time. The requirements for platelet aggregation were investigated using gel-filtered platelets. Fibrinogen and specific anti-ClfB antibodies were found to be sufficient to allow platelet aggregation mediated by the wild-type ClfB protein. It seems that ClfB causes platelet aggregation by a fibrinogen-dependent mechanism. The non-fibrinogen-binding ClfB mutant was unable to stimulate platelet aggregation under these conditions. However, bacteria expressing ClfB Q235A caused platelet aggregation in a complement-dependent manner which required specific anti-ClfB antibodies.

scholarly journals Shear-induced platelet aggregation requires von Willebrand factor and platelet membrane glycoproteins Ib and IIb-IIIa

Blood ◽  
1987 ◽  
Vol 69 (2) ◽  
pp. 625-628 ◽  
Author(s):  
DM Peterson ◽  
NA Stathopoulos ◽  
TD Giorgio ◽  
JD Hellums ◽  
JL Moake
Keyword(s):  
Shear Stress ◽  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Platelet Rich Plasma ◽  
Shear Stresses ◽  
Membrane Glycoprotein ◽  
Membrane Glycoproteins ◽  
Von Willebrand ◽  
Induced Aggregation ◽  
Willebrand Factor

Different types of platelets in various types of plasma were subjected to levels of shear stress that produce irreversible platelet aggregation in normal platelet-rich plasma (PRP). At shear stresses of 90 or 180 dyne/cm2 applied for 30 seconds or five minutes, aggregation was either absent or only transient and reversible using severe von Willebrand's disease (vWD) PRP (less than 1% von Willebrand factor, vWF); Bernard-Soulier syndrome (BSS) PRP (platelets deficient in the membrane glycoprotein Ib, GPIb); normal PRP plus monoclonal antibody (MoAb) to GPIb; thrombasthenic PRP (platelets deficient in membrane glycoprotein IIb-IIIa complex, GPIIb-IIIa); and normal PRP plus MoAb to GPIIb-IIIa. Shear-induced aggregation was inhibited under the above conditions, even though the platelets were activated to release their granular contents. Sheared normal platelets in vWD plasma aggregated in response to added vWF. These studies demonstrate that the formation of stable platelet aggregates under conditions of high shear requires vWF and the availability of both GPIb and GPIIb-IIIa on platelet membranes. The experiments demonstrate that vWF-platelet interactions can occur in the absence of artificial agonists or chemical modification of vWF. They suggest a possible mechanism for platelet aggregation in stenosed or partially obstructed arterial vessels in which the platelets are subjected to relatively high levels of shear stress.

scholarly journals Shear-induced platelet aggregation requires von Willebrand factor and platelet membrane glycoproteins Ib and IIb-IIIa

Blood ◽  
1987 ◽  
Vol 69 (2) ◽  
pp. 625-628 ◽  
Author(s):  
DM Peterson ◽  
NA Stathopoulos ◽  
TD Giorgio ◽  
JD Hellums ◽  
JL Moake
Keyword(s):  
Shear Stress ◽  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Platelet Rich Plasma ◽  
Shear Stresses ◽  
Membrane Glycoprotein ◽  
Membrane Glycoproteins ◽  
Von Willebrand ◽  
Induced Aggregation ◽  
Willebrand Factor

Abstract Different types of platelets in various types of plasma were subjected to levels of shear stress that produce irreversible platelet aggregation in normal platelet-rich plasma (PRP). At shear stresses of 90 or 180 dyne/cm2 applied for 30 seconds or five minutes, aggregation was either absent or only transient and reversible using severe von Willebrand's disease (vWD) PRP (less than 1% von Willebrand factor, vWF); Bernard-Soulier syndrome (BSS) PRP (platelets deficient in the membrane glycoprotein Ib, GPIb); normal PRP plus monoclonal antibody (MoAb) to GPIb; thrombasthenic PRP (platelets deficient in membrane glycoprotein IIb-IIIa complex, GPIIb-IIIa); and normal PRP plus MoAb to GPIIb-IIIa. Shear-induced aggregation was inhibited under the above conditions, even though the platelets were activated to release their granular contents. Sheared normal platelets in vWD plasma aggregated in response to added vWF. These studies demonstrate that the formation of stable platelet aggregates under conditions of high shear requires vWF and the availability of both GPIb and GPIIb-IIIa on platelet membranes. The experiments demonstrate that vWF-platelet interactions can occur in the absence of artificial agonists or chemical modification of vWF. They suggest a possible mechanism for platelet aggregation in stenosed or partially obstructed arterial vessels in which the platelets are subjected to relatively high levels of shear stress.

scholarly journals 3’5’-Adenosine Diphosphate (3′5′ADP):A Physiological Inhibitor of Platelet Aggregation and Platelet Release Reaction

1977 ◽  
Author(s):  
K. Subbarao ◽  
K. Jaya
Keyword(s):  
Platelet Aggregation ◽  
Coenzyme A ◽  
Human Platelet ◽  
Platelet Rich Plasma ◽  
Serotonin Release ◽  
Dependent Manner ◽  
Release Reaction ◽  
Inhibition Of Platelet Aggregation ◽  
Vitro Effect ◽  
Aggregation Of Platelets

Certain analogues of adenosine have been shown to inhibit ADP-induced platelet aggregation. We therefore studied the in vitro effect of 3′5′ADP and coenzyme A on human platelet aggregation and [14C]-serotonin release reaction induced by the addition of ADP, thrombin, collagen and epinephrine to human platelet rich plasma (PRP). It was found that coenzyme A Li3·2H2O at a concentration of 0.12 mM strongly inhibited ADP-induced platelet aggregation of PRP but did not show similar effect on the aggregation of platelets induced by other aggregating agents. The 3′5′ADP which is a part of coenzyme A structure, on the other hand, inhibited both ADP and thrombin induced platelet aggregation. The extent of inhibition of platelet aggregation by coenzyme A and 3′5′ADP was found to depend upon the concentration of the inhibitor and the incubation time. Whereas 3′5′ADP Li2·3H2O at a concentration of 10 μM produced about 70% inhibition of ADP-induced platelet aggregation of human PRP, total inhibition of thrombin induced platelet aggregation was observed when platelets were incubated with 60 μM of 3′5′ADP. The 3′5′ADP also inhibited the [14C]-adeonsine uptake by platelets in a concentration dependent manner. The inhibitory potency of 3′5′ADP on platelet aggregation was found to be 10-fold higher than that of N6-2′-0-dibutyryl-cyclic 3′5′-adenosine monophosphate. The inhibition of platelet aggregation by coenzyme A and 3′5′ADP was always accompanied by the inhibition of [14C]-serotonin release reaction. If coenzyme A and 3′5′ADP are indeed physiological inhibitors of platelet aggregation, then aggregation of platelets should depend on metabolic events that regulate the concentration of these agents in blood.

Effect of SR121566A, a Potent GP IIb-IIIa Antagonist on Platelet-mediated Thrombin Generation In Vitro and In Vivo

1998 ◽  
Vol 79 (02) ◽  
pp. 383-388 ◽  
Author(s):  
J. P. Herault ◽  
V. Peyrou ◽  
P. Savi ◽  
A. Bernat ◽  
J. M. Herbert
Keyword(s):  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Venous Thrombosis ◽  
Thrombin Generation ◽  
Platelet Rich Plasma ◽  
Venous Stasis ◽  
Lag Phase ◽  
Dependent Manner

SummaryThe effect of SR121566A, a new non-peptide GP IIb-IIIa antagonist was studied in vitro with regard to thrombin generation in platelet rich plasma and in vivo on stasis-induced venous thrombosis in the rabbit. SR121566A inhibited ADP-, arachidonic acid- and collagen-induced human platelet aggregation with IC50 values of 46 ± 7.5, 56 ± 6 and 42 ± 3 nM, respectively. In the same experimental conditions, SR121566A strongly inhibited thrombin generation triggered by low concentrations of tissue factor. SR121566A reduced in a dose-dependent manner both the area under the curve and the thrombin peak concentration but did not affect the lag phase (defined as the time until 10 nM thrombin was generated). Aspirin (100 µg/ml) did not affect thrombin generation.One hour after intravenous administration to rabbits, SR121566A exhibited a potent ex vivo inhibitory effect against ADP-, arachidonic acid- and collagen-induced platelet aggregation. The ID50 were 0.6 ± 0.25, 0.7 ± 0.08 and 0.13 ± 0.08 mg/kg, respectively. The ability of aspirin and SR121566A to affect venous stasis was determined in a stasis-induced venous thrombosis model in rabbits under high and low thrombogenic challenges. While aspirin was ineffective in both conditions, SR121566A significantly inhibited thrombus formation under low thrombogenic challenge demonstrating for the first time that a potent non-peptide platelet GP IIb-IIIa antagonist inhibits thrombin generation in vivo and exhibits a strong antithrombotic effect with regard to stasis-induced venous thrombosis. These results therefore confirm the existence of a close relationship between platelet activation and thrombin generation leading to blood coagulation but also emphasise the key role of platelets in the development of venous thrombosis, most likely through activation of the GP IIb-IIIa complex.

scholarly journals Effect of prostaglandin E1 alone and in combination with theophylline or aspirin on collagen-induced platelet aggregation and on platelet nucleotides including adenosine 3′:5′-cyclic monophosphate

1970 ◽  
Vol 120 (4) ◽  
pp. 709-718 ◽  
Author(s):  
G. Ball ◽  
G. G. Brereton ◽  
Mary Fulwood ◽  
D. M. Ireland ◽  
Patricia Yates
Keyword(s):  
Platelet Aggregation ◽  
Cyclic Amp ◽  
Prostaglandin E1 ◽  
Platelet Rich Plasma ◽  
Dependent Manner ◽  
Intracellular Changes ◽  
Induced Aggregation ◽  
Dose Dependent ◽  
Aggregation Of Platelets ◽  
Collagen Treatment

1. Human platelet nucleotides were labelled by incubating platelet-rich plasma with [U-14C]adenine. With such platelets, the effects of prostaglandin E1, theophylline and aspirin were determined on collagen-induced platelet aggregation and release of platelet ATP and ADP. Intracellular changes of platelet radioactive nucleotides, particularly 3′:5′-cyclic AMP, were also determined both with and without collagen treatment. 2. Prostaglandin E1, theophylline and aspirin inhibited collagen-induced aggregation of platelets in a dose-dependent manner. Collagen-induced release of ATP and ADP and breakdown of radioactive ATP were also inhibited in a dose-dependent manner. 3. Prostaglandin E1 stimulated the formation of platelet radioactive 3′:5′-cyclic AMP in a dose-dependent manner. With a given dose of prostaglandin E1, maximum formation of radioactive 3′:5′-cyclic AMP occurred by 10–30s and thereafter the concentrations declined. The degree of inhibition of aggregation produced by prostaglandin E1, however, increased with its time of incubation in platelet-rich plasma before addition of collagen, so that there was an inverse relationship between the radioactive 3′:5′-cyclic AMP concentration measured at the time of collagen addition and the subsequent degree of inhibition of aggregation obtained. 4. Neither theophylline nor aspirin at a concentration in platelet-rich plasma of 1.7mm altered platelet radioactive 3′:5′-cyclic AMP contents. In the presence of prostaglandin E1, theophylline increased the concentration of radioactive 3′:5′-cyclic AMP over that noted with prostaglandin E1 alone, but aspirin did not. 5. Mixtures of prostaglandin E1 and theophylline had a synergistic effect on inhibition of platelet aggregation. The same was true to a lesser extent with mixtures of prostaglandin E1 and aspirin. Such mixtures also inhibited collagen-induced release of platelet ATP and ADP and breakdown of platelet radioactive ATP. 6. Certain concentrations of either theophylline or aspirin and mixtures of small concentrations of prostaglandin E1 with either theophylline or aspirin caused little or no increase of radioactive 3′:5′-cyclic AMP at the time of collagen addition, but inhibited aggregation to a marked degree, whereas higher concentrations of prostaglandin E1 alone caused a much greater increase of radioactive 3′:5′-cyclic AMP at the time of collagen addition but inhibited aggregation to a lesser extent. With these compounds there does not appear to be a correlation between these parameters.

Reaction of Acetaldehyde with Human Platelets

1992 ◽  
Vol 67 (01) ◽  
pp. 126-130 ◽  
Author(s):  
Olivier Spertini ◽  
Jacques Hauert ◽  
Fedor Bachmann
Keyword(s):  
Platelet Aggregation ◽  
Inhibitory Action ◽  
Ex Vivo ◽  
Platelet Rich Plasma ◽  
Shape Change ◽  
Reaction Medium ◽  
Human Platelets ◽  
Membrane Glycoproteins ◽  
Neutral Ph

SummaryPlatelet function defects observed in chronic alcoholics are not wholly explained by the inhibitory action of ethanol on platelet aggregation; they are not completely reproduced either in vivo by short-term ethanol perfusion into volunteers or in vitro by the addition of ethanol to platelet-rich plasma. As acetaldehyde (AcH) binds to many proteins and impairs cellular activities, we investigated the effect of this early degradation product of ethanol on platelets. AcH formed adducts with human platelets at neutral pH at 37° C which were stable to extensive washing, trichloracetic acid hydrolysis and heating at 100° C, and were not reduced by sodium borohydride. The amount of platelet adducts formed was a function of the incubation time and of the concentration of AcH in the reaction medium. At low AcH concentrations (<0.2 mM), platelet bound AcH was directly proportional to the concentration of AcH in the reaction medium. At higher concentrations (≥0.2 mM), AcH uptake by platelets tended to reach a plateau. The amount of adducts was also proportional to the number of exposures of platelets to pulses of 20 pM AcH.AcH adducts formation severely impaired platelet aggregation and shape change induced by ADP, collagen and thrombin. A positive correlation was established between platelet-bound AcH and inhibition of aggregation.SDS-PAGE analysis of AcH adducts at neutral pH demonstrated the binding of [14C]acetaldehyde to many platelet proteins. AcH adduct formation with membrane glycoproteins, cytoskeleton and enzymes might interfere with several steps of platelet activation and impair platelet aggregation.This in vitro study shows that AcH has a major inhibitory action on platelet aggregation and may account for the prolonged ex vivo inhibition of aggregation observed in chronic alcoholics even in the absence of alcoholemia.

Acquired Disorder of Platelet Function Associated with Autoantibodies against Membrane Glycoprotein IIb-IIIa Complex - 1. Glycoprotein Analysis

1991 ◽  
Vol 65 (05) ◽  
pp. 491-496 ◽  
Author(s):  
M Meyer ◽  
C M Kirchmaier ◽  
A Schirmer ◽  
P Spangenberg ◽  
Ch Ströhl ◽  
...  
Keyword(s):  
Platelet Count ◽  
Platelet Aggregation ◽  
Platelet Adhesion ◽  
Bleeding Time ◽  
Membrane Glycoprotein ◽  
Membrane Glycoproteins ◽  
Abnormal Behaviour ◽  
Thrombocytopenic Purpura ◽  
Immunoelectrophoretic Analysis ◽  
Prolonged Bleeding Time

SummaryA patient with idiopathic thrombocytopenic purpura developed after splenectomy a thrombasthenia-like severe haemor-rhagic diathesis characterized by a normal or subnormal platelet count, prolonged bleeding time, strongly reduced platelet adhesion to glass and defective platelet aggregation in response to ADP and collagen. In contrast to hereditary thrombasthenia membrane glycoproteins (GP) lib and Ilia were normally present in the patient’s platelets. Immunoelectrophoretic analysis revealed an abnormal behaviour of the patient’s GP IIb-IIIa complex. Autoantibodies against GP IIb-IIIa were detected in Triton-extracted washed platelets. Incubation of normal platelets with plasma from the patient resulted in a similar immunoelectrophoretic abnormality of the GP IIb-IIIa complex indicating that bound autoantibodies (IgG) are responsible for the abnormal immunoelectrophoretic behaviour of the patient’s GP IIb-IIIa complex. Platelet fibrinogen was severely reduced similar to classical thrombasthenia suggesting that the GP IIb-IIIa complex is involved in platelet fibrinogen storage.

Streptococcus sanguis-induced platelet activation involves two waves of tyrosine phosphorylation mediated by FcγRIIA and αIIbβ3

2005 ◽  
Vol 93 (05) ◽  
pp. 932-939 ◽  
Author(s):  
Caroline Pampolina ◽  
Archibald McNicol
Keyword(s):  
Signal Transduction ◽  
Platelet Aggregation ◽  
Tyrosine Phosphorylation ◽  
Platelet Activation ◽  
Tyrosine Phosphatase ◽  
Protein Tyrosine Phosphatases ◽  
Lag Phase ◽  
Dependent Manner ◽  
Protein Tyrosine ◽  
Streptococcus Sanguis

SummaryThe low-affinity IgG receptor, FcγRIIA, has been implicated in Streptococcus sanguis-induced platelet aggregation. Therefore, it is likely that signal transduction is at least partly mediated by FcγRIIA activation and a tyrosine kinase-dependent pathway. In this study the signal transduction mechanisms associated with platelet activation in response to the oral bacterium, S. sanguis were characterised. In the presence of IgG, S. sanguis strain 2017–78 caused the tyrosine phosphorylation of FcγRIIA 30s following stimulation, which led to the phosphorylation of Syk, LAT, and PLCγ2. These early events were dependent on Src family kinases but independent of either TxA2 or the engagement of the αIIbβ3 integrin. During the lag phase prior to platelet aggregation, FcγRIIA, Syk, LAT, and PLCγ2 were each dephosphorylated, but were re-phosphorylated as aggregation occurred. Platelet stimulation by 2017–78 also induced the tyrosine phosphorylation of PECAM-1, an ITIM-containing receptor that recruits protein tyrosine phosphatases. PECAM-1 co-precipitated with the protein tyrosine phosphatase SHP-1 in the lag phase. SHP-1 was also maximally tyrosine phosphorylated during this phase, suggesting a possible role for SHP-1 in the observed dephosphorylation events. As aggregation occurred, SHP-1 was dephosphorylated, while FcγRIIA, Syk, LAT, and PLCγ2 were rephosphorylated in an RGDS-sensitive, and therefore αIIbβ3-dependent, manner. Additionally, TxA2 release, 5-hydro-xytryptamine secretion and phosphatidic acid formation were all blocked by RGDS. Aspirin also abolished these events, but only partially inhibited αIIbβ3-mediated re-phosphorylation. Therefore, S.sanguis-bound IgG cross links FcγRIIA and initiates a signaling pathway that is down-regulated by PECAM-1-bound SHP-1. Subsequent engagement of αIIbβ3 leads to SHP-1 dephosphorylation permiting a second wave of signaling leading to TxA2 release and consequent platelet aggregation.

FUNDC2 regulates platelet activation through AKT/GSK-3β/cGMP axis

2018 ◽  
Vol 115 (11) ◽  
pp. 1672-1679 ◽  
Author(s):  
Qi Ma ◽  
Weilin Zhang ◽  
Chongzhuo Zhu ◽  
Junling Liu ◽  
Quan Chen
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Platelet Rich Plasma ◽  
Thrombus Formation ◽  
Mitochondrial Protein ◽  
Dependent Manner ◽  
Clot Retraction ◽  
Akt Phosphorylation ◽  
Gsk 3Β

Abstract Aims AKT kinase is vital for regulating signal transduction in platelet aggregation. We previously found that mitochondrial protein FUNDC2 mediates phosphoinositide 3-kinase (PI3K)/phosphatidylinositol-3,4,5-trisphosphate (PIP3)-dependent AKT phosphorylation and regulates platelet apoptosis. The aim of this study was to evaluate the role of FUNDC2 in platelet activation and aggregation. Methods and results We demonstrated that FUNDC2 deficiency diminished platelet aggregation in response to a variety of agonists, including adenosine 5′-diphosphate (ADP), collagen, ristocetin/VWF, and thrombin. Consistently, in vivo assays of tail bleeding and thrombus formation showed that FUNDC2-knockout mice displayed deficiency in haemostasis and thrombosis. Mechanistically, FUNDC2 deficiency impairs the phosphorylation of AKT and downstream GSK-3β in a PI3K-dependent manner. Moreover, cGMP also plays an important role in FUNDC2/AKT-mediated platelet activation. This FUNDC2/AKT/GSK-3β/cGMP axis also regulates clot retraction of platelet-rich plasma. Conclusion FUNDC2 positively regulates platelet functions via AKT/GSK-3β/cGMP signalling pathways, which provides new insight for platelet-related diseases.

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