scholarly journals Heme stimulates platelet mitochondrial oxidant production via the activation of toll-like receptor 4 signaling to mediate targeted granule secretion

2021 ◽  
Author(s):  
Gowtham K Annarapu ◽  
Deirdre Nolfi-Donegan ◽  
Michael Reynolds ◽  
Yinna Wang ◽  
Lauren Kohut ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Vascular Dysfunction ◽  
Human Platelets ◽  
Serine Kinase ◽  
Central Process ◽  
Free Heme ◽  
Oxidant Production ◽  
Granule Release

Hemolysis is a pathological component of many diseases and is associated with thrombosis and vascular dysfunction. Hemolytic products, including cell-free hemoglobin and free heme directly activate platelets. However, the effect of hemolysis on platelet degranulation, a central process in not only thrombosis, but also inflammatory and mitogenic signaling, remains less clear. Our group showed that hemoglobin-induced platelet activation involved the production of mitochondrial reactive oxygen species (mtROS). However, the molecular mechanism by which extracellular hemolysis induces platelet mtROS production, and whether the mtROS regulate platelet degranulation remains unknown. Here, we demonstrate using isolated human platelets that cell free heme is a more potent agonist for platelet activation than hemoglobin, and stimulates the release of a specific set of molecules from the α-granule of platelets, including the glycoprotein thrombospondin-1 (TSP-1). We uncover the mechanism of heme-mediated platelet mtROS production which is dependent on the activation of platelet TLR4 signaling and leads to the downstream phosphorylation of complex-V by the serine kinase Akt. Notably, inhibition of platelet TLR4 or Akt, or scavenging mtROS prevents heme-induced granule release in vitro. Further, heme-dependent granule release is significantly attenuated in vivo in mice lacking TLR4 or those treated with the mtROS scavenger MitoTEMPO. These data elucidate a novel mechanism of TLR4-mediated mitochondrial regulation, establish the mechanistic link between hemolysis and platelet degranulation, and begin to define the heme and mtROS-dependent platelet secretome. These data have implications for hemolysis-induced thrombo-inflammatory signaling and for the consideration of platelet mitochondria as a therapeutic target in hemolytic disorders.

The Bcl-2 Family Protein Inhibitor, ABT-263, Promotes Apoptotic-Like Response in Isolated Platelets.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1122-1122
Author(s):  
Chris Tse ◽  
Haichao Zhang ◽  
Jun Chen ◽  
Ryan M. Fryer ◽  
Richard Nelson ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Reticuloendothelial System ◽  
Human Platelets ◽  
Dense Granule ◽  
Protein Inhibitor ◽  
Cytochrome C Release ◽  
Family Protein ◽  
Granule Release

Abstract Platelets are relatively short-lived, anucleated cells derived from megakaryocytes that are essential for proper hemostasis. Although they lack a nucleus, platelets possess a highly organized structure with multiple cellular organelles including mitochondria and a number of specific granules. Upon recruitment to sites of endothelial injury, platelet activation stimulates their adhesion, aggregation and release of granule components leading to coagulation. Many of the cellular changes observed during platelet activation and platelet senescence resemble those observed during apoptosis in nucleated cells, including loss of mitochondrial membrane potential, caspase activation, phosphatidylserine (PS) externalization, cell shrinkage and microparticle formation (analogous to membrane blebbing). ABT-263 is an orally bioavailable Bcl-2 family protein inhibitor currently in clinical development. This compound is a potent antagonist of the anti-apoptotic proteins Bcl-xL, Bcl-2 and Bcl-w and induces apoptosis in cancer cells dependent on these proteins for survival. ABT-263 induces a unique thrombocytopenia in multiple species that is characterized by a rapid clearance of circulating platelets with recovery to normal platelet counts within several days after treatment cessation. To elucidate the mechanisms underlying this thrombocytopenia, a series of in vitro experiments were performed using freshly isolated platelets comparing ABT-263, its enantiomer (which has significantly lower activity against Bcl-2 family proteins) and known platelet activators. ABT-263, but not the enantiomer control, reduced the viability of dog and human platelets and activated key apoptotic processes, including cytochrome c release, caspase 3 activation, and PS externalization. ABT-263 did not induce aggregation of isolated platelets and did not induce surface expression of GPIIb/IIIa that is essential for platelet adhesion to fibrinogen during clot formation. While ABT-263 had no effect on markers of lysosomal- or alpha-granule release, increases in a marker of dense granule release were apparent at high concentrations. Taken together these data suggest that ABT-263 induces an apoptotic-like response in platelets that is distinct from platelet activation. Furthermore, the lack of effect of the enantiomer control compound suggests this effect is mediated through inhibition of antiapoptotic Bcl-2 family members. Loss of membrane asymmetry and PS externalization has been suggested to be the primary signal for the recognition and clearance of senescent platelets by phagocytes of the reticuloendothelial system. Induction of apoptosis in platelets by Bcl-2 family protein inhibitors such as ABT-263 may coopt this process inducing a premature platelet senescence resulting in a novel type of thrombocytopenia, in vivo.

scholarly journals IκB kinase 2 is not essential for platelet activation

2020 ◽  
Vol 4 (4) ◽  
pp. 638-643
Author(s):  
Manuel Salzmann ◽  
Sonja Bleichert ◽  
Bernhard Moser ◽  
Marion Mussbacher ◽  
Mildred Haase ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Active Site ◽  
Bleeding Time ◽  
Thrombus Formation ◽  
Human Platelets ◽  
Iκb Kinase ◽  
Specific Deletion

Abstract Platelets are small anucleate cells that release a plethora of molecules to ensure functional hemostasis. It has been reported that IκB kinase 2 (IKK2), the central enzyme of the inflammatory NF-κB pathway, is involved in platelet activation, because megakaryocyte/platelet-specific deletion of exons 6 and 7 of IKK2 resulted in platelet degranulation defects and prolonged bleeding. We aimed to investigate the role of IKK2 in platelet physiology in more detail, using a platelet-specific IKK2 knockout via excision of exon 3, which makes up the active site of the enzyme. We verified the deletion on genomic and transcriptional levels in megakaryocytes and were not able to detect any residual IKK2 protein; however, platelets from these mice did not show any functional impairment in vivo or in vitro. Bleeding time and thrombus formation were not affected in platelet-specific IKK2-knockout mice. Moreover, platelet aggregation, glycoprotein GPIIb/IIIa activation, and degranulation were unaltered. These observations were confirmed by pharmacological inhibition of IKK2 with TPCA-1 and BMS-345541, which did not affect activation of murine or human platelets over a wide concentration range. Altogether, our results imply that IKK2 is not essential for platelet function.

scholarly journals An Antithrombotic Strategy by Targeting Phospholipase D in Human Platelets

2018 ◽  
Vol 7 (11) ◽  
pp. 440 ◽  
Author(s):  
Wan Lu ◽  
Chi Chung ◽  
Ray Chen ◽  
Li Huang ◽  
Li Lien ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Phospholipase D ◽  
Thrombus Formation ◽  
Human Platelets ◽  
Platelet Activity ◽  
Clot Retraction ◽  
First Time

Phospholipase D (PLD) is involved in many biological processes. PLD1 plays a crucial role in regulating the platelet activity of mice; however, the role of PLD in the platelet activation of humans remains unclear. Therefore, we investigated whether PLD is involved in the platelet activation of humans. Our data revealed that inhibition of PLD1 or PLD2 using pharmacological inhibitors effectively inhibits platelet aggregation in humans. However, previous studies have showed that PLD1 or PLD2 deletion did not affect mouse platelet aggregation in vitro, whereas only PLD1 deletion inhibited thrombus formation in vivo. Intriguingly, our data also showed that the pharmacological inhibition of PLD1 or PLD2 does not affect mouse platelet aggregation in vitro, whereas the inhibition of only PLD1 delayed thrombus formation in vivo. These findings indicate that PLD may play differential roles in humans and mice. In humans, PLD inhibition attenuates platelet activation, adhesion, spreading, and clot retraction. For the first time, we demonstrated that PLD1 and PLD2 are essential for platelet activation in humans, and PLD plays different roles in platelet function in humans and mice. Our findings also indicate that targeting PLD may provide a safe and alternative therapeutic approach for preventing thromboembolic disorders.

scholarly journals Neutrophil Cathepsin G Enhances Thrombogenicity of Mildly Injured Arteries via ADP-Mediated Platelet Sensitization

2022 ◽  
Vol 23 (2) ◽  
pp. 744
Author(s):  
Abderrahim Nemmar ◽  
Marc F. Hoylaerts
Keyword(s):  
Platelet Activation ◽  
Lung Inflammation ◽  
Platelet Rich Plasma ◽  
Cardiovascular Complications ◽  
Human Platelets ◽  
Cathepsin G ◽  
Blood Concentrations ◽  
The Impact

Inhalation of particulate matter in polluted air causes direct, size-restricted passage in the circulation and pronounced lung inflammation, provoking platelet activation and (non)-fatal cardiovascular complications. To determine potency and mechanism of platelet sensitization via neutrophil enzymes, we performed in vitro aggregation studies in washed human platelets and in murine and human blood, in the presence of elastase, cathepsin G and regular platelet agonists, present in damaged arteries. The impact of both enzymes on in vivo thrombogenicity was studied in the same thrombosis mouse model, previously having demonstrated that neutrophil activation enhances peripheral thrombogenicity. At 0.05 U/mL, cathepsin G activated washed human platelets via PAR1, whereas at 0.35 U/mL, aggregation occurred via PAR4. In Swiss mouse platelet-rich plasma no aggregation occurred by cathepsin G at 0.4 U/mL. In human and murine blood, aggregations by 0.05–0.1 U/mL cathepsin G were similar and not PAR-mediated, but platelet aggregation was inhibited by ADP antagonists, advocating cathepsin G-released ADP in blood as the true agonist of sustained platelet activation. In the mouse thrombosis model, cathepsin G and elastase amplified mild thrombogenicity at blood concentrations that activated platelets in vitro. This study shows that cathepsin G and elastase secreted in the circulation during mild air pollution-induced lung inflammation lyse red blood cell membrane proteins, leading to ADP-leakage into plasma, sensitizing platelets and amplifying their contribution to cardiovascular complications of ambient particle inhalation.

EFFECTS OF SULFINPYRAZONE AND ITS METABOLITE G25671 ON PLATELET ACTIVATION AND DESENSITIZATION AND ON BRONCHOCONSTRICTION INDUCED BY THE PROSTAGLANDIN ENDOPEROXIDE ANALOGUE U46619

1987 ◽  
Author(s):  
M Hatmi ◽  
A Del Maschio ◽  
J Lefort ◽  
G De Gaetano ◽  
B B Varqaftiq ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Platelet Activation ◽  
Ex Vivo ◽  
Oral Treatment ◽  
Human Platelets ◽  
Cyclooxygenase Inhibitors ◽  
Before And After ◽  
A Site

In previous studies we have found (Br. 3. Pharmac. 85, 849, 1985) that a) human platelets pre-exposed to arachidonic acid or to the endoperoxide analogue, U46619 and then washed and resuspended, fail to respond to a second challenge by both arachidonic acid and U46619; b) desensitization by arachidonic acid and U46619 occurs at a site sensitive to endoperoxides / thromboxane (Tx) receptor antagonists; c) the desensitizing effects of U46619 are direct, whereas those of arachidonic acid are mediated by a cyclooxygenase-dependent metabolite. Sulfinpyrazone (100 μM) and its thioether metabolite G25671 (50 μM) are known to suppress arachidonic acid-induced platelet aggregation and TxB2 formation (Eur. 3. Pharmac, 101, 209, 1984). We now demonstrate that the presence of sulfinpyrazone or G25671 during platelet exposure to arachidonic acid or U46619 prevents desensitization. Platelet activation by the endoperoxide analogue U46619 is also prevented by sulfinpyrazone or G25671 (0.3-1 mM). The threshold aggregating concentrations of arachidonic acid and U46619 in healthy subjects before and after oral treatment with sulfinpyrazone were elevated by 2-3 fold and a good correlation between ex vivo and in vitro findings was established. We finally examined the actions of sulfinpyrazone and G25671 on the bronchoconstriction in vivo and parenchymal lung strip contraction in vitro induced by U46619. Neither drug had any preventive effect.Our results demonstrate that sulfinpyrazone and its metabolite G25671 are not only cyclooxygenase inhibitors but can also act as endoperoxide/Tx antagonists and indicate clearly that antagonism of U46619 by both drugs is selective for platelets.

Platelet α-Granules: Content and Characteristics of Secretion

1979 ◽  
Author(s):  
Karen Kaplan ◽  
Johan M. Broekman ◽  
Arthur Chernoff ◽  
Barbara Linder ◽  
DeWitt Goodman
Keyword(s):  
Dienoic Acid ◽  
Arterial Disease ◽  
Human Platelets ◽  
Dense Granule ◽  
Distinct Pattern ◽  
Platelet Factor ◽  
Alternative Pathways ◽  
Granule Release

Platelet α-granule contents and secretion were studied using specific radioimmunoas says and cell culture assays. Washed human platelets were disrupted by nitrogen cavitation and subcellular fractions were separated by ultracentrifugation through sucrose density gradients. α-Granules were found to contain platelet factor 4 (PF4), 8-thromboglobulin (8TG) fibrinogen, and the platelet-derived growth factor: their contents were distinct from those of dense granules and of lysos omes. A distinct pattern of release of the four agranule components was ob served. Release of α- and dense-granule components was induced by ADP and epinephrine; α-granule release was, however, more sensitive than dense granule to low levels of thrombin, collagen, and the endoperoxide analogue (15S)-hydroxy-IIα, 9α (epoxymethano)prosta-5Z, l3E-dienoic acid. Studies with cyclooxygenase (CO) inhibitors suggested that α-granule release in vitro can be mediated both by the CO pathway and by other mechanisms. Thus, aspirin and indomethacin inhibited collagen and thrombin induced release, but “the inhibitory effects were progressively overcome with higher levels of these agents. Studies in vivo showed that normal plasma levels of PF4 and 8TG were only slightly reduced by aspirin, whereas the elevated basal levels of these components found in patients with arterial disease were reduced by aspirin to a greater extent. The in viv data thus support the in vitro findings that both CO mediated and alternative pathways are involved in release of α-granule components.

scholarly journals Submission for Special Issue: The Role of Platelet Activation in the Pathophysiology of HIV, Tuberculosis, and Pneumococcal Disease. Bedaquiline Suppresses ADP-Mediated Activation of Human Platelets In Vitro via Interference With Phosphatidylinositol 3-Kinase

2021 ◽  
Vol 11 ◽  
Author(s):  
Gregory R. Tintinger ◽  
Annette J. Theron ◽  
Helen C. Steel ◽  
Moloko C. Cholo ◽  
Jan G. Nel ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Platelet Rich Plasma ◽  
Statistical Significance ◽  
Human Platelets ◽  
Phosphatidylinositol 3 Kinase ◽  
Inhibitory Effects ◽  
Isolated Cells ◽  
Phosphatidylinositol 3

Although bedaquiline has advanced the treatment of multidrug-resistant tuberculosis (TB), concerns remain about the cardiotoxic potential of this agent, albeit by unexplored mechanisms. Accordingly, we have investigated augmentation of the reactivity of human platelets in vitro as a potential mechanism of bedaquiline-mediated cardiotoxicity. Platelet-rich plasma (PRP) or isolated cells prepared from the blood of healthy, adult humans were treated with bedaquiline (0.625–10 µg/ml), followed by activation with adenosine 5’-diphosphate (ADP), thrombin or the thromboxane A2 receptor agonist (U46619). Expression of platelet CD62P (P-selectin), platelet aggregation, Ca2+ fluxes and phosphorylation of Akt1 were measured using flow cytometry, spectrophotometry, fluorescence spectrometry, and by ELISA procedures, respectively. Exposure to bedaquiline caused dose-related inhibition of ADP-activated, but not thrombin- or U46619-activated, expression of CD62P by platelets, achieving statistical significance at a threshold concentration of 5 µg/ml and was paralleled by inhibition of aggregation and Ca2+ mobilization. These ADP-selective inhibitory effects of bedaquiline on platelet activation were mimicked by wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-K), implicating PI3-K as being a common target of both agents, a contention that was confirmed by the observed inhibitory effects of bedaquiline on the phosphorylation of Akt1 following activation of platelets with ADP. These apparent inhibitory effects of bedaquiline on the activity of PI3-K may result from the secondary cationic amphiphilic properties of this agent. If operative in vivo, these anti-platelet effects of bedaquiline may contribute to ameliorating the risk of TB-associated cardiovascular disease, but this remains to be explored in the clinical setting.

Evidence for an Increased Generation of Prostacyclin in the Microvasculature and an Impairment of the Platelet α-Granule Release in Chronic Renal Failure

1988 ◽  
Vol 60 (02) ◽  
pp. 205-208 ◽  
Author(s):  
Paul A Kyrle ◽  
Felix Stockenhuber ◽  
Brigitte Brenner ◽  
Heinz Gössinger ◽  
Christian Korninger ◽  
...  
Keyword(s):  
Renal Failure ◽  
Chronic Renal Failure ◽  
Blood Clotting ◽  
Normal Subjects ◽  
Chronic Uraemia ◽  
Wall Interaction ◽  
End Stage ◽  
Granule Release

SummaryThe formation of prostacyclin (PGI2) and thromboxane A2 and the release of beta-thromboglobulin (beta-TG) at the site of platelet-vessel wall interaction, i.e. in blood emerging from a standardized injury of the micro vasculature made to determine bleeding time, was studied in patients with end-stage chronic renal failure undergoing regular haemodialysis and in normal subjects. In the uraemic patients, levels of 6-keto-prostaglandin F1α (6-keto-PGF1α) were 1.3-fold to 6.3-fold higher than the corresponding values in the control subjects indicating an increased PGI2 formation in chronic uraemia. Formation of thromboxane B2 (TxB2) at the site of plug formation in vivo and during whole blood clotting in vitro was similar in the uraemic subjects and in the normals excluding a major defect in platelet prostaglandin metabolism in chronic renal failure. Significantly smaller amounts of beta-TG were found in blood obtained from the site of vascular injury as well as after in vitro blood clotting in patients with chronic renal failure indicating an impairment of the a-granule release in chronic uraemia. We therefore conclude that the haemorrhagic diathesis commonly seen in patients with chronic renal failure is - at least partially - due to an acquired defect of the platelet a-granule release and an increased generation of PGI2 in the micro vasculature.

Investigation of the Interaction of Blood Platelets with the Coagulation System at the Site of Plug Formation In Vivo in Man - Effect of Low-Dose Aspirin

1987 ◽  
Vol 57 (01) ◽  
pp. 062-066 ◽  
Author(s):  
P A Kyrle ◽  
J Westwick ◽  
M F Scully ◽  
V V Kakkar ◽  
G P Lewis
Keyword(s):  
Platelet Activation ◽  
Thrombin Generation ◽  
Low Dose ◽  
Bleeding Time ◽  
Blood Platelets ◽  
Dose Aspirin ◽  
Low Dose Aspirin ◽  
Plug Formation ◽  
Granule Release

SummaryIn 7 healthy volunteers, formation of thrombin (represented by fibrinopeptide A (FPA) generation, α-granule release (represented by β-thromboglobulin [βTG] release) and the generation of thromboxane B2 (TxB2) were measured in vivo in blood emerging from a template bleeding time incision. At the site of plug formation, considerable platelet activation and thrombin generation were seen within the first minute, as indicated by a 110-fold, 50-fold and 30-fold increase of FPA, TxB2 and PTG over the corresponding plasma values. After a further increase of the markers in the subsequent 3 minutes, they reached a plateau during the fourth and fifth minute. A low-dose aspirin regimen (0.42 mg.kg-1.day-1 for 7 days) caused >90% inhibition of TxB2formation in both bleeding time blood and clotted blood. At the site of plug formation, a-granule release was substantially reduced within the first three minutes and thrombin generation was similarly inhibited. We conclude that (a) marked platelet activation and considerable thrombin generation occur in the early stages.of haemostasis, (b) α-granule release in vivo is partially dependent upon cyclo-oxygenase-controlled mechanisms and (c) thrombin generation at the site of plug formation is promoted by the activation of platelets.

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