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.

Abstract 13598: The G-protein BetaGamma Subunits Regulate Platelet Function

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Ahmed Alarabi ◽  
Zubair Karim ◽  
Victoria Hinojos ◽  
Patricia A Lozano ◽  
Keziah Hernandez ◽  
...  
Keyword(s):  
G Protein ◽  
Platelet Function ◽  
Thrombus Formation ◽  
Human Platelets ◽  
Inhibitory Effects ◽  
Clot Retraction ◽  
Betagamma Subunits

Platelet activation involves tightly regulated processes to ensure a proper hemostasis response, but when unbalanced, can lead to pathological consequences such as thrombus formation. G-protein coupled receptors (GPCRs) regulate platelet function by interacting with and mediating the response to various physiological agonists. To this end, an essential mediator of GPCR signaling is the G protein Gαβγ heterotrimers, in which the βγ subunits are central players in downstream signaling pathways. While much is known regarding the role of the Gα subunit in platelet function, that of the βγ remains poorly understood. Therefore, we investigated the role of Gβγ subunits in platelet function using a Gβγ (small molecule) inhibitor, namely gallein. We observed that gallein inhibits platelet aggregation and secretion in response to agonist stimulation, in both mouse and human platelets. Furthermore, gallein also exerted inhibitory effects on integrin αIIbβ3 activation and clot retraction. Finally, gallein’s inhibitory effects manifested in vivo , as documented by its ability to modulate physiological hemostasis and delay thrombus formation. Taken together, our findings demonstrate, for the first time, that Gβγ directly regulates GPCR-dependent platelet function, in vitro and in vivo . Moreover, these data highlight Gβγ as a novel therapeutic target for managing thrombotic disorders.

CLEC-2 is an essential platelet-activating receptor in hemostasis and thrombosis

Blood ◽  
2009 ◽  
Vol 114 (16) ◽  
pp. 3464-3472 ◽  
Author(s):  
Frauke May ◽  
Ina Hagedorn ◽  
Irina Pleines ◽  
Markus Bender ◽  
Timo Vögtle ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Thrombus Formation ◽  
Antibody Treatment ◽  
Cellular Activation ◽  
Primary Hemostasis ◽  
Arterial Thrombus ◽  
Normal Adhesion

Abstract Damage to the integrity of the vessel wall leads to exposure of the subendothelial extracellular matrix (ECM), triggering platelet activation and aggregation. This process is essential for primary hemostasis but it may also lead to arterial thrombosis. Although the mechanisms underlying platelet activation on the ECM are well explored, it is less clear which receptors mediate cellular activation in a growing thrombus. Here we studied the role of the recently identified C-type lectin-like receptor 2 (CLEC-2) in this process. We show that anti–CLEC-2 antibody treatment of mice leads to complete and highly specific loss of CLEC-2 in circulating platelets for several days. CLEC-2–deficient platelets displayed normal adhesion under flow, but subsequent aggregate formation was severely defective in vitro and in vivo. As a consequence, CLEC-2 deficiency was associated with increased bleeding times and profound protection from occlusive arterial thrombus formation. These results reveal an essential function of CLEC-2 in hemostasis and thrombosis.

scholarly journals Annexin A7 is a critical regulator of Ca2+ mobilization and lipid metabolism during platelet activation and arterial thrombosis

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
O Borst ◽  
S Geue ◽  
M.C Manke ◽  
B Peng ◽  
P Muenzer ◽  
...  
Keyword(s):  
Lipid Metabolism ◽  
Platelet Activation ◽  
Arterial Thrombosis ◽  
Bleeding Time ◽  
Thrombus Formation ◽  
Vascular Occlusion ◽  
Funding Source ◽  
Annexin A7

Abstract Background Platelet activation after contact to subendothelial collagen following atherosclerotic plaque rupture can lead to arterial thrombosis with acute thrombotic vascular occlusion. Annexin A7 (AnxA7) is an intracellular Ca2+- and phospholipid-binding protein that participates in the regulation of prostaglandin production in inflammatory diseases, but also in cell survival and tumor growth. Objective In the present study, we aimed to determine the role of AnxA7 for platelet Ca2+ signaling and lipid metabolism in platelet activation and arterial thrombosis in gene-targeted mice lacking annexin A7 (Anxa7−/−). Results AnxA7 is strongly expressed in platelets of platelet-rich human coronary thrombi aspirated from patients with acute ST elevation myocardial infarction. Functionally, platelet aggregation and dense granule secretion were significantly abrogated in Anxa7−/− platelets as compared to wildtype platelets (Anxa7+/+) after activation with collagen or collagen-related peptide (CRP), a specific agonist of the major platelet collagen receptor glycoprotein VI (GPVI). Further, in vitro thrombus formation on a collagen-coated surface under high arterial shear rates was significantly diminished in Anxa7-deficient platelets, and thrombotic vascular occlusion after FeCl3-induced injury in vivo was blunted in Anxa7−/−bone marrow chimeric mice, but no prolongation of bleeding time was observed. Moreover, Anxa7−/− platelets showed a significant reduction of IP3 production due to an abolished phospholipase C (PLC) gamma2 phosphorylation resulting in an abolished increase of [Ca2+]i after platelet activation with CRP. Moreover, we could show by quantitative lipidomics analysis that annexin A7 critically affects platelet oxylipid metabolism following activation of GPVI-dependent platelet signalling since Anxa7−/− platelets showed a significant reduction of the bioactive metabolites thromboxane A2 and 12(S)-hydroxy-eicosatetraenoic acid (12(S)-HETE) levels as well as significantly reduced levels of several other prostaglandins following stimulation with collagen or CRP. Finally, defective PLCgamma2 phosphorylation, IP1 production and blunted increase of [Ca2+]i in Anxa7−/− platelets could be rescued by exogenous addition of 12(S)-HETE indicating that AnxA7 is a critical regulator of the platelet oxygenase 12-lipoxygenase (12-LOX) in GPVI-dependent platelet Ca2+ signalling during arterial thrombosis following activation by collagen. Conclusions The present study reveals annexin A7 as a critical regulator of oxylipid metabolism and Ca2+ signaling in GPVI-dependent platelet activation. Anxa7-deficiency further results in decreased in vitro and in vivo thrombus formation, but does not affect bleeding time. In conclusion, annexin A7 plays an important role in platelet signaling during arterial thrombosis and thus, may reflect a promising target for novel antiplatelet strategies. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): German Research Foundation (Deutsche Forschungsgemeinschaft, DFG)

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.

Abstract 52: IkB Kinase beta is a Key Modulator of Platelet Function, and the Remodeling of CARMA1-Bcl10-MALT1 Complex Formation

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Zubair A Karim
Keyword(s):  
Complex Formation ◽  
Platelet Activation ◽  
Platelet Function ◽  
Thrombus Formation ◽  
Clot Retraction ◽  
Functional Responses ◽  
Knockout Mouse Model ◽  
Iκb Kinase

Secretion plays an important role in platelet function, and hence the secretory machinery offers a unique target to modulate thrombogenesis. We previously established that IκB kinase (IKK)-β is phosphorylated upon platelet activation, regulates their SNARE machinery, and involves CBM (CARMA1, Bcl10, and MALT1) complex formation. However, the detailed role of IKKβ in platelet function, the mechanism by which it regulates CBM complex formation and downstream effector activation remain elusive. Using a knockout mouse model system ( IKK β flox/flox -PF4Cre), we first showed that IKKβ plays a vital role in thrombus formation, and that it does so, in part, by regulating platelet functional responses, including dense and alpha granule release, αIIbβ3 activation, and PS exposure. Furthermore, we observed defects in compound fusion, platelet spreading, actin remodeling, and clot retraction. To this end, under clot retraction conditions in the knockout platelets, 7S complex formation was found to be inhibited. In terms of its signaling, using knockout platelets, we observed that IKKβ is required for the recruitment of Bcl10/MALT1 to CARMA1 upon platelet activation, i.e., CBM complex formation, and that this was due to the defective phosphorylation of Bcl10 and the IKKγ polyubiquitination. In conclusion, our data shows that IKKβ is a key regulator of platelet activation, in vitro and in vivo , and the remodeling of the CBM complex. Furthermore, our findings indicate that inducible clustering of signaling mediators and the formation of higher-order multi-protein complexes (i.e., the CBM complex) is a dynamic process, that supports nonlinear signaling networks and is important for platelet activation.

scholarly journals AN IMPORTANT STIMULATORY ROLE FOR THE cGMP-DEPENDENT PROTEIN KINASE II IN PLATELET ACTIVATION, IN VIVO THROMBOSIS AND HAEMOSTASIS

2020 ◽  
Author(s):  
Zhenyu Li ◽  
Ying Liang ◽  
can wang ◽  
Guoying Zhang ◽  
Jens Schlossmann ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Bleeding Time ◽  
Thrombus Formation ◽  
Atp Release ◽  
Downstream Signaling ◽  
Dependent Protein ◽  
Deficient Mice

Background and Purpose: The intracellular second messenger cGMP mediates signals by activating two types of cGMP-dependent protein kinases (PKG), PKG I and PKG II, differentially expressed in different cells. In platelets, cGMP mediates biphasic signals that stimulate and inhibit platelet activation, and the downstream signaling of cGMP is mediated by PKG I, the only PKG known to be expressed in platelets. However, functional defects of PKG I knockout platelets did not fully explain the roles of cGMP and the effect of PKG inhibitors on platelet activation. Experimental Approach: To determine if PKG II is present in platelets and plays a role in platelet activation, we performed RT-PCR and isolation of PKG II protein using cGMP-conjugated beads. We further determined platelet aggregation and ATP release in vitro, and FeCl3-injured carotid artery thrombosis as well as tail bleeding time in vivo. Key Results: PKG II is expressed in platelets and plays an important role in selectively stimulating platelet activation but not in the negative regulatory role of cGMP. Collagen-induced platelet aggregation and ATP secretion were reduced in PKG II-deficient mice but not PKG I-deficient mice. In contrast, low-dose thrombin-induced platelet activation depended on PKG I but not PKG II. Tail bleeding time and FeCl3-induced artery thrombus formation were significantly prolonged in PKG II knockout mice. Conclusion and Implication: PKG II-mediated cGMP signals are important in platelet activation, thrombosis and haemostasis in vitro and in vivo.

scholarly journals Non-alcoholic fatty liver disease in mice with hepatocyte-specific deletion of mitochondrial fission factor

Diabetologia ◽  
2021 ◽  
Author(s):  
Yukina Takeichi ◽  
Takashi Miyazawa ◽  
Shohei Sakamoto ◽  
Yuki Hanada ◽  
Lixiang Wang ◽  
...  
Keyword(s):  
Er Stress ◽  
Mitochondrial Dynamics ◽  
Mitochondrial Fission ◽  
Primary Hepatocytes ◽  
Alcoholic Fatty Liver ◽  
Expression Of Genes ◽  
Specific Deletion

Abstract Aims/hypothesis Mitochondria are highly dynamic organelles continuously undergoing fission and fusion, referred to as mitochondrial dynamics, to adapt to nutritional demands. Evidence suggests that impaired mitochondrial dynamics leads to metabolic abnormalities such as non-alcoholic steatohepatitis (NASH) phenotypes. However, how mitochondrial dynamics are involved in the development of NASH is poorly understood. This study aimed to elucidate the role of mitochondrial fission factor (MFF) in the development of NASH. Methods We created mice with hepatocyte-specific deletion of MFF (MffLiKO). MffLiKO mice fed normal chow diet (NCD) or high-fat diet (HFD) were evaluated for metabolic variables and their livers were examined by histological analysis. To elucidate the mechanism of development of NASH, we examined the expression of genes related to endoplasmic reticulum (ER) stress and lipid metabolism, and the secretion of triacylglycerol (TG) using the liver and primary hepatocytes isolated from MffLiKO and control mice. Results MffLiKO mice showed aberrant mitochondrial morphologies with no obvious NASH phenotypes during NCD, while they developed full-blown NASH phenotypes in response to HFD. Expression of genes related to ER stress was markedly upregulated in the liver from MffLiKO mice. In addition, expression of genes related to hepatic TG secretion was downregulated, with reduced hepatic TG secretion in MffLiKO mice in vivo and in primary cultures of MFF-deficient hepatocytes in vitro. Furthermore, thapsigargin-induced ER stress suppressed TG secretion in primary hepatocytes isolated from control mice. Conclusions/interpretation We demonstrated that ablation of MFF in liver provoked ER stress and reduced hepatic TG secretion in vivo and in vitro. Moreover, MffLiKO mice were more susceptible to HFD-induced NASH phenotype than control mice, partly because of ER stress-induced apoptosis of hepatocytes and suppression of TG secretion from hepatocytes. This study provides evidence for the role of mitochondrial fission in the development of NASH. Graphical abstract

scholarly journals Layer-by-layer coating of stainless steel plates mediated by surface priming treatment to improve antithrombogenic properties

2016 ◽  
Author(s):  
Irene Carmagnola ◽  
Tiziana Nardo ◽  
Francesca Boccafoschi ◽  
Valeria Chiono
Keyword(s):  
Stainless Steel ◽  
Surface Modification ◽  
Platelet Activation ◽  
Colorimetric Assay ◽  
Thrombus Formation ◽  
Human Platelets ◽  
Steel Plates ◽  
Blue Dye ◽  
Layer By Layer

The stainless steel (SS) stents have been used in clinics since 1994. However, typical drawbacks are restenosis and thrombus formation due to limited endothelialisation and hemocompatibility. Surface modification is a smart strategy to enhance antithrombogenicity by promoting endothelialisation. In this work, the layer-by-layer (LbL) technique was applied for coating SS model substrates, after surface priming by functionalisation with 3-aminopropyl triethoxysilane (APTES). A LbL coating made of 14 layers of poly(styrene sulfonate)/poly(diallyldimethylammonium chloride) and heparin as last layer was deposited. FTIR-ATR analysis and contact angle measurements showed that LbL was an effective method to prepare nanostructured coatings. XPS analysis and colorimetric assay employing 1,9-dimethylmethylene blue dye to detect -COOH groups confirmed the successful polyelectrolyte deposition on the coated samples. Preliminary in vitro cell tests, using whole blood and human platelets, were performed to evaluate how surface modification affects platelet activation. Results showed that SS and SS-APTES surfaces induced platelet activation, as indicated by platelet spreading and filopodia formation. After surface modification by LbL coating, the platelets assumed a round shape and no fibrin nets were detected. Data demonstrated that LbL coating is a promising technique to fabricate antithrombogenic surface.

scholarly journals NADPH Oxidases Are Required for Full Platelet Activation In Vitro and Thrombosis In Vivo but Dispensable for Plasma Coagulation and Hemostasis

2020 ◽  
Author(s):  
Dina Vara ◽  
Reiner K. Mailer ◽  
Anuradha Tarafdar ◽  
Nina Wolska ◽  
Marco Heestermans ◽  
...  
Keyword(s):  
Thrombus Formation ◽  
Single Gene ◽  
Coagulation Cascade ◽  
Intracellular Cgmp ◽  
Antithrombotic Effects ◽  
Tail Tip ◽  
Synthase Inhibitor

Objective: Using 3KO (triple NOX [NADPH oxidase] knockout) mice (ie, NOX1 −/− /NOX2 −/− /NOX4 −/− ), we aimed to clarify the role of this family of enzymes in the regulation of platelets in vitro and hemostasis in vivo. Approach and Results: 3KO mice displayed significantly reduced platelet superoxide radical generation, which was associated with impaired platelet aggregation, adhesion, and thrombus formation in response to the key agonists collagen and thrombin. A comparison with single-gene knockouts suggested that the phenotype of 3KO platelets is the combination of the effects of the genetic deletion of NOX1 and NOX2, while NOX4 does not show any significant function in platelet regulation. 3KO platelets displayed significantly higher levels of cGMP—a negative platelet regulator that activates PKG (protein kinase G). The inhibition of PKG substantially but only partially rescued the defective phenotype of 3KO platelets, which are responsive to both collagen and thrombin in the presence of the PKG inhibitors KT5823 or Rp-8-pCPT-cGMPs, but not in the presence of the NOS (NO synthase) inhibitor L-NG-monomethyl arginine. In vivo, triple NOX deficiency protected against ferric chloride–driven carotid artery thrombosis and experimental pulmonary embolism, while hemostasis tested in a tail-tip transection assay was not affected. Procoagulatory activity of platelets (ie, phosphatidylserine surface exposure) and the coagulation cascade in platelet-free plasma were normal. Conclusions: This study indicates that inhibiting NOXs has strong antithrombotic effects partially caused by increased intracellular cGMP but spares hemostasis. NOXs are, therefore, pharmacotherapeutic targets to develop new antithrombotic drugs without bleeding side effects.

scholarly journals Talin is required for integrin-mediated platelet function in hemostasis and thrombosis

2007 ◽  
Vol 204 (13) ◽  
pp. 3103-3111 ◽  
Author(s):  
Brian G. Petrich ◽  
Patrizia Marchese ◽  
Zaverio M. Ruggeri ◽  
Saskia Spiess ◽  
Rachel A.M. Weichert ◽  
...  
Keyword(s):  
Platelet Adhesion ◽  
Ex Vivo ◽  
Focal Adhesions ◽  
Β1 Integrin ◽  
Normal Morphology ◽  
And Function ◽  
Specific Deletion

Integrins are critical for hemostasis and thrombosis because they mediate both platelet adhesion and aggregation. Talin is an integrin-binding cytoplasmic adaptor that is a central organizer of focal adhesions, and loss of talin phenocopies integrin deletion in Drosophila. Here, we have examined the role of talin in mammalian integrin function in vivo by selectively disrupting the talin1 gene in mouse platelet precursor megakaryocytes. Talin null megakaryocytes produced circulating platelets that exhibited normal morphology yet manifested profoundly impaired hemostatic function. Specifically, platelet-specific deletion of talin1 led to spontaneous hemorrhage and pathological bleeding. Ex vivo and in vitro studies revealed that loss of talin1 resulted in dramatically impaired integrin αIIbβ3-mediated platelet aggregation and β1 integrin–mediated platelet adhesion. Furthermore, loss of talin1 strongly inhibited the activation of platelet β1 and β3 integrins in response to platelet agonists. These data establish that platelet talin plays a crucial role in hemostasis and provide the first proof that talin is required for the activation and function of mammalian α2β1 and αIIbβ3 integrins in vivo.

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