Opposing Roles of GSK3α and GSK3β Phosphorylation in Platelet Function and Thrombosis

One of the mechanisms by which PI3 kinase can regulate platelet function is through phosphorylation of downstream substrates, including glycogen synthase kinase-3 (GSK3)α and GSK3β. Platelet activation results in the phosphorylation of an N-terminal serine residue in GSK3α (Ser21) and GSK3β(Ser9), which competitively inhibits substrate phosphorylation. However, the role of phosphorylation of these paralogs is still largely unknown. Here, we employed GSK3α/β phosphorylation-resistant mouse models to explore the role of this inhibitory phosphorylation in regulating platelet activation. Expression of phosphorylation-resistant GSK3α/β reduced thrombin-mediated platelet aggregation, integrin αIIbβ3 activation, and α-granule secretion, whereas platelet responses to the GPVI agonist collagen-related peptide (CRP-XL) were significantly enhanced. GSK3 single knock-in lines revealed that this divergence is due to differential roles of GSK3α and GSK3β phosphorylation in regulating platelet function. Expression of phosphorylation-resistant GSK3α resulted in enhanced GPVI-mediated platelet activation, whereas expression of phosphorylation-resistant GSK3β resulted in a reduction in PAR-mediated platelet activation and impaired in vitro thrombus formation under flow. Interestingly, the latter was normalised in double GSK3α/β KI mice, indicating that GSK3α KI can compensate for the impairment in thrombosis caused by GSK3β KI. In conclusion, our data indicate that GSK3α and GSK3β have differential roles in regulating platelet function.

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

Circulation ◽

10.1161/circ.142.suppl_3.13598 ◽

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.

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Supplemental Fibrinogen Restores Platelet Inhibitor-Induced Reduction in Thrombus Formation without Altering Platelet Function: An In Vitro Study

Thrombosis and Haemostasis ◽

10.1055/s-0040-1715445 ◽

2020 ◽

Vol 120 (11) ◽

pp. 1548-1556

Author(s):

Thomas Bärnthaler ◽

Elisabeth Mahla ◽

Gabor G. Toth ◽

Rufina Schuligoi ◽

Florian Prüller ◽

...

Keyword(s):

Antiplatelet Therapy ◽

Platelet Activation ◽

Platelet Function ◽

Dual Antiplatelet Therapy ◽

Thrombus Formation ◽

In Vitro Study ◽

Coronary Intervention ◽

Calcium Flux ◽

Major Surgery

Abstract Background For patients treated with dual antiplatelet therapy, standardized drug-specific 3-to-7 day cessation is recommended prior to major surgery to reach sufficient platelet function recovery. Here we investigated the hypothesis that supplemental fibrinogen might mitigate the inhibitory effects of antiplatelet therapy. Methods and Results To this end blood from healthy donors was treated in vitro with platelet inhibitors, and in vitro thrombus formation and platelet activation were assessed. Ticagrelor, acetylsalicylic acid, the combination of both, and tirofiban all markedly attenuated the formation of adherent thrombi, when whole blood was perfused through collagen-coated microchannels at physiological shear rates. Addition of fibrinogen restored in vitro thrombus formation in the presence of antiplatelet drugs and heparin. However, platelet activation, as investigated in assays of P-selectin expression and calcium flux, was not altered by fibrinogen supplementation. Most importantly, fibrinogen was able to restore in vitro thrombogenesis in patients on maintenance dual antiplatelet therapy after percutaneous coronary intervention. Conclusion Thus, our in vitro data support the notion that supplementation of fibrinogen influences the perioperative hemostasis in patients undergoing surgery during antiplatelet therapy by promoting thrombogenesis without significantly interfering with platelet activation.

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IκB kinase 2 is not essential for platelet activation

Blood Advances ◽

10.1182/bloodadvances.2019001044 ◽

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.

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

Blood ◽

10.1182/blood-2009-05-222273 ◽

2009 ◽

Vol 114 (16) ◽

pp. 3464-3472 ◽

Cited By ~ 130

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.

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The secreted tyrosine kinase VLK is essential for normal platelet activation and thrombus formation

10.1101/2021.01.24.427281 ◽

2021 ◽

Author(s):

Leila Revollo ◽

Glenn Merrill-Skoloff ◽

Karen De Ceunynck ◽

James R. Dilks ◽

Mattia Bordoli ◽

...

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Phosphorylation ◽

Platelet Activation ◽

Platelet Function ◽

Secretory Pathway ◽

Thrombus Formation ◽

Extracellular Proteins ◽

Normal Platelet

AbstractTyrosine phosphorylation of extracellular proteins is observed in cell cultures and in vivo, but little is known about the functional roles of tyrosine phosphorylation of extracellular proteins. Vertebrate Lonesome Kinase (VLK) is a broadly expressed secretory pathway tyrosine kinase present in platelet ɑ-granules. It is released from platelets upon activation and phosphorylates substrates extracellularly. Its role in platelet function, however, has not been previously studied. In human platelets, we identified phosphorylated tyrosines mapped to luminal or extracellular domains of transmembrane and secreted proteins implicated in the regulation of platelet activation. To determine the role of VLK in extracellular tyrosine phosphorylation and platelet function, we generated mice with a megakaryocyte/platelet-specific deficiency of VLK. Platelets from these mice are normal in abundance and morphology, but have dramatic changes in function both in vitro and in vivo. Resting and thrombin-stimulated VLK-deficient platelets demonstrate a significant decrease of several tyrosine phosphobands. Functional testing of VLK-deficient platelets shows decreased PAR4- and collagen-mediated platelet aggregation, but normal responses to ADP. Dense granule and α-granule release are reduced in these platelets. Furthermore, VLK-deficient platelets exhibit decreased PAR4-mediated Akt (S473) and Erk1/2(T202/Y204) phosphorylation, indicating altered proximal signaling. In vivo, mice lacking VLK in megakaryocytes/platelets demonstrate strongly reduced platelet accumulation and fibrin formation following laser-injury of cremaster arterioles compared to controls. These studies demonstrate that the secretory pathway tyrosine kinase VLK is critical for stimulus-dependent platelet activation and thrombus formation, providing the first evidence that a secreted protein kinase is required for normal platelet function.

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The secreted tyrosine kinase VLK is essential for normal platelet activation and thrombus formation

Blood ◽

10.1182/blood.2020010342 ◽

2021 ◽

Author(s):

Leila Denise Revollo ◽

Glenn Merrill-Skoloff ◽

Karen De Ceunynck ◽

James R Dilks ◽

Shihui Guo ◽

...

Keyword(s):

Tyrosine Kinase ◽

Tyrosine Phosphorylation ◽

Platelet Activation ◽

Platelet Function ◽

Secretory Pathway ◽

Thrombus Formation ◽

Extracellular Proteins ◽

Normal Platelet

Tyrosine phosphorylation of extracellular proteins is observed in cell cultures and in vivo, but little is known about the functional roles of tyrosine phosphorylation of extracellular proteins. Vertebrate Lonesome Kinase (VLK) is a broadly expressed secretory pathway tyrosine kinase present in platelet ɑ-granules. It is released from platelets upon activation and phosphorylates substrates extracellularly. Its role in platelet function, however, has not been previously studied. In human platelets, we identified phosphorylated tyrosines mapped to luminal or extracellular domains of transmembrane and secreted proteins implicated in the regulation of platelet activation. To determine the role of VLK in extracellular tyrosine phosphorylation and platelet function, we generated mice with a megakaryocyte/platelet-specific deficiency of VLK. Platelets from these mice are normal in abundance and morphology, but have significant changes in function both in vitro and in vivo. Resting and thrombin-stimulated VLK-deficient platelets demonstrate a significant decrease of several tyrosine phosphobands. Functional testing of VLK-deficient platelets shows decreased PAR4- and collagen-mediated platelet aggregation, but normal responses to ADP. Dense granule and a-granule release are reduced in these platelets. Furthermore, VLK-deficient platelets exhibit decreased PAR4-mediated Akt (S473) and Erk1/2 (T202/Y204) phosphorylation, indicating altered proximal signaling. In vivo, mice lacking VLK in megakaryocytes/platelets demonstrate strongly reduced platelet accumulation and fibrin formation following laser-injury of cremaster arterioles compared to controls, but normal bleeding times. These studies demonstrate that the secretory pathway tyrosine kinase VLK is critical for stimulus-dependent platelet activation and thrombus formation, providing the first evidence that a secreted protein kinase is required for normal platelet function.

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Role of Caspase in a Subset of Human Platelet Activation Responses

Blood ◽

10.1182/blood.v93.12.4222 ◽

1999 ◽

Vol 93 (12) ◽

pp. 4222-4231 ◽

Cited By ~ 119

Author(s):

Anna Shcherbina ◽

Eileen Remold-O’Donnell

Keyword(s):

Platelet Activation ◽

Caspase 3 ◽

Human Platelet ◽

Thrombus Formation ◽

Shape Change ◽

Vascular Wall ◽

Human Platelets ◽

Granule Secretion ◽

Platelets Function

Abstract Platelets function to protect the integrity of the vascular wall. A subset of platelet activation responses that are especially important for thrombus formation include exposure of phosphatidylserine and release of microparticles, which generate procoagulant surfaces. The resemblance of these platelet activation processes to events occurring in nucleated cells undergoing apoptosis suggests a possible role for caspases, which are major effector enzymes of nucleated cell apoptosis. We demonstrate here the presence of caspase-3 in human platelets and its activation by physiological platelet agonists. Using cell-permeable specific inhibitors, we demonstrate a role for a caspase-3–like protease in the agonist-induced (collagen plus thrombin or Ca2+ ionophore) platelet activation events of phosphatidylserine exposure, microparticle release, and cleavage of moesin, a cytoskeletal-membrane linker protein. The role of caspase-3 in platelet activation is restricted rather than global, because other activation responses,  granule secretion, shape change, and aggregation were unaffected by caspase-3 inhibitors. Experiments with two classes of protease inhibitors show that caspase-3 function is distinct from that of calpain, which is also involved in late platelet activation events. These findings show novel functions of caspase and provide new insights for understanding of platelet activation.

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Abstract 391: Platelet CD40L Affects Thrombus Formation via Phosphatidylinositol 3-kinase ß, But Not Via CD40 or IKKα

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.35.suppl_1.391 ◽

2015 ◽

Vol 35 (suppl_1) ◽

Author(s):

Marijke J Kuijpers ◽

Nadine J Mattheij ◽

Lina Cipolla ◽

Johanna P van Geffen ◽

Toby Lawrence ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Thrombus Formation ◽

Aggregate Formation ◽

Phosphatidylinositol 3 Kinase ◽

Akt Phosphorylation ◽

Soluble Cd40l ◽

Granule Secretion ◽

Collagen Receptor

Objective: To investigate the roles and signaling pathways of CD40L and CD40 in platelet activation and thrombus formation under atherothrombotic conditions. Approach and Results: Mouse platelets lacking CD40L (Cd40lg -/- Apoe -/- ) showed diminished αIIbβ3 activation and α-granule secretion in response to collagen receptor (GPVI) stimulation, while CD40 deficient platelets (Cd40 -/- Apoe -/- ) showed increased responses. ADP- or thrombin-evoked activation was unaffected. In both Cd40lg -/- Apoe -/- and Cd40 -/- Apoe -/- mice, formation of multi-layered thrombi was decreased on both atherosclerotic plaque material and collagen, in comparison to controls. Addition of CD40L prior to perfusion over collagen or plaque material enhanced dense aggregate formation in Apoe -/- , Cd40lg -/- Apoe -/- and Cd40 -/- Apoe -/- blood. CD40L or low GPVI stimulation separately did not cause platelet aggregation. But when combined, aggregation was potentiated, even in the absence of CD40. This potentiation was antagonized by inhibiting PI3Kβ, as well as in platelets from Pik3cb R/R mice. CD40L enhanced Akt phosphorylation at low GPVI stimulation, which was again antagonized by PI3Kβ inhibition and absent in platelets from Pik3cb R/R mice. Finally, Chuk1 A/A Apoe -/- mice, deficient in IKKα, displayed no differences in platelet aggregation - with or without CD40L - nor in thrombus formation in whole blood, indicating that these effects are not mediated via IKKα/NFkB. Conclusions: Under atherothrombotic conditions, CD40L enforces collagen-dependent platelet activation, by supporting integrin αIIbβ3 activation, secretion and dense thrombus formation via PI3Kβ, but not IKKα. Since shedding of CD40L starts minutes after activation, these results point to a joint role of both platelet-bound and soluble CD40L in controlling the size of rapidly formed thrombi.

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Non-genomic effects of the Pregnane X Receptor negatively regulate platelet functions, thrombosis and haemostasis

Scientific Reports ◽

10.1038/s41598-019-53218-x ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Gagan D. Flora ◽

Khaled A. Sahli ◽

Parvathy Sasikumar ◽

Lisa-Marie Holbrook ◽

Alexander R. Stainer ◽

...

Keyword(s):

Platelet Function ◽

Thrombus Formation ◽

Pregnane X Receptor ◽

Human Platelets ◽

Regulatory Effects ◽

Granule Secretion ◽

Species Specific ◽

Platelet Functions

AbstractThe pregnane X receptor (PXR) is a nuclear receptor (NR), involved in the detoxification of xenobiotic compounds. Recently, its presence was reported in the human vasculature and its ligands were proposed to exhibit anti-atherosclerotic effects. Since platelets contribute towards the development of atherosclerosis and possess numerous NRs, we investigated the expression of PXR in platelets along with the ability of its ligands to modulate platelet activation. The expression of PXR in human platelets was confirmed using immunoprecipitation analysis. Treatment with PXR ligands was found to inhibit platelet functions stimulated by a range of agonists, with platelet aggregation, granule secretion, adhesion and spreading on fibrinogen all attenuated along with a reduction in thrombus formation (both in vitro and in vivo). The effects of PXR ligands were observed in a species-specific manner, and the human-specific ligand, SR12813, was observed to attenuate thrombus formation in vivo in humanised PXR transgenic mice. PXR ligand-mediated inhibition of platelet function was found to be associated with the inhibition of Src-family kinases (SFKs). This study identifies acute, non-genomic regulatory effects of PXR ligands on platelet function and thrombus formation. In combination with the emerging anti-atherosclerotic properties of PXR ligands, these anti-thrombotic effects may provide additional cardio-protective benefits.

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Protein Palmitoylation in Platelet Function: Role in G Protein-Mediated Platelet Activation and Platelet Recruitment into Thrombi.

Blood ◽

10.1182/blood.v106.11.650.650 ◽

2005 ◽

Vol 106 (11) ◽

pp. 650-650

Author(s):

Robert Flaumenhaft ◽

James R. Dilks ◽

Derek S. Sim

Keyword(s):

Signal Transduction ◽

G Protein ◽

Platelet Activation ◽

Platelet Function ◽

Thrombus Formation ◽

Dependent Manner ◽

Platelet Protein ◽

Protein Palmitoylation ◽

Calcein Am ◽

Granule Secretion

Abstract Protein palmitoylation represents the covalent linkage of a 16-carbon saturated fatty acid to a protein. This reversible post-translational modification directs protein-protein interactions as well as protein association with membranes and lipid rafts. Protein palmitoylation participates in ligand-induced signal transduction in several nucleated cells. Its role in platelet activation, however, has not previously been evaluated. We have found that platelets contain the palmitoyl transfer proteins GODZ and HIP14 as well as the palmitoyltransferase, acyl-protein thioesterase 1 (APT1). Thus, platelets possess the basic machinery for regulated palmitoylation. Studies using [3H]-labeled platelets identified several platelet proteins that were palmitoylated following exposure to the protease-activated receptor 1 (PAR-1) ligand, SFLLRN. To determine whether protein palmitoylation functions in activation-induced platelet functions, we infused recombinant APT1 into permeabilized platelets prior to activation with SFLLRN. Infusion of APT1 inhibited platelet protein palmitoylation and completely blocked platelet α-granule secretion induced by SFLLRN. Similarly, the protein palmitoylation inhibitor cerulenin blocked SFLLRN-induced platelet protein palmitoylation, α-granule secretion, and platelet aggregation in intact platelets. To assess the mechanism by which protein palmitoylation affects platelet function, we evaluated the effect of inhibitors of protein palmitoylation on G protein activity. Gαq is essential to PAR-1-mediated platelet activation and is palmitoylated in an activation-dependent manner in nucleated cells. Immunoprecipitation of Gαq from [3H]-labeled platelets showed that it is palmitoylated following activation of platelets with SFLLRN. Both APT1 and cerulenin inhibited SFLLRN-induced palmitoylation of Gαq. In addition, APT1 and cerulenin inhibited SFLLRN-induced GTPase activity as detected using [γ-32P]GTP-labeled platelet lysates. These results show that palmitoylation of Gαq participates in PAR-1-mediated signal transduction. We next used intravital microscopy to determine if protein palmitoylation functions in thrombus formation in vivo. For these experiments, platelets from a donor mouse were incubated with cerulenin and labeled with calcein-AM (green) or incubated with vehicle alone and labeled with calcein-AM red-orange (red). Equal numbers of green and red labeled platelets were then infused into a recipient mouse. The accumulation of cerulenin- and vehicle-treated platelets into thrombi following laser-induced injury of the mouse cremaster muscle was quantified using high speed, digital videomicroscopy. Incubation of platelets with cerulenin resulted in an approximately 50% reduction in their ability to accumulate into thrombi. These studies show that platelet protein palmitoylation is required for thrombus formation as well as for normal platelet function.

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