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

Blood ◽  
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.

Gβ1 a Component Of The Heterotrimeric G Protein Is a New Protein Phosphatase 1c Interacting Protein That Regulates Platelet Activation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3508-3508
Author(s):  
Subhashree Pradhan ◽  
Tanvir Khatlani ◽  
Satya P. Kunapuli ◽  
K. Vinod Vijayan
Keyword(s):  
Signal Transduction ◽  
G Protein ◽  
Platelet Activation ◽  
Protein Interactions ◽  
Protein Phosphatase ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Adaptor Protein ◽  
Gpcr Signaling

Abstract Platelet activation at the site of injury is tied to signal transduction events that are mediated by protein kinases and phosphatases. Reversible tyrosine, serine/threonine (Ser/Thr) phosphorylation-dependent assembly and/or disassembly of effector (cytoskeletal, signaling and adaptor) protein complexes propagate signaling downstream of G protein coupled receptors (GPCRs). Compared to kinases, the contribution of Ser/Thr phosphatases and its effectors in GPCR signaling studies is not well explored. Our previous studies had revealed that the catalytic subunit of protein phosphatase 1γ (PP1cγ) support GPCR signaling and thrombus formation. Since cell signaling networks are dependent on protein-protein interactions, we sought to identify the potential effectors of PP1cγ. We employed yeast two-hybrid interaction studies with the full length PP1cγ fused to GAL4 activating domain as bait and screened human bone marrow library. A novel interaction of PP1cγ with a protein called Gβ1 (GNB1) was identified. Gβ1 is a component of the heterotrimeric G proteins like the Gα and couple to GPCR. However, unlike Gα subunits, Gβ1 is unexplored in platelets. Co-immunoprecipitation (co-IP) studies validated PP1cγ-Gβ1 interaction in 293 cells expressing PP1cγ-HA and Gβ1-FLAG. Importantly, Gβ1 interacted with all the PP1c isoforms, suggesting that Gβ1 could target all PP1c isoforms to the GPCR complex. Purified PP1c bound to recombinant Gβ1-GST protein but not to GST protein, indicating that the in vitro interaction of PP1c with Gβ1 was direct and independent of Gα and Gγ subunits. A small molecule inhibitor of G protein βγ, gallein decreased thrombin-induced human platelet aggregation and adhesion to immobilized fibrinogen. There is a paucity of Gβ1-/- platelets because Gβ1-/- mice die within 2 days of birth due to microencephaly. siRNA mediated depletion of Gβ1 in murine megakaryocytes reduced PAR4-activating peptide induced soluble fibrinogen binding to αIIbβ3. These studies suggest a functional role for Gβ1 in GPCR signaling. PP1c co-immunoprecipitated with Gβ1 in resting platelets and agonist (thrombin and ADP) treatment under non-stirring conditions induced dissociation of PP1c from Gβ1. These studies indicate that Gβ1-PP1c complex in platelets is responsive to agonist. Furthermore, PP1c and Gβ1 associated with P2Y12 receptor in resting but not agonist activated platelets in a co-IP assay, suggesting a role for this complex in G protein signaling. Finally, agonist induced dissociation of PP1c from Gβ1 correlated with the association of PP1c with the downstream GPCR effector phospholipase C β3 (PLCβ3) with a concomitant dephosphorylation of PLCβ3 at Ser1105. Since previous studies have revealed that PLCβ3 activity is inhibited by Ser1105 phosphorylation, our observation suggest that agonist-induced association of PP1c with PLCβ3 facilitates dephosphorylation and activation of PLCβ3. These studies highlight a coupling of GPCR signaling with the phosphatase driven signal transduction during platelet activation. Disclosures: No relevant conflicts of interest to declare.

Targeting The PI3K/AKT Pathway To Inhibit Platelet Activation and Thrombus Formation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3513-3513
Author(s):  
Wenxiu Yi ◽  
Wei Li ◽  
Lijie Ren ◽  
Xinliang Mao ◽  
Li Zhu
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Platelet Function ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Venous Blood ◽  
Pi3k Pathway ◽  
Dependent Manner ◽  
Clot Retraction ◽  
Occlusion Time

Abstract The phosphatidylinositol 3' –kinase (PI3K)-Akt signaling pathway has been shown to be critical in modulating platelet function and increasing number of studies have been focusing on the development of PI3K inhibitors to modulate platelet function. We recently identified a novel small molecule compound S14161, namely 8-ethoxy-2-(4-fluorophenyl)-3-nitro-2H-chromene, displaying potent antileukemia and antimyeloma activity via inhibition of the PI3K pathway (Mao et al, Blood, 2011, 117:1986). In the present study, we evaluated the effect of S14161 on platelet activation and the underlying mechanisms. Gel-filtered human platelets were isolated from venous blood of healthy adults and the effect of S14161 on platelet aggregation in response to agonists was determined. Results showed that S14161 inhibited platelet aggregation induced by collagen, convulxin, thrombin, PAR1 agonist peptide SFLLRN, and U46619 in a dose dependent manner (2.5-10μM) with the most striking inhibition for collagen by 89.8% (P<0.001, n=3) and for U46619 by 94.3% (P<0.001, n=3), respectively compared to vehicle-treated samples when 10μM S14161 was used. Flow cytometry studies showed that S14161 inhibits convulxin- or thrombin-induced P-selectin expression and fibrinogen binding of single platelet. S14161 also inhibited platelet spreading on fibrinogen and clot retraction, processes mediated by outside-in signaling. Using a microfluidic chamber we demonstrated that incubation of S14161 decreases platelet adhesion on collagen-coated surface by about 80% at various time points of blood flow in the chambers. Western blot showed that similar to LY294002, the classic PI3K inhibitor, S14161 inhibited phosphorylation of Akt Ser473 and Akt Thr308 in response to collagen, thrombin, or U46619, implying the involvement of PI3K pathway. Additionally, S14161 inhibited MAPK/ERK1/2 phosphorylation. Finally, the effects of S14161 on thrombus formation in vivo were measured using a ferric chloride-induced carotid artery injury model in mice. The intraperitoneal injection of S14161 (2mg/kg) to male C57BL6/J mice significantly extended the first occlusion time (5.05±0.99 min, N=9) compared to the vehicle controls (3.72±0.95 min, N=8) (P<0.05), but did not increase the bleeding time (P>0.05). Taken together, our data showed that S14161 inhibits platelet activation and thrombus formation, and may be developed as a novel therapeutic agent for the prevention of thrombotic disorders. (This study was supported by National Natural Science Foundation of China 81170132 to Li Zhu) Disclosures: No relevant conflicts of interest to declare.

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

2021 ◽  
Vol 22 (19) ◽  
pp. 10656
Author(s):  
Samantha F. Moore ◽  
Ejaife O. Agbani ◽  
Andreas Wersäll ◽  
Alastair W. Poole ◽  
Chris M. Williams ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Platelet Function ◽  
Glycogen Synthase ◽  
Thrombus Formation ◽  
Resistant Mouse ◽  
Function Expression ◽  
Substrate Phosphorylation ◽  
Granule Secretion

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.

Acyl-Protein Thioesterase 1 Functions in Palmitoylation/Depalmitoylation Cycles of G Proteins and Regulates Platelet Activation

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1062-1062
Author(s):  
Louisa M. Dowal ◽  
James R. Dilks ◽  
Nathalie A. Fadel ◽  
Omozuanvbo R. Aisiku ◽  
Glenn Merrill-Skoloff ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Signaling Pathways ◽  
G Protein ◽  
Platelet Activation ◽  
Conflicts Of Interest ◽  
G Protein Signaling ◽  
Dependent Manner ◽  
Protein Signaling ◽  
Granule Secretion ◽  
Proximal Signaling

Abstract Abstract 1062 Protein palmitoylation is a reversible post-translational modification that regulates both lipid-protein and protein-protein interactions. During the palmitoylation cycle, palmitoylation occurs through a thioester linkage to a cysteine residue. Depalmitoylation occurs primarily through cleavage of this bond by acyl-protein thioesterase 1 (APT1). We have previously demonstrated the presence of APT1 in platelets and showed that APT1 translocates to membranes in an activation-dependent manner. However, the function of APT1 in platelet activation is not known. To determine whether APT1 functions in platelet signal transduction we evaluated the effect of palmostatins, novel small molecule inhibitors of APT1, on platelet function. Palmostatins B and M both inhibited platelet aggregation and α -granule secretion induced through protease-activated receptor (PAR) 1 with an IC50 of 15 μM. To assess which signaling pathways were affected by APT1 inhibition, we screened palmostatins for their ability to inhibit activation induced by several agonists. Palmostatins blocked platelet aggregation induced by a PAR1 agonist, a PAR4 agonist, TxA2, or epinephrine. In contrast, palmostatins failed to inhibit aggregation induced by collagen, PMA, or ionophore. Palmostatins also inhibited α -granule exocytosis induced by a PAR1 agonist or TxA2, but not exocytosis induced by PMA or ionophore. These results suggested that palmostatins blocked proximal signaling events mediated through G protein coupled receptors (GPCRs). To evaluate this supposition, we tested the effect of palmostatin B on PAR1-mediated [Ca2+]i flux. Palmostatin B inhibited PAR1-induced Ca2+ signaling with and IC50 of 15 μM, the same concentration required for inhibition of platelet aggregation and α -granule secretion. We have recently described the platelet palmitoylome (Dowal et al., Blood, 118:e62-73) and found several components of the proximal G protein signaling pathway that are palmitoylated, including many Gα subunits. To directly assess the effect of APT1 inhibition on palmitoylation/depalmitoylation cycles on a target Gα subunit, we evaluated Gα q palmitoylation using acyl biotin exchange chemistry. Total Gα q palmitoylation decreased substantially with activation of platelets through PAR1. In the presence of palmostatin B, however, Gα q palmitoylation increased following PAR1 activation. These results demonstrate that Gα q is a substrate for APT1. Our studies demonstrate a role for palmitoylation/depalmitoylation cycles in proximal signaling pathways downstream of GPCRs and implicate APT1 as an essential regulator of G protein signaling in platelets. Disclosures: No relevant conflicts of interest to declare.

Protein Palmitoylation Participates in PAR1-Mediated Platelet Activation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1560-1560
Author(s):  
Derek S. Sim ◽  
Blake R. Peterson ◽  
Steffen P. Creaser ◽  
Robert Flaumenhaft
Keyword(s):  
Platelet Activation ◽  
Inhibitory Effect ◽  
Serotonin Release ◽  
Scintillation Counting ◽  
Dependent Manner ◽  
Critical Determinant ◽  
Potent Inducer ◽  
Platelet Protein ◽  
Protein Palmitoylation

Abstract Platelet accumulation at sites of vascular injury is the primary event in arterial thrombosis. We have performed a screen of 16,320 small molecules to identify compounds that inhibit platelet activation mediated by the PAR1 specific agonist, SFLLRN. JF081204{5C} (9-methylene-4-pentyl-2,3,4,9-tetrahydro-1H-cyclopenta(b)quinoline) was identified to be a potent antiplatelet and antithrombotic compound. We have previously shown that JF0812405{5C} inhibited thrombosis in a murine model by up to 80% with an IC50 of 2 mg/kg. Assays of structure activity relationships revealed that length of the alkyl chain of the compound is a critical determinant of its activity. Initial target-directed studies suggested that this compound inhibited protein palmitoylation. We therefore sought to clarify the effects of this compound on platelet protein palmitoylation and determine the role of palmitoylation in signaling induced by PAR1. We first analyzed the effects of JF081204{5C} on palmitoylation in resting platelets. Resting platelets were incubated with a cell-permeable peptide (MyrGCK(NBD)) that renders cells fluorescent upon palmitoylation. By confocal microscopy, the peptide was observed within platelets primarily localized around the periphery. Incubation of resting platelets with JF081204{5C} inhibited palmitoylation-induced fluorescence of the MGC peptide. The inhibitory effect of JF081204{5C} on palmitoylation was confirmed by 3H-palmitate labeling experiments. Resting platelets were incubated with 3H-palmitate in the presence or absence of JF081204{5C} and proteins were extracted by acetone precipitation. 3H-palmitate incorporation was decreased in platelets incubated with JF081204{5C} as evidenced by scintillation counting. We next analyzed the effects of JF081204{5C} on PAR1-mediated activation. JF081204{5C} inhibited SFLLRN-induced P-selectin expression, 14C-serotonin release, aggregation, and αIIbβ3 activation with an IC50 of 5 μM. In contrast, JF081204{5C} failed to inhibit platelet activation induced by ADP, PMA, A23187, or collagen. However, JF081204{5C} inhibited epinephrine-induced platelet aggregation with an IC50 of 5 μM, indicating that JF081204{5C} acts on an intracellular target. Stimulation with SFLLRN resulted in increased 3H-palmitate-incorporation into platelet proteins as evidenced by scintillation counting and autoradiography. In contrast, this increase was not observed following stimulation with U46619 or epinephrine. Autoradiography of the labeled proteins separated by SDS-PAGE demonstrated that JF081204{5C} inhibited SFLLRN-induced 3H-palmitate incorporation into select bands by up to 50%, while not affecting 3H-palmitate incorporation into other bands. We next sought to assess the role of palmitoylation in signaling through PAR1 using alternative inhibitors. 2-Bromopalmitate, a palmitic acid analog, abolished 3H-palmitate-incorporation into platelet protein and inhibited SFLLRN-induced platelet activation with an IC50 of 30 μM. Acyl-protein thioesterase 1 (APT1) is an enzyme that specifically depalmitoylates proteins. Infusion of APT1 into permeabilized platelets inhibited SFLLRN-induced P-selectin expression in a dose-dependent manner with an IC50 of 350 nM. The observations that SFLLRN is a more potent inducer of protein palmitoylation than other platelet agonists and that inhibitors of protein palmitoylation block SFLLRN-mediated platelet activation demonstrate that protein palmitoylation plays a prominent role in signaling downstream of PAR1.

scholarly journals Matrix Metalloproteases and PAR Activation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. SCI-43-SCI-43
Author(s):  
Athan Kuliopulos
Keyword(s):  
Platelet Aggregation ◽  
G Protein ◽  
Platelet Activation ◽  
Platelet Function ◽  
Plaque Rupture ◽  
Thrombus Formation ◽  
Matrix Metalloproteases ◽  
Morbidity And Mortality ◽  
Arterial Blood Flow ◽  
Platelet Thrombus

Abstract Abstract SCI-43 Myocardial infarction due to rupture of atherosclerotic plaques is a leading contributor to morbidity and mortality in the United States, Europe, and other industrialized nations. Although pathoanatomic studies of human atherosclerotic lesions suggest that large plaques can cause ischemic symptoms, a key contributing factor to the morbidity and mortality associated with atherosclerosis is excessive platelet thrombus formation on exposed collagen surfaces following acute plaque rupture. Following their initial tethering to subendothelial collagen and matrix proteins, activation of transiently adhered platelets by autocrine mediators is critical for propagation of the platelet thrombus. Reinforcement of the transient adhesive contacts by activating G protein-dependent shape change, granule release, and integrins permits growth of a stable thrombus that is resistant to the high shear stress of arterial blood flow. Drugs that target the secondary autocrine mediators of platelet thrombus formation such as aspirin and thienopyridines have proven to be beneficial; however, many patients taking these drugs still sustain thrombotic events and might benefit from new therapeutics that interfere with matrix-dependent platelet activation. Matrix metalloproteases (MMPs) have recently emerged as important mediators of platelet function and vascular biology. Initially described as matrix remodeling enzymes involved in tissue repair and cancer invasion, a renewed focus has centered on MMPs and the related metalloprotease disintegrins because of their prominence in vascular wall inflammation and thrombotic thrombocytopenic purpura. Endogenous platelet metalloproteases have been shown to damage platelet function by cleaving cell surface receptors and broad-spectrum metalloprotease inhibitors improve post-transfusion recovery of platelet concentrates. Platelets express several metalloproteases including MMP-1, MMP-2, MMP-3, and MMP-14 on their surface but their roles in platelet aggregation are not well understood. It was recently shown that the G protein-coupled receptor, PAR1, is directly cleaved and activated on the surface of cancer cells by fibroblast-derived MMP-1. PAR1 is the major thrombin receptor of human platelets and is an important mediator of platelet aggregation following tissue factor (TF)-dependent generation of thrombin. However, under pathophysiologic conditions of acute plaque rupture, exposed collagen is the most efficient stimulus of the critical early events of platelet recruitment and propagation under arterial flow, which could trigger metalloprotease activation on the platelet surface. We found that exposure of platelets to collagen caused activation of MMP-1, which in turn directly cleaved PAR1 on the surface of platelets. Unexpectedly, MMP-1 cleaved the N-terminal extracellular domain of PAR1 at a distinct site from the thrombin cleavage site. This cleavage event generated a longer tethered peptide ligand, which was an agonist of platelet activation and PAR1 signaling. Blocking the MMP1-PAR1 pathway inhibited collagen-dependent thrombogenesis, arterial thrombosis and clot retraction, suggesting that therapeutics that target this metalloprotease-receptor system could be a new strategy in the treatment of patients with acute coronary syndromes. Disclosures: No relevant conflicts of interest to declare.

Abstract 56: Coagulation Factor XI Promotes Distal Platelet Activation and Single Platelet Consumption in the Bloodstream Under Shear Flow

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Jenya Zilberman-Rudenko ◽  
Chantal Wiesenekker ◽  
Asako Itakura ◽  
Owen J McCarty
Keyword(s):  
Shear Flow ◽  
Platelet Activation ◽  
Platelet Adhesion ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Coagulation Factor ◽  
Thrombin Inhibitor ◽  
Dependent Manner ◽  
Factor Xi ◽  
Primate Model

Objective: Coagulation factor XI (FXI) has been shown to contribute to thrombus formation on collagen or tissue factor (TF)-coated surfaces in vitro and in vivo by enhancing thrombin generation. Whether the role of the intrinsic pathway of coagulation is restricted to the local site of thrombus formation is unknown. This study was designed to determine whether FXI could promote both proximal and distal platelet activation and aggregate formation in the bloodstream. Approach and Results: Pharmacological blockade of FXI activation or thrombin activity in blood did not affect local platelet adhesion, yet reduced local platelet aggregation, thrombin localization and fibrin formation on immobilized collagen and TF under shear flow, ex vivo . Downstream of the thrombus formed on immobilized collagen or collagen and 10 pM TF, platelet CD62P expression and microaggregate formation and progressive platelet consumption were significantly reduced in the presence of FXI-function blocking antibodies or a thrombin inhibitor in a shear rate- and time-dependent manner. In a non-human primate model of thrombus formation, we found that inhibition of FXI reduced single platelet consumption in the bloodstream distal to a site of thrombus formation. Conclusions: This study demonstrates that the FXI-thrombin axis contributes to distal platelet activation and procoagulant microaggregate formation in the blood flow downstream of the site of thrombus formation. Our data highlights FXI as a novel therapeutic target for inhibiting distal platelet activation without affecting proximal platelet adhesion.

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.

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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