Integrin αIIbβ3 outside-in signaling activates human platelets through serine 24 phosphorylation of Disabled-2

Abstract Background Bidirectional integrin αIIbβ3 signaling is essential for platelet activation. The platelet adaptor protein Disabled-2 (Dab2) is a key regulator of integrin signaling and is phosphorylated at serine 24 in eukaryotic cells. However, the mechanistic insight and function of Dab2-serine 24 phosphorylation (Dab2-pSer24) in platelet biology are barely understood. This study aimed to define whether and how Dab2 is phosphorylated at Ser24 during platelet activation and to investigate the effect of Dab2-pSer24 on platelet function. Results An antibody with confirmed specificity for Dab2-pSer24 was generated. By using this antibody as a tool, we showed that protein kinase C (PKC)-mediated Dab2-pSer24 was a conservative signaling event when human platelets were activated by the platelet agonists such as thrombin, collagen, ADP, 12-O-tetradecanoylphorbol-13-acetate, and the thromboxane A2 activator U46619. The agonists-stimulated Dab2-pSer24 was attenuated by pretreatment of platelets with the RGDS peptide which inhibits integrin outside-in signaling by competitive binding of integrin αIIb with fibrinogen. Direct activation of platelet integrin outside-in signaling by combined treatment of platelets with manganese dichloride and fibrinogen or by spreading of platelets on fibrinogen also resulted in Dab2-pSer24. These findings implicate that Dab2-pSer24 was associated with the outside-in signaling of integrin. Further analysis revealed that Dab2-pSer24 was downstream of Src-PKC-axis and phospholipase D1 underlying the integrin αIIbβ3 outside-in signaling. A membrane penetrating peptide R11-Ser24 which contained 11 repeats of arginine linked to the Dab2-Ser24 phosphorylation site and its flanking sequences (RRRRRRRRRRR19APKAPSKKEKK29) and the R11-S24A peptide with Ser24Ala mutation were designed to elucidate the functions of Dab2-pSer24. R11-Ser24 but not R11-S24A inhibited agonists-stimulated Dab2-pSer24 and consequently suppressed platelet spreading on fibrinogen, with no effect on platelet aggregation and fibrinogen binding. Notably, Ser24 and the previously reported Ser723 phosphorylation (Dab2-pSer723) occurred exclusively in a single Dab2 molecule and resulted in distinctive subcellular distribution and function of Dab2. Dab2-pSer723 was mainly distributed in the cytosol of activated platelets and associated with integrin inside-out signaling, while Dab2-pSer24 was mainly distributed in the membrane fraction of activated platelets and associated with integrin outside-in signaling. Conclusions These findings demonstrate for the first time that Dab2-pSer24 is conservative in integrin αIIbβ3 outside-in signaling during platelet activation and plays a novel role in the control of cytoskeleton reorganization and platelet spreading on fibrinogen.

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A novel role for the fibrinogen Asn-Gly-Arg (NGR) motif in platelet function

Thrombosis and Haemostasis ◽

10.1160/th14-04-0366 ◽

2015 ◽

Vol 113 (02) ◽

pp. 290-304 ◽

Cited By ~ 5

Author(s):

Róisín Moriarty ◽

Ciara A. McManus ◽

Matthew Lambert ◽

Thea Tilley ◽

Marc Devocelle ◽

...

Keyword(s):

Platelet Activation ◽

Platelet Function ◽

Platelet Adhesion ◽

Recognition Sequence ◽

Integrin Αiibβ3 ◽

Recognition Motif ◽

Activated Platelets ◽

Fibrinogen Binding ◽

Platelet Spreading

SummaryThe integrin αIIbβ3 on resting platelets can bind to immobilised fibrinogen resulting in platelet spreading and activation but requires activation to bind to soluble fibrinogen. αIIbβ3 is known to interact with the general integrin-recognition motif RGD (arginine–glycine–aspartate) as well as the fibrinogen-specific γ-chain dodecapeptide; however, it is not known how fibrinogen binding triggers platelet activation. NGR (asparagine–glycine–arginine) is another integrin-recognition sequence present in fibrinogen and this study aims to determine if it plays a role in the interaction between fibrinogen and αIIbβ3. NGR-containing peptides inhibited resting platelet adhesion to fibrinogen with an IC50 of 175 μM but failed to inhibit the adhesion of activated platelets to fibrinogen (IC50 > 500 μM). Resting platelet adhesion to mutant fibrinogens lacking the NGR sequences was reduced compared to normal fibrinogen under both static and shear conditions (200 s-1). However, pre-activated platelets were able to fully spread on all types of fibrinogen. Thus, the NGR motif in fibrinogen is the site that is primarily responsible for the interaction with resting αIIbβ3 and is responsible for triggering platelet activation.

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Talin distribution and phosphorylation in thrombin-activated platelets

Journal of Cell Science ◽

10.1242/jcs.106.4.1189 ◽

1993 ◽

Vol 106 (4) ◽

pp. 1189-1199 ◽

Cited By ~ 1

Author(s):

M.E. Bertagnolli ◽

S.J. Locke ◽

M.E. Hensler ◽

P.F. Bray ◽

M.C. Beckerle

Keyword(s):

Platelet Activation ◽

Subcellular Distribution ◽

Proteolytic Cleavage ◽

Human Platelets ◽

Fourfold Increase ◽

Activated Platelets ◽

Thrombin Activation ◽

And Function ◽

Adhesion Plaque

We have previously demonstrated that the subcellular distribution of the adhesion plaque protein, talin, changes dramatically in human platelets in response to platelet activation (Beckerle et al., J. Cell Biol. 109, 3333–3346, 1989). Talin is uniformly distributed throughout the cytoplasm of resting platelets. However, when platelets are stimulated to become activated and adhesive, a significant amount of the talin population rapidly redistributes to a peripheral, submembranous location. In the present study we have examined talin phosphorylation and proteolytic cleavage as possible mechanisms by which talin's subcellular distribution could be regulated in platelets. We have found that thrombin activation of platelets leads to a fourfold increase in talin phosphorylation. Proteolytic cleavage of talin, however, is not detected in washed platelets activated with thrombin for as long as 30 minutes. Because talin moves to a submembranous location upon platelet activation and has been shown to interact with integrins in vitro, we also investigated whether the major platelet integrin, GPIIb-IIIa, is required for talin redistribution. Using Glanzmann thrombasthenic platelets, which are deficient in GPIIb-IIIa, we found that talin redistribution occurs even in the absence of GPIIb-IIIa. Collectively, our studies suggest that neither proteolytic cleavage of talin nor interactions between talin and GPIIb-IIIa is required for the regulated redistribution of talin in thrombin-activated platelets. Phosphorylation of talin in response to thrombin activation may, however, be one mechanism utilized by platelets to regulate talin distribution and function in human platelets.

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Immunological Evidence for Prothrombin in Human Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661534 ◽

1986 ◽

Vol 55 (02) ◽

pp. 268-270

Author(s):

R J Alexander

Keyword(s):

Electrophoretic Mobility ◽

Platelet Activation ◽

Double Diffusion ◽

Human Platelets ◽

Secreted Proteins ◽

Platelet Surface ◽

Activated Platelets ◽

Similar Protein ◽

Diffusion Assay ◽

Supernatant Solution

SummaryAn attempt was made to isolate from plasma the platelet surface substrate for thrombin, glycoprotein V (GPV), because a GPV antigen was reported to be present in plasma (3). Plasma fractionation based on procedures for purification of GPV from platelets revealed a thrombin-sensitive protein with appropriate electrophoretic mobility. The protein was purified; an antiserum against it i) reacted with detergent-solubilized platelet proteins or secreted proteins in a double diffusion assay, ii) adsorbed a protein from the supernatant solution of activated platelets, and iii) inhibited thrombin-induced platelet activation, but the antiserum did not adsorb labeled GPV. The purified protein was immunochemically related to prothrombin rather than to GPV. Other antibodies against prothrombin were also able to adsorb a protein from platelets. It is concluded that 1) plasma does not contain appreciable amounts of GPV, and 2) platelets contain prothrombin or an immunochemically similar protein.

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Neutrophil P-selectin-glycoprotein-ligand-1 binding to platelet P-selectin enhances metalloproteinase 2 secretion and platelet-neutrophil aggregation

Thrombosis and Haemostasis ◽

10.1160/th05-05-0344 ◽

2005 ◽

Vol 94 (12) ◽

pp. 1230-1235 ◽

Cited By ~ 21

Author(s):

Haissam Abou-Saleh ◽

Jean-François Théorêt ◽

Daniel Yacoub ◽

Yahye Merhi

Keyword(s):

Platelet Activation ◽

Human Platelets ◽

Vascular Remodelling ◽

Adhesive Interaction ◽

Platelet Binding ◽

Enhanced Secretion ◽

Thrombin Activation ◽

Neutrophil Aggregation ◽

And Function ◽

The Impact

SummaryPlatelets and neutrophils constitute a high source of metalloproteinases (MMPs), and their interactions via P-selectin and Pselectin- glycoprotein-ligand-1 (PSGL-1) are involved in thrombosis, vascular remodelling, and restenosis. We investigated the impact of these interactions on platelet MMP-2 secretion and function in platelet and neutrophil aggregation. The secretion of MMP-2 from human platelets was significantly increased threefold after thrombin activation, and enhanced two-fold in the presence of neutrophils. Neutrophil supernatant had no effect on platelet MMP-2 secretion. While no MMP-2 was detected in the supernatant of neutrophils, a high amount of MMP-9 was released by neutrophils, and remained unchanged upon thrombin activation or in the presence of platelets. Platelet P-selectin, which increased significantly after activation, triggered platelet binding to neutrophils that was completely inhibited by P-selectin or PSGL-1 antagonists, and was reduced by 50% with a GPIIb/ IIIa antagonist. P-selectin or PSGL-1 antagonism abolished the enhanced secretion of platelet MMP-2 in the presence of neutrophils and reduced platelet-neutrophil aggregation. Platelet activation and binding to neutrophils enhance the secretion of platelet MMP-2 via an adhesive interaction between P-selectin and PSGL-1, which contribute to increase platelet-neutrophil aggregation.

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Prostaglandin E2 potentiates platelet aggregation by priming protein kinase C

Blood ◽

10.1182/blood.v82.9.2704.2704 ◽

1993 ◽

Vol 82 (9) ◽

pp. 2704-2713 ◽

Cited By ~ 53

Author(s):

R Vezza ◽

R Roberti ◽

GG Nenci ◽

P Gresele

Keyword(s):

Protein Kinase C ◽

Platelet Aggregation ◽

Protein Kinase ◽

Prostaglandin E2 ◽

Platelet Activation ◽

Human Platelets ◽

Platelet Surface ◽

Kinase C ◽

Activated Platelets ◽

Induced Aggregation

Abstract Prostaglandin E2 (PGE2) is produced by activated platelets and by several other cells, including capillary endothelial cells. PGE2 exerts a dual effect on platelet aggregation: inhibitory, at high, supraphysiologic concentrations, and potentiating, at low concentrations. No information exists on the biochemical mechanisms through which PGE2 exerts its proaggregatory effect on human platelets. We have evaluated the activity of PGE2 on human platelets and have analyzed the second messenger pathways involved. PGE2 (5 to 500 nmol/L) significantly enhanced aggregation induced by subthreshold concentrations of U46619, thrombin, adenosine diphosphate (ADP), and phorbol 12-myristate 13-acetate (PMA) without simultaneously increasing calcium transients. At a high concentration (50 mumol/L), PGE2 inhibited both aggregation and calcium movements. PGE2 (5 to 500 nmol/L) significantly enhanced secretion of beta-thromboglobulin (beta TG) and adenosine triphosphate from U46619- and ADP-stimulated platelets, but it did not affect platelet shape change. PGE2 also increased the binding of radiolabeled fibrinogen to the platelet surface and increased the phosphorylation of the 47-kD protein in 32P- labeled platelets stimulated with subthreshold doses of U46619. Finally, the amplification of U46619-induced aggregation by PGE2 (500 nmol/L) was abolished by four different protein kinase C (PKC) inhibitors (calphostin C, staurosporine, H7, and TMB8). Our results suggest that PGE2 exerts its facilitating activity on agonist-induced platelet activation by priming PKC to activation by other agonists. PGE2 potentiates platelet activation at concentrations produced by activated platelets and may thus be of pathophysiologic relevance.

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Junctional Adhesion Molecule a Helps Maintain Integrin αIIbβ3 in Resting State

Blood ◽

10.1182/blood.v112.11.111.111 ◽

2008 ◽

Vol 112 (11) ◽

pp. 111-111 ◽

Cited By ~ 1

Author(s):

Meghna Ulhas Naik ◽

Timothy J. Stalker ◽

Lawrence F. Brass ◽

Ulhas Pandurang Naik

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Adhesion Molecule ◽

Resting State ◽

Human Platelets ◽

Artery Occlusion ◽

In Vivo Model ◽

Platelet Surface ◽

Integrin Αiibβ3

Abstract Under physiological conditions, fibrinogen receptor integrin αIIbβ3 on the circulating platelets is in a low-affinity, or resting state, unable to bind soluble ligands. During platelet activation by agonists, a cascade of signaling events induces a conformational change in the extracellular domain of αIIbβ3, thereby converting it into a high-affinity state capable of binding ligands through a process known as “inside-out signaling”. What maintains this integrin in a low-affinity state is not well understood. We have previously identified JAM-A, junctional adhesion molecule A, on the platelet surface. We have shown that an antibody blockade of JAM-A dose-dependently activates platelets. To understand the molecular mechanism through which JAM-A regulates platelet aggregation, we used Jam-A null mice. Interestingly, the mouse bleeding times were significantly shortened in Jam-A null mice compared to wildtype littermates. Furthermore, the majority of these mice showed a rebleeding phenotype. This phenotype was further confirmed by FeCl3-induced carotid artery occlusion, a well-accepted in vivo model for thrombosis. Platelets derived from Jam-A-null mice were used to evaluate the role of JAM-A in agonist-induced platelet aggregation. We found that Jam-A null platelets showed enhanced aggregation in response to physiological agonists such as PAR4 peptide, collagen, and ADP as compared to platelets from wildtype littermates. JAM-A was found to associate with αIIbβ3 in unactivated human platelets, but this association was disrupted by both agonist-induced platelet aggregation and during outside-in signaling initiated upon platelet spreading on immobilized Fg. We also found that in resting platelets, JAM-A is phosphorylated on a conserved tyrosine 280 in its cytoplasmic domain, which was dephosphorylated upon platelet activation. Furthermore, JAM-A is rapidly and transiently phosphorylated on serine 284 residue during platelet activation by agonists. Interestingly, JAM-A also formed a complex with Csk, a tyrosine kinase known to be inhibitory to Src activation, in resting platelets. This complex was dissociated upon activation of platelets by agonists. These results suggest that tyrosine-phosphorylated JAM-A recruits Csk to αIIbβ3 in resting platelets, thus maintaining a low-affinity state of integrin αIIbβ3. Agonist–induced activation of platelets results in rapid dephosphorylation of JAM-A on Y280 and phosphorylation on S284 residues. This causes dissociation of JAM-A from integrin αIIbβ3 facilitating platelet aggregation.

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Integrin-αIIbβ3-mediated outside-in signalling activates a negative feedback pathway to suppress platelet activation

Thrombosis and Haemostasis ◽

10.1160/th16-02-0096 ◽

2016 ◽

Vol 116 (11) ◽

pp. 918-930 ◽

Cited By ~ 8

Author(s):

Baiyun Dai ◽

Peng Wu ◽

Feng Xue ◽

Renchi Yang ◽

Ziqiang Yu ◽

...

Keyword(s):

Flow Cytometry ◽

Platelet Activation ◽

Negative Feedback ◽

Human Platelets ◽

Wild Type ◽

Integrin Αiibβ3 ◽

Atp Secretion ◽

Platelet Disaggregation ◽

Feedback Pathway

SummaryIntegrin-αIIbβ3-mediated outside-in signalling is widely accepted as an amplifier of platelet activation; accumulating evidence suggests that outside-in signalling can, under certain conditions, also function as an inhibitor of platelet activation. The role of integrin-αIIbβ3-mediated outside-in signalling in platelet activation is disputable. We employed flow cytometry, aggregometry, immunoprecipitation, and immunoblotting to investigate the role of integrin-αIIbβ3-mediated outside-in signalling in platelet activation. Integrin αIIbβ3 inhibition enhances agonist-induced platelet ATP secretion. Human platelets lacking expression of αIIbβ3 exhibited more platelet ATP secretion than their wild-type counterparts. Moreover, integrin-αIIbβ3-mediated outside-in signals activate SHIP-1, which in turn mediates p-Akt dep-hosphorylation, leading to inactivation of PI3K/Akt signalling. Furthermore, 3AC (SHIP-1 inhibitor) inhibits platelet disaggregation, and promotes platelet ATP secretion. Upon ADP stimulation, Talin is recruited to αIIbβ3, and it is dissociated from αIIbβ3 when platelets disaggregate. In addition, treatment with RUC2, an inhibitor of αIIbβ3, which blocks αIIbβ3-mediated outside-in signalling, can markedly prevent the dissociation of talin from integrin. SHIP1 Inhibitor 3AC inhibits the dissociation of talin from integrin-β3. These results suggest that integrin-αIIbβ3-mediated outside-in signalling can serve as a brake to restrict unnecessary platelet activation by activated SHIP-1, which mediated the disassociation of talin from β3, leading to integrin inactivation and blocking of PI3K/Akt signalling to restrict platelet ATP secretion.

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Recent Advances in the Discovery and Function of Antimicrobial Molecules in Platelets

International Journal of Molecular Sciences ◽

10.3390/ijms221910230 ◽

2021 ◽

Vol 22 (19) ◽

pp. 10230

Author(s):

Alba S. Aquino-Domínguez ◽

María de los A. Romero-Tlalolini ◽

Honorio Torres-Aguilar ◽

Sergio R. Aguilar-Ruiz

Keyword(s):

Antimicrobial Properties ◽

Human Platelets ◽

Rabbit Serum ◽

Functional Evaluation ◽

Host Defense Peptides ◽

Vascular Integrity ◽

Activated Platelets ◽

And Function ◽

Identification And Characterization

The conventional function described for platelets is maintaining vascular integrity. Nevertheless, increasing evidence reveals that platelets can additionally play a crucial role in responding against microorganisms. Activated platelets release molecules with antimicrobial activity. This ability was first demonstrated in rabbit serum after coagulation and later in rabbit platelets stimulated with thrombin. Currently, multiple discoveries have allowed the identification and characterization of PMPs (platelet microbicidal proteins) and opened the way to identify kinocidins and CHDPs (cationic host defense peptides) in human platelets. These molecules are endowed with microbicidal activity through different mechanisms that broaden the platelet participation in normal and pathologic conditions. Therefore, this review aims to integrate the currently described platelet molecules with antimicrobial properties by summarizing the pathways towards their identification, characterization, and functional evaluation that have promoted new avenues for studying platelets based on kinocidins and CHDPs secretion.

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Platelet Proinflammatory and Hemostatic Function Is Differentially Regulated in Cystic Fibrosis.

Blood ◽

10.1182/blood.v108.11.3948.3948 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3948-3948

Author(s):

Alexander Sturm ◽

Helge Hebestreit ◽

Ralf Grossmann

Keyword(s):

Cystic Fibrosis ◽

Chronic Inflammation ◽

Platelet Activation ◽

Platelet Function ◽

Healthy Controls ◽

Soluble Cd40 Ligand ◽

Integrin Αiibβ3 ◽

Markers Of Inflammation ◽

Activated Platelets

Abstract Introduction: Platelet function and mechanisms of platelet-leukocyte interactions have been investigated in several vascular und inflammatory disorders. In most studies, platelet activation and an increase of platelet-leukocyte-aggregates (PLA) could be observed. We investigated platelet function in clinically stable patients with cystic fibrosis (CF). Methods: In addition to routine markers of inflammation (e. g. CRP, IgG, ESR) parameters of platelet function were measured in 54 clinically stable CF patients and 55 healthy controls (age range 3 to 41 years): The percentage of P-selectin (CD62P) and PAC-1 (activated integrin αIIbβ3) positive resting and activated platelets (in-vitro activation with the thrombin receptor activating peptide 6) and the number of PLA were determined by flow cytometry. The plasma markers of platelet activation soluble P-selectin (sCD62P) and soluble CD40 ligand (sCD40L) were measured by ELISA. Furthermore, 15 CF patients and 14 healthy controls were investigated to determine CD41a-expression (integrin αIIbβ3) on resting and activated platelets as well as leukocyte expression of P-selectin-glycoprotein ligand 1 (PSGL-1, receptor of CD62P) and integrin αMβ2. Results: Chronic inflammation leads to a decrease of PAC-1-binding to resting and activated platelets. The effects were stronger in patients with higher markers of inflammation. CD41a expression was reduced on in-vitro-activated CF platelets. In contrast, proinflammatory platelet functions remained unchanged (CD62P-expression on resting and activated platelets) or increased (sCD62P, sCD40L, PLA). Leukocyte integrin αMβ2 expression was increased and PSGL-1 expression remained unchanged in CF. Discussion: Platelet function in clinically stable patients with CF is differentially regulated: Chronic inflammation leads to an upregulation of platelet proinflammatory function. In the presence of several procoagulatory mechanisms in inflammation there is a compensatory loss of platelet hemostatic function, shown by the decreased activation and exocytosis of the platelet major integrin αIIbβ3.

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The Roles of Akt1 and Akt2 in GPIb-IX-Mediated Platelet Activation Signaling.

Blood ◽

10.1182/blood.v110.11.3635.3635 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3635-3635

Author(s):

Hong Yin ◽

Aleksandra Stojanovic ◽

Nissim Hay ◽

Xiaoping Du

Keyword(s):

Shear Stress ◽

Signaling Pathway ◽

Platelet Activation ◽

Platelet Adhesion ◽

Thromboxane A2 ◽

Human Platelets ◽

Integrin Activation ◽

Akt Inhibitor ◽

Platelet Spreading ◽

Inside Out

Abstract The platelet von Willebrand factor (VWF) receptor, glycoprotein Ib-IX (GPIb-IX), mediates platelet adhesion and induces signaling leading to integrin activation. Phosphoinositol 3-kinase (PI3K) is important in GPIb-IX-mediated signaling. PI3K-dependent signaling mechanisms, however, are unclear. To understand the downstream signaling pathway of GPIb-IX signaling, we investigated the roles of PI3K effector kinases, Akt1 and Akt2, in VWF/GPIb-IX-induced platelet activation. VWF/GPIb-IX-induced platelet aggregation was impaired in Akt1- or Akt2-knockout mouse platelets and in human and mouse platelets treated with an Akt inhibitor, SH-6. GPIb-IX-mediated platelet stable adhesion to VWF under shear stress was also inhibited in mouse platelets deficient in Akt1 or Akt2, and in human platelets treated with SH-6. Interestingly, while deficiency of Akt1 or Akt2 caused nearly complete inhibition of stable platelet adhesion to VWF under shear stress, stable platelet adhesion was only partially reduced in platelets treated with both P2Y1 and P2Y12 ADP receptor antagonists, A3P5P and 2MeSAMP or thromboxane A2 pathway inhibitor, aspirin or Syk inhibitor, piceatannol. Therefore, Akt1 and Akt2 are important in early GPIb-IX signaling independent of Syk, ADP or thromboxane A2 (TXA2), in addition to their recognized roles in ADP- and TXA2-dependent secondary amplification pathways. Knockout of either Akt1 or Akt2 diminished platelet spreading on VWF, but not on immobilized fibrinogen. Thus, Akt1 and Akt2 are both required only in the GPIb-IX-mediated integrin activation (inside-out signaling). In contrast, PI3K inhibitors abolished platelet spreading on both VWF and fibrinogen, indicating a role for PI3K in integrin outside-in signaling distinct from that in GPIb-IX-mediated inside-out signaling. Furthermore, Akt1 or Akt2 deficiency diminished VWF-induced cGMP elevation, and their inhibitory effects on GPIb-IX-dependent platelet adhesion were reversed by low concentration of exogenous cGMP, indicating that Akt1 and Akt2 mediate GPIb-IX signaling via the cGMP-dependent signaling pathway. In conclusion, both Akt1 and Akt2 mediate VWF/GPIb-IX-induced signaling pathway leading to platelet activation and the consequent stable platelet adhesion, spreading and aggregation.

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