Adaptor Protein CIN85 Supports Platelet Integrin αIIbβ3 outside-in Signaling and Thrombus Formation

Abstract Activation of platelets in the setting of an atherosclerotic rupture contributes to thrombosis. Platelet responses at these sites are dependent on agonist induced inside-out and integrin αIIbβ3 mediated outside-in signaling processes, which in turn are regulated by protein kinases and phosphatases. Reversible tyrosine and/or serine/threonine phosphorylation dependent assembly of effector proteins propagate signaling downstream of platelet receptors. Adaptor proteins are key effectors in signal transmission because they are endowed with multiple structural domains, which enable them to engage with a wide variety of proteins in a spatial and temporal fashion to fine tune signaling. Emerging studies in the field suggest a potential for targeting integrin-induced outside-in signaling processes to attenuate thrombotic responses. During the course of our studies to understand how the catalytic subunit of protein phosphatase 2A (PP2Ac) regulates platelet integrin outside-in signaling, we previously identified a new complex of PP2Ac with an adaptor protein CIN85 (Cbl-interacting protein of 85kDa) in human platelets. Disruption of an endogenous PP2Ac-CIN85 complex with a cell permeable myristylated P3 synthetic peptide decreased platelet integrin αIIbβ3-dependent signaling functions. However, since the adaptor protein CIN85 has not been characterized in platelets, the contribution of CIN85 in integrin signaling is unknown. To explore the potential role of CIN85 in integrin function, we generated a platelet specific CIN85-/- mice by crossing the CIN85 flox/flox mice with a PF4Cre mice. Immunoblotting studies confirmed that platelets but not the non-megakaryocytic tissues from CIN85-/- mice lost the ~85kDa CIN85 protein. Loss of CIN85 did not significantly alter agonist-induced primary aggregation response, suggesting comparable inside-out signaling response. In contrast, integrin αIIbβ3 outside-in signaling responses such as spreading on immobilized fibrinogen and fibrin-mediated clot retraction was decreased in CIN85-/- platelets. Activation of Src that promotes integrin outside-in signaling was decreased in fibrinogen engaged CIN85-/- platelets. Perfusion of whole blood from the CIN85-/- mice on collagen at a shear rate of 1000 s-1 revealed significantly decreased platelet adhesion and thrombus formation. PLCγ2 is activated downstream of platelet collagen receptor engagement, and CIN85-/- platelets showed decreased PLCγ2 Tyr 529 phosphorylation. These studies indicate that the adaptor protein CIN85 supports platelet integrin outside in signaling functions. To extend these findings in human platelets, we disrupted CIN85-PP2A complex with the myristylated P3 peptide. P3 peptide but not the scrambled peptide significantly decreased human platelet adhesion and thrombus formation on collagen at a shear rate of 1000 s-1. Thus, loss of CIN85 in murine platelets or disruption of CIN85-PP2A complex in human platelets attenuates integrin outside-in signaling and thrombus formation. These studies suggest that CIN85 may represent a new potential anti-thrombotic target. Disclosures No relevant conflicts of interest to declare.

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CRGT Peptide In Cytoplasm Regulates Thrombus Formation Via Dissociating c-Src-β3 Interaction

Blood ◽

10.1182/blood.v122.21.3501.3501 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3501-3501

Author(s):

Jiansong Huang ◽

Xiaofeng Shi ◽

Wenda Xi ◽

Ping Liu ◽

Xiaodong Xi

Keyword(s):

Molecular Mechanisms ◽

Platelet Adhesion ◽

Thrombus Formation ◽

Human Platelets ◽

Flow Conditions ◽

Cho Cell ◽

Integrin Αiibβ3 ◽

Shear Rates ◽

Platelet Adhesion And Aggregation

Abstract The RGT sequences of the integrin β3 tail directly and constitutively bind the inactive c-Src, regulating integrin αIIbβ3 signaling and platelet function. Previous work has shown that disrupting the interaction of c-Src with β3 via myristoylated RGT peptide or deletion of the RGT sequences in β3 selectively inhibits integrin αIIbβ3 outside-in signaling in platelets. However, the precise molecular mechanisms by which the Src-β3 association regulates integrin αIIbβ3 signaling need to be clarified. We found that active c-Src phosphoylated the Y747 and Y759 residues of β3 directly at the in vitro protein/protein level or in CHO cell models bearing Tac-β3 chimeras, which were devoid of the intact β3 signal transduction. Furthermore, data from mass spectrometry, [γ-32P] ATP incorporation assays and CHO cell/Tac-β3 chimeras demonstrated that the direct phosphorylation of Y747 and Y759 by active c-Src did not depend on the binding of c-Src to the RGT sequences of the β3 tail. To further investigate the biological functions of Src-β3 association in signal transduction we employed a cell-permeable and reduction-sensitive peptide (myr-AC∼CRGT), which disrupted the Src-β3 association in platelets independent of membrane-anchorage, and found that when platelets were stimulated by thrombin the c-Src activation and the phosphorylation of the tyrosine residues of the β3 tail were substantially inhibited by the presence of the peptide. These results suggest that one of the crucial biological functions of Src-β3 association is to serve as a “bridge” linking integrin signaling with the c-Src full activation and phosphorylation of the tyrosines of the β3 tail. To answer whether the RGT peptide binding to Src is able to alter the enzymatic activity of c-Src, we examined the Src-Csk association, the phosphorylation status of Y416 and Y527 of c-Src and the c-Src kinase catalytic activity. Results showed that myr-AC∼CRGT did not dissociate Csk from c-Src in resting platelets and the phosphorylation level of Y416 and Y527 of c-Src remained unaltered. Consistent data were also obtained from in vitro analysis of the c-Src kinase catalytic activity in the presence of CRGT peptide. These results suggest that myr-AC∼CRGT peptide per se does not fully activate c-Src. Myr-AC∼CRGT was also found to inhibit integrin αIIbβ3 outside-in signaling in human platelets. To examine the effect of the myr-AC∼CRGT on platelet adhesion and aggregation under flow conditions, we measured the platelet thrombus formation under different shear rates. Myr-AC∼CRGT did not affect the platelet adhesion at a wall shear rate of 125 s-1. The inability of myr-AC∼CRGT to affect platelet adhesion and aggregation remained at 500 s-1 shear rates. At 1,500 s-1, or 5,000 s-1 rates, myr-AC∼CRGT partially inhibited platelet adhesion and aggregation. These observations indicate that the Src-regulated outside-in signaling plays a pivotal role in the stable thrombus formation and the thrombus growth under flow conditions. The present study reveals novel insights into the molecular mechanisms by which c-Src regulates integrin αIIbβ3 signaling, particularly the phorsphorylation of the β3 cytoplasmic tyrosines, and provides first evidence in human platelets that the RGT peptide or derivatives regulate thrombus formation through dissociating the Src-β3 interaction. The data of this work allow us to anticipate that intracellular delivery of the RGT peptide or its analogues may have potential in the development of a new antithrombotic strategy where only the Src-β3 interaction is specifically interrupted so as to provide an effective inhibition on thrombosis together with a decent hemostasis. Disclosures: No relevant conflicts of interest to declare.

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Role and regulation of phosphatidylinositol 3-kinase β in platelet integrin α2β1 signaling

Blood ◽

10.1182/blood-2011-07-364992 ◽

2012 ◽

Vol 119 (3) ◽

pp. 847-856 ◽

Cited By ~ 47

Author(s):

Alessandra Consonni ◽

Lina Cipolla ◽

Gianni Guidetti ◽

Ilaria Canobbio ◽

Elisa Ciraolo ◽

...

Keyword(s):

Knockout Mice ◽

Type I Collagen ◽

Thrombus Formation ◽

Human Platelets ◽

Time Dependent ◽

Type I ◽

Integrin Αiibβ3 ◽

Akt Phosphorylation ◽

Inside Out ◽

Stimulation Of

Abstract Integrin α2β1–mediated adhesion of human platelets to monomeric type I collagen or to the GFOGER peptide caused a time-dependent activation of PI3K and Akt phosphorylation. This process was abrogated by pharmacologic inhibition of PI3Kβ, but not of PI3Kγ or PI3Kα. Moreover, Akt phosphorylation was undetectable in murine platelets expressing a kinase-dead mutant of PI3Kβ (PI3KβKD), but occurred normally in PI3KγKD platelets. Integrin α2β1 failed to stimulate PI3Kβ in platelets from phospholipase Cγ2 (PLCγ2)–knockout mice, and we found that intracellular Ca2+ linked PLCγ2 to PI3Kβ activation. Integrin α2β1 also caused a time-dependent stimulation of the focal kinase Pyk2 downstream of PLCγ2 and intracellular Ca2+. Whereas activation of Pyk2 occurred normally in PI3KβKD platelets, stimulation of PI3Kβ was strongly reduced in Pyk2-knockout mice. Neither Pyk2 nor PI3Kβ was required for α2β1–mediated adhesion and spreading. However, activation of Rap1b and inside-out stimulation of integrin αIIbβ3 were reduced after inhibition of PI3Kβ and were significantly impaired in Pyk2-deficient platelets. Finally, both PI3Kβ and Pyk2 significantly contributed to thrombus formation under flow. These results demonstrate that Pyk2 regulates PI3Kβ downstream of integrin α2β1, and document a novel role for Pyk2 and PI3Kβ in integrin α2β1 promoted inside-out activation of integrin αIIbβ3 and thrombus formation.

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A Mechanism For Switch Of Integrin Signaling Direction and a New Anti-Thrombotic Strategy Through Selective Outside-In Signaling Inhibition

Blood ◽

10.1182/blood.v122.21.2295.2295 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2295-2295

Author(s):

Bo Shen ◽

Xiaojuan Zhao ◽

Kelly A O'Brien ◽

Aleksandra Stojanovic-Terpo ◽

Michael Keegan Delaney ◽

...

Keyword(s):

Side Effect ◽

Early Phase ◽

Conflicts Of Interest ◽

Thrombus Formation ◽

Late Phase ◽

Integrin Signaling ◽

Clot Retraction ◽

Integrin Antagonists ◽

Inside Out

Abstract Antagonists of platelet integrin alphaIIbbeta3 are potent anti-thrombotics due to critical roles of integrins in thrombosis. However, integrins are also important in hemostasis, and thus integrin antagonists have potentially life-threatening bleeding side effect. It would be ideal if we can develop integrin antagonists without bleeding side effect. Integrins transmit signals bidirectionally. Intracellular signals activate integrin alphaIIbbeta3, leading to talin-dependent integrin ligation, which is required for platelet adhesion and initial hemostatic thrombus formation. Integrin ligation in turn mediates Galpha13/Src-dependent outside-in signaling that stabilizes and amplify thrombi, which is crucial for occlusive arterial thrombosis. Here we show that talin and Galpha13 bind to mutually exclusive but distinct sites in integrin beta3, and their bindings are dynamically regulated during integrin signaling. The first talin binding wave mediates inside-out signaling and also “ligand-induced integrin activation”, but is not required for early phase outside-in signaling. Integrin ligation induces talin dissociation and Galpha13 binding, which selectively mediates early phase outside-in signaling. The second talin binding wave is associated with late phase outside-in signaling and clot retraction. Based on these findings, we have designed a selective inhibitor of Galpha13-integrin interaction, which specifically abolished outside-in signaling without affecting inside-out signaling and integrin ligation. Strikingly, this inhibitor potently inhibits occlusive thrombosis in vivo, but has no effect on tail bleeding time, in contrast to the current integrin antagonists. Thus, we have discovered a mechanism for switching the direction of integrin signaling and a new anti-thrombotic that does not cause bleeding. Disclosures: No elevant conflicts of interest to declare.

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JAM-A protects from thrombosis by suppressing integrin αIIbβ3-dependent outside-in signaling in platelets

Blood ◽

10.1182/blood-2011-12-397398 ◽

2012 ◽

Vol 119 (14) ◽

pp. 3352-3360 ◽

Cited By ~ 48

Author(s):

Meghna U. Naik ◽

Timothy J. Stalker ◽

Lawrence F. Brass ◽

Ulhas P. Naik

Keyword(s):

Platelet Activation ◽

Ex Vivo ◽

Thrombus Formation ◽

Receptor Activation ◽

Clot Retraction ◽

Integrin Αiibβ3 ◽

Genetic Ablation ◽

Fibrinogen Receptor ◽

Inside Out

Abstract Mounting evidence suggests that agonist-initiated signaling in platelets is closely regulated to avoid excessive responses to injury. A variety of physiologic agonists induce a cascade of signaling events termed as inside-out signaling that culminate in exposure of high-affinity binding sites on integrin αIIbβ3. Once platelet activation has occurred, integrin αIIbβ3 stabilizes thrombus formation by providing agonist-independent “outside-in” signals mediated in part by contractile signaling. Junctional adhesion molecule A (JAM-A), a member of the cortical thymocyte marker of the Xenopus (CTX) family, was initially identified as a receptor for a platelet stimulatory mAb. Here we show that JAM-A in resting platelets functions as an endogenous inhibitor of platelet function. Genetic ablation of Jam-A in mice enhances thrombotic function of platelets in vivo. The absence of Jam-A results in increase in platelet aggregation ex vivo. This gain of function is not because of enhanced inside-out signaling because granular secretion, Thromboxane A2 (TxA2) generation, as well as fibrinogen receptor activation, are normal in the absence of Jam-A. Interestingly, integrin outside-in signaling such as platelet spreading and clot retraction is augmented in Jam-A–deficient platelets. We conclude that JAM-A normally limits platelet accumulation by inhibiting integrin outside-in signaling thus preventing premature platelet activation.

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Effect of Aspirin and Dipyridamole on the Interaction of Human Platelets with Sub-Endothelium: Studies Using Citrated and Native Blood

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650150 ◽

1981 ◽

Vol 45 (02) ◽

pp. 136-141 ◽

Cited By ~ 24

Author(s):

Harvey J Weiss ◽

Vincent T Turitto ◽

William J Vicic ◽

Hans R Baumgartner

Keyword(s):

Shear Rate ◽

Platelet Adhesion ◽

Inner Core ◽

Thrombus Formation ◽

Human Subjects ◽

Rabbit Aorta ◽

Blood Platelet ◽

Inhibitory Effect ◽

Human Platelets ◽

Platelet Thrombi

SummaryThe effect of aspirin and dipyridamole ingestion on the interaction of platelets with the subendothelium was studied using both citrated blood and directly sampled (native) blood. After obtained control studies, normal human subjects ingested 0.6 g of aspirin, 150 mg of dipyridamole, or a placebo and studies were repeated 1½ hrs later. Subjects continued on placebo, aspirin (0.6 g b.i.d.) or dipyridamole (100 mg q.i.d.) for 6 days and studies were obtained 1½ hrs after the last dose. Blood was circulated through an annular chamber on whose inner core were mounted everted segments of de-endothelialized rabbit aorta. The wall shear rate was 2,600 sec-1. Surface coverage with adherent platelets and platelet thrombi, as well as several parameters of thrombus dimensions, were evaluated morphometrically. Aspirin ingestion markedly reduced platelet thrombi in citrated blood, – but had a much lesser inhibitory effect in native blood. Platelet adhesion was unaffected in native blood; it was slightly decreased in citrated blood, in contrast to previous findings in which a lower shear rate (800 sec-1) was used. Ingestion of dipyridamole did not inhibit platelet adhesion or thrombi in either citrated or native blood. The studies indicate that, with the flow conditions used, aspirin is a relatively weak inhibitor of platelet thrombus formation in directly sampled human blood.

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Abstract 27: Dok-1 Negatively Regulates Integrin aIIbß3 Outside-in Signaling and Inhibits Thrombosis

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.33.suppl_1.a27 ◽

2013 ◽

Vol 33 (suppl_1) ◽

Author(s):

Masaru Niki ◽

Hong Jin ◽

Anil K Chauhan ◽

Steven R Lentz

Keyword(s):

Carotid Artery ◽

Adaptor Protein ◽

Clot Retraction ◽

Thromboxane A2 Receptor ◽

Integrin Αiibβ3 ◽

Fibrinogen Binding ◽

Platelet Signaling ◽

Platelet Spreading ◽

Inside Out

Background Platelet agonists activate integrin αIIbβ3 to allow the binding of soluble fibrinogen (a process known as inside-out signaling). Subsequent platelet aggregation leads to αIIbβ3 outside-in signaling, which results in tyrosine phosphorylation of β3 and other proteins, cytoskeletal reorganization, and platelet spreading. It has been reported that the adapter protein Dok-1 binds to the cytoplasmic tail of β3 and inhibits αIIbβ3 activation, but the specific role of Dok-1 in regulating inside-out or outside-in platelet signaling remains undefined. Methods We assessed the effects of Dok-1 on platelet signaling and thrombosis in Dok-1 null (Dok-1-/-) mice. Inside-out signaling was assessed by measuring αIIbβ3 activation (using the JON/A antibody) and fibrinogen binding by flow cytometry after stimulation of platelets with thrombin, ADP, and/or the thromboxane A2 receptor agonist U46619. Outside-in signaling was examined by measuring platelet spreading and clot retraction. Tail-transection bleeding time and susceptibility to thrombotic occlusion of the carotid artery in response to photochemical injury (rose bengal) were also measured. Results No significant differences in JON/A or fibrinogen binding were detected between wild-type (Dok-1+/+) and Dok-1-/- platelets, suggesting that Dok-1 does not regulate inside-out signaling. In contrast, Dok-1-/- platelets exhibited increased clot retraction and enhanced spreading on fibrinogen upon thrombin stimulation compared to Dok-1+/+ platelets (P<0.05), suggesting that Dok-1 negatively regulates outside-in signaling. Compared with Dok-1+/+ mice, Dok-1-/- mice had shorter bleeding times (181±168 vs. 379±193 seconds; P<0.001) and Dok-1-/- mice had shorter times to stable occlusion times of the carotid artery after photochemical injury compared with Dok-1+/+ mice (16.4±6.1 vs. 25.0±8.1 minutes; P<0.05). Conclusions The adaptor protein Dok-1 functions to negatively regulate integrin αIIbβ3 outside-in signaling. Deficiency of Dok-1 results in a prothrombotic phenotype, with shorten bleeding times and enhanced arterial thrombosis.

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Integrin αIIb cytoplasmic Tail Is Essential for Integrin Outside-in Signaling and Regulation of in Vivo Thrombosis

Blood ◽

10.1182/blood.v124.21.4160.4160 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4160-4160

Author(s):

Meghna Ulhas Naik ◽

Ulhas P Naik

Keyword(s):

Platelet Aggregation ◽

Conflicts Of Interest ◽

Cytoplasmic Tail ◽

Cytoplasmic Domain ◽

P38 Map Kinase ◽

Clot Retraction ◽

Integrin Αiibβ3 ◽

Signaling Events ◽

Inside Out

Abstract Platelet aggregation plays an important role in physiological hemostasis and pathological thrombosis. Platelet agonists induce a series of events called inside-out signaling that lead to the activation of integrin αIIbβ3. Fibrinogen binding to the activated integrin relay signals termed as outside-in signaling that regulate thrombus growth and stability. Talin and kindlin bind to the integrin β3 cytoplasmic tail to induce inside-out signaling. Although, integrin αIIb cytoplasmic domain also bind to a number of proteins, its importance in hemostasis or thrombosis is not well understood. Previously, we have identified a calcium- and integrin-binding protein (CIB1) that specifically interacts with the integrin αIIb cytoplasmic tail. Using a novel technique to inhibit interaction of CIB1 with integrin αIIb in intact human platelets, we have shown that CIB1 regulates outside-in signaling through integrin αIIbβ3. Recently, using Cib1-/- mice, we showed that CIB1 is a key regulator of thrombosis in vivo. Interestingly, agonist-induced platelet aggregation and secretion was normal in Cib1-/- platelets. Furthermore, expression or activation of integrin αIIbβ3 was also not affected by Cib1 deficiency, suggesting that integrin inside-out signaling is not affected in Cib1-/- platelets. However, adhesion and spreading on immobilized fibrinogen (Fg) was severely affected in Cib1-/- platelets. When we analyzed the rate of clot retraction, we found that significantly (P<0.001) delayed clot retraction was observed in Cib1-/- platelets compared to WT littermates, suggesting that integrin outside-in signaling is impaired in the absence of Cib1. To delineate the molecular mechanism regulated by CIB1 during platelet spreading and clot retraction, we analyzed the known signaling events activated during outside-in signaling. We found that Fg-dependent activation of ERK1 and p38 MAP kinase was significantly reduced in Cib1-/- null platelets. Furthermore, phosphorylation of the myosin light chain was also blocked in Cib1-/- platelets adhered to immobilized Fg. Furthermore, outside-in signaling-dependent tyrosine phosphorylation of β3 was greatly reduced in Cib1-/- platelets. When analyzed for the candidate tyrosine kinase responsible for reduced β3 phosphorylation, both Src and FAK activation was significantly reduced in Cib1-/- platelets. Furthermore, downstream signaling events such as activation of PAK1, PI3K, PDK1, as well as Akt were significantly affected in Cib1-/- platelets adhered to immobilized Fg. To test if impaired inhibition of GSK3-β is the cause of defective outside in signaling in Cib1-/- platelets we treated Cib1-/- platelets with SB216763, a specific GSK3-β inhibitor. We found that inhibition of GSK3-β rescued defective platelet adhesion and clot retraction observed in Cib1-/- platelets. It also rescued activation of p38 and Erk2 activation as well as MLC phosphorylation. However, activation of FAK, Src, PAK1, PI3K, PDK1, and Akt was not rescued, suggesting that these are upstream of GSK3-β. Furthermore, we found that outside-in dependent recruitment of FAK to the integrin-c-Src complex is greatly reduced in the absence of Cib1 suggesting that integrin αIIb cytoplasmic domain serves as a docking site for CIB1 so that it can recruit FAK to the integrin-c-Src complex and propagate outside-in signaling leading to GSK3-β inhibition, which is crucial for thrombus growth and stability. These in vivo and in vitro results clearly show that CIB1 regulates thrombosis by regulating outside-in signaling without affecting inside-out signaling through integrin αIIbβ3. Our results highlight an essential function to integrin αIIb cytoplasmic tail in regulating integrin outside-in signaling and thus thrombus growth and stability. Disclosures No relevant conflicts of interest to declare.

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The Antithrombotic Agent Pterostilbene Interferes with Integrin αIIbβ3-Mediated Inside-Out and Outside-In Signals in Human Platelets

International Journal of Molecular Sciences ◽

10.3390/ijms22073643 ◽

2021 ◽

Vol 22 (7) ◽

pp. 3643

Author(s):

Wei-Chieh Huang ◽

Kao-Chang Lin ◽

Chih-Wei Hsia ◽

Chih-Hsuan Hsia ◽

Ting-Yu Chen ◽

...

Keyword(s):

Platelet Activation ◽

Arterial Thrombosis ◽

Fibrin Clot ◽

Human Platelets ◽

Clot Retraction ◽

Antithrombotic Agent ◽

Strong Activity ◽

Integrin Αiibβ3 ◽

Occlusion Time ◽

Inside Out

Platelets play a crucial role in the physiology of primary hemostasis and pathological processes such as arterial thrombosis; thus, developing a therapeutic target that prevents platelet activation can reduce arterial thrombosis. Pterostilbene (PTE) has remarkable pharmacological activities, including anticancer and neuroprotection. Few studies have reported the effects of pterostilbene on platelet activation. Thus, we examined the inhibitory mechanisms of pterostilbene in human platelets and its role in vascular thrombosis prevention in mice. At low concentrations (2–8 μM), pterostilbene strongly inhibited collagen-induced platelet aggregation. Furthermore, pterostilbene markedly diminished Lyn, Fyn, and Syk phosphorylation and hydroxyl radical formation stimulated by collagen. Moreover, PTE directly hindered integrin αIIbβ3 activation through interfering with PAC-1 binding stimulated by collagen. In addition, pterostilbene affected integrin αIIbβ3-mediated outside-in signaling, such as integrin β3, Src, and FAK phosphorylation, and reduced the number of adherent platelets and the single platelet spreading area on immobilized fibrinogen as well as thrombin-stimulated fibrin clot retraction. Furthermore, pterostilbene substantially prolonged the occlusion time of thrombotic platelet plug formation in mice. This study demonstrated that pterostilbene exhibits a strong activity against platelet activation through the inhibition of integrin αIIbβ3-mediated inside-out and outside-in signaling, suggesting that pterostilbene can serve as a therapeutic agent for thromboembolic disorders.

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The Small GTPase Rap1b: A Bidirectional Regulator of Platelet Adhesion Receptors

Journal of Signal Transduction ◽

10.1155/2012/412089 ◽

2012 ◽

Vol 2012 ◽

pp. 1-9 ◽

Cited By ~ 20

Author(s):

Gianni Francesco Guidetti ◽

Mauro Torti

Keyword(s):

Cell Adhesion ◽

Signaling Pathways ◽

Platelet Adhesion ◽

Thrombus Formation ◽

Small Gtpases ◽

Small Gtpase ◽

Complex Pattern ◽

Adhesive Properties ◽

Adhesion Receptors ◽

Inside Out

Integrins and other families of cell adhesion receptors are responsible for platelet adhesion and aggregation, which are essential steps for physiological haemostasis, as well as for the development of thrombosis. The modulation of platelet adhesive properties is the result of a complex pattern of inside-out and outside-in signaling pathways, in which the members of the Rap family of small GTPases are bidirectionally involved. This paper focuses on the regulation of the main Rap GTPase expressed in circulating platelets, Rap1b, downstream of adhesion receptors, and summarizes the most recent achievements in the investigation of the function of this protein as regulator of platelet adhesion and thrombus formation.

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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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