Characterization of the Platelet Phenotype Caused by a Germline RUNX1 Variant in a CRISPR/Cas9-Generated Murine Model

2021 ◽  
Author(s):  
Ana Marín-Quílez ◽  
Ignacio García-Tuñón ◽  
Cristina Fernández-Infante ◽  
Luis Hernández-Cano ◽  
Verónica Palma-Barqueros ◽  
...  
Keyword(s):  
Murine Model ◽  
Genetic Diagnosis ◽  
Adenosine Diphosphate ◽  
Pathogenic Potential ◽  
Related Disorder ◽  
Integrin Αiibβ3 ◽  
Fibrinogen Binding ◽  
Runx1 Gene ◽  
Granule Secretion ◽  
Platelet Spreading

Abstract RUNX1-related disorder (RUNX1-RD) is caused by germline variants affecting the RUNX1 gene. This rare, heterogeneous disorder has no specific clinical or laboratory phenotype, making genetic diagnosis necessary. Although international recommendations have been established to classify the pathogenicity of variants, identifying the causative alteration remains a challenge in RUNX1-RD. Murine models may be useful not only for definitively settling the controversy about the pathogenicity of certain RUNX1 variants, but also for elucidating the mechanisms of molecular pathogenesis. Therefore, we developed a knock-in murine model, using the CRISPR/Cas9 system, carrying the RUNX1 p.Leu43Ser variant (mimicking human p.Leu56Ser) to study its pathogenic potential and mechanisms of platelet dysfunction. A total number of 75 mice were generated; 25 per genotype (RUNX1WT/WT, RUNX1WT/L43S, and RUNX1L43S/L43S). Platelet phenotype was assessed by flow cytometry and confocal microscopy. On average, RUNX1L43S/L43S and RUNX1WT/L43S mice had a significantly longer tail-bleeding time than RUNX1WT/WT mice, indicating the variant's involvement in hemostasis. However, only homozygous mice displayed mild thrombocytopenia. RUNX1L43S/L43S and RUNX1WT/L43S displayed impaired agonist-induced spreading and α-granule release, with no differences in δ-granule secretion. Levels of integrin αIIbβ3 activation, fibrinogen binding, and aggregation were significantly lower in platelets from RUNX1L43S/L43S and RUNX1WT/L43S using phorbol 12-myristate 13-acetate (PMA), adenosine diphosphate (ADP), and high thrombin doses. Lower levels of PKC phosphorylation in RUNX1L43S/L43S and RUNX1WT/L43S suggested that the PKC-signaling pathway was impaired. Overall, we demonstrated the deleterious effect of the RUNX1 p.Leu56Ser variant in mice via the impairment of integrin αIIbβ3 activation, aggregation, α-granule secretion, and platelet spreading, mimicking the phenotype associated with RUNX1 variants in the clinical setting.

Conformation-Specific Blockade of αIIbβ3 by a Non-RGD Peptide to Inhibit Platelet Activation without Causing Significant Bleeding and Thrombocytopenia

2020 ◽  
Vol 120 (10) ◽  
pp. 1432-1441 ◽  
Author(s):  
Chuanbin Shen ◽  
Ming Liu ◽  
Huiwen Tian ◽  
Jiameng Li ◽  
Runjia Xu ◽  
...  
Keyword(s):  
Side Effects ◽  
Adenosine Diphosphate ◽  
Rgd Peptide ◽  
Active Conformation ◽  
Integrin Αiibβ3 ◽  
Fibrinogen Binding ◽  
Skin Secretions ◽  
Platelet Spreading ◽  
Carotid Arterial

AbstractBleeding and thrombocytopenia to readministration are the most serious side effects of clinical integrin αIIbβ3 antagonists such as RGD-containing peptides. Here we show that a non-RGD peptide ZDPI, identified from skin secretions of Amolops loloensis, inhibited platelet aggregation induced by agonists, such as adenosine diphosphate, collagen, arachidonic acid, PAR1AP, and integrin αIIbβ3 allosteric activator, and reduces soluble fibrinogen binding to activated platelets without perturbing adhesion numbers on immobilized fibrinogen. Further study showed that ZDPI preferred to bind to the active conformation of integrin αIIbβ3, and thus inhibited c-Src-mediated integrin signaling transduction. In contrast to currently used clinical blockers of integrin αIIbβ3, which are all conformation-unspecific blockers, ZDPI conformation specifically binds to activated integrin αIIbβ3, subsequently suppressing platelet spreading. In vivo study revealed that ZDPI inhibited carotid arterial thrombosis with limited bleeding and thrombocytopenia. A non-RGD peptide which targets the active conformation of integrin αIIbβ3, such as ZDPI, might be an excellent candidate or template to develop antithrombotics without significant bleeding and thrombocytopenia side effects.

Fibrinogen binding to the integrin αIIbβ3 modulates store-mediated calcium entry in human platelets

Blood ◽  
2001 ◽  
Vol 97 (9) ◽  
pp. 2648-2656 ◽  
Author(s):  
Juan A. Rosado ◽  
Else M. Y. Meijer ◽  
Karly Hamulyak ◽  
Irena Novakova ◽  
Johan W. M. Heemskerk ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Polymerization ◽  
Adenosine Diphosphate ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Integrin Αiibβ3 ◽  
Fibrinogen Binding

Abstract Effects of the occupation of integrin αIIbβ3 by fibrinogen on Ca++signaling in fura-2–loaded human platelets were investigated. Adding fibrinogen to washed platelet suspensions inhibited increases in cytosolic [Ca++] concentrations ([Ca++]i) evoked by adenosine diphosphate (ADP) and thrombin in a concentration-dependent manner in the presence of external Ca++ but not in the absence of external Ca++ or in the presence of the nonselective cation channel blocker SKF96365, indicating selective inhibition of Ca++entry. Fibrinogen also inhibited store-mediated Ca++ entry (SMCE) activated after Ca++ store depletion using thapsigargin. The inhibitory effect of fibrinogen was reversed if fibrinogen binding to αIIbβ3 was blocked using RDGS or abciximab and was absent in platelets from patients homozygous for Glanzmann thrombasthenia. Fibrinogen was without effect on SMCE once activated. Activation of SMCE in platelets occurs through conformational coupling between the intracellular stores and the plasma membrane and requires remodeling of the actin cytoskeleton. Fibrinogen inhibited actin polymerization evoked by ADP or thapsigargin in control cells and in cells loaded with the Ca++ chelator dimethyl BAPTA. It also inhibited the translocation of the tyrosine kinase p60src to the cytoskeleton. These results indicate that the binding of fibrinogen to integrin αIIbβ3 inhibits the activation of SMCE in platelets by a mechanism that may involve modulation of the reorganization of the actin cytoskeleton and the cytoskeletal association of p60src. This action may be important in intrinsic negative feedback to prevent the further activation of platelets subjected to low-level stimuli in vivo.

Impaired “outside-in” integrin αIIbβ3 signaling and thrombus stability in TSSC6-deficient mice

Blood ◽  
2006 ◽  
Vol 108 (6) ◽  
pp. 1911-1918 ◽  
Author(s):  
Matt W. Goschnick ◽  
Lai-Man Lau ◽  
Janet L. Wee ◽  
Yong S. Liu ◽  
P. Mark Hogarth ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Fluorescein Isothiocyanate ◽  
Clot Retraction ◽  
In Vivo Evaluation ◽  
Integrin Αiibβ3 ◽  
Normal Platelet ◽  
Granule Secretion ◽  
Platelet Spreading ◽  
Deficient Mice

AbstractWe investigated the role of the hematopoietic-specific tetraspanin superfamily member, TSSC6, in platelet function using wild-type mice and TSSC6-deficient mice. TSSC6 is expressed on the surface of murine platelets and is up-regulated by thrombin stimulation, indicating an intracellular pool of TSSC6. Immunoprecipitation/Western blot studies reveal a constitutive physical association of TSSC6 with the integrin αIIbβ3 complex under strong detergent conditions. In vivo evaluation of hemostasis by tail bleeding revealed increased bleeding time, volume of blood lost, and evidence of tail rebleeds in TSSC6 null mice, indicating unstable hemostasis. Using ex vivo techniques, we showed that TSSC6-deficient platelets exhibited impaired kinetics of clot retraction, platelet aggregation at lower doses of PAR-4, and collagen and platelet spreading on fibrinogen in the presence of normal integrin αIIbβ3 expression. TSSC6-deficient platelets showed normal alpha granule secretion, normal “insideout” integrin αIIbβ3 signaling (fluorescein isothiocyanate [FITC]–fibrinogen and JON/A binding), and normal platelet adhesion on fibrinogen. Furthermore, we show that absence of platelet TSSC6 affects the secondary stability of arterial thrombi in vivo upon vascular injury. These data demonstrate that TSSC6 appears to regulate integrin αIIbβ3 “outside-in” signaling events in platelets and is necessary for stability of arterial thrombi in vivo.

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

2015 ◽  
Vol 113 (02) ◽  
pp. 290-304 ◽  
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.

scholarly journals Coenzyme Q10 Attenuates Platelet Integrin αIIbβ3 Outside-in Signaling through Targeting cAMP/PKA Pathway and Inhibits Atherosclerosis

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2423-2423
Author(s):  
Yan Yang ◽  
Xiaohong Ruby Xu ◽  
Heyu Ni ◽  
Liping Ma ◽  
Wenhua Ling ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Clot Retraction ◽  
Integrin Αiibβ3 ◽  
Granule Secretion ◽  
Platelet Spreading ◽  
Pka Pathway ◽  
Inside Out

Abstract Introduction: Platelet integrin αIIbβ3 outside-in signaling is crucial for platelet adhesion and aggregation, and contributes to atherogenesis. Coenzyme Q10 (CoQ10) has been implicated as a protective factor against cardiovascular diseases (CVDs), particularly atherosclerosis. However, whether CoQ10 attenuates atherosclerosis through inhibiting platelet function and αIIbβ3 outside-in signaling is unknown. The aim of this study was to explore whether CoQ10 affects platelet function and αIIbβ3 outside-in signalling and thus inhibiting the progress of atherosclerosis in vivo and the underlying mechanisms in vitro. Methods: In vitro study, The murine platelet rich plasma (PRP) from C57BL/6J wild-type (WT) mice or human PRP and gel-filtered platelets were incubated with different concentrations (1, 10 or 100 μM) of CoQ10 or the vehicle control for 50 min. Platelet aggregation, spreading on fibrinogen (Fg) and clot retraction were determined. In addition, the effects of CoQ10 on platelet integrin αIIbβ3 inside-out signalling (e.g., talin-1 and kindlin-3 binding to integrin β3) were determined by immunoprecipitation, and outside-in signalling (e.g., phosphorylation of sarcoma tyrosine-protein kinase (c-Src), focal adhesion kinase (FAK), and β3 cytoplasmic tail, myosin light chain (MLC)) were determined by Western blotting. The levels of platelet ATP and cAMP were measured by ELISA assays. In vivo study, male homozygous apolipoprotein E-deficient (apoE-/-) mice (C57BL/6 genetic background) were fed either a standard normal AIN-93G diet (NC group), a Western-type diet (HFD group) or a Western-type diet supplemented with CoQ10 (1800 mg/kg diet) (CoQ10 group) for 12 weeks. Platelet aggregation, granule secretion, platelet spreading, clot retraction, integrin αIIbβ3 outside-in signalling, platelet-leukocyte interactions and carotid artery plaque area were also examined. In our randomized, double-blind, placebo-controlled trial, 101 hypercholesterolemic subjects were randomly administrated to 120 mg CoQ10 or placebo daily for 24 weeks. Platelet intracellular CoQ10 levels, platelet aggregation in PRP, platelet platelet factor 4 (PF-4) and C-C motif ligand 5 (CCL5) release, and platelet integrin αIIbβ3 outside-in signalling were also evaluated before and after 24 weeks of intervention. Results: We found that CoQ10 inhibited human and WT mouse platelet aggregation, platelet spreading, granule secretion, and clot retraction in vitro and apoE-/- mice on a high fat diet. CoQ10 also reduced atherosclerosis and platelet-monocyte aggregation in apoE-/- mice. The inhibitory effects of CoQ10 is mediated by attenuated αIIbβ3 outside-in signalling pathway (e.g., attenuation of phosphorylation of c-Src, FAK, and β3 cytoplasmic tail, and MLC in thrombin-activated platelets or platelets exposed to immobilized Fg), which requires up-regulation of the cAMP/PKA pathway, where CoQ10 inhibited phosphodiesterase 3A activity and activated the A2A adenosine receptor. However, CoQ10 did not affect platelet integrin αIIbβ3 inside-out signalling pathway, platelet cellular ATP, or platelet apoptosis (the mitochondrial membrane potential and phosphatidylserine exposure). Moreover, our clinical trial in dyslipidemic patients demonstrated that CoQ10 supplementation attenuated platelet aggregation, which was positively correlated with the increased platelet CoQ10 concentrations, inhibited αIIbβ3 outside-in signalling and decreased platelet PF-4 and CCL5 secretion. Conclusions: We present new data to suggest that CoQ10 plays a novel role in attenuating platelet function and integrin αIIbβ3 outside-in signalling though targeting cAMP/PKA signalling cascade and thus inhibiting the progress of atherosclerosis. CoQ10 is therefore a promising agent for the prevention and/or treatment for cardiovascular disease. Disclosures No relevant conflicts of interest to declare.

CIB1 Positively Regulates Outside-In Signaling through αIIbβ3 by Enhancing FAK and Src Activity.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3917-3917
Author(s):  
Meghna U. Naik ◽  
Ulhas P. Naik
Keyword(s):  
Cytochalasin D ◽  
Blocking Antibody ◽  
Integrin Αiibβ3 ◽  
Cytoskeletal Rearrangement ◽  
Essential Step ◽  
Fibrinogen Receptor ◽  
Src Inhibitor ◽  
Initial Sequence ◽  
Fibrinogen Binding ◽  
Platelet Spreading

Abstract Integrin αIIbβ3, the platelet fibrinogen receptor, mediates outside-in signaling upon fibrinogen binding. Calcium- and integrin-binding protein 1 (CIB1) specifically interacts with the cytoplasmic domain of αIIb and is involved in platelet spreading. Here we show that CIB1 binding to αIIb is an essential step in the propagation of outside-in signaling through integrin αIIbβ3. Incorporation of BAPTA-AM completely blocked outside-in signaling and CIB-1 binding to aIIb, suggesting that an intracellular Ca2+ rise induced by fibrinogen binding is required for CIB1 interaction with αIIb. When CIB1-binding to αIIb was inhibited by introduction of a function blocking antibody or a synthetic peptide corresponding to αIIb tail, CIB1 localization at the tip of the filopodia, but filopodia formation was not blocked. This result suggests that interaction of CIB1 with αIIb is not required for filopodia formation, but is needed for CIB1 accumulation at the filopodia, as well as platelet spreading. Immunoprecipitation experiments showed that during outside-in signaling, CIB1 associates with FAK. Although association of FAK and CIB1 does not require dynamic rearrangement of the cytoskeleton, their accumulation at the filopodia and the activation of FAK is dependent on cytoskeletal rearrangement, as treatment with cytochalasin D after the platelets form filopodia affects CIB1 localization. Interestingly, the interaction of CIB1 with αIIb upon fibrinogen binding is also necessary for FAK and Src activation. However, Src activity is not required for CIB1 accumulation at the filopodia since this accumulation was not stopped by the Src inhibitor PP2, despite blocking platelet spreading. Our results suggest that during outside-in signaling an intracellular Ca2+ rise occurs that facilitates CIB1 interaction with αIIb. CIB1 recruits FAK to this complex and is localized to the filopodia dependent upon dynamic cytoskeletal rearrangement. Furthermore, activation of Src and FAK requires interaction of CIB1 with αIIb. Although Src activity is not required for the accumulation of CIB1 at the filopodia, it is required for platelet spreading on immobilized fibrinogen. Thus we provide evidence for initial sequence of outside-in signaling in platelets.

CIB1 Is Required for FAK Activation during Outside-in Signaling through Platelet Integrin αIIbβ3

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2873-2873 ◽  
Author(s):  
Ulhas Pandurang Naik ◽  
Meghna Ulhas Naik
Keyword(s):  
Resting State ◽  
Ex Vivo ◽  
Clot Retraction ◽  
Integrin Αiibβ3 ◽  
High Affinity ◽  
Fibrinogen Binding ◽  
Platelet Spreading ◽  
Inside Out ◽  
Dependent Interaction

Abstract Platelets play an important role in the processes of hemostasis and thrombosis. Platelet integrin αIIbβ3 mediates bi-directional signaling during these processes. Agonist-dependent activation of integrin αIIbβ3 through inside-out signaling results in high-affinity binding of soluble ligands, such as fibrinogen. Fibrinogen binding induces a cascade of signaling through the integrin, termed outside-in signaling that results in platelet aggregation and clot retraction. Previously, we have characterized CIB1, a calcium- and integrin-binding protein that specifically interacts with the cytoplasmic domain of αIIb. Previous reports using in vitro and ex vivo studies implicated that CIB1 is involved in maintaining αIIbβ3 in its resting state, agonist-induced activation of the integrin, and outside-in signaling resulting in platelet spreading. Here, we show that platelet filopodia formation induced by fibrinogen binding to integrin αIIbβ3 needs Ca2+, but is independent of the Ca2+-dependent interaction of CIB1 with αIIb. Additionally, dynamic rearrangement of the cytoskeleton is required for the recruitment of FAK to the CIB1-αIIb complex at the filopodia and FAK activation. Moreover, disruption of the association of CIB1 and αIIb by incorporation of αIIb peptide or CIB1 antibody inhibited FAK activation. Furthermore, Cib1 null platelets acquired a spiky morphology and failed to fully spread on immobilized fibrinogen. Interestingly, FAK activation was significantly reduced in Cib1 null platelets exposed to immobilized fibrinogen. Our results suggest that during outside-in signaling, a rise in the intracellular Ca2+ level and filopodia formation occurs prior to the interaction of CIB1 with αIIb. Additionally, Ca2+ bound CIB1 recruits FAK to the αIIbβ3 complex at the filopodia, where FAK is activated, resulting in platelet spreading. Thus, our results have provided a mechanism through which CIB1 regulates outside-in signaling through integrin αIIbβ3.

Caspase-12: a developmental link between G-protein–coupled receptors and integrin αIIbβ3 activation

Blood ◽  
2004 ◽  
Vol 104 (5) ◽  
pp. 1327-1334 ◽  
Author(s):  
Steven W. Kerrigan ◽  
Meenakshi Gaur ◽  
Ronan P. Murphy ◽  
Sanford J. Shattil ◽  
Andrew D. Leavitt
Keyword(s):  
G Protein ◽  
Adenosine Diphosphate ◽  
G Protein Coupled Receptors ◽  
Free Calcium ◽  
Wild Type ◽  
Integrin Αiibβ3 ◽  
Caspase 12 ◽  
Fibrinogen Binding ◽  
G Protein Coupled ◽  
Inside Out

Abstract Fibrinogen binding by integrin αIIbβ3 is promoted by platelet agonists that increase the affinity and avidity of αIIbβ3 for fibrinogen through a process called “inside-out” signaling. Having previously demonstrated that inside-out activation of αIIbβ3 is defective in murine megakaryocytes that lack the transcription factor NF-E2, we screened for NF-E2–regulated genes that affect αIIbβ3 activation. Caspase-12 is the most down-regulated gene we identified in NF-E2–/– megakaryocytes. Therefore, the role of this protein in αIIbβ3 activation was determined using platelets from caspase-12–/– mice. Despite wild-type levels of αIIbβ3, caspase-12–/– platelets exhibit reduced fibrinogen binding to αIIbβ3 following stimulation by adenosine diphosphate (ADP) or protease-activated receptor 4 (PAR4) receptor-activating peptide. The defect in αIIbβ3 activation is associated with decreased cytosolic free calcium and inositol triphosphate levels, and with reduced aggregation, despite wild-type phospholipase Cβ expression levels. In contrast, agonist-induced surface expression of P-selectin, suppression of cAMP levels following ADP stimulation, and spreading on immobilized fibrinogen are unimpaired. Moreover, although caspase-12 is highly expressed in mature megakaryocytes, it is undetectable in platelets. Taken together, these studies establish that caspase-12 expression in murine megakaryocytes is regulated, directly or indirectly, by NF-E2, and suggest that caspase-12 participates in the development of fully functional signaling pathways linking some G-protein–coupled receptors to αIIbβ3 activation.

scholarly journals Antithrombotic effects of targeting αIIbβ3 signaling in platelets

Blood ◽  
2009 ◽  
Vol 113 (15) ◽  
pp. 3585-3592 ◽  
Author(s):  
Ararat J. Ablooglu ◽  
Jian Kang ◽  
Brian G. Petrich ◽  
Mark H. Ginsberg ◽  
Sanford J. Shattil
Keyword(s):  
Tyrosine Phosphorylation ◽  
Adenosine Diphosphate ◽  
Spontaneous Bleeding ◽  
Glycoprotein Vi ◽  
Fibrinogen Binding ◽  
Vessel Injury ◽  
Antithrombotic Effects ◽  
Platelet Spreading

Abstract αIIbβ3 interaction with fibrinogen promotes Src-dependent platelet spreading in vitro. To determine the consequences of this outside-in signaling pathway in vivo, a “β3(Δ760-762)” knockin mouse was generated that lacked the 3 C-terminal β3 residues (arginine-glycine-threonine [RGT]) necessary for αIIbβ3 interaction with c-Src, but retained β3 residues necessary for talin-dependent fibrinogen binding. β3(Δ760-762) mice were compared with wild-type β3+/+ littermates, β3+/− heterozygotes, and knockin mice where β3 RGT was replaced by β1 C-terminal cysteine-glycine-lysine (EGK) to potentially enable signaling by Src kinases other than c-Src. Whereas β3+/+, β3+/− and β3/β1(EGK) platelets spread and underwent tyrosine phosphorylation normally on fibrinogen, β3(Δ760-762) platelets spread poorly and exhibited reduced tyrosine phosphorylation of c-Src substrates, including β3 (Tyr747). Unlike control mice, β3(Δ760-762) mice were protected from carotid artery thrombosis after vessel injury with FeCl3. Some β3(Δ760-762) mice exhibited prolonged tail bleeding times; however, none demonstrated spontaneous bleeding, excess bleeding after surgery, fecal blood loss, or anemia. Fibrinogen binding to β3(Δ760-762) platelets was normal in response to saturating concentrations of protease-activated receptor 4 or glycoprotein VI agonists, but responses to adenosine diphosphate were impaired. Thus, deletion of β3 RGT disrupts c-Src–mediated αIIbβ3 signaling and confers protection from arterial thrombosis. Consequently, targeting αIIbβ3 signaling may represent a feasible antithrombotic strategy.

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

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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