scholarly journals ADAP interactions with talin and kindlin promote platelet integrin αIIbβ3 activation and stable fibrinogen binding

Blood ◽  
2014 ◽  
Vol 123 (20) ◽  
pp. 3156-3165 ◽  
Author(s):  
Ana Kasirer-Friede ◽  
Jian Kang ◽  
Bryan Kahner ◽  
Feng Ye ◽  
Mark H. Ginsberg ◽  
...  
Keyword(s):  
Integrin Αiibβ3 ◽  
Fibrinogen Binding ◽  
Key Points ◽  
Molecular Machinery

Key Points ADAP interacts with talin and kindlin-3 in platelets. ADAP is a hematopoietic component of the molecular machinery that promotes activation of and stable fibrinogen binding to αIIbβ3.

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.

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.

Targeted Gene Correction of Glanzmann Thrombasthenia Induced Pluripotent Stem Cells Restores Surface Expression and Fibrinogen Binding of Integrin αIIbβ3,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4173-4173
Author(s):  
Spencer Sullivan ◽  
Jason A. Mills ◽  
Li Zhai ◽  
Prasuna Paluru ◽  
Guohua Zhao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Cell Lines ◽  
Surface Expression ◽  
Ips Cells ◽  
Ips Cell ◽  
Glanzmann Thrombasthenia ◽  
Integrin Αiibβ3 ◽  
Fibrinogen Binding ◽  
Induced Pluripotent

Abstract Abstract 4173 Glanzmann Thrombasthenia (GT) is a rare, autosomal recessive disorder resulting from an absence of functional platelet integrin αIIbβ3, leading to impaired platelet aggregation and clinically presenting with severe bleeding. It is a model of an inherited platelet disorder that might benefit from corrective gene therapy. Treatment options for GT are limited and largely supportive. They include anti-fibrinolytics, activated factor VII, platelet transfusions, and bone marrow transplantation. Recent gene therapy research in a canine model for GT demonstrated that lentiviral transduction of mobilized hematopoietic stem cells could restore 6% αIIbβ3 receptors in thrombasthenic canine platelets relative to wild type (WT) canine platelets. As an alternative gene therapy strategy, we generated induced pluripotent stem (iPS) cell lines from the peripheral blood of two patients with GT and examined whether a megakaryocyte-specific promoter driving αIIb cDNA expression within the AAVS1 safe harbor locus could ameliorate the GT phenotype in iPS cell-derived megakaryocytes. Patient 1 is a compound heterozygote for αIIb with the following two missense mutations: exon 2 c.331T>C (p.L100P) and exon 5 c.607G>A (p.S192N). Patient 2 is homozygous for a c.818G>A (p.G273D) mutation adjacent to the first calcium-binding domain of αIIb, leading to impaired intracellular transport of αIIbβ3. Both patients express <5% αIIbβ3 on the surface of their platelets. Peripheral blood mononuclear cells from both GT patients and WT controls were efficiently reprogrammed to pluripotency using a doxycycline-inducible polycistronic lentivirus containing OCT4, KLF4, SOX2, and CMYC. Transgene constructs using a murine GPIbα promoter driving either a green fluorescent protein (GFP) reporter or αIIb cDNA were inserted into a gene-targeting vector specific for the first intron of AAVS1, a locus amenable to gene targeting and resistant to transgene silencing in human iPS cells. The GPIbα-driven GFP transgene was efficiently targeted into AAVS1 in WT iPS cells using zinc finger nuclease-mediated homologous recombination, as was the αIIb construct into GT iPS cell lines. PCR and Southern blot analyses confirmed single, non-random, transgene integrations. The iPS cells were differentiated into megakaryocytes using a feeder-free/serum-free adherent monolayer protocol and analyzed by flow cytometry. GFP, along with endogenous CD41 (αIIb), was initially expressed in primitive WT hematopoietic progenitor cells. GFP expression was lost in erythrocytes and myeloid cells, but maintained in CD41+/CD42+ megakaryocytes, demonstrating that this transgenic construct mirrors endogenous CD41 expression. The GT phenotype was confirmed in megakaryocytes derived from patient iPS cells, showing loss of αIIbβ3 expression. When compared to WT iPS cell-derived megakaryocytes, gene-corrected GT iPS cell-derived megakaryocytes showed >50% and >70% αIIbβ3 surface expression for patients 1 and 2, respectively. Both patients' iPS cell-derived megakaryocytes also demonstrated fibrinogen binding upon thrombin activation. This is the first report of the generation and genetic correction of iPS cell lines from patients with a disease affecting platelet function. These findings suggest that this GPIbα-promoter construct targeted to the AAVS1 locus drives megakaryocyte-specific expression at a therapeutically significant level, which offers the possibility of correcting severe inherited platelet disorders beginning with iPS cells derived from these affected individuals. Disclosures: Lambert: Cangene: Honoraria.

Integrin αIIbβ3 Reconstituted into Lipid Bilayers Is Nonclustered in Its Activated State but Clusters after Fibrinogen Binding†

Biochemistry ◽  
1997 ◽  
Vol 36 (24) ◽  
pp. 7395-7402 ◽  
Author(s):  
Eva-Maria Erb ◽  
Kirsten Tangemann ◽  
Bernd Bohrmann ◽  
Beate Müller ◽  
Jürgen Engel
Keyword(s):  
Lipid Bilayers ◽  
Integrin Αiibβ3 ◽  
Fibrinogen Binding ◽  
Activated State

scholarly journals Primary Megakaryocytes Reveal a Role for Transcription Factor Nf-E2 in Integrin αiibβ3 Signaling

1999 ◽  
Vol 147 (7) ◽  
pp. 1419-1430 ◽  
Author(s):  
Masamichi Shiraga ◽  
Alec Ritchie ◽  
Sallouha Aidoudi ◽  
Veronique Baron ◽  
David Wilcox ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Sindbis Virus ◽  
Genetic Manipulation ◽  
Focal Adhesions ◽  
Surface Expression ◽  
Normal Surface ◽  
Integrin Αiibβ3 ◽  
Fibrinogen Binding ◽  
Intracellular Signals ◽  
Bone Marrow Cultures

Platelet integrin αIIbβ3 responds to intracellular signals by binding fibrinogen and triggering cytoskeletal reorganization, but the mechanisms of αIIbβ3 signaling remain poorly understood. To better understand this process, we established conditions to study αIIbβ3 signaling in primary murine megakaryocytes. Unlike platelets, these platelet precursors are amenable to genetic manipulation. Cytokine-stimulated bone marrow cultures produced three arbitrary populations of αIIbβ3-expressing cells with increasing size and DNA ploidy: small progenitors, intermediate-size young megakaryocytes, and large mature megakaryocytes. A majority of the large megakaryocytes bound fibrinogen in response to agonists, while almost none of the smaller cells did. Fibrinogen binding to large megakaryocytes was inhibited by Sindbis virus-mediated expression of isolated β3 integrin cytoplasmic tails. Strikingly, large megakaryocytes from mice deficient in the transcription factor NF-E2 failed to bind fibrinogen in response to agonists, despite normal surface expression of αIIbβ3. Furthermore, while megakaryocytes from wild-type mice spread on immobilized fibrinogen and exhibited filopodia, lamellipodia and Rho-dependent focal adhesions and stress fibers, NF-E2–deficient megakaryocytes adhered poorly. These studies establish that agonist-induced activation of αIIbβ3 is controlled by NF-E2–regulated signaling pathways that mature late in megakaryocyte development and converge at the β3 cytoplasmic tail. Megakaryocytes provide a physiologically relevant and tractable system for analysis of bidirectional αIIbβ3 signaling.

scholarly journals Platelet Integrin αIIbβ3 Activation is Associated with 25-Hydroxyvitamin D Concentrations in Healthy Adults

2020 ◽  
Vol 120 (05) ◽  
pp. 768-775
Author(s):  
Floor E. Aleva ◽  
Rahajeng N. Tunjungputri ◽  
Lisa N. van der Vorm ◽  
Yang Li ◽  
Yvonne F. Heijdra ◽  
...  
Keyword(s):  
Vitamin D ◽  
Cardiovascular Events ◽  
Nucleotide Polymorphisms ◽  
Integrin Αiibβ3 ◽  
Hydroxyvitamin D ◽  
Fibrinogen Binding ◽  
Vitamin D Levels ◽  
Pearson's R ◽  
25 Hydroxyvitamin D ◽  
Pearson’S R

Abstract Background Cardiovascular events are associated with low circulating vitamin D concentrations, although the underlying mechanisms are poorly understood. This study investigated associations between 25-hydroxyvitamin D concentrations, platelet function, and single-nucleotide polymorphisms (SNPs) in genes influencing vitamin D biology in the 500 Functional Genomics (500FG) cohort. Methods In this observational study, platelet activation and function were measured by flow cytometry by binding of fibrinogen to the activated fibrinogen receptor integrin αIIbβ3 and expression of P-selectin, markers of platelet aggregation and degranulation, respectively. These parameters were correlated to serum 25-hydroxyvitamin D and genotyping was performed to investigate SNPs in genes important for vitamin D biology. Results Circulating 25-hydroxyvitamin D concentrations correlated inversely with baseline platelet binding of fibrinogen to integrin αIIbβ3 (Pearson's r= –0.172, p = 0.002) and platelet responses to platelet agonist cross-linked collagen-related peptide (CRP-XL) (Pearson's r= –0.196,p = 0.002). This effect was due to circulating vitamin D levels ≤50nmol/L, since no differences in platelet fibrinogen binding were observed between subjects with normal 25-hydroxyvitamin D concentrations (>75nmol/L) and a 25-hydroxyvitamin D insufficiency (50–75 nmol/L). No correlations between 25-hydroxyvitamin D concentrations and platelet P-selectin expression were found. Several SNPs in the GC region of the vitamin D binding proteingene were associated with platelet responses to CRP-XL. Conclusion Low circulating vitamin D concentrations are associated with increased platelet fibrinogen binding to integrin αIIbβ3 in unstimulated samples and after stimulation with CRP-XL. These findings may contribute to the increased incidence of cardiovascular events in vitamin D deficient adults and its seasonal variation. Further studies are needed to investigate causality.

scholarly journals The peptide LSARLAF causes platelet secretion and aggregation by directly activating the integrin αIIbβ3

1997 ◽  
Vol 325 (2) ◽  
pp. 309-313 ◽  
Author(s):  
Jerry M. DERRICK ◽  
Donald B. TAYLOR ◽  
Robert G. LOUDON ◽  
T. Kent GARTNER
Keyword(s):  
Platelet Aggregation ◽  
Conformational Change ◽  
Prostaglandin E1 ◽  
Agonist Peptide ◽  
Integrin Αiibβ3 ◽  
Fibrinogen Binding ◽  
Platelet Secretion

A novel peptide (designed to bind to αIIbβ3) caused platelet aggregation and aggregation-independent secretion of the contents of α-granules in an αIIbβ3-dependent fashion. The agonist peptide induced secretion in the presence of prostaglandin E1. In cell-free assays, αIIbβ3 bound specifically to immobilized agonist peptide and the peptide enhanced the binding of fibrinogen to immobilized αIIbβ3. The agonist peptide apparently activates αIIbβ3 by directly inducing a conformational change in the receptor. This change activates the platelets and causes secretion in a manner independent of fibrinogen binding.

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 Novel whole blood assay for phenotyping platelet reactivity in mice identifies ICAM-1 as a mediator of platelet-monocyte interaction

Blood ◽  
2015 ◽  
Vol 126 (10) ◽  
pp. e11-e18 ◽  
Author(s):  
Paul C. J Armstrong ◽  
Nicholas S. Kirkby ◽  
Melissa V. Chan ◽  
Michaela Finsterbusch ◽  
Nancy Hogg ◽  
...  
Keyword(s):  
High Throughput ◽  
Whole Blood ◽  
Platelet Reactivity ◽  
Research Application ◽  
Whole Blood Assay ◽  
Blood Assay ◽  
Fibrinogen Binding ◽  
Key Points ◽  
Low Volume ◽  
Whole Blood Aggregometry

Key PointsLow-volume, high-throughput whole blood aggregometry will facilitate future mouse platelet function research. Application of this approach identifies ICAM-1 as a novel mediator of platelet-monocyte interaction through fibrinogen binding.

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