Functional Comparison of Induced Pluripotent Stem Cell- and Blood-Derived GPIIbIIIa Deficient Platelets

Human induced pluripotent stem cells (hiPSCs) represent a versatile tool to model genetic diseases and are a potential source for cell transfusion therapies. However, it remains elusive to which extent patient-specific hiPSC-derived cells functionally resemble their native counterparts. Here, we generated a hiPSC model of the primary platelet disease Glanzmann thrombasthenia (GT), characterized by dysfunction of the integrin receptor GPIIbIIIa, and compared side-by-side healthy and diseased hiPSC-derived platelets with peripheral blood platelets. Both GT-hiPSC-derived platelets and their peripheral blood equivalents showed absence of membrane expression of GPIIbIIIa, a reduction of PAC-1 binding, surface spreading and adherence to fibrinogen. We demonstrated that GT-hiPSC-derived platelets recapitulate molecular and functional aspects of the disease and show comparable behavior to their native counterparts encouraging the further use of hiPSC-based disease models as well as the transition towards a clinical application

Current Strategies in Diagnosis of Inherited Storage Pool Defects

Inherited platelet defects lead to bleeding symptoms of varying severity. Typically, easy bruising, petechiae, epistaxis, and mucocutaneous bleeding are observed in affected patients. The platelet defects are classified into disorders affecting either platelet surface receptors or intracellular organelles of platelets. The latter are represented by platelet storage pool diseases (SPD) which share a defect of platelet granules. Platelet α-granules, δ-granules, or both may be affected resulting in the clinical picture of α-SPD (e.g. Gray platelet syndrome, Quebec platelet disorder, arthrogryposis, renal dysfunction, and cholestasis syndrome), δ-SPD (e.g. Hermansky-Pudlak syndrome, Chediak-Higashi syndrome, Griscelli syndrome), or αδ-SPD (e.g. X-linked thrombocytopenia, Wiskott-Aldrich syndrome). Diagnosis of SPD is very extensive and requires platelet aggregation and flow cytometry analyses with interpretation from a specialist. Many of these disorders share common treatments, however, efficacy can vary between different patients. Therapy regiments with tranexamic acid, DDAVP, activated FVIIa, and platelet transfusions have been published. Stem cell or bone marrow transplantations are preserved for severe defects. Here, we describe the pathophysiology, clinical manifestations, and diagnosis of the major human SPDs

Novel Mutation in Bernard-Soulier Syndrome

Background: Bernard-Soulier syndrome (BSS) is a severe congenital bleeding disorder characterized by thrombocytopenia, thrombocytopathy and decreased platelet adhesion. BSS results from genetic alterations of the glycoprotein (GP) Ib/IX/V complex. Methods: We report on a patient demonstrating typical BSS phenotype (thrombocytopenia with giant platelets, bleeding symptoms). However, BSS was not diagnosed until he reached the age of 39 years. Results: Flow cytometry of the patient’s platelets revealed absence of GPIb/IX/V receptor surface expression. In addition, immunofluorescence analysis of patient’s platelets demonstrated very faint staining of GPIX. A novel homozygous deletion comprising 11 nucleotides starting at position 1644 of the GPIX gene was identified using molecular genetic analysis. Conclusions: The novel 11-nucleotide deletion (g.1644_1654del11) was identified as causing the bleeding disorder in the BSS patient. This homozygous deletion includes the last 4 nucleotides of the Kozak sequence as well as the start codon and the following 4 nucleotides of the coding sequence. The Kozak sequence is a region indispensable for the initiation of the protein translation process, thus preventing synthesis of functional GPIX protein in the case of deletion.Hintergrund: Das Bernard-Soulier-Syndrom (BSS) ist eine angeborene Blutungsstörung, die mit Thrombozytopenie, Thrombozytopathie und verminderter Thrombozytenadhäsion assoziiert ist. BSS wird durch genetische Veränderungen des Glykoprotein(GP)-Ib/IX/V-Komplexes verursacht. Methoden: Wir berichten über einen Patienten mit typischem BSS-Phänotyp (Thrombozytopenie mit Riesenthrombozyten, Blutungssymptome). Dennoch wurde die Diagnose BSS erst im Alter von 39 Jahren gestellt. Ergebnisse: Die Durchflusszytometrie der Thrombozyten des Patienten ergab eine fehlende Oberflächenexpression des GPIb/IX/V-Rezeptors. Zusätzlich zeigten Immunfluoreszenz-Analysen der Thrombozyten eine nur sehr schwache Anfärbung von GPIX. In der molekulargenetischen Analyse wurde eine noch nicht bekannte homozygote Deletion von 11 Nukleotiden (beginnend an Position 1644 im GPIX-Gen) identifiziert. Schlussfolgerungen: Diese neue Deletion von 11 Nukleotiden (g.1644_1654del11) wurde als Ursache für die vermehrte Blutungsneigung bei dem BSS-Patienten identifiziert. Von der homozygoten Deletion betroffen sind die letzten 4 Nukleotide der Kozak-Sequenz sowie das Startkodon und weitere 4 Nukleotide des kodierenden Bereichs. Die Kozak-Sequenz ist unerlässlich für die Initiation der Translation in der Proteinbiosynthese, so dass die bei dem Patienten nachgewiesene Deletion die Synthese des funktionellen GPIX-Proteins verhindert.Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich

Functional comparison of induced pluripotent stem cell- and blood-derived GPIIbIIIa deficient platelets.

Human induced pluripotent stem cells (hiPSCs) represent a versatile tool to model genetic diseases and are a potential source for cell transfusion therapies. However, it remains elusive to which extent patient-specific hiPSC-derived cells functionally resemble their native counterparts. Here, we generated a hiPSC model of the primary platelet disease Glanzmann thrombasthenia (GT), characterized by dysfunction of the integrin receptor GPIIbIIIa, and compared side-by-side healthy and diseased hiPSC-derived platelets with peripheral blood platelets. Both GT-hiPSC-derived platelets and their peripheral blood equivalents showed absence of membrane expression of GPIIbIIIa, a reduction of PAC-1 binding, surface spreading and adherence to fibrinogen. We demonstrated that GT-hiPSC-derived platelets recapitulate molecular and functional aspects of the disease and show comparable behavior to their native counterparts encouraging the further use of hiPSC-based disease models as well as the transition towards a clinical application

Deletion of human GP1BB and SEPT5 is associated with Bernard-Soulier syndrome, platelet secretion defect, polymicrogyria, and developmental delay.

The bleeding disorder Bernard-Soulier syndrome (BSS) is caused by mutations in the genes coding for the platelet glycoprotein GPIb/IX receptor. The septin SEPT5 is important for active membrane movement such as vesicle trafficking and exocytosis in non-dividing cells (i.e. platelets, neurons). We report on a four-year-old boy with a homozygous deletion comprising not only glycoprotein Ibβ (GP1BB) but also the SEPT5 gene, located 5' to GP1BB. He presented with BSS, cortical dysplasia (polymicrogyria), developmental delay, and platelet secretion defect. The homozygous deletion of GP1BB and SEPT5, which had been identified by PCR analyses, was confirmed by Southern analyses and denaturing HPLC (DHPLC). The parents were heterozygous for this deletion. Absence of GPIbβ and SEPT5 proteins in the patient's platelets was illustrated using transmission electron microscopy. Besides decreased GPIb/IX expression, flow cytometry analyses revealed impaired platelet granule secretion. Because the bleeding disorder was extremely severe, the boy received bone marrow transplantation (BMT) from a HLA-identical unrelated donor. After successful engraftment of BMT, he had no more bleeding episodes. Interestingly, also his mental development improved strikingly after BMT. This report describes for the first time a patient with SEPT5 deficiency presenting with cortical dysplasia (polymicrogyria), developmental delay, and platelet secretion defect

Flow cytometry of PAC-1 binding and integrin surface expression after platelet activation.

<p>(A) Cells were stained with PAC-1 (x-axis) and anti-CD42b (y-axis) antibodies in the absence (top) or presence (bottom) of ADP/TXA₂. (B) Flow cytometry of CD41 (GPIIb) and CD61 (GPIIIa) surface expression on hiPSC-derived and peripheral blood platelets in the presence of ADP/TXA₂. After activation with ADP/TXA₂, cells were stained with anti-CD42b, anti-CD41/CD61, anti-CD41 and anti-CD61 antibodies. The FSC/SSC log gate of peripheral platelets was applied and further gated for CD42b+ cells.</p

Surface spreading, adhesion and cytoskeleton rearrangement of peripheral blood and hiPSC-derived platelets.

<p>(A) Platelets spreading on fibrinogen-coated wells were imaged with DICM in the presence of ADP/TXA₂ at the indicated time points. For complete movie sequence see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115978#pone.0115978.s012" target="_blank">S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115978#pone.0115978.s015" target="_blank">S4</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115978#pone.0115978.s016" target="_blank">S5</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115978#pone.0115978.s018" target="_blank">S7</a> Movies. (B) Quantification of fibrinogen binding of MKs and platelets. The percentages of attached cells are shown as scatter columns with horizontal bars representing the mean. In the hiPSC-derived groups, red and black symbols indicate results from the two hiPSC clones. A total mean of 1991±262.6 cells per sample was counted before washing. P-values from unpaired t-tests; peripheral blood platelets from GT (n = 10) and CTR (n = 10); hiPSC-derived platelets from GT (n = 5) and CTR (n = 6). (C) Rhodamine-conjugated phalloidin was used to stain actin filaments after permeabilization of platelets spreading on fibrinogen. All cells shown stained positive for CD42b and negative for DAPI. All scale bars represent 10 μm. Representative images for each sample.</p

Generation and integrin surface expression of hiPSC-derived platelets from GT and CTR.

<p>(A) Immunofluorescence (top) and flow cytometry (bottom) of hiPSC-derived and peripheral blood platelets. The cells were stained with anti-CD42b (red / y-axis), anti-CD41/CD61 (green / x-axis) antibodies and DAPI (blue). (B) Flow cytometry of CD41 (GPIIb) and CD61 (GPIIIb) surface expression on hiPSC-derived and peripheral blood platelets. The cells were stained with anti-CD42b, anti-CD41/CD61, anti-CD41 and anti-CD61 antibodies. The FSC/SSC log gate of peripheral platelets was applied and further gated for CD42b+ cells. All scale bars represent 10 μm. Representative images for each sample.</p