The Critical Role of Kindlin-3 in Intiation of Physiological Thrombus Formation ~ Analysis of Kindlin-3 Deficient Patient

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1062-1062
Author(s):  
Hisashi Kato ◽  
Shigenori Honda ◽  
Hirokazu Kashiwagi ◽  
Nobuko Nishiura ◽  
Keigo Akuta ◽  
...  
Keyword(s):  
Cell Line ◽  
Nonsense Mutation ◽  
Thrombus Formation ◽  
Severe Bleeding ◽  
Sequencing Analysis ◽  
Aggregate Formation ◽  
Hel Cells ◽  
Healthy Control ◽  
Inside Out

Background: Kindlin-3 which is expressed mainly in hematopoietic cells, is essential for platelet fibrinogen receptor integrin αIIbβ3 (GPIIb-IIIa) activation and kindlin-3 deficiency causes severe bleeding problems. αIIbβ3 activation is tightly regulated by inside-out signaling, and the direct interaction of talin and kindlin-3 with β3-cytoplasmic tail following CalDAG-GEFI-induced Rap1 activation is critical for αIIbβ3 activation. We have reported that immediate and sustained αIIbβ3 activation by inside-out signaling is important for thrombus formation under physiological conditions (Blood 2016). However, the details of inside-out signaling are not still fully understood. Recently we identified patient with kindlin-3 deficiency as the first Japanese patient with kindlin-3 deficiency caused by novel p.W277X nonsense mutation. To clarify the role of kindlin-3 and the molecular mechanism of inside-out signaling, we analyzed single platelet behavior adhered on collagen under physiological flow conditions, and established kindlin-3 deficient human erythroleukemia HEL cell line. Case: The patient was 8-month old female, born to Japanese consanguineous parents. She has been suffering from bleeding tendency shortly after birth. Her peripheral blood showed slightly decreased platelet count (95x103/L), anemia (Hb 6.9 g/L), and elevated leukocyte count of 37.2x106/L with 1.0% blast. Although the surface expressions of glycoproteins were comparable to healthy control, her platelet aggregations and αIIbβ3 activation were strongly impaired in all agonist stimulations due to defective kindlin-3 expression. The sequencing analysis revealed the homozygous novel nonsense mutation, p.W277X (c.918G>A) in kindlin-3. Methods: Human platelets were obtained from healthy control subjects (CT) and patients with kindlin-3 deficiency and Glanzmann thrombasthenia (GT). Whole blood was perfused at a shear rate 1,250s-1 on collagen surface and shear-induced in vitro thrombus formation during 10 minutes was observed. To evaluate the single platelet behavior after the initial attachment on collagen, each single platelet was analyzed by CellTracker software (Pccinini F. et al. Bioinformatics 2015). To further determine the role of kindlin-3 in inside-out signaling, Kindlin-3 was knocked out by CRISPR-Cas9 system and established kindlin-3 deficient HEL cell line. Results: First, we compared shear-induced thrombus formation between CT, GT, and kindlin-3 deficiency. In contrast to the stable and large platelet aggregate formation in CT after 10 minutes blood perfusion, almost no aggregate was observed in both GT and Kindlin-3 deficiency. In kindlin-3 deficiency, the initial platelet attachment on collagen seemed comparable to that of CT and GT. However, the single adherent platelets looked unstable. Next, we determined the behavior of initially attached platelets. Between CT, GT, and kindlin-3 deficiency, the numbers of platelets attached on collagen during 10 seconds were comparable. Between the initially attached platelets, 31.25% of CT platelets formed stable adhesion followed by platelet aggregation. Similar to CT, 33% of GT platelets adhered stably. However, these GT platelets did not proceed to aggregate formation due to αIIbβ3 deficiency. In contrast to GT, kindlin-3 deficient platelets showed increased detachment, only 9% of initially attached platelets stably adhered. These results suggest that kindlin-3 is indispensable for platelet initial adhesion to collagen by integrin α2β1 activation and explains severe bleeding symptoms in kindlin-3 deficiency than GT. To further investigate how kindlin-3 contributes in initial platelet adhesion to collagen, we established kindlin-3 deficient HEL cell line. In contrast to parental HEL cells, PMA stimulation did not induce αIIbβ3 activation in kindlin-3 deficient HEL cells suggesting impaired inside-out signaling. The introduction of wild type kindlin-3 cDNA, but not W227X mutant, rescued αIIbβ3 activation. Conclusion: The detailed analyses by tracking single adherent platelet on collagen confirmed the role of kindlin-3 in initial step of physiological thrombus formation. Newly established kindlin-3 deficient HEL cell line is useful for further exploration of the role of kindlin-3 and inside-out signaling in platelets and expected contribution for development of new antiplatelet therapy. Disclosures Tomiyama: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Chugai: Honoraria; Kyowa-Kirin: Honoraria.

scholarly journals Platelet Integrin αIIbβ3 Activation Kinetics in Inherited Platelet Functional Disorders ∼ the Role of ADP Receptor P2Y12, Caldag-GEFI and Kindlin-3 αIIbβ3 Activation By inside-out Signaling

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1136-1136
Author(s):  
Hisashi Kato ◽  
Nobuko Nishiura ◽  
Keigo Akuta ◽  
Hirokazu Kashiwagi ◽  
Koichi Kokame ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Time Course ◽  
Thrombus Formation ◽  
Severe Bleeding ◽  
Activation Kinetics ◽  
Healthy Control ◽  
Initial Activation ◽  
Adp Receptor ◽  
Inside Out

Abstract Background and objectives Activation of platelet fibrinogen receptor integrin αIIbβ3 (GPIIb-IIIa) by inside-out signaling is essential for platelet aggregate formation. In αIIbβ3 activation, it has been established that CalDAG-GEFI-induced Rap1 activation is necessary to induce the direct interaction of β3 cytoplasmic tail with talin and kindlin-3. Agonist induced platelet and αIIbβ3 activation are generally assessed using platelet aggregation assay and flow cytometric analysis of monoclonal antibody specific for activated αIIbβ3, PAC-1, binding. However, we found that the results of PAC-1 binding assay were not associated with severity of bleeding symptoms in patient with CalDAG-GEFI (Blood 2016) or ADP receptor P2Y12 (J Thromb Haemost 2005) deficiency. To further determine the role of molecules in inside-out signaling on αIIbβ3 activation and the impact on hemostasis, we studied the αIIbβ3 activation kinetics in patient's platelets deficient for αIIbβ3, P2Y12, CalDAG-GEFI, or kindlin-3 using the αIIbβ3 velocity assay (Exp Hematol 2013). Methods Human platelets were obtained from healthy control subjects and patients with inherited platelet disorders (Glanzmann thrombasthenia, P2Y12 deficiency, CalDAG-GEFI deficiency, and kindlin-3 deficiency). Agonist induced platelet and αIIbβ3 activation were assessed by light transmission aggregometer and PAC-1 binding on flow cytometry. In addition to the conventional PAC-1 binding assay which is simultaneous incubation of stimulated platelets with FITC-PAC-1 for 20 minutes, the time course of αIIbβ3 activation was determined by αIIbβ3 velocity assay. In brief, to measure the activation state of αIIbβ3 at the time of interest (0, 30, 60, 120 and 300 seconds), FITC-labeled PAC-1 was added after the PAR1-AP stimulation. After 30 seconds incubation with FITC-PAC-1, the bound FITC-PAC-1 was immediately assessed by flow cytometry. To determine the thrombus formation under the physiological condition, shear-induced thrombus formation on collagen coated surface was observed. Results In the patient with kindlin-3 deficiency, strongly impaired platelet aggregation and αIIbβ3 activation determined by conventional PAC-1 binding were correlated to the patients severe bleeding problems appeared from early infancy. The αIIbβ3 activation kinetics of kindling-3 deficient platelets showed no PAC-1 binding during 300 seconds after the thrombin receptor agonist PAR1-AP stimulation like platelets of Granzmann thrombasthenia, which suggests the essential role of Kindlin-3 in αIIbβ3 activation. In contrast, in spite of the strongly impaired PAC-1 binding in conventional assay for P2Y12 deficient platelets, the 67 year-old patient showed only minor bleeding symptoms and no transfusion was required during delivery. The αIIbβ3 activation observed in P2Y12 deficiency was transient and the level of initial activation at 30 seconds was the same as healthy control platelets. Under the flow conditions, P2Y12 deficient platelets could form thrombus albeit unstable and fragile. In the 16-yaer-old patient with CalDAG-GEFI deficiency who has been suffering from severe nose bleed and menorrhagia, her platelet aggregation and conventional PAC-1 binding were only modestly affected. The time course of αIIbβ3 activation showed delayed activation. Although αIIbβ3 activation in CalDAG-GEFI deficient platelets reached to similar to that of control at 300 seconds, the initial activation at 30 seconds was significantly decreased to only 19% of control. Her platelets could adhere on collagen under the flow condition. However, almost no aggregate formation was observed which explains her severe bleeding symptoms. Conclusion The analyses of αIIbβ3 activation kinetics in platelets with inherited platelet disorder revealed the importance of immediate and sustained αIIbβ3 activation for hemostasis in physiological conditions. In addition to well established platelet functional assay, the analysis of αIIbβ3 activation kinetics contributes to understand the patient's bleeding symptoms and also clarifies the role of each molecule in inside-out αIIbβ3 activation, CalDAG-GEFI for the immediate activation, P2Y12 for the maintenance of activation, and Kindlin-3 at the last step of αIIbβ3 activation. Disclosures Kanakura: Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding. Tomiyama:Kyowa Hakko Kirin Co., Ltd.: Honoraria; Novartis Pharma Co., Ltd.: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sysmex Corporation: Consultancy; Chugai Pharmaceutical Co., Ltd.: Honoraria.

Abstract 391: Platelet CD40L Affects Thrombus Formation via Phosphatidylinositol 3-kinase ß, But Not Via CD40 or IKKα

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Marijke J Kuijpers ◽  
Nadine J Mattheij ◽  
Lina Cipolla ◽  
Johanna P van Geffen ◽  
Toby Lawrence ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Thrombus Formation ◽  
Aggregate Formation ◽  
Phosphatidylinositol 3 Kinase ◽  
Akt Phosphorylation ◽  
Soluble Cd40l ◽  
Granule Secretion ◽  
Collagen Receptor

Objective: To investigate the roles and signaling pathways of CD40L and CD40 in platelet activation and thrombus formation under atherothrombotic conditions. Approach and Results: Mouse platelets lacking CD40L (Cd40lg -/- Apoe -/- ) showed diminished αIIbβ3 activation and α-granule secretion in response to collagen receptor (GPVI) stimulation, while CD40 deficient platelets (Cd40 -/- Apoe -/- ) showed increased responses. ADP- or thrombin-evoked activation was unaffected. In both Cd40lg -/- Apoe -/- and Cd40 -/- Apoe -/- mice, formation of multi-layered thrombi was decreased on both atherosclerotic plaque material and collagen, in comparison to controls. Addition of CD40L prior to perfusion over collagen or plaque material enhanced dense aggregate formation in Apoe -/- , Cd40lg -/- Apoe -/- and Cd40 -/- Apoe -/- blood. CD40L or low GPVI stimulation separately did not cause platelet aggregation. But when combined, aggregation was potentiated, even in the absence of CD40. This potentiation was antagonized by inhibiting PI3Kβ, as well as in platelets from Pik3cb R/R mice. CD40L enhanced Akt phosphorylation at low GPVI stimulation, which was again antagonized by PI3Kβ inhibition and absent in platelets from Pik3cb R/R mice. Finally, Chuk1 A/A Apoe -/- mice, deficient in IKKα, displayed no differences in platelet aggregation - with or without CD40L - nor in thrombus formation in whole blood, indicating that these effects are not mediated via IKKα/NFkB. Conclusions: Under atherothrombotic conditions, CD40L enforces collagen-dependent platelet activation, by supporting integrin αIIbβ3 activation, secretion and dense thrombus formation via PI3Kβ, but not IKKα. Since shedding of CD40L starts minutes after activation, these results point to a joint role of both platelet-bound and soluble CD40L in controlling the size of rapidly formed thrombi.

Cadherin-6 Forms a Functional Adhesion Complex with Alpha-Catenin and Beta-Catenin in Platelets and Is Required for Platelet Aggregation and Thrombus Formation

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2232-2232 ◽  
Author(s):  
Michele Mumaw ◽  
Maria de la Fuente ◽  
Carolyn Aldana ◽  
Wei Li ◽  
Marvin T Nieman
Keyword(s):  
Platelet Aggregation ◽  
Adhesion Molecules ◽  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Bleeding Complications ◽  
Severe Bleeding ◽  
Beta Catenin ◽  
Adhesion Complex

Abstract The regulation of hemostasis and thrombus formation is a tightly controlled event that has catastrophic consequences when it is deregulated. One of the hallmarks of the thrombus is aggregated platelets. Upon platelet stimulation, adhesion molecules become activated and mediate multiple cell-cell interactions. Therapeutically, blocking platelet adhesion is a proven method for preventing pathological arterial thrombus formation. However, targeting the primary adhesion receptor, integrin αIIbβ3, results in severe bleeding complications. Therefore, identifying novel proteins or uncovering novel functions for known proteins in platelets is a necessary first step to facilitate the development of safer anti-platelet therapeutics. We have identified that the cell adhesion molecule cadherin-6 forms a functional adhesion complex with α-catenin and β-catenin in platelets. The goal of our project was to determine the mechanism of cadherin-6 mediated adhesion in platelets. Our initial experiments demonstated that cadherin-6 and β-catenin co-localize at the plasma membrane in platelets using confocal immunofluorescence microscopy. We determined that α-catenin and β-catenin co-immunoprecipitate with cadherin-6 from platelet lysates. To examine the functional role of cadherin-6 on platelet aggregation we used a cadherin-6 blocking antibody (10 μg/ml). Blocking cadherin-6 inhibited mouse platelet aggregation induced by PAR4 peptide. We next determined the role of cadherin-6 in vivo by examining carotid artery thrombosis after 7.5% FeCl3 treatment. C57Bl6 mice were injected with cadherin-6 antibody IV and labeled with rhodamine 6G by jugular vein injection. Thrombus formation was imaged in real time by fluorescent intravital microscopy. Blocking cadherin-6 prevented thrombosis for the duration of the experiment (30 min). To verify that the effects that we observed were specific to cadherin-6 expressed on platelets, we isolated platelets from donor mice and treated with cadherin-6 antibody or control IgG ex vivo. The treated platelets were perfused into recipient mice that were irradiated with 11 Gy to make the animals thrombocytopenic. The cadherin-6 antibody treated platelets formed an occlusion at 26.4 ± 3.6 min vs. 13.7 ± 2.0 min for the IgG (p=0.03). Importantly, the cadherin-6 antibody did not affect platelet counts compared to IgG controls 2.97 ± 0.40 (×108) vs. 3.02 ± 0.20 (×108). These combined studies show that caderhin-6 forms a complex with the necessary proteins required to mediate adhesion in platelets. Our results demonstrate that platelet cadherin-6 has a physiologically important role during platelet activation and thrombus formation in vivo. In summary, we have identified a novel adhesion complex in platelets that may provide a mechanism to limit platelet aggregation therapeutically. On going studies will determine the regulation of the cadherin-6/catenin complex and how cadherin-6 cooperates with other platelet adhesion molecules. Disclosures No relevant conflicts of interest to declare.

Critical Role of Endogenous ADP Via P2Y12 Receptor in Maintenance of αIibβ3 Activation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1655-1655
Author(s):  
Tsuyoshi Kamae ◽  
Masamichi Shiraga ◽  
Hirokazu Kashiwagi ◽  
Hisashi Kato ◽  
Seiji Tadokoro ◽  
...  
Keyword(s):  
Binding Capacity ◽  
Thrombus Formation ◽  
Critical Role ◽  
Adenosine Diphosphate ◽  
P2y12 Receptor ◽  
Platelet Suspension ◽  
High Affinity ◽  
Static Conditions ◽  
Inside Out

Abstract Platelet αIIbβ3 (GPIIb-IIIa), a noncovalently associated heterodimer, is a prototypic integrin that functions as a physiologic receptor for fibrinogen and von Willebrand factor. αIIbβ3 plays a crucial role in platelet aggregation, a key event of hemostatic plug formation and pathologic thrombus formation. During thrombogenesis, the affinity of αIIbβ3 for macromolecular ligands is dynamically regulated. After exposure to subendothelial matrix, several mediators such as ADP and thromboxane A2, or shear stress, platelets becomes activated and inside-out signaling that induces a high-affinity state of αIIbβ3 for soluble ligands (αIIbβ3 activation) are generated. Previous studies revealed that activation of αIIbβ3 is a reversible process. When platelets are stimulated with weak agonists such as adenosine diphosphate (ADP) and epinephrine in the absence of fibrinogen, αIIbβ3 gradually looses its binding capacity. In contrast, thrombin can induce long-lasting αIIbβ3 activation in the absence of fibrinogen. Although much attention has been directed to the nature of inside-out signaling, the mechanisms by which αIIbβ3 keep in the high-affinity state still remains elusive. In this study, we have demonstrated a critical role of endogenous ADP via its P2Y12 receptor in the maintenance of αIIbβ3 activation. Washed platelets adjusted to 50 x 106/ml were stimulated with 0.2 U/ml thrombin or 5 mM U46619 under static conditions. After the 15-min stimulation, 1 mM AR-C69931MX (a P2Y12 antagonist), 1 mM A3P5P (a P2Y1 antagonist) or buffer alone was added to the suspensions for additional 5 min. The platelet suspensions were then incubated with FITC-PAC1 and PE-anti-CD62P for 30 min and analyzed in a flow cytometer. AR-C69931MX and A3P5P attenuated the number of activated αIIbβ3 about 95% and 45%, respectively on the already activated platelets with thrombin- or U46619 without inhibiting CD62P expression. In an another set of experiments, platelets stimulated with thrombin or U46619 for 15min were then diluted to 1:100 with buffer containing the same agonist concentration (0.5 x 106/ml). In these conditions the number of activated αIIbβ3 was also markedly decreased (~85% reduction). Furthermore, the reduction in activated αIIbβ3 by the dilution was reversed by the addition of exogenous ADP in a dose-dependent fashion. HPLC analysis revealed that the amounts of ADP released from thrombin- and U46619-stimulated platelets were 2.6 and 0.75 mmol/1011platelets, respectively, and these values were comparable with ADP doses required for sustained αIIbβ3 activation in the diluted platelet suspension. Thus, released endogenous ADP plays a critical role in the maintenance of αIIbβ3 activation, and certain platelet concentrations are needed for this action. We also examined Rap 1b activation during the maintenance of αIIbβ3 activation. Thrombin induced sustained Rap 1b activation in the absence of ligand. However, AR-C69931MX disrupted the sustained Rap 1b activation. Thus, there was a close relationship between the maintenance αIIbβ3 activation and Rap 1b activation. Our data provide that the continuous interaction between released ADP and P2Y12 receptor is critical for the maintenance of αIIbβ3 activation.

scholarly journals Human NANOS1 Represses Apoptosis by Downregulating Pro-Apoptotic Genes in the Male Germ Cell Line

2020 ◽  
Vol 21 (8) ◽  
pp. 3009
Author(s):  
Damian M. Janecki ◽  
Erkut Ilaslan ◽  
Maciej J. Smialek ◽  
Marcin P. Sajek ◽  
Maciej Kotecki ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Germ Cell ◽  
Cell Line ◽  
Germ Cells ◽  
Male Germ Cell ◽  
Seminiferous Tubules ◽  
Sequencing Analysis ◽  
Germ Cell Line ◽  
Apoptotic Genes

While two mouse NANOS paralogues, NANOS2 and NANOS3, are crucial for maintenance of germ cells by suppression of apoptosis, the mouse NANOS1 paralogue does not seem to regulate these processes. Previously, we described a human NANOS1 p.[(Pro34Thr);(Ser83del)] mutation associated with the absence of germ cells in seminiferous tubules of infertile patients, which might suggest an anti-apoptotic role of human NANOS1. In this study, we aimed to determine a potential influence of human NANOS1 on the maintenance of TCam-2 model germ cells by investigating proliferation, cell cycle, and apoptosis. Constructs encoding wild-type or mutated human NANOS1 were used for transfection of TCam-2 cells, in order to investigate the effect of NANOS1 on cell proliferation, which was studied using a colorimetric assay, as well as apoptosis and the cell cycle, which were measured by flow cytometry. RNA-Seq (RNA sequencing) analysis followed by RT-qPCR (reverse transcription and quantitative polymerase chain reaction) was conducted for identifying pro-apoptotic genes repressed by NANOS1. Here, we show that overexpression of NANOS1 downregulates apoptosis in TCam-2 cells. Moreover, we found that NANOS1 represses a set of pro-apoptotic genes at the mRNA level. We also found that the infertility-associated p.[(Pro34Thr);(Ser83del)] mutation causes NANOS1 to functionally switch from being anti-apoptotic to pro-apoptotic in the human male germ cell line. Thus, this report is the first to show an anti-apoptotic role of NANOS1 exerted by negative regulation of mRNAs of pro-apoptotic genes.

scholarly journals From Patients to Platelets and Back Again: Pharmacological Approaches to Glycoprotein VI, a Thrilling Antithrombotic Target with Minor Bleeding Risks

2019 ◽  
Vol 119 (11) ◽  
pp. 1720-1739 ◽  
Author(s):  
Augusto Martins Lima ◽  
Ana C. Martins Cavaco ◽  
Rodrigo A. Fraga-Silva ◽  
Johannes A. Eble ◽  
Nikolaos Stergiopulos
Keyword(s):  
Thrombus Formation ◽  
Biological Effects ◽  
Bleeding Risk ◽  
Severe Bleeding ◽  
Minor Bleeding ◽  
Pharmacological Agents ◽  
Glycoprotein Vi ◽  
Potential Benefits ◽  
High Bleeding Risk

AbstractDespite significant advances in the treatment of thrombogenic diseases, antiplatelet therapies are still associated with a high bleeding risk. Consequently, potential benefits of preventing thromboembolic events by pharmacological agents need to be balanced with the potential harm of inducing hemorrhage. Glycoprotein VI (GPVI) is a platelet-specific receptor, which plays a crucial role in thrombus formation. GPVI deficiency has been identified in patients who suffer from significant reduction of collagen-induced thrombus formation, with a slight tendency for mild bleeding. However, an isolated GPVI deficiency can reduce thrombus formation while not resulting in severe bleeding. Together, these observations strongly suggest that physiological hemostasis does not require GPVI, but pharmacological GPVI modulation may provide novel “bleeding-free” antithrombotic therapies. In this review, we discuss recent findings regarding the biological role of GPVI in platelet-related disorders and highlight the efforts to develop potential therapeutic strategies based on its structure, signaling pathways, and biological effects.

Single channel recordings of potassium currents in an insulin-secreting cell line

1986 ◽  
Vol 109 (2) ◽  
pp. 201-207 ◽  
Author(s):  
N. C. Sturgess ◽  
M. L. J. Ashford ◽  
C. A. Carrington ◽  
C. N. Hales
Keyword(s):  
Cell Line ◽  
Single Channel ◽  
Dependent Manner ◽  
Patch Clamp Technique ◽  
Pancreatic Islet Cell ◽  
Secreting Cell ◽  
Dose Dependent ◽  
Inside Out ◽  
Rat Pancreatic Islet

ABSTRACT Using the patch-clamp technique we observed three distinct classes of K+ channels which were spontaneously active in excised 'inside-out' membrane patches from an insulin-secreting rat pancreatic islet cell line (CRI-G1). Two of these occurred infrequently, one with a conductance of approximately 7 pS, and the other a conductance of 220 pS. The activation of the 220 pS K+ channel was dependent upon the membrane voltage and was sensitive to the concentration of calcium ions at the cytoplasmic surface of the membrane. The third, and by far the most common class of K+ channel, was characterized by its sensitivity to ATP. Application of ATP to the cytoplasmic side of the membrane reversibly inhibited this K+ channel in a dose-dependent manner, but had no effect when applied to the external side. The properties of the ATP-sensitive K+ channel appear to be indistinguishable from those of a channel found in rat neonatal β cells. Thus this insulin-secreting cell line should prove valuable in the investigation of the role of K+ channels in the regulation of insulin secretion. J. Endocr. (1986) 109, 201–207

Defects in Glanzmann thrombasthenia and LAD-III (LAD-1/v) syndrome: the role of integrin β1 and β3 in platelet adhesion to collagen

Blood ◽  
2012 ◽  
Vol 119 (2) ◽  
pp. 583-586 ◽  
Author(s):  
Edith van de Vijver ◽  
Iris M. De Cuyper ◽  
Anja J. Gerrits ◽  
Arthur J. Verhoeven ◽  
Karl Seeger ◽  
...  
Keyword(s):  
Leukocyte Adhesion ◽  
Thrombus Formation ◽  
Diagnostic Methods ◽  
Severe Bleeding ◽  
Bleeding Tendency ◽  
Aggregation Assay ◽  
Glanzmann Thrombasthenia ◽  
Integrin Αiibβ3 ◽  
Fibrinogen Receptor

Abstract Patients with Glanzmann thrombasthenia or Leukocyte Adhesion Deficiency-III syndrome (LAD-III or LAD-1/variant) present with increased bleeding tendency because of the lack or dysfunction of the fibrinogen receptor GPIIb/IIIa (integrin αIIbβ3), respectively. Although the bleeding disorder is more severe in LAD-III patients, classic aggregometry or perfusion of Glanzmann or LAD-III platelets over collagen-coated slides under physiologic shear rate does not discriminate between these 2 conditions. However, in a novel flow cytometry-based aggregation assay, Glanzmann platelets were still capable of forming small aggregates upon collagen stimulation, whereas LAD-III platelets were not. These aggregates required functional GPIa/IIa (integrin α2β1) instead of integrin αIIbβ3, thus explaining the clinically more severe bleeding manifestations in LAD-III patients, in which all platelet integrins are functionally defective. These findings provide genetic evidence for the differential requirements of platelet integrins in thrombus formation and demonstrate that correct integrin function assessment can be achieved with a combination of diagnostic methods.

Morphogenesis of a New Human Herpes-Type Virus Isolate (Sasha Virus)

1973 ◽  
Vol 31 ◽  
pp. 592-593
Author(s):  
R. F. Zeigel ◽  
W. Munyon
Keyword(s):  
Virus Isolate ◽  
Calf Serum ◽  
Uranyl Acetate ◽  
Human Herpes ◽  
Human Embryonic Lung ◽  
Human Herpes Virus ◽  
Intracytoplasmic Inclusions ◽  
Hel Cells ◽  
And Control

In continuing studies on the role of viruses in biochemical transformation, Dr. Munyon has succeeded in isolating a highly infectious human herpes virus. Fluids of buccal pustular lesions from Sasha Munyon (10 mo. old) uiere introduced into monolayer sheets of human embryonic lung (HEL) cell cultures propagated in Eagles’ medium containing 5% calf serum. After 18 hours the cells exhibited a dramatic C.P.E. (intranuclear vacuoles, peripheral patching of chromatin, intracytoplasmic inclusions). Control HEL cells failed to reflect similar changes. Infected and control HEL cells were scraped from plastic flasks at 18 hrs. of incubation and centrifuged at 1200 × g for 15 min. Resultant cell packs uiere fixed in Dalton's chrome osmium, and post-fixed in aqueous uranyl acetate. Figure 1 illustrates typical hexagonal herpes-type nucleocapsids within the intranuclear virogenic regions. The nucleocapsids are approximately 100 nm in diameter. Nuclear membrane “translocation” (budding) uias observed.

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