bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Phosphorylation-Dependent Binding of 14-3-3ζ to the Central Region of the GPIbalpha Cytoplasmic Domain.
Authors: Yuan Yanhong , Zhang Weilin , Yan Rong , Liao Yi , Zhao Lili , Ruan Changgeng , Du Xiaoping , Dai Kesheng 
Published: October 2019

Abstract
Abstract 2995
Poster Board II-973

Introduction:
The interaction of glycoprotein (GP) Ib-IX with subendothelial-bound von Willebrand factor (VWF) initiates circulated platelet transient adhesion on the injured vascular wall under flow conditions. VWF conformational changes in response to high shear stress are thought to be critical for initiating platelet adhesion, there is increasing evidences indicate that the interactions of intraplatelet proteins 14-3-3ζ and filamin A with the cytoplasmic domain of GPIbalpha also play key roles in the regulation of VWF binding function of GPIb-IX, whereas it is unclear whether their structural linkage has functional implication. This study was to explore the mechanism underlying the roles of 14-3-3ζ and filamin A in the regulation of the VWF binding function of GPIb-IX.

Methods and Results:
A truncation mutant of GPIbalpha (at residue 565) deleting the C-terminal 14-3-3ζ binding sites retains 14-3-3ζ binding function, in contrast, deletion of the C-terminal residues 551-610 of GPIbalpha totally abolished 14-3-3ζ binding, indicating that the residues 551-564 of GPIbalpha is important in the interaction between 14-3-3ζ and GPIb-IX. An antibody recognizing phosphorylated R557GpSLP561 sequence reacted with GPIbalpha suggesting phosphorylation of a population of GPIbalpha molecules at Ser559, and a membrane permeable phosphopeptide (MP-P), M-R557GpSLP561 corresponding to residues 557-561 of GPIbalpha eliminated the association of 14-3-3ζ with the truncation mutant of GPIbalpha . MP-P also promoted GPIb-IX association with the membrane skeleton, and inhibited ristocetin-induced platelet agglutination, VWF binding to platelets and platelet adhesion to immobilized VWF. Furthermore, a GPIb-IX mutant replacing Ser559 of GPIbalpha with alanine showed an enhanced association with the membrane skeleton, reduced ristocetin-induced VWF binding and diminished ability to mediate cell adhesion to VWF under flow conditions.

Conclusions:
These data suggest a phosphorylation-dependent binding of 14-3-3ζ to central filamin A binding site of GPIbalpha, and the dimeric 14-3-3ζ binding to both the C-terminal site and central RGpSLP site inhibits GPIb-IX association with the membrane skeleton and promotes the VWF binding function of GPIb-IX.

Disclosures:
No relevant conflicts of interest to declare.
The Interaction of Von Willebrand Factor with GPIb-IX Initiates Platelet Apoptosis.
Authors: Li Suping , Wang Zhicheng , Liao Yi , Liu Guanglei , Zhang Weilin , Shi Quanwei , Yan Rong , Ruan Changgeng , Dai Kesheng 
Published: October 2019

Abstract
Abstract 3002
Poster Board II-979

Background:
The interaction of glycoprotein (GP) Ibalpha with von Willebrand factor (VWF) initiates the adherence of platelets to sites of vascular injury, simultaneously triggers intracellular signaling events such as elevation of cytoplasmic calcium and activations of multiple protein kinase pathways which result in the activation of the ligand binding function of GPIIb/IIIa, leading to platelet activation and thrombus formation. The intracellular signaling protein 14-3-3ζ and the membrane skeleton protein filamin A have been confirmed to interact with the intracellular domain of GPIbalpha and play important roles in the regulation of platelet function. The signaling events elicited by GPIbalpha-VWF interaction, such as calcium mobilization and phosphatidylserine (PS) exposure are similar to those occurring during apoptosis. Particularly, the 14-3-3ζ binding domain of GPIbalpha has been reported to involve in the regulation of cell proliferations. However, it is still unclear whether the GPIbalpha-VWF interaction induces platelet apoptosis.

Objectives:
To investigate whether the GPIbalpha-VWF interaction induces platelet apoptosis and the role of 14-3-3ζ in apoptotic signaling.

Methods:
Apoptotic events were assessed in platelets or Chinese hamster ovary (CHO) cells expressing GPIb-IX (1b9) interacted with VWF by flow cytometry or Western blotting.

Results:
Ristocetin induced GPIbalpha-VWF interaction elicited apoptotic events in platelets, including phosphatidylserine exposure, elevations of Bax and Bak, gelsolin cleavage, and depolarization of mitochondrial inner transmembrane potential. Apoptotic events were also elicited in platelets exposed to pathologic shear stresses in the presence of VWF, however, the shear-induced apoptosis was eliminated by anti-GPIbalpha antibody AK-2. Furthermore, apoptotic events occurred in 1b9 cells stimulated with VWF and ristocetin, but were significantly diminished in two CHO cell lines expressing mutant GPIb-IX with truncation of the cytoplasmic domain of GPIbalpha or a serine-to-alanine mutation at 14-3-3ζ binding site in GPIbalpha.

Conclusions:
This study demonstrates that the GPIbalpha-VWF interaction induces apoptotic events in platelets and association of 14-3-3ζ with the cytoplasmic domain of GPIbalpha is essential for apoptotic signaling. The finding may suggest a novel mechanism for platelet clearance or some thrombocytopenic diseases. The reagents that block 14-3-3ζ-GPIbalpha interaction might be potentially useful in platelet storage or anti-thrombocytopenia.

Disclosures:
No relevant conflicts of interest to declare.
P2Y12 Inhibitors Restore the Ability of GPIb-IX to Inhibit Outside-in Signaling Across alphaIIbbeta3.
Authors: Fox Joan B. , Zhang Yi , Bialkowska Katarzyna 
Published: October 2019

Abstract
Abstract 3006
Poster Board II-982
P2Y12 inhibitors decrease the reactivity of platelets. Since the P2Y12 receptor acts through Gαai-induced signals, which inhibit adenyl cyclase, P2Y12 inhibitors are thought to act through phosphorylation of protein kinase A (PKA) substrates. One PKA substrate is serine166 (S166) of GPIbβ. Using a phospho-specific antibody, Western blots, and FACS of whole blood, we found that ADP and epinephrine, which activate P2Y12 receptors caused a dose-dependent dephosphorylation of S166 while apyrase and a P2Y12 inhibitor prevented this dephosphorylation. Since S166 functions with S609 of GPIbαa to bind 14-3-3, we determined whether i) dephosphorylation was associated with release of 14-3-3 from GPIb, and ii) 14-3-3 regulated αaIIbβ3 function. GPIb bound ∼91 ± 5% of the 14-3-3, as determined by its insolubility in streptolysin O-permeabilized platelets and its solubilization by phospho-S609 and -S166 peptides. Quantitation of 14-3-3 in GPIb immunoprecipitates showed that graded Gαai-induced dephosphorylation of S166 was accompanied by graded release of 14-3-3 from GPIb. Evidence that 14-3-3 participates in αaIIbβ3 signaling came from its co-immunoprecipitation with αaIIbβ3 from sheared platelets. Moreover, when CHO/αaIIbβ3 cell 14-3-3 was sequestered by expression of GPIb, αaIIbβ3-induced activation of RhoGTPases and cell spreading were inhibited; overexpression of 14-3-3 restored these functions. These studies: i) show that 14-3-3 is critical for αaIIbβ3-induced spreading; ii) describe a novel function of GPIb in dampening the transition to firm adhesion through the regulated sequestration of 14-3-3; and iii) suggest that the availability of 14-3-3 for αaIIbβ3-mediated adhesion is determined by the Gai-modulated level of GPIb phosphorylation.

Disclosures:
No relevant conflicts of interest to declare.
Regulation of Human Sickle Erythrocyte Hydration Status by Endothelin-1 Is Mediated by Casein Kinase II.
Authors: Romero Jose R. , Hoehn Corey , Alves Jessica , Rivera Alicia 
Published: October 2019

Abstract
Abstract 3020
Poster Board II-996
In Sickle Cell Disease, K+ efflux and osmotically induced water loss leads to erythrocyte dehydration, an important step in the formation of hemoglobin S polymers and irreversible sickle cells. We have previously reported that activation of sickle erythrocyte endothelin-1 (ET-1) receptors is partially responsible for dense irreversible sickle cell formation thru mechanisms that are still undefined. Casein Kinase II (CK2), a serine/threonine kinase that phosphorylates acidic proteins, has been shown to regulate calmodulin activity and cytoskeletal proteins. Thus, we posit that CK2 may regulate cellular hydration status. CK2 activity is modulated by apigenin, emodin, heparin, and ornithine decarboxylase. Our results indicate that CK2 activity in erythrocyte membranes increases in the presence of ET-1 (2.8 ± 0.1 to 4.9 ± 0.01 nmol/min/mL * 106 cell) an event that is blunted by pre-incubation with the ET-1 B receptor blocker, BQ788 (2.5 ± 0.1 nmol/min/mL * 106 cell). Our data also indicate that 20 μM apigenin blocks ET-1 induced CK2 activity in sickle erythrocytes (2.8 ± 0.1 to 2.7 ± 0.4 nmol/min/mL * 106 cell). We then examined the role of CK2 activation in cellular dehydration. We incubated sickle erythrocytes for 3 hours in deoxygenation-oxygenation cycles in the presence or absence of 20μM apigenin or 2μM 4,5,6,7-tetrabromobenzotriazole (TBB), another specific CK2 inhibitor, and measured the changes in erythrocyte density by the phthalate oil method. We observed that inhibition of CK2 led to the inhibition of deoxygenation-stimulated cellular dehydration in sickle erythrocytes by apigenin (D50= 1.106 to 1.100 g/mL) or TBB (D50 =1.097 g/mL). Apigenin also inhibited the volume-stimulated K+ efflux in a dose-dependent manner (IC50 = 18 ± 1 μM). These results strongly implicate CK2 as an intermediate of the ET-1 stimulated cellular volume regulatory systems in erythrocytes and suggest that regulation of CK2 activity may lead to reduction of hemoglobin S polymerization in vivo. Supported by NIH R01-HL09632 to AR.

Disclosures
No relevant conflicts of interest to declare.
The Role of gas6 in Venous Thrombosis In Vivo.
Authors: Robins Richard , Carmeliet Peter , Blostein Mark 
Published: October 2019

Abstract
Abstract 3057
Poster Board II-1033
Gas6 is the vitamin-K dependent protein product of growth arrest specific gene 6. A genetic deficiency of this protein protects mice against experimentally induced thrombosis without causing a bleeding diathesis. Protection from thrombosis results from a deficiency in platelet aggregation and secretion. In addition to being expressed by platelets, Gas6 and its receptors are also expressed by vascular cells including the endothelium, an organ known to play a role in the hemostatic balance. While endothelial Gas6 has been shown to promote inflammation and cell survival, it remains unknown if it contributes to the pathophysiology of venous thrombosis. To answer this question, we employed a bone marrow transplantation (BMT) strategy using wild type and Gas6 null mice to create chimeric mice with combined genotypes in the vascular and platelet compartments. Mice were exposed to a dose of radiation optimized to maximize both survival and ablation of recipient marrow. Irradiated mice were then infused with bone marrow cells isolated from the femurs and tibias of donor mice and were allowed a one month recovery period for hematologic reconstitution. Success of marrow uptake was confirmed by PCR. They were then subjected to the Ferric Chloride model of venous thrombosis in the Inferior Vena Cava (IVC). Four groups of transplanted mice were studied. Results from these BMT experiment show a contributing effect by both endothelial as well as platelet Gas6 to thrombus formation (n=8, p<0.01). Mice with combined genotypes (Gas6-/- into WT and WT into Gas6 -/-) show an intermediate thrombus weight suggesting that both vascular and platelet derived Gas6 are both responsible for thrombosis pathology. Therefore, Gas6 at both sites could be potential targets in treating venous thrombosis.

Disclosures
No relevant conflicts of interest to declare.
Components of the Metabolic Syndrome in 500 Adult Long-Term Survivors of Childhood Cancer.
Authors: van Waas Marjolein , Neggers Sebastian , Pieters Rob , Van Den Heuvel M.M. 
Published: October 2019

Abstract
Abstract 3068
Poster Board III-5

Introduction
Adult survivors of childhood cancer have been reported to have an increased risk of late sequels. A cluster of abnormalities that contribute to the metabolic syndrome may be expressed at a higher level and therefore result in an increased risk for diabetes mellitus and cardiovascular diseases.

Patients and Methods
We investigated a single centre cohort of 500 adult survivors (228 females) of childhood cancer, median age 28 years (range 18-59 years) and median follow-up time 19 years (range 6–49 years). This cohort included 164 acute lymphoblastic leukaemia (ALL) survivors (75 females). We measured total cholesterol, high-density lipoprotein-cholesterol (HDL), systolic and diastolic blood pressure, body mass index and the prevalence of diabetes mellitus. Data from the Dutch epidemiologic MORGEN-study were used to calculate standard deviation scores as normative values.

Results
The criteria of the metabolic syndrome were met in 13% of the total cohort. ALL survivors treated with cranial irradiation had an increased risk of developing the metabolic syndrome compared to ALL survivors not treated with cranial irradiation (23% vs. 7%, P=0.011). ALL survivors who received CRT had higher total cholesterol levels compared to ALL survivors who did not (mean SDS 0.38 vs. mean SDS –0.05, P=0.027), whereas their HDL levels did not differ. Also, ALL survivors treated with CRT were more often hypertensive compared to ALL survivors not treated with CRT (22% vs. 10%, P=0.036) and more often overweight (59% vs. 34%, P=0.003), however they were not more often obese (12% vs. 9%, ns).

Conclusions
Adult survivors of childhood cancer, especially ALL survivors treated with cranial irradiation, are at increased risk of developing the metabolic syndrome. This increased risk is probably determined by higher prevalence of overweight and hypertension in ALL survivors.

Disclosures
No relevant conflicts of interest to declare.
Pharmacogenetic Risk Factors for Altered Bone Mineral Density and Body Composition in Pediatric Acute Lymphoblastic Leukemia.
Authors: te Winkel Mariël L. , van Beek Robert D , de Muinck Keizer-Schrama Sabine M.P.F. , Uitterlinden André G. , Hop Wim C.J. , Pieters Rob , Van Den Heuvel M.M. 
Published: October 2019

Abstract
Abstract 3071
Poster Board III-8

Introduction
As the survival of pediatric acute lymphoblastic leukemia (ALL) improves, research on treatment-related morbidity, like disturbance of body composition and bone mineral density (BMD), is required. This study investigates pharmacogenetic risk factors for BMD and body composition in pediatric ALL.

Methods
We determined the influence of SNPs in 4 genes (vitamin-D receptor (VDR: BsmI/ ApaI/ TaqI and Cdx-2/ GATA), collagen type I alpha 1 (SpI), estrogen receptor 1 (ESR1: PvuII/ XbaI) and the glucocorticoid receptor (BclI)) on body composition, BMD and fracture risk during dexamethasone-based pediatric ALL treatment. Anthropometry data of 69 patients (mean age 7.4 (range: 1.6-16.8) year) treated according to the DCOG-ALL9 protocol were evaluated. In patients aged >4 years body composition and BMD of the total body (BMD-TB) and the lumbar spine (BMD-LS) were measured repeatedly using dual energy X-ray absorptiometry. To correct for bone size we calculated bone mineral apparent density of the lumbar spine (BMAD-LS) with the model BMAD-LS = BMD-LS x (4/ (n × width)). All values were expressed as standard deviation scores (SDS). Repeated measurements analysis (ANOVA) was used.

Results
Non-carriers of VDR 5'-end (Cdx-2/ GATA) haplotype 3 revealed a significant larger fat gain than carriers (Δ%fat: non-carriers: +1.76 SDS, carriers: +0.77 SDS, p<0.001). At diagnosis and during therapy, BMD-LS was significant higher in non-carriers of VDR 5'-end (Cdx-2/ GATA) haplotype 3 than in carriers. The other SNPs did not influence BMD or fracture risk during or after treatment. The year after treatment discontinuation, lean body mass increased in non-carriers of ESR1 (PvuII/ XbaI) haplotype 3 and decreased in carriers (Δ lean body mass: non-carriers: +0.28 SDS, carriers: -0.55 SDS, p<0.01).

Conclusion
This is the first study investigating the influence of genetic variation of the VDR, COLIA1, ESR1 and GR on body composition, BMD and fracture risk in pediatric ALL. We found the VDR 5'-end (Cdx-2/GATA) haplotype 3 as a protective factor for excessive fat gain during therapy. Moreover, this haplotype 3 of the VDR 5'-promoter was determined as risk factor for a lower BM(A)D-LS at diagnosis that remained during ALL treatment. Carriage of ESR1 (PvuII/XbaI) haplotype 3 negatively influenced recovery of lean body mass after treatment discontinuation.

Disclosures
No relevant conflicts of interest to declare.
Prognosis of Secondary Acute Myeloid Leukemia Is Affected by the Type of the Preceding Hematologic Disorders and the Presence of Trisomy 8.
Authors: Koh Youngil , Kim Inho , Kim Hyun-Kyung , Yoon Sung-Soo , Lee Dong Soon , Park Seonyang , Kim Byoung Kook 
Published: October 2019

Abstract
Abstract 3117
Poster Board III-54
Differences in the clinical course of secondary acute myeloid leukemia (s-AML) according to the type of the preceding disorders are not defined. We compared the outcomes of therapy-related AML (t-AML), AML following myelodysplastic syndrome (MDS-AML) and AML following myeloproliferative disorder (MPD-AML). We also intended to find prognostic factors in s-AML overall.
Retrospective medical record review at Seoul National University Hospital was performed. We assessed response to induction chemotherapy and overall survival (OS).
95 s-AML patients (median age 56.4 years) were analyzed. 26, 57 and 12 patients had t-AML, MDS-AML and MPD-AML respectively. For patients receiving induction chemotherapy, complete remission (CR) rate was 47.5% and CR rate was different according to the type of the preceding disorders (p=0.004). Compared to t-AML (p=0.027) and MDS-AML (p=0.050), MPD-AML had shorter OS. In s-AML, presence of trisomy 8 had a prognostic impact (p=0.003) along with cytogenetic risk group (p=0.016). In multivariate analysis, the type of the preceding disorders (p=0.026), 5q deletion (p=0.015) and trisomy 8 (p=0.040) were independent prognostic factors.
In conclusion, prognosis of s-AML was different according to the type of the preceding disorders with the worst prognosis in MPD-AML. Along with cytogenetic risk grouping, trisomy 8 had a prognostic impact in s-AML.
Table 1 Characteristics of s-AML patients according to preceding disorders Characteristics t-AML AML-MDS AML-MPD p-value Age (median) 54.1 57.2 65.5 0.888 Gender 0.262 Male 13 39 8 Female 13 18 4 Latency period 31.2 6.7 92.6 0.008 White blood cell (median) (/μL) 5615 2905 12785 0.002 Hemoglobin (median) (g/dL) 8.0 8.4 8.7 0.543 Platelet (median) (/μL) 51500 32000 100500 0.509 Risk group 0.001 Favorable 6 0 0 Intermediate 12 37 10 Poor 5 12 2 5q deletion 0.731 Present 5 7 2 Absent 18 42 10 -7/7q deletion 0.107 Present 1 11 1 Absent 22 38 11 Trisomy 8 0.777 Present 6 13 2 Absent 18 36 10 Induction chemotherapy 0.619 Yes 23 47 11 No 3 10 1 Complete remission after induction 0.004 Yes 17 19 2 No 6 27 9 Stem cell transplantation 0.137 Yes 9 4 1 No 8 15 2
Figure 1 CR-1 (A) and OS (B) according to preceding disorders CR-1 was not affected by preceding disorders, however OS was significantly the shorter in AML evolving from MPD (black line) compared to AML evolving from MDS (orange line) and t-AML (green line). Figure 1. CR-1 (A) and OS (B) according to preceding disorders CR-1 was not affected by preceding disorders, however OS was significantly the shorter in AML evolving from MPD (black line) compared to AML evolving from MDS (orange line) and t-AML (green line).

Disclosures
No relevant conflicts of interest to declare.
Atorvastatin Reduces Collagen-Induced Platelet Function in Aspirin-Treated Patients with Acute Myocardial Infarction: Mechanisms Involved.
Authors: Teresa Santos Maria , Fuset Maria Paz , Ruano Miguel , Moscardo Antonio , Cortina Belen , Dasi Maria Angeles , Valles Juana 
Published: October 2019

Abstract
Abstract 3126
Poster Board III-63
Treatment with statin at the onset of acute myocardial infarction (AMI) has demonstrated a clinical benefit. The effects of statins on platelet TXA2 synthesis or platelet function could be involved in the clinical benefit, but has not been previously evaluated in patients with AMI. Collagen-induced (1μg/mL)-platelet aggregation, activation (14C-5HT release, TXA2 synthesis) and platelet recruitment (proaggregatory activity of activated platelets cell-free releasate) (1, 2) were evaluated in 184 patients with AMI within 48h of the onset of the acute event. Patients were divided into 2 groups: treated with aspirin (ASA) alone (n=139) and treated with both ASA and atorvastatin (AT; n=45). Insufficient inhibition of TXA2 (&lt;95% vs. ASA-free normal subjects) was detected in 25% of patients treated with ASA vs. 9% in the group treated with ASA+AT (p&lt;0.05), and persistent TXA2 synthesis was reduced by 87.5% in patients treated with ASA+AT vs. ASA alone. Reduction of TXA2 by AT was associated with significant inhibitions of collagen-induced: aggregation (30%), 14C-5HT release (39%) and recruitment (33%). To evaluate the biochemical mechanisms involved in the AT effect on TXA2 synthesis, experiments in vitro with washed platelets from normal subjects were performed. Incubation of AT alone (1-20μM) or AT plus a suboptimal ASA concentration (1μM) resulted in a significant inhibition of collagen-induced (1μg/mL) TXA2 synthesis and aggregation, confirming the results in the patients. In contrast, AT did not influence arachidonic acid-induced aggregation, suggesting an effect of the statin on the cytosolic phospholipase A2 (cPLA2) activity rather than on cyclooxygenase-1. cPLA2 activity is known to be regulated by its phosphorylation by MAPK (p38 or erk 1/2) and the increase in cytosolic Ca2+. In kinetic studies we found that AT reduced collagen-induced cPLA2 and erk 1/2 phosphorylation, but did not modify p38 phosphorylation. In addition, AT almost blocked collagen-induced increase in cytosolic Ca2+. In conclusion, AT associated with ASA significantly reduced collagen-induced persistent TXA2 synthesis and platelet reactivity early at the onset of AMI, reducing aspirin resistance in these patients. This effect is mediated by the AT control of cPLA2 activity via both erk1/2-mediated cPLA2 phosphorylation and blockade of collagen-induced elevation of cytosolic calcium. 1. Santos MT et.al. J Clin Invest 1991;87:571-80; 2. Vallés J et.al. Blood 2002;99:3978-84.FIS07/0463/MMA2006/RD06/0026

Disclosures
No relevant conflicts of interest to declare.