bluuebunny/crossref_metadata_2025 huggingface dataset:
Thrombopoietin Initiates Demethylation-Based Transcription of GP6 during Megakaryocyte Differentiation.
Authors: Kanaji Sachiko , Jacquelin Beatrice , Chang Mei , Nugent Diane J. , Komatsu Norio , Izuhara Kenji , Kunicki Thomas J.
Published: October 2019

Glycoprotein VI (GPVI) is an essential platelet receptor for collagens that is exclusively expressed in the megakaryocytic lineage. Transcription of the human gene GP6 is driven largely by GATA-1, Sp1 and Fli-1. However, the mere presence of these is not sufficient to initiate transcription during megakaryocyte differentiation, and other mechanisms are suggested to be involved in the regulation of megakaryocyte-specific expression of GPVI. In this study, we show that GPVI expression during megakaryocytic differentiation is dependent on CpG demethylation that can be initiated by thrombopoietin (TPO). Sodium bisulfite genomic sequencing established that a CpG-rich island within the GP6 promoter region is fully methylated at 10 CpG sites in GPVI non-expressive cell lines, such as UT-7/EPO and C8161, but completely unmethylated in GPVI expressive cell lines, including UT-7/TPO and CHRF288-11. To further confirm the relationship between CpG demethylation and expression of GPVI in primary cells, we treated human cord blood cells with TPO. The GP6 promoter is highly methylated in cord blood mononuclear cells (progenitors) but not in CD41+enriched cells obtained after TPO differentiation. Furthermore, when UT-7/EPO-Mpl cells, which stably express human c-mpl, were treated with TPO, demethylation of the GP6 promoter was induced. In every case, demethylation of the GP6 promoter correlated with an increase in mRNA level. Thus, megakaryocyte-specific expression of the GP6 gene is regulated, in part, by CpG demethylation which can be directly initiated by TPO. Our results establish, for the first time, a role for TPO in dynamic changes in CpG methylation status that are involved in the epigenetic regulation of megakaryocyte-specific gene expression.
A Role for Bile Salt-Dependent Lipase in Platelet Activation and in Thrombus Formation in Vivo.
Authors: Panicot-Dubois Laurence , Dubois Christophe , Furie Barbara C. , Furie Bruce , Lombardo Dominique
Published: October 2019

Bile Salt Dependent Lipase (BSDL) is an enzyme secreted by pancreatic acinar cells. BSDL, in the presence of primary bile salts, participates in the hydrolysis of dietary lipid esters in the duodenum lumen. This 105 kDa N and O-glycosylated protein has been detected in the plasma of normal subjects. Recent in vitro and in vivo studies demonstrated that pancreatic BSDL reaches the blood via transcytosis through enterocytes. Other studies showed that pancreatic human BSDL is captured by human umbilical vein endothelial cells and induces the proliferation of smooth muscle cells in vitro at BSDL concentrations found in blood, suggesting that this enzyme may play a role in hemostasis and thrombosis. However the specific role of circulating BSDL is unknown. The goal of this study was to determine the possible involvement of circulating BSDL in thrombus formation. We investigated the participation of circulating mouse BSDL in thrombus formation using widefield intravital microscopy in the cremaster muscle of living mice. Thrombi were formed following laser injury of the vessel wall of an arteriole in the cremaster muscle. Pancreatic mouse BSDL, a 74 kDa glycoprotein, was detected using several antibodies directed against either the whole human BSDL (pAbL64, pAbL32) or a peptide based on a sequence in the N-terminal domain of BSDL (Ser326-Thr350; pAbAntipeptide). Mouse and human BSDL share about 80% sequence homology, the main difference localized in the C-terminal domain, which is truncated to the mouse BSDL compared with the human enzyme. All the antibodies are able to specifically recognize the mouse pancreatic BSDL. Using antibodies pAbL64, pAbL32, or pAbAntipeptide we observed specific accumulation of circulating mouse BSDL into the growing thrombus. The circulating BSDL co-localized with platelets present in the thrombus. These results suggest that circulating BSDL is involved in thrombus formation in vivo. In order to determine if BSDL plays a role in platelet activation and aggregation, we performed in vitro studies on human washed platelets. BSDL increased both the amount of phosphatidylserine exposure on the surface of platelets and the activation of αIIbβ3 induced by thrombin. These results indicate that this enzyme can amplify the activation of platelets in vitro. While BSDL alone cannot induce the aggregation of platelets, this enzyme significantly increases the amount of platelet aggregation induced by SFLLRN peptide or thrombin. Altogether, these data suggeste that circulating BSDL participates in the thrombus formation after laser injury of the arterial wall and can amplify both the activation of platelets and the phosphatidylserine exposure, increasing the thrombotic response after vessel injury. This mechanism may be operative in the development of venous thromboembolic disease in pancreatic cancer.
Human Platelets Contain and Release an Active ADAMTS-13-Like Metalloprotease.
Authors: Liu Li , Choi Huiwan , Aubrey Bernardo , Bergeron Angila , Nolasco Leticia , Moake Joel F. , Ruan Changgeng , Dong Jing-Fei
Published: October 2019

The adhesion ligand von Willebrand factor (VWF) is synthesized and stored in vascular endothelial cells and megakaryocytes/platelets. These cells release VWF in response to similar stimulations. However, a longstanding observation stated that, unlike endothelial cells, platelets do not release the ultra-large form (UL) of VWF upon activation. The lack of ULVWF release may be attributed to the difference in multimerization process in the storage granules or the intrinsic proteolysis of the hyperreactive ULVWF. To examine these possibilities, we analyzed the multimer compositions of VWF stored in and released from platelets. We found that ULVWF was detected by immunobloting of washed platelet lysates in the presence, but not in the absence of 5 mM EDTA. Similarly, when stimulated with 5 μM of thrombin receptor-activating peptide (TRAP), platelets released only VWF that was similar in sizes to plasma VWF, suggesting that platelet ULVWF may have been cleaved before or during its release. In support of this hypothesis, we found that platelets expressed ADAMTS-13-like molecule by western blot of platelet lysates and flow cytometry using two different ADAMTS-13 antibodies. The surface expression of ADAMTS-13-like molecule increased significantly upon platelet activation by TRAP and ADP. Consistent with these observations, the Triton-X-100 lysates of washed platelets and supernatant of washed platelets activated by TRAP contained VWF-cleaving activity, as demonstrated by the cleavage of ULVWF strings formed on histamine-stimulated endothelial cells under fluid shear stress. Our finding is consistent with a previous report, but further showed that this ADAMTS-13-like molecule exists not only in cytoplasma, but also on the surface of platelets. More importantly, the ADAMTS-13-like molecule increased its expression on platelet surface and was released upon platelet activation. The ADAMTS-13-like molecule either in platelets or released upon activation was active in cleaving ULVWF. This intrinsic mechanism for ULVWF proteolysis may be physiologically important. It may prevent the sudden release of hyper-reactive ULVWF from platelets and serve as the second pool of ADAMTS-13 to encounter the increase in ULVWF release from endothelial cells during conditions such as severe infection. The role of the platelet membrane-bound ADAMTS-13 in hemostasis remains to be further determined.
Mechanisms for Thrombopoietin-Induced Potentiation of Platelet Aggregation.
Authors: Lova Paolo , Campus Francesca , Bertoni Alessandra , Sinigaglia Fabiola , Balduini Cesare , Torti Mauro
Published: October 2019

Thrombopoietin (TPO) is the major cytokine regulating megakaryocytes proliferation and thrombopoiesis. Binding of TPO to the c-Mpl receptor expressed on human platelets promotes tyrosine phosphorylation of a number of proteins and potentiates platelet aggregation induced by low concentrations of a variety of agonists including ADP. Nevertheless, TPO alone is not able to induce any platelet functional response, such as shape change, granule secretion, or aggregation. It is now clear that platelet aggregation induced by many different agonists results from the concomitant activation of both Gi-and Gq-dependent signaling pathways. In particular, activation of a Gi-dependent pathway is essential, albeit not sufficient, to elicit complete platelet aggregation. Recently, it has been proposed that the small GTPase Rap1B is an important element in the Gi-dependent pathway for platelet aggregation. ADP activates Gq and Gi-dependent pathways by binding to the P2Y1 and the P2Y12 receptors, respectively. In this study we investigated the contribution of TPO in platelet activation induced by ADP. We found that TPO could restore ADP-induced platelet aggregation when the Gq-coupled receptor was blocked, but failed to restore ADP-induced aggregation in absence of Gi stimulation. Similarly, TPO was unable to restore U46619-induced platelet aggregation, when the Gi-coupled P2Y12 receptor for ADP was blocked, but caused irreversible platelet aggregation when added in combination with epinephrine, which binds to a Gi-coupled receptor. TPO did not cause any detectable calcium movements or pleckstrin phosphorylation in human platelets. Moreover, TPO failed to restore ADP-induced cytosolic calcium incresases and pleckstrin phosphorylation when the Gq-coupled P2Y1 receptor was blocked, although aggregation was completely restored. Moreover, we found that TPO induced activation of Rap1B in platelets, and could completely restore ADP-induced Rap1B activation, which was partially reduced upon blockade of the P2Y1 receptor. Finally, ADP-induced binding of both fibrinogen and PAC-1 monoclonal antibody to integrin αIIbβ3 was prevented by the blockade of the Gq-coupled P2Y1 receptor, but completely restored by the simultaneous addition of TPO. These findings indicate that TPO can complement Gi-, but not Gq-dependent pathways for integrin αIIbβ3 activation and platelet aggregation, through a new mechanism that does not involve activation of phospholipase C, but may involve the small GTPase Rap1B.
Phosphoinositide3-Kinase Independent Regulation of Bruton’s Tyrosine Kinase in Platelet Immunoreceptor Signaling.
Authors: Watanabe Naohide , Nakajima Hideaki , Oda Atsushi , Ikeda Yasuo , Handa Makoto
Published: October 2019

Phosphoinositide 3-kinase (PI3K)-dependent activation of Bruton’s tyrosine kinase (Btk) is an indispensable step of B cell antigen receptor (BCR)-mediated signaling leading to cell development and function. Btk is a cytosolic tyrosine kinase and its recruitment to the plasma membrane is a necessary step for its function. In the BCR pathway, class 1A PI3K is though to play a major role in Btk recruitment by generating the D3 phosphoinositide as a docking site for the pleckstrin homology (PH) domain of this effecter kinase. This widely accepted hypothesis has been tested in platelets from gene knockout or mutant mice, since the cells utilize sets of transducers in collagen-induced GP VI signaling similar to those involved in immunoreceptor tyrosine-based activation motif-mediated signaling cascades activated by BCR and T cell antigen receptor (TCR) ligation. GP VI stimulation by collagen or collagen related peptide induces cellular responses including aggregation, granular secretion and adhesion, and Btk/phospholipase C (PLC) γ2 activation. As compared with control mice, these cellular responses and PLCγ2 tyrosine phosphorylation of either Btk or PI3K p85α−/− platelets were readily impaired, but the defect was greater in Btk−/− than p85α−/− platelets. Most strikingly, platelets from double-deficiency mice showed a most severely compromised phenotype implying the existence of a PI3K-independent pathway for Btk activation. Moreover, unlike B cells, as compared with Btk−/− platelets, only subtle functional defect was observed in X-linked immunodeficiency (Xid) platelets in which PI3K-dependent Btk activation is selectively lacking due to a naturally occurring point mutation of the gene encoding the PH domain of the kinase. In the TCR pathway, an adaptor complex formed by LAT, Gads and SLP-76 proteins that is membrane-bound via LAT palmitoylation readily recruits Itk, which is a counterpart Btk/Tec family kinase specific for TCR. Indeed, Btk was found to be associated with LAT/Gads/SLP-76 complex in platelets in a GP VI-stimulation dependent manner, and this phenomenon was unaffected by either PI3K defect or PI3K inhibitor. These results indicate that in platelet immunoreceptor signaling, Btk function is under control, at least in part, by a mechanism independent of PI3K engagement.
Reversible Exposure of Phosphatidylserine on Thrombin-Stimulated Platelets Detected by Binding of Lactadherin.
Authors: Shi Jialan , Rasmussen Jan T. , Heegaard Christian W. , Gilbert Gary E.
Published: October 2019

Platelets stimulated by thrombin support activity of the phosphatidylserine (PS)-dependent prothrombinase and factor Xase complexes. However, annexin V, a PS-detecting protein, does not bind to thrombin-stimulated platelets. Thus, the exposure of PS and its necessity for the prothrombinase and factor Xase complexes on platelets has been questioned. We hypothesized that platelets expose limited quantities of plasma membrane PS at levels that remain insufficient to support annexin V binding. We tested the hypothesis by investigating the capacity of platelets to support binding of lactadherin, a PS-binding milk protein. Binding of fluorescein-labeled lactadherin vs. fluorescence-labeled annexin V to synthetic membranes and to platelets was measured by flow cytometry. Lactadherin binding was proportional to PS content over the 0–4% range while annexin V required a threshold of approx. 2.5% PS. Platelets stimulated with 0.1–0.5 μM A23187 bound lactadherin but not annexin V. Binding of lactadherin was Ca++-independent, inhibited by PS-containing phospholipid vesicles and by antibodies to the C2 domain of lactadherin indicating that binding occurs via the phospholipid-binding rather than the integrin-binding motif of lactadherin. Platelets stimulated by thrombin or via the PAR-1 thrombin receptor also bound lactadherin but not annexin V. The KD was 84 ± 1 nM and there were 380 binding sites/platelet. Experiments in which platelets were labeled with fluorescent NBD-PS indicated that PS exposure occurred over 2 min and involved less than 15% of platelet PS. Exposed PS subsequently decreased, with more than 75% loss over 2 hr. Lactadherin inhibited greater than 98% of prothrombinase and factor Xase activity on platelets stimulated by A23187 or by thrombin. These results indicate that thrombin-stimulated platelets support the prothrombinase and factor Xase complexes through reversible exposure of a small fraction of membrane PS. Exposure of PS can be detected with Ca++-independent lactadherin binding in dilute platelet rich plasma, a method suitable for clinical studies.
CpG Binding Protein Is Required for Hematopoiesis in Zebrafish.
Authors: Young Suzanne R.L. , Doyle Christine , Marrs James , Skalnik David G.
Published: October 2019

CpG binding protein (CGBP) is a transcriptional activator that binds unmethylated CpG motifs. CGBP is ubiquitously expressed, and deletion of the CGBP gene in mice results in an embryonic lethal phenotype (E3.5–E6) indicating that CGBP is required for embryonic mammalian development. CGBP-null murine embryonic stem (ES) cells are viable but unable to differentiate and have a 60% decrease in CpG methylation compared to wild type ES cells. Based upon these results, it is hypothesized that CGBP is required for normal stem cell function. RT-PCR was used to identify a homologue of CGBP in zebrafish which is ~70% identical to the mouse CGBP. It was determined that CGBP is expressed in the zebrafish as early as 2 hpf. Morpholino oligonucleotides were designed to the zebrafish CGBP and injected into 1–2 cell embryos. Approximately 80% of the zebrafish treated with the CGBP morpholino oligonucleotides had little or no circulating red blood cells and had abnormal yolk sac morphology at 48 hpf. More than half of the CGBP treated zebrafish also exhibited cardiac edema, and ~14% were dead at 24 hpf. A similar phenotype was observed with a second morpholino designed to an independent site of the zebrafish CGBP cDNA. Zebrafish treated with a control morpholino oligonucleotide at the 1–2 cell stage exhibited normal yolk sac morphology and normal red blood cell circulation at 48 hpf. The control treated zebrafish did not have cardiac edema, and only 2–3% of these fish were dead at 24 hpf. The specificity of phenotypes observed following treatment with the CGBP morpholino oligonucleotide was assessed by co-injection of mouse CGBP mRNA with the CGBP morpholino oligonucleotide at the 1–2 cell stage. Only 37% of these fish exhibited a decrease in circulating red blood cells compared to 80% of zebrafish treated with CGBP morpholino alone. While 14% of the CGBP morpholino treated zebrafish were dead at 24 hpf, only 4% of the co-injected zebrafish were dead at 24 hpf. This experiment demonstrates that the mouse CGBP is able to rescue the phenotypes induced by a reduction of the endogenous zebrafish CGBP. These data indicate that CGBP is essential for normal zebrafish hematopoiesis and establishes the importance of CGBP in post-gastrulation development.
Carrier-Independent Nuclear Import of the Transcription Factor PU.1.
Authors: Yaseen Nabeel R. , Takeda Akiko , Nazari Reza , Shio Helen , Blobel Gunter , Zhong Hualin
Published: October 2019

PU.1 is a transcription factor of the Ets family with important functions in hematopoietic cell differentiation. Using GFP-PU.1 fusions, we show that the Ets DNA-binding domain of PU.1 is necessary and sufficient for its nuclear localization. Fluorescence and ultrastructural nuclear import assays showed that PU.1 nuclear import requires energy but not soluble carriers. PU.1 interacted with the FG repeats of nucleoporins Nup62 and Nup153. The binding of PU.1 to Nup153, but not to Nup62, dramatically increased in the presence of RanGMPPNP, indicating the formation of a PU.1/RanGTP/Nup153 complex. The Ets domain accounted for the bulk of the interaction of PU.1 with Nup153 and RanGMPPNP. Since Nup62 is located close to the midplane of the nuclear pore complex (NPC) while Nup153 is at its nuclear side, these findings suggest a model whereby RanGTP propels PU.1 towards the nuclear side of the NPC by increasing its affinity for Nup153. This notion was confirmed by ultrastructural studies using gold-labeled PU.1 in permeabilized cells.
Inhibition of Hb S Polymerization In Vitro by a Novel EF Helix ß73 His Containing Peptide.
Authors: Akbar Mohammed G.K. , Ding Min , Surrey Saul , Adachi Kazuhiko
Published: October 2019

Sickle hemoglobin polymerization involves not only ß6 Val in a largely hydrophobic acceptor pocket but other contact sites. We recently found that the ß73 His Hb S variant (ß6 Val and ß73 His) promoted polymerization compared to deoxy Hb S, while ß73 Leu Hb S (ß6 Val and ß73 Leu) inhibited polymerization like deoxy Hb C-Harlem (ß6 Val and ß73 Asn). In fact, ß73 Asp in Hb S makes a hydrogen bond with ß4 Thr in deoxy Hb S polymers, and this is a unique position to promote or inhibit polymerization by amino acid change [Adachi et al., Biochemistry (2003), 42, 4476]. Kinetics of polymerization, solubility and minimum concentration required for polymerization of the ß73 Hb S variants were affected by ß73 amino acid (inhibition of polymerization: His < <Asp <<Asn < Leu). Inhibition of Hb S ß73 Leu polymerization compared to Hb S may be caused by weakening of the hydrogen bond interaction between the hydroxyl group of ß4 Thr and the ß73 amino acid like Hb C-Harlem. Furthermore, kinetics of polymerization of 1:1 Hb S/Hb A ß73 His mixtures were enhanced compared to AS mixtures, while Hb S/Hb A ß73 Leu mixtures showed inhibitory effects similar to FS mixtures. These results suggest that the Hb A ß73His variant promotes Hb S polymerization almost as efficiently as Hb S ß73 His, and that the ß73 His in Hb A and Hb S variants strengthens the hydrogen bond with ß4 Thr, which helps facilitate formation of domains and 14-stranded fibers.

Based on these results, we chemically synthesized a 15-mer EF helix peptide containing ß73 His (Lys-Lys-Val-Leu-Gly-Ala-Phe-Ser-His-Gly-Leu-Ala-His-Leu-Asp) and evaluated effects of this peptide on Hb S polymerization. DIC analysis in 1.0 M phosphate buffer showed peptide-induced inhibition of fiber elongation rate for deoxy-Hb S which increased linearly with increasing amounts of peptide (e.g., a 5-fold molar excess of peptide resulted in a 6-fold decrease in fiber elongation rate). Solubility of deoxy Hb S increased linearly with increasing peptide (e.g., a 5-fold molar excess of peptide increased solubility by 1.2-fold). The delay time prior to polymerization of deoxy Hb S in a high phosphate buffer also was increased significantly in the presence of this peptide. In contrast, the same 15-mer peptide containing ß73 Leu instead of His showed no effect on solubility or kinetics of polymerization, suggesting that the ß73 His peptide specifically affects the A-helix of Hb S containing ß4 Thr just like the ß73 His Hb S variant. This and possibly other peptides interrupting the ß73-ß4 interaction which result in inhibition of deoxy Hb S polymerization may be good candidates for inhibitors of Hb S polymerization.
A Sickle Transgenic Mouse Model of Acute Chest Syndrome.
Authors: Fabry Mary E. , Alleyne Dawn , Klings Elizabeth S. , Farber Harrison , Zhang Yingyi , Resnick Robert , Aldrich Thomas K. , Steinberg Martin H. , Nagel Ronald L.
Published: October 2019

Acute chest syndrome (ACS), the most common cause of death among adults with sickle cell disease and a significant threat to children, is often triggered by bone marrow embolization, especially among adults. We have recently developed a sickle transgenic mouse model for acute chest that consists of injecting a mixture of bone marrow and mouse fat into the tail vein of a sickle transgenic partial knockout mouse. The mouse used was a NY1 partial KO expressing 100% human α and 55% βS globins. Mice were injected with bone marrow (BM) from two femurs (about 15 mg) and purified peritoneal fat (about 15 mg, as murine BM lacks fat), and examined at 0, 6, 24, and 48 hours post-injection. Creatine kinase (CK), predominantly the MM form, increased above baseline levels at 6, 24, and 48 hours in NY1 partial KO mice; whereas, in control C57Bl mice, there was an initial increase at 6 hours with a return to baseline values at 24 and 48 hours. sVCAM levels were not significantly different between C57Bl and NY1 partial KO mice at baseline: 717±203 in C57Bl mice and 752±147 in NY1 partial KO mice (N=15 and 11, respectively); after injection of fat/BM sVCAM levels nearly doubled in NY1 partial KO mice and remained elevated at 48 hours. In fat/BM injected C57Bl mice, sVCAM levels remained essentially constant from 0 to 48 hours. Mouse lungs were harvested at 48 hours after saline perfusion and inflated with 10% phosphate buffered formalin. The hematoxylin and eosin stained lung sections were normal among the C57Bl control mice examined 48 hours after fat/BM injection, but among NY1 partial KO mice edema, inflammation, and congestion were found with numerous red cells entrapped, despite saline perfusion. In summary, this preparation shows promise as an animal model of ACS. Pulmonary edema and elevated levels of sVCAM are characteristic features of ACS in sickle cell patients and are found, after fat/BM injection, in our sickle mice but not in control mice. The mice survived the procedure, indicating that it will be possible to study the natural course of lung injury and interventions that may ameliorate injury.
Adult Bone Marrow-Derived Cell Fusion with Cardiomyocytes and Purkinje Neurons in Response to Irradiation but Not in Steady State.
Authors: Nygren Jens M. , Stott Simon , Liuba Karina , Breitbach Martin , Röll Willhelm , Fleischmann Bernd , Kirik Deniz , Björklund Anders , Jacobsen Sten Eirik W.
Published: October 2019

Several recent studies have suggested that bone marrow (BM) cells can contribute to non-hematopoietic cell lineages through cell fusion rather than transdiffentiation. As this phenomenon has been observed in multiple organs, including the brain and heart, without prior infliction of organ-specific insults, it has been proposed that BM cells might contribute to replacement of non-hematopoietic cell lineages during steady state, and that BM transplantation might be developed as a therapeutic modality in diseases of these organs. However, as all observations of BM-derived cell fusion in vivo have been made in lethally irradiated mice reconstituted with genetically marked BM cells, we addressed to what degree cell fusion occurs normally and/or in response to whole body irradiation. To be able to distinguish between these possibilities we used c-kit deficient (w41/w41) mice, which unlike wild type mice do not require irradiation-induced myeloablation to facilitate reconstitution of transplanted BM cells. Noteworthy, no BM-derived cell fusion events were observed in the brain (purkinje neurons) or heart (cardiomyocytes) when unconditioned w41/w41 mice were reconstituted with beta actin GFP transgenic BM cells. In striking contrast, following whole body irradiation (875 rad), BM-derived cell fusion was observed in recipient cardiomyocytes and purkinje neurons of all BM transplanted mice. Thus, spontaneous adult BM-derived cell fusion does not occur in steady state but is potently facilitated by irradiation-induced injuries to the organs in which cell fusion occurs.
Decreased Serum Pro-Hepcidin in Thalassaemia Intermedia; a Mechanism for Increased Iron Absortion Mediated Though Ineffective Erthropoiesis and NTBI?.
Authors: Shah Farrukh T. , Rafique Roozina , Evan Patricia , Porter John B.
Published: October 2019

Background: Pro-hepcidin is 10 kDa peptide localized at the basolateral membrane of hepatocytes which is also present in plasma and is believed to be a hepcidin precursor (Kulaksis et al, Gut2004;53). Experimental hepatic iron loading in mice up-regulates hepcidin mRNA while iron deficiency has the reverse effect (Pigeon et al, JBC2001;276). Paradoxically mice with a thalassaemia intermedia phenotype had reduced liver hepcidin mRNA. Furthermore, presentation of non-transferrin bound iron (NTBI) to human liver cells (Nemeth, Blood2003; 101)(Rafique, 2004 in preparation) decreased hepcidin mRNA. Patients and methods: 3 groups of patients were compared (thalassaemia major (TM), thalassaemia intermedia (TI) and sickle cell anaemia (HbSS)) to examine if variations in pro-hepcidin expression could account for the differences in iron loading. Pro-hepcidin measurements were performed at 1:1 dilutions in duplicate with a Pro-hepcidin ELISA (DRG diagnostics) using a previously published method (Kulakis, 2004). Iron status was evaluted by liver CMR , serum ferritin and NTBI; C reactive protein (CRP) was measured to exclude inflammation. All patients with a raised creatinine were excluded from the analysis. Results: TI patients (n=20) had a significantly lower serum pro-hepcidin (77 ± 40ng/ml) ( p<0.001) compared to HbSS (n=26) (111 ± 53 ng/ml), TM (n=27) (107 ± 44.5ng/ml) or control subjects (n=9) (119 ± 45ng/ml). The difference in serum pro-hepicidin between TI and HbSS persisted when patients were matched for serum ferritin (p = 0.02) or liver iron (p < 0.001). NTBI was significantly lower in HbSS (1.06 ± 0.27umol/l) than TI (2.49 ± 2.01 umol/l) even when patients were matched for serum ferritin (p <0.001) or for liver iron (p <0.05). Hb values were not significantly different between TI (8.6 g/dl) and HbSS patients (8.9g/dl). When all patients with a Hb <8g/dl were compared to those with a Hb > 10g/dl serum pro-hepcidin was not significantly different. No correlation between serum pro-hepcidin and liver iron, serum ferritin, CRP or Hb in any of the three groups was seen. Conclusion: Pro-hepcidin is significantly lower in TI than TM or HbSS patients and this cannot be accounted for by levels of anemia. We propose that NTBI, present early in TI secondary to high ineffective erythropoiesis, may lead to a similar paradoxical decrease in hepcidin synthesis reported in model systems and that this is reflected by reduced serum pro-hepcidin. Although levels of NTBI and liver iron did not differ significantly in TI and TM , transfusion and chelation therapy in TM may overide the NTBI mediated down regulation of pro-hepcidin. In HbSS, NTBI is lower than in TI or TM at matched levels of iron loading, resulting in less inhibition of pro-hepcidin synthesis and less duodenal iron absorption than in TI.
Early Hepatitis C Viral Response (EVR) to Peginterferon Alfa 2a and Ribavirin in Patients with β Thalassemia.
Authors: Harmatz Paul , Olivieri Nancy F. , Vichinsky Elliott , Kwiatkowski Janet , Giardina Patricia , Shah Farrukh , Elizabeth Wright C. , Cunningham Melody , Maureen Jonas
Published: October 2019

Hepatitis C virus (HCV) infection is an important cause of liver failure and death in patients with thalassemia who received frequent blood transfusions before HCV screening became routine. Severe transfusion-related iron overload may accelerate the progression of liver disease, influence response to therapy, and increase the risk of antiviral treatment in these patients. An open-label, single arm trial of peginterferon alfa 2a (180 ug weekly) and ribavirin (1200 mg/day for > 75 kg, 1000 mg/day 50 to 75 kg, 800 mg for < 50kg) was initiated in August, 2003 in the NIH-sponsored Thalassemia Clinical Trials Network to examine safety and efficacy in adult patients. Although the study is still enrolling patients, we are presenting viral response data for the first 16 patients (mean age: 33 yrs, range 22 to 44, 50% female, 75% genotype 1). Iron burden was assessed by chemical analysis of liver biopsy and HCV RNA levels were measured using the Roche COBAS HCV Tests (qualitative and quantitative). These analyses were performed by a central laboratory. HCV levels were measured at baseline, weeks 1, 2, 3, 4, 12 and 24.

Early Virologic Responses

Week 1 Week 2 Week 3 Week 4 Week 12 Week 24 *number with result available; **non-detectable; ***greater than 2 log decrease N* 16 16 16 16 11 6 Non-d** 1 2 2 3 7 5 >2-log 5 6 7 7 3 1 % >2 log 38% 50% 56% 63% 91% 100%

Four patients discontinued between weeks 4 through 12 (1 death, 1 persistent cough, 1 extreme fatigue, and 1 pregnancy (spouse). These patients had all achieved > 2 log drop by week 4. The mean liver iron at baseline was 10.1 mg/gm dry wt (range: 1.1 to 21.2) and the mean log HCV RNA level was 5.8 (range 4.0 to 6.8). The median time to > 2 log decrease was 1 week for patients with genotype 2 and 4 weeks for patients with genotype 1 (p=0.004). There was no correlation either between baseline iron or baseline HCV level and time to > 2 log decrease. Transfusion requirement increased 52% as assessed at 12 weeks and did not predict earlier viral response. In summary, there is EVR (>2 log drop) in 63% at 4 weeks and 91 % at 12 weeks, comparable to those seen in patients without thalassemia. Quantitative hepatic iron concentration at baseline was not a predictor of EVR. Completion of the study is planned to determine whether the EVR predicts sustained viral response and whether the iron overload or increased transfusion requirements during therapy pose significant safety issues.
Hamster Prions Are a Suitable Model for Partitioning of Human CJD Prions during Plasma Processing Steps.
Authors: Groener Albrecht , Schäfer Wolfram , Baron Henry , Vey Martin
Published: October 2019

Prion removal evaluation of plasma processing procedures is one important basis to assess the margin of safety of plasma protein therapeutics. Currently, in these evaluation studies to assess the removal capacity of selected manufacturing steps for human prions mainly prions derived from scrapie-infected hamsters or mice are used in spiking studies. In order to test the validity of hamster prions instead of different human prion strains as spiking reagents, we compared the partitioning of these prion preparations at two purification steps common to the manufacturing of several human plasma-derived therapeutic proteins at ZLB Behring. The glycine precipitation (inherent in the manufacture of e.g., vWF/Factor VIII) and the 25% ethanol precipitation step (inherent in the manufacture of e.g., alpha-1-proteinase inhibitor and albumin) were down scaled to mimic the production process. A microsomal membrane preparation as well as purified full-length pathogenic prion (PrPSc; without membrane association) prepared (a) from hamster brains infected with the model scrapie strain Sc237, (b) from brains of transgenic mice infected with human sporadic CJD prions, and (c) from the brain of a patient who died of vCJD were used for spiking experiments. The different prion preparations were spiked to the starting material of the manufacturing steps studied and the manufacturing step was performed in a dedicated laboratory. The amount of PrPSc in all fractions was determined quantitatively utilizing an ELISA-formatted Conformation Dependent Immunoassay [Safar et al., Nat Med1998; 41157–1165]. The 25% ethanol precipitation removed very effectively all prion preparations equally regardless of their origin (reduction factor ≥ 3 log10). The glycine precipitation removed the microsomal as well as the purified prion preparations from all species equally whereby prions of the purified prion preparation were removed to a considerably higher degree than the membrane preparation (reduction factor ≥ 3 log10 vs. 1.6 to 1.8 log10). In parallel, prion infectivity of each sample is currently tested in animal bioassays involving transgenic mice susceptible for the tested prion strain. It can be concluded, up to now based on immunological data, that spiked hamster prions are a reliable model to confirm the safety of human plasma-derived products also for human derived prions.
GPIbα Is Essential for Platelet Adhesion during Thrombus Formation: Studies with Mutant Mice Deficient in the Extracellular Domain of GPIbα.
Authors: Bergmeier Wolfgang , Piffath Crystal L. , Ruggeri Zaverio M. , Ware Jerry L. , Wagner Denisa D.
Published: October 2019

GPIbα binding to von Willebrand factor (vWf) exposed at a site of vascular injury is the first step in the formation of a hemostatic plug. In addition, GPIbα binding to platelet-bound vWf has previously been shown to play a key role in the incorporation of circulating platelets into a growing thrombus. However, studies in vWf-deficient mice demonstrated delayed but not absent arterial thrombus formation, suggesting that GPIbα may bind a ligand other than vWf to facilitate platelet adhesion, or that a platelet surface receptor other than GPIbα can mediate platelet adhesion in arterioles in the absence of vWf. Here we studied thrombus formation in transgenic mice expressing GPIbα in which the extracytoplasmic sequence has been replaced by an isolated domain of the human interleukin 4 receptor (IL4R-tg mice). Early platelet adhesion to ferric chloride-treated mesenteric arterioles in IL4R-tg mice was decreased by >98% when compared with controls. As a consequence, thrombus formation was completely inhibited in all of the mutant mice. To study the role of GPIbα in platelet adhesion to already adherent platelets, we studied thrombus formation in wild-type mice infused with wild-type platelets labeled with calcein-green and IL4R-tg platelets labeled with calcein-orange/red. Upon ferric chloride-induced injury, wild-type but not IL4R-tg platelets incorporated into the growing thrombus. A similar result was observed with wild-type platelets treated with O-sialoglycoprotein endopeptidase to remove the 45 kD N-terminal domain of GPIbα. In summary, our studies in GPIbα mutant mice provide additional evidence that under arterial flow conditions GPIbα is the only receptor expressed on the platelet surface that mediates initial platelet adhesion to the subendothelium as well as to already adherent platelets. Our data further suggest that a ligand other than vWf may contribute to GPIbα-dependent platelet adhesion to subendothelium and to a growing thrombus.
Polynitroxyl Albumin Prevents Vaso-Occlusion in Transgenic Sickle Mice.
Authors: Mahaseth Hemchandra , Belcher John D. , Welch Thomas E. , Sonbol Khalid M. , Bowlin Paul R. , Hebbel Robert P. , Ma Li , Hsia Carleton J.C. , Vercellotti Gregory M.
Published: October 2019

Sickle cell disease is characterized by excessive oxidative stress. Sickle patients have enhanced rates of endogenous reactive oxygen species (ROS) production and impaired antioxidant defense mechanisms. We hypothesize that excessive production of ROS promotes activation of vascular endothelium and vaso-occlusion in sickle cell disease. Intravascular ROS production has been shown to trigger the activation of vascular endothelium and increase leukocyte-endothelium interactions. We investigated whether inhibition of intravascular ROS production by treatment with polynitroxyl albumin (PNA), a superoxide dismutase and catalase mimetic agent, could modulate endothelial cell activation and vaso-occlusion in S+S Antilles transgenic sickle mice after hypoxia-reoxygenation. Nuclear factor-kappa B (NF-kB), an oxidant sensitive transcription factor critical for endothelial cell activation, was elevated in lungs and livers of sickle mice and was reduced 2.5 (p=0.043) and 1.5 (p=0.043) fold respectively, by treatment with PNA after hypoxia-reoxygenation. Similarly, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) were elevated 3 to 4.7 fold in the same organs (p<0.05). After treatment with PNA, VCAM-1 expression was reduced 2.3 fold in lungs (p=0.014) and 1.5 fold in livers (p=0.006), ICAM-1 expression decreased 1.7 fold in lungs (p=0.014) and 2 fold in livers (p=0.007). Control studies using human serum albumin had no effect on NF-kB activation or adhesion molecule expression in sickle mice. These anti-inflammatory effects of PNA on endothelium corresponded with changes in blood flow in dorsal skin venules. Intravital microscopy revealed that PNA significantly inhibited hypoxia-reoxygenation-induced leukocyte rolling (3.8 fold inhibition, p<0.001) and completely inhibited vaso-occlusion (p<0.001) in venules in the dorsal skin of transgenic sickle mice. We speculate that therapies that reduce ROS will result in improved organ perfusion in sickle cell disease.
Childhood Paroxysmal Nocturnal Hemoglobinuria (PNH), a Report of 11 Cases in the Netherlands.
Authors: Van Den Heuvel-Eib M.M. , Bredius R.G.M. , te Winkel M.L. , Tamminga R. , de Kraker J. , Meeteren N. Schouten-van , Bruin M. , Korthof E.T.
Published: October 2019

PNH, a rare disease in children, is caused by an acquired somatic mutation of the phosphatidyl-inositol glycan (GPI)class A (PIG-A) gene, followed by a survival-advantage of the PNH clone. This results in a deficiency of GPI-anchored proteins on the hematopoietic cells, making them more susceptible for attacks of the complement system, causing hemolysis and pancytopenia. Binding-impairment of coagulation-system-regulators increases thrombosis-risk. Until now, world-wide only 40 cases of pediatric PNH have been reported. Clinical data from Dutch cases from 1983–2003 were collected nation-wide and studied retrospectively. Diagnosis was confirmed by a decrease of GPI-linked anchor proteins on granulocytes and/or erythrocytes (CD24, CD66b/67, CD59, CD16).

All 11 PNH patients (4 boys, 7 girls), with a median age of 11 years (range 6–14 years) presented with pancytopenia-related symptoms. Two cases showed clinical hemoglobinuria, and one developed thrombosis during the course of the disease. BM biopsies showed hypoplasia/aplasia (AA)(n=7) or MDS (n=4). All cases showed decreased percentages of the GPI-linked proteins, 3/8 showed positive acid-Ham tests, 5/6 showed positive sucrose-lysis tests. Seven patients received prednisolone, in 5 combined with androgens and in 4 with ATG. After initial treatment, 3 patients reached complete remission (CR), and 2 showed partial response (PR), 1 subsequently relapsed and was transplanted. Three died of disease related mortality, one showed spontaneous recovery of blood counts.In total 5 patients underwent BMT (5MUD/2MSD). Conditioning regimens were Busulphan/Melphalan/Cyclophosphamide (n=2) in MDS, and Cyclophosphamide/ low dose TBI (n=3) in AA. Three patients received additional anti-Tcell antibody therapy. Of the 5 BMT patients 4 in CCR (median follow-up: 6 years, 10 months (range 7 months-14 years)). One patient died post-BMT of intracranial hemorrhage. PNH, a rare but serious disease in children, should be considered in every child with aplastic anemia/hypoplastic MDS. Hemoglobinuria is not an obligatory presenting symptom. GPI-linked anchor protein analysis provides the most reliable diagnostic tool. If prednisolone/ATG/androgens is not effective, BMT should be considered even if no matched sibling donor is available.
Intravenous Infusion of Liposomal Clodronate Blocks Clearance of Opsonized Erythrocytes in Dogs.
Authors: Mathes Mark A. , Dow Steven W. , Jordan Michael
Published: October 2019

Immune-Mediated Hemolytic Anemia (IMHA) is a spontaneously occurring disease of man and dogs in which immunoglobulin-coated erythrocytes are destroyed in the spleen. Immunosuppressive therapies and splenectomy have been previously used to combat premature destruction of erythrocytes. Liposomal clodronate has been previously shown to be a potent anti-macrophage agent when administered intravenously in a murine model. Splenic macrophages were selectively depleted through the induction of apoptosis following phagocytosis of liposomes containing clodronate. We hypothesized that such an approach might be useful in blocking clearance of erythrocytes in dogs with spontaneous IMHA.

Clodronate (dichloromethyl bisphosphonate) was formulated in liposomes (phosphatidyl choline, cholesterol and mannose), according to a previously published technique. The liposomal clodronate was evaluated in vitro for activity using canine and murine histiocytic cell lines, as assessed by cell killing and blocking of erythrophagia. Cell killing was monitored using microscopic evaluation of cell change, MTT and by propidium iodide staining. Blocking of erythophagia was monitored using flow cytometry. In vivo in normal dogs, increasing doses of liposomal clodronate were administered intravenously and evaluated to determine efficacy in blocking erythrocyte clearance and toxicity. Twenty-four hours post infusion, dye-labeled erythrocytes were opsonized using rabbit anti-canine erythrocyte antibody. Erythrocytes clearance was monitored using flow cytometry. The safe and effective dose of liposomal clodronate was determined to be 0.5 ml/kg administered IV via CRI over a 90 minute time period. After administration of intravenous liposomal clodronate in normal dogs, clearance of opsonized erythrocytes was nearly completely blocked at the 0.5 ml/kg dose. In vitro assays indicated that liposomal clodronate induced death of canine macrophage and dendritic cell lines. Five dogs with spontaneous IMHA were treated once with liposomal clodronate at the 0.5 ml/kg dose. Though erythrocyte clearance was not completely blocked, the drug was well-tolerated and all 5 dogs survived to leave the hospital. These studies suggest that liposomal clodronate may be an effective agent for temporary suppression of erythrocyte destruction in IMHA. Additional studies, including evaluation of higher doses of liposomal clodronate, are warranted in dogs with spontaneous IMHA.
Safety of Liver Biopsies in Children with Transfusional Iron Overload.
Authors: Mueller Brigitta Ursula , Williams Jennifer , Finegold Milton J. , Teruya Jun , Karpen Saul J.
Published: October 2019

Secondary iron overload, caused by chronic transfusions, is common among patients who have chronic hemolytic disorders, especially sickle cell disease and thalassemias. Accurate assessment of the hypertransfused patient’s body iron burden is important in deciding when and whether to start chelation therapy in order to prevent iron -related toxicities such as heart failure and endocrine abnormalities. The gold standard to measure iron content is through a liver biopsy. Though shown to be safe, many physicians are still reluctant to use this invasive approach in children to evaluate liver iron content.

Study Design: A retrospective chart review of chronically transfused patients at Texas Children’s Hospital (1998–2003) was performed to assess possible biopsy complications, including pain, hemorrhage, bilious peritonitis, puncturing of other organs, and bacterial infections, in patients with hemolytic disorders. The study was approved by the Institutional Review Board.

Results: There were 65 biopsies, 43 patients had one biopsy and 11 patients had two biopsies. 56 % were male, 44 % were female; 52% (28/54) were African American, 15 % (8/54) were Oriental/Asian, and 22 % (12/54) were Hispanic. 30 patients (56%) had sickle cell disease, 16 (30%) thalassemias, 5 (14%) other forms of anemia, 1 patient had leukemia, and 1 had lymphoma with the sickle cell trait. Eight of these patients (15%) are not on or have never been on chelation therapy, 11 (20%) used to be on chelation therapy, 35 (65%) are currently on chelation therapy. The average age when the first biopsy was performed was 11 years (range 2 to 18.9 years); and the average age at the time of the second biopsy was 12.7 years (range, 5.8 to 21.1 years). The reasons for their biopsies included possible iron overload due to hypertransfusions, pre-bone marrow transplant evaluation, and elevated liver enzymes. The average serum ferritin level before biopsy was 2681.8 ng/mL ranging from 348 to 35,332 ng/mL, and the average liver iron content (LIC) determined through biopsy was 12,278 ug/g dry wt. Ultrasound guidance was used for all 65 biopsies; 46 (71%) biopsies were done percutaneously, 8 (12%) laparoscopically, and 6 (10%) through an open procedure. There were no complications during the biopsy. There were no major and only 5 minor complications after the biopsy: 3 patients (5%) had transient abdominal pain at the biopsy site which subsided over a couple of days, and 2 (3%) had a fever with other (non-biopsy-related) complications requiring an increased hospital stay.

Conclusions: Liver biopsies in the pediatric population are safe and feasible and they should be considered for the accurate assessment of iron content in chronically transfused patients. Certain factors contributing to the success of the biopsies include having an experienced physician, using ultrasound guidance, and fully informing the patient or patient’s parents about the biopsy before the procedure.