bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of Apolipoprotein E Receptor 2′ as the Low-Density Lipoprotein Receptor on Platelets.
Authors: Akkerman Jan-Willem N. , Korporaal Suzanne J.A. , Relou Ingrid A.M. , Van Eck Miranda , Bezemer Martineke , Gorter Gertie , Van Berkel Theo J.C. , Nimf Johannes , Lenting Peter J.
Published: October 2019

The interaction of platelets with low-density lipoprotein (LDL) plays an important role in the pathogenesis of atherosclerosis and thrombosis. Previously, we have shown that native LDL (nLDL) is a mild activator of platelets increasing their sensitivity to aggregation-inducing agents. Binding of nLDL to platelets was saturable, reversible and initiated signal transduction to p38MAPK suggesting the involvement of a receptor. A peptide mimic of the B-site in apoB100 resembled nLDL in its platelet-activating properties, suggesting that the receptor is a member of the LDL-receptor family. Platelets from familial hypercholesterolemia patients, who lack or have a defective apoB/E receptor, responded normally to nLDL and an antibody against this receptor left nLDL-induced activation of normal platelets undisturbed, excluding the involvement of the classical LDL (apoB/E)-receptor. In this study, we provide evidence that nLDL initiates platelet signaling to p38MAPK via a splice variant of the LDL-receptor family member Apolipoprotein E Receptor 2 (ApoER2′). This conclusion is based on (i) blockade of nLDL-induced p38MAPK activation by receptor-associated protein (RAP), an inhibitor of ligand binding to members of the LDL-receptor family, (ii) confocal microscopy showing a high degree of co-localization of nLDL and ApoER2′ at the platelet surface, (iii) binding of both nLDL and the B-site peptide to soluble ApoER2′, (iv) activation of p38MAPK by an anti-ApoER2 antibody with similar kinetics as nLDL, and (v) tyrosine phosphorylation of ApoER2′ upon binding of nLDL. The nLDL-induced phosphorylation of ApoER2′ could be abolished by PP1, an inhibitor of Src-like tyrosine kinases. In the absence of PP1, ApoER2′ phosphorylation was accompanied by co-association with the Src-family member Fgr. We conclude that binding of nLDL to platelets involves ApoER2′. Upon nLDL binding, the receptor is phosphorylated which induces the recruitment of Fgr, a kinase known to activate p38MAPK. The ApoER2′-Fgr complex subsequently activates p38MAPK, an upstream element in the formation of thromboxane A2 that primes the platelets to further stimulation by aggregation-inducing agents.
A Soluble Truncated Form of the Mer Tyrosine Kinase Inhibits Gas6 Activity in Platelets and Monocytes.
Authors: Graham Douglas K. , Sather Susan
Published: October 2019

Several receptor tyrosine kinases generate soluble ligand binding domains either by proteolytic cleavage of the extracellular domain or differential splicing yielding in a truncated RNA transcript. Mer is a member of a receptor tyrosine kinase family which includes Axl and Tyro-3. All three members of this family have an extracellular domain which includes two immunoglobulin and two fibronectin type III motifs. Mer, Axl, and Tyro-3 are transforming in vitro in numerous cell types and are overexpressed in several different human cancers. In addition, Mer, Axl, and Tyro-3 and the common ligand Gas6 are all expressed in platelets and directly affect platelet activation and clot stability. We have found that the 205 kD Mer protein in platelets and monocytes is post-translationally processed by cleavage of the extracellular domain via a TACE-like metalloprotease. The cleavage results in a soluble extracellular domain protein (approximately 150kD) and a membrane-bound kinase domain. The proteolytic cleavage of Mer can be enhanced by LPS and PMA, and specifically inhibited by a TAPI, a TACE inhibitor. Significant amounts of the soluble Mer protein are present in human and mouse serum. The soluble Mer protein directly binds the ligand Gas6, and inhibits Gas6 stimulation of full-length Mer. Gas6, related to anticoagulant protein S, is also a ligand for Axl and Tyro-3. The cleavage of Mer may represent a mechanism of directly regulating the numerous functions of Gas6 including promoting platelet adhesion and clot stability, stimulating cell proliferation, inducing cell adhesion and chemotaxis, and preventing apoptosis. Furthermore, the ability of soluble Mer to inhibit Gas6 activity in platelets may provide a potential novel therapeutic intervention in the treatment of clotting disorders.
The Genetic Basis of Human Erythrocyte Pyridoxal Kinase Activity Variation.
Authors: Flanagan Jonathan M. , Beutler Ernest
Published: October 2019

Previous studies have shown that there is a striking difference in the erythrocyte activity of the human pyridoxal kinase (PdxK) enzyme, with African-Americans having approximately half the mean activity of their Caucasian counterparts. This difference was observed only in erythrocytes, with leukocyte and skin fibroblast PdxK enzyme activities being similar in both ethnic groups (Science187: 1084–1086, 1975). At the time of the original report it was impossible to elucidate the mechanism by which evolution had selectively lowered the enzyme activity in one cell type but not in another. To now determine the genetic basis for this variation, the entire PdxK gene was sequenced to identify candidate mutations in a sample of Caucasian and African-Americans. A number of variations were discovered, with one interestingly being a deletion located in the promoter region. This deletion was found to occur at a gene frequency of 42.7% in Caucasians (n=56), 25.3% in Asians (n=162) and 56.6% in African-Americans (n=334). Sequence analysis of the PdxK promoter using the software program MatInspector (Nucleic Acids Research23: 4878–4884, 1995) revealed that the deletion removed a putative Core-Promoter Binding Promoter (CPBP) transcription factor binding site. The CPBP transcription factor is known to enhance transcriptional activity by at least four fold and is thought to be a general regulator of TATA box-less genes (Journal of Biological Chemistry272: 9573–9580, 1997). The potential CPBP site was located -300bp to the PdxK start codon and was adjacent to a binding site for the erythroid specific transcription factor, Erythroid Krueppel Like Factor (EKLF). This suggested that the presence of the deletion would cause decreased transcription of PdxK in erythroid precursors only. The presence of the deletion element was found to be significantly associated with a reduced erythrocyte PdxK activity (p=0.0017). Although other modifiers of PdxK activity, such as diet, were found to play a role, the identified promoter deletion allows altered erythrocyte specific transcription to exclusively influence erythrocyte PdxK enzyme activity.
Ammonium Transport by the Erythrocyte Rh-Associated Protein.
Authors: Westhoff Connie M. , Dang Binh , Daniel Mak , Kevin Foskett J.
Published: October 2019

The erythrocyte Rh and Rh-associated (RhAG) proteins have distant sequence identity to a family of ammonium transporters found in yeast and bacteria. We previously showed that RhAG mediates movement of ammonium when expressed in yeast and in Xenopus oocytes. Importantly, these are the first mammalian proteins found to transport ammonium as a principal substrate. Elucidation of the mechanism and actual substrate(s) transported (protonated NH4+ or unprotonated NH3, or both) is important to understand their role in elimination of ammonium, proton recycling, and their impact on cellular pH and acid/base regulation. Functional characterization revealed that uptake was independent of the membrane potential and the Na+ gradient, but was dramatically stimulated by raising extracellular pH or lowering intracellular pH. This suggested that uptake was coupled to an outwardly directed H+ gradient and led us to hypothesize that RhAG might function by an H+-coupled, counter-transport mechanism. To further define the mechanism and actual substrate transported, RhAG-expressing oocytes were exposed to varying concentrations of NH4+ with constant NH3, and vice versa, by manipulation of the NH4Cl concentration and the pH of the buffer. A voltage-ramping protocol was used to evaluate changes in membrane conductance and reverse potential to measure membrane depolarization. Radioactive flux uptake of 14C-methylammonium, an analogue of ammonium, was used to measure transport. In the presence of substrate in the physiologic range (20 uM-500 uM), RhAG-mediated transport responded to the concentration of protonated NH4+ rather than the amount of unprotonated NH3 present. Currents in RhAG-expressing oocytes did not differ from water-injected controls. No significant changes in membrane conductance or membrane depolarization and reverse potential were observed. Taken together these data support a role for RhAG in the electronuetral transport of NH4+ by exchange with H+, and for erythrocytes in the maintenance of total blood ammonia levels. Sequestration of ammonium by erythrocytes would keep blood plasma levels low, preventing exposure of cells to toxic levels. Erythrocytes are ideally postioned to then transport ammonium to be exchanged in the liver and kidney, where other Rh-related proteins (RhBG and RhCG) are expressed.
L-arginine Administration Reverses Anemia of Renal Disease.
Authors: Imagawa Shigehiko , Tarumoto Takahisa , Kobayashi Makoto , Ozawa Keiya
Published: October 2019

Erythropoietin (EPO) gene expression is positively regulated by hypoxia-inducible factor-1, which binds to the 3′ enhancer of the EPO gene. EPO expression is negatively regulated by GATA, which binds to the GATA site in the 5′ promoter. Anemia of renal disease is believed to be caused by damage to the EPO-producing cells in the kidney as a result of renal failure. However, even mild renal failure can reduce the serum concentrations of EPO, suggesting that, in addition to the damage to EPO-producing kidney cells, uremic toxin(s) may play an important role in the pathogenesis of the anemia of renal disease. NG-monomethyl-L-arginine (L-NMMA), which competes with L-arginine as a substrate for nitric oxide synthase (NOS), is undetectable in nonuremic subjects, but is markedly elevated in patients with chronic renal failure. We have previously shown in the Hep3B cell line that L-NMMA suppresses EPO expression by upregulating GATA through the suppression of NOS activity, and that inhibition of EPO production with NG -nitro-L-arginine methyl ester (L-NAME) is reversed by pretreatment with L-arginine in mice. In this study, eight patients with anemia of renal disease, who were in the predialysis state of chronic renal failure, were enrolled in a nonrandomized study. All patients were anemic, and had hemoglobin (Hb) levels ranging between 7.9 and 10.8 g/dL. Patients with iron deficiency anemia, hemolytic anemia, and hematological malignancies were excluded. Blood urea nitorgen (BUN) levels were 29.7–90.1 mg/dL, and creatinine (Cre) levels were 1.1–3.8 mg/dL. Eight patients with anemia of renal disease were treated orally with L-arginine at 1.3 g/day for 8 to 22 wks. During this period, patients did not receive blood transfusions, or treatment with iron, vitamins or recombinant human EPO. Six patients responded to the treatment with increased levels of Hb and EPO, and three of these showed improved renal function. The EPO concentrations decreased in two nonresponders. There were no significant adverse effects. These limited data suggest that oral administration of 1.3 g/day of L-arginine can significantly improve EPO production and reverse anemia without adverse effects in patients with anemia of renal disease who are in the predialysis state of chronic renal failure. More studies including younger patients with severe renal dysfunction are in progress.
Incompatibilities of HA-1 and CD62L Polymorphic Adhesion Molecule Induce Graft-versus-Leukemia Effect Rather Than GVHD Resulted in Long-Term Survival in HLA Identical Myeloablative Stem Cell Transplantation.
Authors: Katagiri Takamasa , Shiobara Shintaro , Furukawa Tatsuo , Tsukada Jyunichi , Nakao Shinji , Wakano Miki , Muranaka Eriko , Harada Mine
Published: October 2019

Mismatches of minor histocompatibility antigen ( mHag) between HLA identical stem cell donor and host are known as a major risk factor for graft-versus-host disease (GVHD). We determined alleles of 5 polymorphic molecules including HA-1 and 4 adhesion molecules for 107 patients stem cell transplanted from HLA identical donors and investigated the association of their mismatches with relapse rate and GVHD. All of 107 recipients received stem cell transplantation ( SCT) after myeloablative conditioning regimen from 1978 to 2002, 89 received marrow, 15 received PBSC and 3 received both. Stage of the disease at SCT was 63 in standard risk and 43 in high risk. After SCT, 37 developed acute GVHD( ≥2), 50 developed chronic GVHD and 31 relapsed during at least 2 years follow up period.

We observed 16.1% of relapse rate in patients with at least one or more incompatibilities and 39.3% of relapse rate without incompatibilities (P= 0.018). Relapse rate of patients with CD62L, HA-1, CD31 at codon 563, CD31 at codon 125 and 49b incompatibilities are as followed 6.1%, 14.3%, 11.7%, 20% and 40% respectively. No difference of acute GVHD was observed in patients with and without incompatibilities. Among standard risk patients, 11 patients with HA-1 incompatibility showed 0% of relapse rate which was better than 45% (P= 0.003) in 47 patients without HA-1 incompatibility. In addition, 10 year survival rate was 100% in the former patients which was better than 52% of survival rate (p=0.03) in the latter patients. These data suggests that polymorphic molecules of HA-1 and CD62L contribute to GVL effect rather than the developing of GVHD which resulted in long term survivor after HLA identical stem cell transplantation. Mhag analysis of donor and host in addition to HLA antigen will help a patient to find a suitable donor in HLA identical family and voluntary donor.

Figure Figure
Genetic Modifiers of Cerebrovascular Large Vessel Stenosis in Sickle Cell Anemia.
Authors: Driscoll M. Catherine , Devaney Joseph , Gordish Heather , Minniti Caterina , Hoffman Eric P.
Published: October 2019

Cerebrovascular disease is a common complication in sickle cell anemia (SCA) with overt stroke occuring in 10% of patients and silent stroke in 24% of patients by age 20 years. Etiologies of overt stroke in SCA are heterogenous and include stenosis of large arteries, hemorrhage, hypertensive and hypoxic encephalopathy. Candidate genes identified as possible modifiers of overt stroke in SCA include adenylate cyclase 9 (ADCY9, rs731471 intron C/T), endothelin-converting enzyme (ECE-1, rs212527, intron A/G), Klotho (KL, rs480780, intron C/A), plasminogen activator inhibitor-1 (PAI-1, rs1799768, − 675 4/5,), transforming growth factor beta receptor 3 (TGFBR3, rs284157, intron G/A), tumor necrosis factor alpha (TNF, rs1800629,-308 G/A), and interlukin 4 receptor (IL4R, rs1805015, 503S/P). We examined the association of these candidate genes in a cohort of patients with SCA and stenosis of large cerebral vessels who are followed at a single institution. Patients with stenosis (n = 42, male = 22) were identified as having overt stroke (n = 29) or positive transcranial Doppler (TCD) (n = 13). The mean age at time of stroke or (+) TCD was 7.8 years. All had large vessel stenosis on magnetic resonance angiogram imaging (MRA). A control group of patients with SCA and normal TCD were identified (n = 71, males = 46, mean age = 14.8 years).This control group has passed through the peak years of stroke risk, ages 2–10 years. Single nucleotide polymorphisms (SNPs) were genotypic using novel Taqman assays. Genotypic associations were determined using logistic regression analysis with odds ratios (OR), 95% confidence intervals, and p-values reported. Among the candidate genes only ADYC9 (OR = 2.75, CI 1.2–6.3, p = 0.017) and TGFBR3 (OR = 2.33, CI 1.0–5.3, p = 0.043) had significant association with stenosis of large vessels in SCA. ADYC9 is a membrane-associated enzyme that catalyzes cAMP formation, which is critical for neuronal signaling and survival. TGFBR3 has a role in cardiac endothelial cell transformation to mesenchyme. Genotypic studies of a complex trait such as stroke in SCA will eventually lead to early diagnosis and new or early interventions. However, such genetic studies require clear definitions of phenotypic subtypes based on neuroimaging studies, including MRA.
Plasma ‘Free’ HB Is Related to Red Cell Derived Vesicle Numbers in Sickle Cell Anemia and Thalassemia Intermedia: Implications for Nitric Oxide (NO) Scavenging and Pulmonary Hypertension.
Authors: Westerman Maxwell , Pizzey Arnold , Hirschman Jocelyn , Cerino Mario , Eze Ada , Ramoton Priya , Weil-Weiner Yonit , Porter John
Published: October 2019

Plasma hemoglobin (Hb) is a scavenger of nitric oxide (NO), which is a likely contributor to the pathogenesis and treatment of clinical abnormalities such as pulmonary hypertension and vasocclusive episodes in sickle cell anemia (SCA) and possibly in thalassemia syndromes, where pulmonary hypertension has been described most frequently in splenectomised patients with thalassemia intermedia (TI). Since plasma Hb consists of both free Hb and RBC-derived microvesicle Hb (Greenwalt. Vox Sang1991;61:14), the relationship between plasma Hb and circulating vesicles may be significant. We have measured plasma Hb and plasma vesicle levels in adults with SCA and thalassemia intermedia (TI). Patients with SCA were all untransfused and TI patients had no transfusions during the previous 3 months. Plasma Hb values in healthy adult controls (1.77±0.2, n=7) were significantly lower than in SCA (11.21±2.08mg/dl, n=15, p=0.003) or in splenectomised TI patients (48.46 ±3.66mg/dl, n=5, p=0.0038). Plasma Hb levels were significantly greater in TI as compared to levels in SCA (p=0.001). Vesicle numbers in SCA were 12.59±3.65 x103/ul (n=21, p< 0.001, SCA v control) and in TI were 24.19 ±12.23 (n=7, p< 0.001, TI v control). Thus both plasma Hb and circulating vesicle levels in SCA and TI were significantly greater than healthy controls. Plasma Hb was significantly greater in TI than in SCA and circulating vesicles markedly greater in TI than in SCA. Furthermore there was a significant correlation between plasma Hb and vesicle numbers in SCA, TI and normal controls (n=26, R2=0.59, p=0.0015). An analysis of splenectomised versus non-splenectomised TI patients revealed a further trend; both plasma Hb and vesicle numbers were significantly higher in splenectomised (n=5, n=7 respectively) than in non-splenectomised patients (n=4, n=4 respectively). Plasma Hb and vesicle numbers were respectively 48.5±3.6 and 24.19±12.2 in splenectomised patients compared to 17.18±5.58 (p=0.014) and 4.36±0.81 (p=0.008) in non-splenectomized TI patients. These findings show that raised plasma Hb levels are related to increments in vesicle numbers in TI and SCA. Splenectomy in TI, which is associated with increased risk of pulmonary hypertension and thrombosis, is associated with increased vesicle numbers and plasma Hb. We suggest that the scavenger characteristics of Hb containing vesicles for NO may differ from the scavenger characteristics of free Hb and thus require detailed study..
A Genetic Screen for Zebrafish Mutants Affecting Early Hematopoiesis.
Authors: English Milton A. , Lei Lin , Giannetti Margot , Lyons Susan E. , Liu Paul P.
Published: October 2019

Hematopoiesis, or blood cell formation, starts from hematopoietic stem cells and goes through many rounds of cell proliferation and a number of commitment steps that progressively restrict cells to a particular lineage. The generation and maintenance of stem cell pool and the regulation of lineage commitment are poorly understood. Right now there is only one reported zebrafish mutant, cloche, that has defects in early hematopoiesis. We have undertaken a chemical mutagenesis screen using ENU in the zebrafish to uncover mutants in early hematopoiesis. Haploid embryos from F1 females were screened by RNA in situ hybridization using a stem cell marker, core binding factor ? (cbfb), and a myeloid-specific marker, leukocyte-specific plastin (l-plastin). We have screened 510 F1 females and 47 putative mutants have been identified. Of these, 22 showed decreased expression of l-plastin, 3 for cbfb, 2 for both l-plastin and cbfb and the remainder were mutated for l-plastin and other non-hematopoietic markers. Right now, we have focused our efforts on two mutants.

The first mutant line, L32, displayed a blood-less phenotype and showed a significant decrease in the expression of the hemangioblast marker scl. In addition to the marked reduction in the expression of the myeloid specific markers l-plastin, c/ebp1 and mpo, these embryos also showed decreased expression of the erythroid specific marker gata-1. Linkage analysis has placed this mutant to the proximal region of linkage group 12. Currently, we are in the process of testing candidate genes to identify the mutated gene. The second mutant line, L63 also has a blood less phenotype and showed decreased expression of both l-plastin and mpo. Like L32, L63 homozygous embryos have significant developmental defects and die 3 dpf. Low resolution mapping has placed this mutant to LG 8. Through this approach, we hope to identify novel genes that play critical roles in the developmental process regulating early hematopoiesis.
Fibroblast Growth Factors Regulate Stem Cell Functioning In Vitro and in Vivo.
Authors: Yeoh Joyce S.G , van Os Ronald , Weersing Ellen , Dontje Bert , Vellenga Edo , Bystrykh Leonid , de Haan Gerald
Published: October 2019

Fibroblast Growth Factors (FGF) are a large family of signaling molecules widely involved in tissue development, maintenance and repair. Little is known about the role of FGF/FGF-receptor signaling in the regulation of adult hematopoietic stem cells (HSC). In this study, we assessed the potential of exogenously added FGF-1/2, or retrovirally overexpressed FGF-1 to preserve HSC function in vitro and in vivo.

First, we demonstrate that in vitro culture of unfractionated mouse bone marrow cells, in serum-free medium, supplemented with FGF-1 or FGF-2 or FGF-1 + 2 resulted in the robust generation of long-term repopulating (LTR) HSCs. Cultures were maintained for 12 weeks and during that time in vivo competitive reconstitution assays were performed. Stem cell activity was detectable at 3, 5, and 8 weeks after initiation of culture, but lost after 12 weeks. However, whereas 3 and 5 week cultured cells provided radioprotection in non-competitive assays, animals transplanted with 8 or 12 week cultured cells succumbed due to bone marrow failure. So far, we have been unable to expand single, highly purified Lin−Sca-1+c-Kit+ using FGF-1 + 2. Consequently, we speculated that essential intermediate cell populations or signals are required for FGF-induced stem cell conservation. To test this we cultured highly purified CD45.1 Lin−Sca-1+c-Kit+ cells in a co-culture with CD45.2 unfractionated BM. Co-cultured cells were transplanted after 5 weeks in lethally irradiated recipients, and CD45.1 chimerism levels were assessed. High levels of CD45.1 chimerism confirmed that Lin−Sca-1+c-Kit+ cells require an accessory signal in addition to FGF to induced stem cell activity in vitro. We subsequently tested stem cell potential of cells cultured in FGF-1 + 2 for 5 weeks, with the addition of SCF + IL-11 + Flt3L for the last 2, 4 or 7 days. Cell numbers increased with increasing time of growth factor presence. However, only when growth factors were present for 2 days engraftment of cultured cells in a competitive repopulation assay was increased 3.5-fold.

Finally, we show by immunohistochemistry that ~10% of freshly isolated Lin−Sca-1+c-Kit+ expresses high levels of FGF-1. Retroviral overexpression of FGF-1 in stem cells resulted in increased growth potential and sustained clonogenic activity in vitro. Upon transplantation of transduced stem cells, FGF-1 overexpression resulted in increased white blood cell counts 4 weeks post-transplant compared to control animals. Most notable was a marked granulocytosis in FGF-1 overexpressing recipients Our results reveal FGF as an important regulator of HSC signaling and homeostasis. Importantly, in the presence of FGF stem cells can be maintained in vitro for 2 months. These findings open novel avenues for in vitro manipulation of stem cells for future clinical therapies.
Factor Xa Binding Sites on Factor Va in the Prothrombinase Complex.
Authors: Gale Andrew J. , Griffin John H.
Published: October 2019

Factor Va (FVa), a non-enzymatic cofactor for proteolytic activation of prothrombin by factor Xa (FXa), interacts with FXa via both its heavy and light chains. Phospholipids can bind both FVa and FXa and enhance prothrombin activation. The action of the prothrombinase complex (FXa:FVa) on its substrate, prothrombin, involves stepwise binding and enzymatic interactions, including FVa interactions with exosites on FXa that are topologically distinct from its active site. Synthetic peptides used as inhibitors of the prothrombinase complex implicated FVa residues 311–335 and 493–506 as probable FXa binding sites. Analysis of sequence alignments of FV with factor FVIII and of our 3-dimensional FVa model lead us to hypothesize that residues 312–315, 319–323, and 499–505, which delineate a remarkable compact triangular surface on the A2 domain are involved in FXa binding. Hence, we used homologous loop mutagenesis and substituted these three FV sequences with FVIII sequences, replacing 312-TREQ-315 with RSVA, 319-MKRWE-323 with PKTWV, and 499-KSRSLDR-505 with YKESVDQ. Each recombinant FV mutant was purified, characterized and compared to control wild type (wt)-FVa using ELISA and functional assays. Prothrombinase assays using thrombin-activated FVa and purified components were used to determine functional apparent Kd values of FVa for FXa by varying FXa concentrations in the absence or presence of saturating phospholipid (PL) vesicles (20%PS/80%PC). In the absence of PL vesicles, the kinetically determined apparent Kd values of each FVa mutant were greatly increased, such that mutation of either 312–315 or 319–323 in FVa decreased the apparent affinity for FXa by greater than 80-fold while mutation of 499–505 resulted in a 20-fold lower apparent affinity for FXa. However, in the presence of PL vesicles, these three FVa mutants each showed only a moderately reduced affinity for FXa (2.0–2.5 fold increased apparent Kd). None of the three FVa mutations significantly altered the Km for prothrombin. Therefore, each of the three mutated sequences in FVa is critical for normal functional interactions with FXa. Furthermore, these results illustrate the importance of each different kind of interactions, namely protein-protein and protein-PL, for the overall formation of the prothrombinase complex. Binding of both FXa and FVa to PL surfaces can help optimize the relative orientation of FVa to FXa to facilitate enzyme:cofactor exosite interactions and/or can promote conformational changes in one or both of FXa and FVa that affects exosite interactions. In the absence of PL, the effects of the A2 domain mutations on protein-protein interactions are magnified whereas when FXa and FVa are PL-bound, the observable effects of these mutations are greatly reduced. In summary, these mutagenesis studies support the hypothesis that three, closely located sequences on the surface of the FVa A2 domain comprising residues 312–315, 319–323, and 499–505 contribute significantly to FXa binding.
Contribution of the 60 Loop To Substrate and Inhibitor Specificity of Thrombin.
Authors: Rezaie Alireza R.
Published: October 2019

Relative to chymotrypsin, the 60-loop of thrombin contains 8–9 insertion residues which are believed to be partly responsible for the restricted substrate and inhibitor specificity of thrombin. Previous deletion of 3–4 residues of this loop (des-PPW and des-YPPW) dramatically impaired the activity of thrombin toward antithrombin, protein C and fibrinogen, implicating a key role for the productive interaction of these residues with the target macromolecules. To further investigate the role of this loop, we expressed a mutant of thrombin in which all 8 insertion residues (Tyr-Pro-Pro-Trp-Asp-Lys-Asn-Phe) of the 60-loop were deleted (des-60-loop). In contrast to the partially deleted loop mutants, we discovered that des-60-loop thrombin cleaved small synthetic substrates, clotted purified fibrinogen, and activated protein C with a near normal catalytic efficiency; however, its activity toward cofactors V and VIII was impaired ~2–4-fold. Further studies revealed that the reactivity of des-60-loop with antithrombin is not impaired, but rather improved ~2-fold. Remarkably, the mutant could also activate prothrombin to thrombin. These results suggest that the 60-loop plays a key role in regulating the specificity of thrombin by shielding the active-site pocket; however, its productive interaction with the target molecules may not be as critical for the catalytic function of thrombin as has been speculated in previous reports.
T Cells Genetically Modified to Be CD19-Specific with Improved Potential for Trafficking and Activation.
Authors: Al-Kadhimi Zaid , Serrano Lisa Marie , Olivares Simon , Gonzalez Sergio , Pfeiffer Timothy , Vishwanath Reena , Jensen Michael C. , Forman Steven J. , Cooper Laurence J.N.
Published: October 2019

The safety and feasibility of adoptive immunotherapy using ex vivo-expanded differentiated human effector T cells that express tumor-specific chimeric receptors are being evaluated in clinical trials. Typically, these T cells are CCR7neg and bear a T-cell receptor of unknown specificity. To improve the therapeutic potential of genetically engineered T cells in general, and CD19-specific T cells in particular, strategies are needed to improve their ability traffic to sites of residual/macroscopic disease where infused T cells can be specifically activated for proliferation, cytokine secretion, and tumor-lysis. To accomplish these goals we have generated a selection process that uses genetically modified T cells, expressing influenza A matrix protein 1 (MP1) or CMV pp65, to act as antigen presenting cells (T-APC) in order to expand autologous viral-specific T cells in vitro and in vivo. The viral-specific effector T cells can then be genetically modified with a CD19-specific chimeric immunoreceptor (CD19R), which recognizes CD19 on malignant B cells, independent of MHC. By using these viral-specific T cells as a platform for the introduction of CD19R, we now demonstrate that bi-specific T cells express the chemokine receptor CCR7, which is implicated in the trafficking of T cells to lymph nodes. We demonstrate that this chemokine receptor functions to directionally chemotax the genetically modified bi-specific T-cells along concentration gradients of CCL19 or CCL21. We further demonstrate that both the endogenous and introduced chimeric immunoreceptor continue to function in CCR7+ bi-specific T cells. Indeed, the bi-specific T cells are capable of augmented cytokine production and proliferation upon docking with both CD19 and MP1 antigens, compared with these same T cells interacting with either CD19 or MP1 alone. This enhanced activation is an explanation for the enhanced in vivo anti-tumor activity demonstrated by bi-specific T-cells when stimulated with MP1+ T-APC in treating CD19+ lymphoma in NOD/scid mice. An advantage of this methodology is that the CCR7+ bi-specific T cells and T-APC can be genetically modified and expanded in compliance with current good manufacturing practice (cGMP) for 2nd generation Phase I/II clinical trials to test their ability to traffic to sites of lymphoma providing potent regional/local T-cell activation.

Legend: (A) CCR7+ viral- and CD19-bi-specific T cells migrate along recombinant CCL19 and CCL21 concentration gradients, whereas CCR7neg CD19-specific T cells do not. (B) Stimulation of both introduced chimeric immunoreceptor and endogenous T-cell receptor on CD19- and MP1- bi-specific T-cells, using artificial APC, results in augmented cytokine production.

Figure Figure
Analysis of Vector Integration with LM-PCR after Retroviral HSV-Tk/ΔLNGFR Gene Transfer.
Authors: Wehnert Stefanie , Krüger Tillmann , von Neuhoff Nils , Hertenstein Bernd , Ganser Arnold , Weissinger Eva-Maria
Published: October 2019

The development of secondary leukemia as an unexpected complication after retroviral transfer of potentially therapeutic genes was recently reported. In the pathogenesis of this complication, the upregulation of proto-oncogenes due to random vector integration played a crucial role. Therefore, the analysis of vector integration after retroviral gene transfer could become an interesting tool for risk assessment and safety measurement. In this study, 4 patients diagnosed with chronic myelogenous leukemia (CML) and 2 patients suffering from acute myelogenous leukemia (AML) were transplanted with T-cell-depleted CD34+ enriched stem cells (HSCT) from their HLA-identical siblings without further immunosuppression. After day +60 following HSCT, they received gene-modified donor T-cells infected with the replication-deficient retrovirus SFCMM-3, expressing the herpes simplex thymidine kinase (HSV-Tk) as a suicide gene, and the truncated low affinity nerve growth factor (ΔLNGFR) to control graft versus leukemia (GvL) effects and graft versus host disease (GvHD). In order to assess the viral integration sites in the transfused gene-modified donor T-cells, we performed a ligation-mediated PCR (LM-PCR) to amplify the vector flanking sequences. Final PCR products were visualized by gel electrophoresis and silver staining. Afterwards, sequence analysis (CEQ 8000, Beckman Coulter GmbH, Krefeld, Germany) of the clonal products was carried out. Standard National Center for Biotechnology Information (NCBI) blast searches were done to identify the flanking sequences (http://www.ncbi.nlm.nih.gov/BLAST). Within the investigated gene-modified T-cells of our study population, we found several integration sites. Up to now there has been no evidence for retroviral integration in the vicinity of proto-oncogene promoters after sequence analysis and database search. Furthermore, we were able to monitor the presence of the transduced T-cells at several time points after HSCT by detecting the internal retroviral band. In conclusion, the LM-PCR is a specific method to amplify and identify the flanking sequences after retroviral gene transfer, contributing significantly to the safety of gene therapy.
Increased Incidence of Major Bleeding in Children Receiving Unfractionated Heparin for Clinical Management: A Prospective Cohort Study.
Authors: Mitchell Lesley G. , Kuhle Stefan , Massicotte Patricia M. , Vegh Patricia
Published: October 2019

BACKGROUND: Unfractionated heparin (UFH) is one of the most frequently prescribed drugs in paediatric tertiary care centres and is used in a diverse group of disorders including cardiopulmonary bypass, extra corporeal membrane oxygenation, dialyses and maintenance of both venous and arterial catheter patency. Dosing of UFH in children is extrapolated from adults and is assessed by either a chromogenic Anti-Xa assay or a clot-based activated partial thromboplastin time (aPTT). The overall objective of the study was to assess safety of current standard of practice in the use of therapeutic UFH in children.

Objective #1: The primary objective was to determine the incidence of bleeding and the incidence of recurrent thrombosis in children receiving UFH.

Objective #2: To assess the monitoring UFH by assessing the relationship of the aPTT and Anti-Xa heparin levels to heparin dose.

STUDY DESIGN: A prospective cohort study in nonselected children in a intensive care setting. The primary outcomes were major bleeding events and recurrent thrombosis. The secondary outcomes were assessing the APTT and Anti-Xa levels. Inclusion Criteria: Patients 〉 36 weeks gestation and 〈18 years of age requiring therapeutic doses of UFH. Exclusion Criteria: patients who received UFH for less than 1 day. Major bleeding was defined aprior as any of the following: CNS bleeding, retroperitoneal bleeding, and/or bleeding that results in stopping UFH infusion.

RESULTS: Patient Population 39 patients were enrolled, 22 (56%) male, 32 (82%) < 1 year of age and 90% of which where cardiac patients. Major Bleeding events: 11/39 patients had a major bleeding event 28.2% (95% CI 15.0–44.9%). No patient had recurrent thrombosis. Relationship of aPPT and Anti-Xa to heparin dose; A total of 188 paired aPTTs and anti-Xa levels were performed. There was little correlation between aPTT and anti-Xa levels (r2=0.205) and APTT and UFH dose (r2=0.054). There was no relationship between anti-Xa levels and UFH dose (r2=0.0089). (Figure 1 and 2)

Figure Figure

CONCLUSIONS:. There is an unacceptably high rate of bleeding in children receiving UFH for clinical care. There is little or no relationship of aPPT and Anti-Xa to heparin dose. Clinical trials are needed to assess the appropriate use of UFH therapy in children.
Prevention of Venous Thromboembolism and Pregnancy Complications using Low-Molecular-Weight Heparin during Pregnancy: A Systematic Review of Safety and Thromboprophylactic Efficacy.
Authors: Greer Ian A. , Nelson-Piercy Catherine
Published: October 2019

Introduction Low-molecular-weight heparin (LMWH) is used in pregnancy for venous thromboembolism (VTE) prophylaxis and for prevention of pregnancy complications, because of efficacy greater than or equal to unfractionated heparin, and a lower rate of side effects observed in non-pregnant patients. Due to the lack of data from large randomized controlled trials to guide physician’s practices, there is a need for increased data on safety and efficacy of LMWH for these indications. Our aim was to evaluate the safety and efficacy of using LMWH during pregnancy for preventing VTE and pregnancy complications by performing a systematic review of data from published literature.

Methods Data from published studies on the use of LMWH during pregnancy as VTE prophylaxis or LMWH for prevention of pregnancy complications were identified by searching MEDLINE and EMBASE databases up to the end of 2003. The reference lists from identified articles were also hand searched. Data on the LMWH regime, incidence of VTE, pregnancy complications, clinical outcomes and side effects were extracted and entered into pre-piloted forms.

Results Fifty studies reporting 2,322 pregnancies were included in this analysis. LMWH was received antenatally in 1883 (81%) of cases, and only peri or postpartum in 389 (17%) of cases. Dalteparin and enoxaparin were the most commonly used LMWH, but certoparin, nadroparin, rivaparin, and tinzaparin were also used. There were no maternal deaths. VTE was reported in 26 (1.1%) pregnancies. Severe maternal bleeding occurred in 46 (2%) pregnancies and was generally associated with obstetric causes. Thrombocytopenia occurred in 10 (0.4%) pregnancies and was not associated with thrombosis. Minor allergic skin reactions occurred in 23 (1%) pregnancies, and osteoporosis in two (0.09%) pregnancies.

Conclusion Data from this systematic review of the literature suggest that LMWH is both safe and effective for use as VTE prophylaxis during pregnancy.
Effect of Covalently-Bound Heparin on Insertion of the Reactive Centre Loop in Antithrombin.
Authors: Mewhort Tracy Anne , Berry Leslie R. , Chan Anthony K.C.
Published: October 2019

Heparin exerts its anticoagulant effects through its interaction with antithrombin (AT), leading to the inhibition of thrombin, a major player in the coagulation cascade. AT exists in equilibrium between a latent and an active state. In latent AT, the reactive centre loop (RCL) of AT is inserted into the central b-sheet (A-Sheet), forming a six-stranded A-Sheet. In active AT, residues P10-P14 of the RCL of AT are inserted into the A-sheet, retaining inhibitory activity. We have produced a molecule of covalently linked AT and heparin (ATH). Intrinsic fluorescence studies of ATH have shown that the AT is permanently activated in the covalent molecule. This study was designed to examine degree of the insertion of the RCL into the A-sheet of ATH. The tetradecapeptide P1-P14 was used to compete with the RCL of AT for insertion into the A-sheet. Peptides corresponding to the RCL residues P7-14, P3-P14 and P1-P14 of human AT were radiolabelled using reductive alkylation with formaldehyde + [3H]-NaBH4. Varying concentrations of radiolabelled peptide (0-3000 nM) were incubated with 1000 nM AT in the presence or absence of unfractionated heparin (UFH), and with ATH in 0.05 M Tris HCl buffer (pH 7.3) at 37°C. A 24-hour equilibrium dialysis against 0.05 M Tris HCl buffer followed. The amount of peptide bound to the AT or ATH was quantified following dialysis using liquid scintillation counting of samples taken from inside and outside the dialysis tubing. Results were expressed as bound vs. free peptide in moles. No binding to AT in the presence or absence of UFH, nor to ATH was seen using the P7-P14 peptide, while some binding to AT was seen for both the P3-P14 and P1-P14 peptides. There was a trend towards increased binding with AT in the presence of UFH over AT alone or ATH with the P3-P14 peptide. Binding to AT was lost in the presence of UFH, and no binding was seen with ATH in the P1-P14 condition, consistent with the literature. Furthermore, binding to AT was greatly reduced compared to that seen with the P3-P14 peptide. These data suggest that binding of heparin creates a conformational change in AT preventing the insertion of the RCL peptide into the molecule. Furthermore, results with the P3-P14 peptide may infer that it is the P1-P2 region that is most affected by the conformational change. This is consistent with the AT being in a partially activated conformation in the covalently linked ATH molecule.
Supression of FLT3-Expressing Leukemia by a Monoclonal Antibody-Auristatin Conjugate.
Authors: Li Yiwen , Li Hongli , Wang Mei-Nai , Bassi Rajiv , Ludwig Dale , Witte Larry , Zhu Zhenping , Bohlen Peter , Meyer Damon , Larkin Angelyn , Senter Peter , Hicklin Daniel
Published: October 2019

The receptor tyrosine kinase FLT3 is overexpressed in blasts of ~90% of acute myelogenous leukemia (AML) and the majority of B-lymphoid leukemia patients. Internal tandem duplications (ITDs) in the juxtamembrane region and point mutations in the kinase domain of FLT3 are found in ~37% of AML patients and are associated with a poor prognosis. We have recently developed a fully human monoclonal antibody (IMC-EB10) which binds with high affinity to FLT3 receptor on human leukemia cells. In the present study, a novel auristatin conjugate of the anti-FLT3 antibody (EB10-MMAF) was prepared using a dipeptide linker that allows for drug release inside the lysosomes of antigen-positive cells. The MMAF conjugates were stable in buffers and plasma. EB10-MMAF (drug/antibody raito = 8) was highly potent, and selectively inhibited the growth of FLT3-expressing leukemia cells with an IC50 of 0.19 nM and 0.08 nM for MV4;11 and BaF3-ITD cells (both positive for FLT3-ITD), 1.11 nM, 6.18 nM and 1.82 nM for REH , EOL-1, EM3 cells (all three positive for wild-type FLT3), and 135 nM for JM1 (negative for FLT3). An MMAF conjugate with a control antibody was not active in these cell lines (IC50s > 5.9 uM). Flow cytometric analysis with annexin V indicated that EB10-MMAF treatment induced apoptosis of leukemia cells in vitro. In vivo treatment with EB10-MMAF strongly inhibited leukemia growth and prolonged survival of mice in both EOL-1 and BaF3-ITD leukemia models. In summary, immunoconjugates composed of a fully human anti-FLT3 antibody and a potent auristatin drug may provide a valuable therapeutic approach for AML and other FLT3-positive leukemias.
Assessment of Heparins, Heparinoids and Direct Thrombin Inhibitors Using a New FXa-and RVV-V-Based Assay.
Authors: Calatzis Andreas , Reininger Armin J. , Spannagl Michael
Published: October 2019

UFH and direct thrombin inhibitors are usually monitored using the activated partial thromboplastin time (aPTT), which has many known limitations. These include non-linear-dose response-relationships as well as a high variability among different reagents. In addition the aPTT has a low sensitivity towards low molecular weight heparin (LMWH). Newer methods such as chromogenic substrate analysis, ecarin clotting time or the Heptest assay have provided improved sensitivity and specificity, but still have not reached broad application in routine or emergency laboratories. The performance of a new universal monitoring method (Pefakit® PiCT®, Pentapharm, Basel) in detecting anticoagulant effects of anti-Xa and / or arti-IIa agents was evaluated.

Methods: Measurements were performed by addition of platelet poor plasma to a reagent containing a combination of FXa, RVV-V (FV activating snake venom) and phospholipids. After 180 s the samples were recalcified and the clotting time was recorded. Plasma samples from 30 healthy blood donors and 50 patients receiving low molecular weight heparin were analyzed. To evaluate the assay precision 30 samples were analyzed in triplicate. Plasma pools from healthy volunteers and patients under oral anticoagulant treatment were spiked with unfractionated heparin, low molecular weight heparin, argatroban and pentasaccharide.

Results: PiCT® clotting times of the healthy volunteers were 20.6 ± 1.2 sec. Clotting times of the patient samples ranged 29–195 sec. Correlation of anti-Xa activity to PiCT® clotting times was good (r=0.94). The mean variation coefficient of the repeated determinations was 2.6%. Addition of LMWH, UFH or Argatroban to normal plasma showed a steep increase and almost linear dose-response of the PiCT clotting times. Addition of pentasaccharide showed a non-linear dose-response with lesser increase of clotting times at concentrations above 0.75 μg/ml plasma. The addition of LMWH or UFH to plasma of patients under warfarin therapy showed moderate additive effects when compared to normal plasma, while stronger additive effects of warfarin and heparin therapy were found for argatroban.

Conclusion: The prothrombinase induced clotting test (PiCT ®) is a new clotting assay, which sensitively assesses LMWH, UFH and direct thrombin inhibitors with almost linear dose-response-relationships. The results revealed a wide measuring range which allows for assessment of prophylactic, therapeutic and supratherapeutic doses of these compounds. As a clotting test with pipetting volumes and an incubation regimen identical with the aPTT, the test can be used in routine or emergency laboratories, thus facilitating 24-hour availability of the method with short turn-around times. Allowing the assessment of heparins, heparinoids as well as direct thrombin inhibitor therapy with one method may simplify their management in clinical practice.