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1.
Pediatr Blood Cancer ; 69(3): e29545, 2022 03.
Article in English | MEDLINE | ID: mdl-34957695

ABSTRACT

BACKGROUND: Factor XI (FXI) deficiency is a rare autosomal recessive bleeding disorder. Only scarce publications address its clinical features in children. The increased prevalence of FXI deficiency in Israel enabled data collection for this large multicenter cohort study. OBJECTIVE: Some hemostatic challenges may be unique or more common in children, such as bleeding in the neonatal period or trauma-related injury. The current study was designed to explore the potential impact of these differences in children with severe FXI deficiency. METHODS: Medical files of all children with FXI level under 15% followed at five tertiary centers were evaluated. The retrieved data comprised demographic and clinical characteristics, including bleeding episodes, surgical interventions, treatment strategies, as well as laboratory features. RESULTS: Sixty children, whose median age at diagnosis was 4.2 years and their median FXI level was 4%, were included. Three children experienced triggered intracranial hemorrhage (ICH) and two children had major bleeds. No bleeding complications occurred in surgeries in which hemostatic treatment consisting mostly of tranexamic acid or fresh frozen plasma was applied (n = 45). In contrast, excessive bleeding was noted in 25% of surgical procedures performed without hemostatic preparation (p = .002). CONCLUSION: This study's findings confirm the generally favorable outcome of this rare bleeding disorder, with no spontaneous bleeds or cases of perinatal ICH. Nonetheless, proper diagnosis and adequate hemostasis in the surgical setting are imperative. Unlike previous studies in adults, our pediatric study suggests an association between the severity of FXI deficiency and bleeding tendency.


Subject(s)
Factor XI Deficiency , Hemorrhagic Disorders , Hemostatics , Adult , Child , Cohort Studies , Factor XI/therapeutic use , Factor XI Deficiency/complications , Factor XI Deficiency/therapy , Female , Hemorrhage/complications , Hemostatics/therapeutic use , Humans , Infant, Newborn , Intracranial Hemorrhages , Pregnancy
2.
Blood Cells Mol Dis ; 89: 102560, 2021 07.
Article in English | MEDLINE | ID: mdl-33711653

ABSTRACT

INTRODUCTION: Glanzmann thrombasthenia (GT) is a severe inherited platelet function disorder (IPFD), presenting with bleeding diathesis and impaired platelet aggregation, is caused by mutations in the genes ITGA2B or ITGB3. AIM: We aimed to study the genetic cause of IPFD mimicking GT. METHODS: During 2017-2019, 16 patients were referred to our tertiary center with bleeding symptoms, impaired platelet aggregation and normal platelet count and size. RESULTS: Using flow cytometry, 13/16 patients were diagnosed with GT, yet three patients displayed normal surface expression of the integrins αIIbß3 and αvß3, as well as normal integrin αIIbß3 activation following incubation with the activating monoclonal antibody anti-LIBS6, while platelet activation following ADP or epinephrine was impaired. Whole exome sequencing detected 2 variants in RASGRP2 gene in all 3 patients. DISCUSSION: Both RASGRP2 mutations predicted frameshift, premature stop codon (p. I427Mfs*92 and p. R494Afs*54, respectively) and truncated calcium-sensing guanine nucleotide exchange factor [CalDAG-GEFI]- the major signaling molecule that regulates integrin-mediated aggregation and granule secretion, causing IPFD-18. CONCLUSION: Patients who suffer from bleeding diathesis without immune dysregulation, may be mistakenly diagnosed as GT. Further studies are required to confirm the diagnosis of specific IPFD.


Subject(s)
Diagnostic Errors , Guanine Nucleotide Exchange Factors/genetics , Thrombasthenia/diagnosis , Thrombasthenia/genetics , Adult , Female , Frameshift Mutation , Humans , Infant , Male , Pedigree , Platelet Aggregation , Point Mutation , Exome Sequencing , Young Adult
3.
Blood Coagul Fibrinolysis ; 31(3): 225-228, 2020 Apr.
Article in English | MEDLINE | ID: mdl-31990752

ABSTRACT

: Evaluation of bleeding risk before operation includes history of bleeding, complete blood count and basic coagulation tests, prothrombin time and activated partial thromboplatin time (aPTT). In this article, we present a patient with colon cancer who presented with asymptomatic prolonged aPTT of 72-100 s, while past a PTT values were within normal limits. aPTT was corrected in vitro by mixing with normal plasma. Further laboratory workup excluded coagulation factors deficiencies or an acquired inhibitor to coagulation factors. The patient underwent uncomplicated laparoscopic anterior resection of a recto-sigmoid carcinoma after receiving fresh frozen plasma and correction of aPTT. Further investigation revealed a rare disorder of an acquired prekallikrein deficiency. We describe the patient's clinical presentation, laboratory workup and review the literature of contact phase proteins deficiency.


Subject(s)
Partial Thromboplastin Time/adverse effects , Aged , Humans , Male
4.
Acta Haematol ; 143(3): 266-271, 2020.
Article in English | MEDLINE | ID: mdl-31610537

ABSTRACT

INTRODUCTION: Patients treated with direct Xa inhibitors may require urgent surgery. Administration of prothrombin complex concentrate (PCC) in this setting is common; however, it is based on limited experience in healthy volunteers. OBJECTIVE: To characterize the population receiving PCC for apixaban/rivaroxaban reversal prior to an urgent surgery and evaluate its efficacy and safety. METHODS: This was a retrospective study in 2 tertiary hospitals. Bleeding was evaluated based on surgical reports, hemoglobin drop, and the use of blood products or additional PCC during 48 h. Safety measures were thrombotic complications and 30-day mortality. RESULTS: Sixty-two patients aged 80.7 ± 9 years, treated with apixaban (39.63%) or rivaroxaban (23.37%), received PCC before an urgent surgery/procedure. Most underwent abdominal operation (61%), orthopedic surgery (13%), or transhepatic cholecystostomy insertion (10%). Bleeding during surgery was reported in 3 patients (5%), no patient required additional PCC, and 16 patients (26%) received packed cells (median: 1 unit, range: 1-5). The 30-day mortality and thrombosis rates were 21% (n = 13) and 3% (n = 2), respectively. The cause of death was related to the primary disease, most commonly sepsis. No patient died due to bleeding/thrombosis. CONCLUSIONS: Our results support the use of PCC to achieve hemostasis in patients treated with Xa inhibitors prior to an urgent surgery.


Subject(s)
Blood Coagulation Factors/therapeutic use , Blood Loss, Surgical/prevention & control , Emergencies , Factor Xa Inhibitors/adverse effects , Postoperative Hemorrhage/prevention & control , Preoperative Care/methods , Pyrazoles/adverse effects , Pyridones/adverse effects , Rivaroxaban/adverse effects , Academic Medical Centers/statistics & numerical data , Aged , Aged, 80 and over , Atrial Fibrillation/complications , Blood Coagulation Factors/adverse effects , Blood Component Transfusion , Factor Xa Inhibitors/therapeutic use , Female , Hemostatics/therapeutic use , Humans , Male , Postoperative Hemorrhage/chemically induced , Pyrazoles/therapeutic use , Pyridones/therapeutic use , Retrospective Studies , Rivaroxaban/therapeutic use , Surgical Procedures, Operative , Tertiary Care Centers/statistics & numerical data , Thrombophilia/drug therapy , Thrombophilia/etiology , Thrombosis/etiology , Tranexamic Acid/therapeutic use
5.
Drugs Aging ; 36(2): 165-177, 2019 02.
Article in English | MEDLINE | ID: mdl-30460518

ABSTRACT

BACKGROUND: There is a paucity of data on apixaban levels among octogenarians with non-valvular atrial fibrillation (NVAF). We aimed to compare apixaban levels between octogenarians (with and without dose reduction) and younger patients, to assess the frequency of high and above-range drug levels. METHODS: A cross-sectional, prospective study of 80 patients treated with apixaban for NVAF was conducted. Apixaban levels were compared among octogenarians treated with 5 mg twice daily (bid), octogenarians with appropriately reduced dose (2.5 mg bid), octogenarians with inappropriately reduced dose and younger patients (age < 70 years). Trough and peak levels were measured by a chromogenic assay calibrated for apixaban and compared to predicted manufacturer levels. RESULTS: A significant proportion of the cohort had above-range trough [n = 11 (13.8%)] and peak [n = 16 (20%)] levels, especially octogenarians with the 5-mg bid dosage [n = 6 (30%) for trough and n = 8 (40%) for peak]. No significant differences were found in the trough or peak geometric mean (GM) levels among the groups, apart from the peak GM levels between the 5-mg octogenarian group and the other two 2.5-mg bid octogenarian groups (p = 0.0004). The frequency of apixaban peak levels within the upper quartile was significantly higher in the 5-mg octogenarian group compared to the other groups [n = 12 (60%) of measurements, p = 0.019), whereas trough levels were comparable between groups. CONCLUSION: High and above-range peak apixaban steady-state levels are highly prevalent in octogenarians receiving the appropriate dosage of 5 mg bid for NVAF stroke prevention. Age above 80 strongly affects apixaban levels. TRIAL REGISTRATION: ClinicalTrials.gov Identifier number NCT02623049.


Subject(s)
Atrial Fibrillation/blood , Pyrazoles/blood , Pyridones/blood , Age Factors , Aged , Aged, 80 and over , Anticoagulants/blood , Anticoagulants/therapeutic use , Atrial Fibrillation/drug therapy , Cohort Studies , Cross-Sectional Studies , Factor Xa Inhibitors/blood , Factor Xa Inhibitors/therapeutic use , Female , Humans , Male , Middle Aged , Prospective Studies , Pyrazoles/therapeutic use , Pyridones/therapeutic use , Stroke/prevention & control
6.
Platelets ; 28(7): 676-681, 2017 Nov.
Article in English | MEDLINE | ID: mdl-28060550

ABSTRACT

Platelet function disorders (PFDs) are a common cause of mild bleeding tendency. However, they cannot be recognized by standard screening studies. The gold standard test for PFD is platelet aggregation, performed by light transmission aggregometry (LTA). A newer and less validated method is the closure time (CT), performed by the platelet function Analyzer 100 (PFA-100). Data regarding the validity of these tests in children are limited. The aim of this study was to evaluate the usefulness of LTA and PFA-100 for the diagnosis of pediatric patients with bleeding tendency. This retrospective study included patients one month-18 year old that had LTA tests performed at the coagulation laboratory of Rabin Medical Center between the years 2006-2015. Bleeding severity was assessed using a pediatric bleeding score. Patients were excluded from analysis if they had thrombocytopenia, thrombocytosis or coagulation factors deficiencies. One hundred and thirty-seven (137) patients were included in the analysis. The median age was 7.5 years (range one month-18 years). Most patients (93%) had a bleeding score of 2 or more. Abnormal LTA was found in 40% and prolonged CT in 23% of the patients. Abnormal LTA was significantly more common in patients with a bleeding score of 2 or more compared to patients with a lower bleeding scores (P = 0.04). No significant correlation was found between the bleeding severity and the number of agonists which induced abnormal responses (p = 0.52) or the CT (p = 0.35). Furthermore, no correlation was found between abnormal LTA and prolonged CT. To conclude, we were able to diagnose 40% of children who presented with bleeding tendency with platelet aggregation defects by LTA. Abnormal LTA was significantly more prevalent in patients with a bleeding score of 2 and above. In contrast, CT was not found to be sensitive as a screening tool for PFD. Therefore, our data extend the validity of the use of LTA for the evaluation of pediatric patients with bleeding tendency.


Subject(s)
Blood Platelets/pathology , Hemorrhage/diagnosis , Platelet Aggregation/drug effects , Adenosine Diphosphate/pharmacology , Adolescent , Arachidonic Acid/pharmacology , Automation, Laboratory , Blood Platelets/drug effects , Blood Platelets/metabolism , Child , Child, Preschool , Epinephrine/pharmacology , Female , Hemorrhage/blood , Humans , Infant , Infant, Newborn , Male , Platelet Function Tests , Retrospective Studies , Severity of Illness Index
8.
Am J Respir Cell Mol Biol ; 46(3): 313-22, 2012 Mar.
Article in English | MEDLINE | ID: mdl-21997486

ABSTRACT

Death-associated protein kinase (DAPk) is a tumor suppressor thought to inhibit cancer by promoting apoptosis and autophagy. Because cancer progression is linked to inflammation, we investigated the in vivo functions of DAPk in lung responses to various acute and chronic inflammatory stimuli. Lungs of DAPk knockout (KO) mice secreted higher concentrations of IL-6 and keratinocyte chemoattractant (or chemokine [C-X-C motif] ligand 1) in response to transient intranasal administrations of the Toll-like receptor-4 (TLR4) agonist LPS. In addition, DAPk-null macrophages and neutrophils were hyperresponsive to ex vivo stimulation with LPS. DAPk-null neutrophils were also hyperresponsive to activation via Fc receptor and Toll-like receptor-3, indicating that the suppressive functions of this kinase are not restricted to TLR4 pathways. Even after the reconstitution of DAPk-null lungs with DAPk-expressing leukocytes by transplanting wild-type (WT) bone marrow into lethally irradiated DAPk KO mice, the chimeric mice remained hypersensitive to both acute and chronic LPS challenges, as well as to tobacco smoke exposure. DAPk-null lungs reconstituted with WT leukocytes exhibited elevated neutrophil content and augmented cytokine secretion in the bronchoalveolar space, as well as enhanced epithelial cell injury in response to both acute and chronic inflammatory conditions. These results suggest that DAPk attenuates a variety of inflammatory responses, both in lung leukocytes and in lung epithelial cells. The DAPk-mediated suppression of lung inflammation and airway injury may contribute to the tumor-suppressor functions of this kinase in epithelial carcinogenesis.


Subject(s)
Apoptosis Regulatory Proteins/metabolism , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Lung/enzymology , Pneumonia/prevention & control , Animals , Apoptosis Regulatory Proteins/deficiency , Apoptosis Regulatory Proteins/genetics , Bone Marrow Transplantation , Calcium-Calmodulin-Dependent Protein Kinases/deficiency , Calcium-Calmodulin-Dependent Protein Kinases/genetics , Chemokine CXCL1/metabolism , Death-Associated Protein Kinases , Disease Models, Animal , Epithelial Cells/enzymology , Epithelial Cells/immunology , Inflammation Mediators/metabolism , Interleukin-6/metabolism , Lipopolysaccharides , Lung/immunology , Macrophages/enzymology , Macrophages/immunology , Male , Mice , Mice, Knockout , Neutrophils/enzymology , Neutrophils/immunology , Pneumonia/chemically induced , Pneumonia/enzymology , Pneumonia/immunology , Receptors, Fc/metabolism , Time Factors , Tobacco Smoke Pollution , Toll-Like Receptor 3/metabolism , Toll-Like Receptor 4/immunology , Transplantation Chimera
9.
Nephrol Dial Transplant ; 25(3): 931-9, 2010 Mar.
Article in English | MEDLINE | ID: mdl-19861313

ABSTRACT

BACKGROUND: Adenosine, a potent regulator of inflammation, is produced under stressful conditions due to degradation of ATP/ADP by the ectoenzymes CD39 and CD73. Adenosine is rapidly degraded by adenosine deaminase (ADA) or phosphorylated in the cell by adenosine kinase (AK). From four known receptors to adenosine, A(1) (A(1)R) promotes inflammation by a G(i)-coupled receptor. We have previously shown that A(1)R is up-regulated in the first hours following bacterial inoculation. The aim of the current study is to characterize the inflammatory mediators that regulate adenosine-metabolizing enzymes and A(1)R at the onset of peritonitis. METHODS: Peritonitis was induced in CD1 mice by intraperitoneal injection of Escherichia coli. TNFalpha and IL-6 levels were determined in peritoneal fluid by enzyme-linked immunosorbent assay. Adenosine-metabolizing enzymes and the A(1)R mRNA or protein levels were analyzed by quantitative PCR or by Western blot analysis, respectively. RESULTS: We found that CD39 and CD73 were up-regulated in response to bacterial stimuli (6-fold the basal levels), while AK and ADA mRNA levels were down-regulated. Cytokine production and leukocyte recruitment were enhanced (2.5-fold) by treatment with an A(1)R agonist (2-chloro-N(6)-cyclopentyladenosine, 0.1 mg/kg) and reduced (2.5-3-fold) by the A(1)R antagonist (8-cyclopentyl-1, 3-dipropylxanthine, 1 mg/kg). In contrast to lipopolysaccharide, IL-1, TNF and IFNgamma, only low IL-6 levels (0.01 ng/ml), in the presence of its soluble IL-6R (sIL-6R), were found to promote A(1)R expression on mesothelial cells. In mice, administration of neutralizing antibody to IL-6R or soluble gp130-Fc (sgp130-Fc) blocked peritoneal A(1)R up-regulation following inoculation. CONCLUSION: Bacterial products induce the production of adenosine by up-regulation of CD39 and CD73. Low IL-6-sIL-6R up-regulates the A(1)R to promote efficient inflammatory response against invading microorganisms.


Subject(s)
Adenosine Deaminase/metabolism , Adenosine Kinase/metabolism , Adenosine/metabolism , Peritonitis/metabolism , Receptors, Purinergic P1/metabolism , 5'-Nucleotidase/metabolism , Animals , Antigens, CD/metabolism , Apyrase/metabolism , Disease Models, Animal , Escherichia coli , Injections, Intraperitoneal , Interleukin-6/metabolism , Mice , Mice, Inbred Strains , Peritonitis/microbiology , Receptors, Interleukin-6/metabolism , Tumor Necrosis Factor-alpha/metabolism
10.
Nephrol Dial Transplant ; 24(8): 2392-9, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19221177

ABSTRACT

BACKGROUND: Long-term peritoneal dialysis (PD) is associated with peritoneal fibrosis and loss of function. It has been shown that activation of the adenosine A(2A) receptor (A(2A)R) promotes tissue repair, wound healing and extracellular matrix (ECM) production. We have previously shown that adenosine is a potent regulator of inflammation in the peritoneum. In the current study, we explored the role of adenosine and the A(2A)R in two experimental models. METHODS: Collagen deposition was evaluated in primary peritoneal fibroblasts following treatment with an A(2A)R agonist and antagonist. In addition, peritoneal fibrosis was induced by i.p. injection of either chlorhexidine gluconate for 2 weeks or 4.25% glucose peritoneal dialysis fluid (PDF) for 1 month. The development of fibrosis was compared between wild-type (WT) and WT mice treated with caffeine (an A(2A)R antagonist) in drinking water or between (A(2A)R(+/+)) mice and A(2A)R-deficient mice (A(2A)R(-/-)). RESULTS: Adenosine or the A(2A)R agonist CGS21680 stimulated collagen production by peritoneal fibroblasts in vitro and A(2A)R antagonists (ZM241385 and caffeine) blocked this effect. Consistent with these results, caffeine-treated WT or A(2A)R(-/-) mice had reduced submesothelial thickness, collagen deposition and mRNA levels of fibroblast-specific protein (FSP-1) and connective tissue growth factor (CTGF). In addition, treatment with caffeine in vitro and in vivo diminished A(2A)R and A(2B)R mRNA levels induced by CG or PDF while it upregulated A(1)R levels. CONCLUSION: Our data suggest that adenosine through its A(2A)R promotes peritoneal fibrosis and therefore should be considered as a target for pharmacological intervention.


Subject(s)
Adenosine A2 Receptor Antagonists , Disease Models, Animal , Peritoneal Cavity/pathology , Adenosine/analogs & derivatives , Adenosine/pharmacology , Adenosine A2 Receptor Agonists , Animals , Caffeine/pharmacology , Cells, Cultured , Central Nervous System Stimulants/pharmacology , Chlorhexidine/analogs & derivatives , Chlorhexidine/toxicity , Collagen/metabolism , Fibroblasts/metabolism , Fibrosis/prevention & control , Mice , Mice, Knockout , Phenethylamines/pharmacology , Vasodilator Agents/pharmacology
11.
Mol Immunol ; 46(2): 250-7, 2008 Dec.
Article in English | MEDLINE | ID: mdl-18849075

ABSTRACT

CD40, a TNF receptor (TNFR) family member, is composed of four cysteine rich domains (CRDs) followed by a transmembrane domain and a signaling intracellular C-terminus. CD154 ligation to CD40 regulates major inflammatory and immune processes. A natural soluble form of CD40 was detected in uremic patient's serum which might be alternative splicing product. Our aim was to identify CD40 isoforms produced by primary human cells and to evaluate their biological activity. By RT-PCR, we isolated from primary human cells three major products differing from the expected wild type (WT) transcript which lacks exon 5 (CD40-5), exon 6 (CD40-6) and both exons 5 and 6 (CD40-5 + 6). The first two were predicted computationally to be soluble decoy receptors with various CRDs numbers. Recombinant soluble CD40 (sCD40) isoforms containing either three or four CRDs are able to bind coated surfaces or membranous CD154 while the sCD40 isoform containing 2 CRDs did not. sCD40 proteins inhibited RANTES secretion, but did not block B cell proliferation induced by soluble CD154. These data suggest that sCD40 natural isoforms encompassing either three or four CRDs might exert different antagonistic effects from known CD154 antibodies by recognition of only membranous CD154.


Subject(s)
Alternative Splicing/immunology , CD40 Antigens/immunology , CD40 Ligand/immunology , CD40 Antigens/biosynthesis , CD40 Antigens/genetics , CD40 Ligand/genetics , CD40 Ligand/metabolism , Cells, Cultured , Exons/immunology , Humans , Protein Isoforms/biosynthesis , Protein Isoforms/genetics , Protein Isoforms/immunology , Protein Structure, Tertiary/physiology , Solubility
12.
PLoS One ; 3(5): e2107, 2008 May 07.
Article in English | MEDLINE | ID: mdl-18461129

ABSTRACT

BACKGROUND: Adenosine levels rise during inflammation and modulate inflammatory responses by engaging with four different G protein-coupled receptors. It is suggested that adenosine exhibits pro-inflammatory effects through its A(1) receptor (A(1)R), and anti-inflammatory effects through A(2A) receptor (A(2A)R). Therefore, understanding of the mechanisms that govern adenosine receptor regulation may advance treatment of various inflammatory disorders. We previously reported that peak A(1)R expression during leukocyte recruitment, is followed by a peak in A(2A)R during inflammation resolution. PRINCIPAL FINDINGS: Here, we examined whether A(1)R activation sequentially induces A(2A)R expression and by this reverses inflammation. The effect of adenosine on A(1)R mediated A(2A)R expression was examined in peritoneal macrophages (PMPhi) and primary peritoneal mesothelial cells (PMC) in vitro. Induction of A(2A)R was inhibited by pertussis toxin (PTX) and partly dependent on A(2A)R stimulation. Administration of A(1)R agonists to healthy mice reduced A(1)R expression and induced A(2A)R production in PMC. Mice that were preconditioned with A(1)R agonists 24 hours before E. coli inoculation exhibited decreased TNFalpha and IL-6 sera levels and reduced leukocytes recruitment. Preconditioning was blocked by pretreatment with A(1)R antagonist, as well as, or by late treatment with A(2A)R antagonist, and was absent in A(2A)R(-/-) mice. CONCLUSIONS: Our data suggest that preconditioning by an A(1)R-agonist promotes the resolution of inflammation by inducing the production of A(2A)R. Future implications may include early treatment during inflammatory disorders or pretreatment before anticipated high risk inflammatory events, such as invasive surgery and organ transplantation.


Subject(s)
Adenosine A1 Receptor Agonists , Anti-Inflammatory Agents/pharmacology , Adenosine/metabolism , Animals , Epithelium/physiology , Female , Inflammation/genetics , Inflammation/physiopathology , Ischemic Preconditioning , Mice , Mice, Knockout , Peritonitis/physiopathology , Pertussis Toxin/pharmacology , Receptor, Adenosine A2A/deficiency , Receptor, Adenosine A2A/drug effects , Receptor, Adenosine A2A/genetics , Receptor, Adenosine A2A/physiology
13.
Mol Cell Endocrinol ; 283(1-2): 83-95, 2008 Feb 13.
Article in English | MEDLINE | ID: mdl-18179861

ABSTRACT

The gonadotrophins LH, FSH and human (h) CG are non-covalent heterodimers composed of a common alpha and the hormone-unique beta subunit. LH regulates the production of androgens and progestins in the follicle, and the levels of these steroids are critical for the ovarian function. Structural features of the gonadotrophins involved in the steroidogenic response of the ovary are not completely understood. As an approach to address how the topology of the ligand affects steroidogenesis we exploited the single-chain (SC) gonadotrophin methodology because manipulating the relative position of the tethered subunit domains in SC hCG analogs enabled to change in the conformation, secretion, receptor binding and adenylyl cyclase activity. We genetically engineered a SC bovine LH analog with a linker derived from the CTP domain of the hCGbeta subunit, NH2-alpha-CTP-LHbeta-COOH (denoted as alphaCTPLHbeta; AB configuration) and evaluated the secretion form transfected CHO cells and steroidogenesis in follicular derived cells in comparison to the variant NH2-LHbeta-CTP-alpha-COOH (LHbetaCTPalpha; BA configuration). The secretion of the analogs from CHO cells was quantitative, and that of alphaCTPLHbeta was more efficient than that of LHbetaCTPalpha The experiments suggested that both variants were N- and O- glycosylated, though the posttranslational modifications are likely to be non-identical in the AB and BA analogs. The analogs stimulated progesterone secretion by immortalized rat granulosa cells that express the rat LH receptor but the EC50 of alphaCTPLHbeta (AB orientation) was higher by 20 fold, as compared to LHbetaCTPalpha (BA). In primary cultures of bovine theca cells, alphaCTPLHbeta stimulated progesterone release with a reduced sensitivity (by at least 50 folds) and smaller magnitude over the basal levels (about 3 folds) relative to LHbetaCTPalpha. In contrast, the accumulation of androstenedione in the media of the same primary cultures appeared to be nearly identical. As a result, the androstenedione/progesterone ratio for the alphaCTPLHbeta analog was significantly increased relative to LHbetaCTPalpha (2-3 folds). This unequal response suggests a distinct regulation of progesterone and androstenedione biosynthesis. Our data demonstrate major differences in steroid balance following stimulation of the receptor with structural LH analogs and provide further insight into gonadotrophin regulation of ovarian steroid production.


Subject(s)
Glycoprotein Hormones, alpha Subunit/chemistry , Glycoprotein Hormones, alpha Subunit/metabolism , Luteinizing Hormone, beta Subunit/analogs & derivatives , Luteinizing Hormone, beta Subunit/metabolism , Protein Subunits/chemistry , Protein Subunits/metabolism , Androstenedione/metabolism , Animals , Biological Assay , CHO Cells , Cattle , Cells, Cultured , Cricetinae , Cricetulus , Female , Glycosylation/drug effects , Granulosa Cells , Humans , Luteinizing Hormone, beta Subunit/chemistry , Mutant Proteins/metabolism , Progesterone/metabolism , Protein Structure, Tertiary , Rats , Structure-Activity Relationship , Theca Cells/drug effects , Theca Cells/metabolism , Transfection , Tunicamycin/pharmacology
14.
Mol Cell Endocrinol ; 252(1-2): 136-41, 2006 Jun 27.
Article in English | MEDLINE | ID: mdl-16644100

ABSTRACT

Single-chain gonadotropin analogs had been constructed for the purpose of structure-function studies and analog design. Incorporation of a spacer derived from the carboxyl terminal peptide (CTP) of the choriogonadotropin (CG) beta subunit between the tethered subunit domains of the human gonadotropins is beneficial for the secretion of the single-chain variants without compromising biocactivity. Although the CGbeta subunit containing the CTP domain is expressed only in primates and equids, a CTP-like sequence exists in the untranslated region of the LHbeta gene of several mammalian species, including the bovine species. The CTP encrypted in the bovine LHbeta DNA (designated as 'boCTP') and the CTP derived from the human CGbeta subunit (denoted as 'huCTP') served as a linker sequence in the design of bovine single-chain luteinizing hormone (LH) analogs. The purpose of the present study was to evaluate steroidogenesis in cultured bovine theca cells following stimulation with these single-chain analogs. The concentration of the LHbetaboCTPalpha and LHbetahuCTPalpha analogs in the conditioned media of the expressing CHO cells was three- to six-fold higher than that of the "linkerless" LHbetaalpha and LHbeta111alpha variants. The four analogs induced androstenedione and progesterone secretion from the primary theca cells in a dose-dependent manner, but differences in the steroidogenic response were observed. The LHbetaboCTPalpha analog (10 ng/ml) effectively induced androstenedione and progesterone secretion over unstimulated levels (4.0- and 4.4-fold increase for androstenedione and progesterone, respectively). The response to the pituitary bovine LH standard (10 ng/ml) was less pronounced for both steroids (two- to three-fold increase over basal levels). The activities of LHbetahuCTPalpha, LHbetaalpha and LHbeta111alpha were comparable and sightly reduced relative to the LHbetaboCTPalpha activity. The data suggested that LHbetaboCTPalpha was ranked as the most potent and this was even more prominent when analogs were used at a lower dose (1 ng/ml). These data suggest that the design, including the huCTP or boCTP linker, is favorable for the production of single-chain bovine LH analogs. Furthermore, spacing of the tethered subunit domains with the cryptic boCTP sequence that originated from the bovine LHbeta gene appears advantageous for the purpose of stimulating steroid production in the species-specific bioassay. Thus, an effective strategy to produce bioactive single-chain LH analogs in non-primate, non-equid species would be the mutatation of the LHbeta genes with the aim of expressing the cryptic CTP sequence as a spacer derived from the DNA of the same organism.


Subject(s)
Androstenedione/biosynthesis , Luteinizing Hormone/genetics , Luteinizing Hormone/pharmacology , Ovarian Follicle/physiology , Progesterone/biosynthesis , Steroids/biosynthesis , Animals , CHO Cells , Cattle , Cricetinae , Female , Genetic Variation , Kinetics , Ovarian Follicle/drug effects , Structure-Activity Relationship , Transfection
15.
J Biol Chem ; 280(17): 16676-84, 2005 Apr 29.
Article in English | MEDLINE | ID: mdl-15723833

ABSTRACT

The expression of a previously untranslated carboxylterminal sequence is associated with the ancestral lutropin (LH) beta to the beta-subunit gene evolution of choriogonadotropins (CG). The peptide extension (denoted as CTP) is rich in mucin-type O-glycans and confers new hormonal properties on CG relative to the LH. Although the LHbeta gene is conserved among mammals and only a few frameshift mutations account for the extension, it is merely seen in primates and equids. Bioinformatics identified a CTP-like sequence that is encrypted in the LHbeta gene of several mammalian species but not in birds, amphibians, or fish. We then examined whether or not decoding of the cryptic CTP in the bovine LHbeta gene (boCTP) would be sufficient to generate the LHbeta species of a ruminant with properties typical to the CGbeta subunit. The mutated bovine LHbeta-boCTP subunit was expressed and N-glycosylated in transfected Chinese hamster ovary cells. However, unlike human (h) CGbeta CTP, the cryptic boCTP was devoid of mucin O-glycans. This deficiency was further confirmed when the boCTP domain was substituted for the natural CTP in the human CGbeta subunit. Moreover, when expressed in polarized Madin-Darby canine kidney cells, this hCGbeta-boCTP chimera was secreted basolaterally rather than from the apical compartment, which is the route of the wild type hCGbeta subunit, a sorting function attributed to the O-glycans attached to the CTP. This result shows that the cryptic peptide does not orientate CG to the apical face of the placenta, to the maternal circulation as seen in primates. The absence of this function, which distinguishes CG from LH, provides an explanation as to why the LHbeta to CGbeta evolution did not occur in ruminants. We propose that in primates and equids, further natural mutations in the progenitor LHbeta gene resulted in the efficient O-glycosylation of the CTP, thus favoring the retention of an elongated reading frame.


Subject(s)
Chorionic Gonadotropin/chemistry , Luteinizing Hormone, beta Subunit/chemistry , Luteinizing Hormone, beta Subunit/physiology , Mucins/chemistry , Oligosaccharides/chemistry , Amino Acid Sequence , Animals , Base Sequence , CHO Cells , Cattle , Codon, Terminator , Computational Biology/methods , Cricetinae , DNA/metabolism , Dogs , Glycosylation , Humans , Immunoprecipitation , Molecular Sequence Data , Mutation , Open Reading Frames , Peptides/chemistry , Placenta/metabolism , Polysaccharides/chemistry , Protein Structure, Tertiary , Recombinant Fusion Proteins/chemistry , Sequence Homology, Amino Acid , Time Factors , Transfection
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