COVID‐19 and acute coagulopathy in pregnancy Vlachodimitropoulou Koumoutsea, Evangelia; Vivanti, Alexandre J.; Shehata, Nadine ...
Journal of thrombosis and haemostasis,
July 2020, Volume:
18, Issue:
7
Journal Article
Peer reviewed
Open access
We present a putative link between maternal COVID‐19 infection in the peripartum period and rapid maternal deterioration with early organ dysfunction and coagulopathy. The current pandemic with ...SARS‐CoV‐2 has already resulted in high numbers of critically ill patients and deaths in the non‐pregnant population, mainly due to respiratory failure. During viral outbreaks, pregnancy poses a uniquely increased risk to women due to changes to immune function, alongside physiological adaptive alterations, such as increased oxygen consumption and edema of the respiratory tract. The laboratory derangements may be reminiscent of HELLP (hemolysis, elevated liver enzymes, low platelet count) syndrome, and thus knowledge of the COVID‐19 relationship is paramount for appropriate diagnosis and management. In addition to routine measurements of D‐dimers, prothrombin time, and platelet count in all patients presenting with COVID‐19 as per International Society on Thrombosis and Haemostasis (ISTH) guidance, monitoring of activated partial thromboplastin time (APTT) and fibrinogen levels should be considered in pregnancy, as highlighted in this report. These investigations in SARS‐CoV‐2‐positive pregnant women are vital, as their derangement may signal a more severe COVID‐19 infection, and may warrant pre‐emptive admission and consideration of delivery to achieve maternal stabilization.
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FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Summary
Iron chelators are increasingly combined clinically but the optimal conditions for cellular iron mobilization and mechanisms of interaction are unclear. Speciation plots for iron(III) binding ...of paired combinations of the licensed iron chelators desferrioxamine (DFO), deferiprone (DFP) and deferasirox (DFX) suggest conditions under which chelators can combine as ‘shuttle’ and ‘sink’ molecules but this approach does not consider their relative access and interaction with cellular iron pools. To address this issue, a sensitive ferrozine‐based detection system for intracellular iron removal from the human hepatocyte cell line (HuH‐7) was developed. Antagonism, synergism or additivity with paired chelator combinations was distinguished using mathematical isobologram analysis over clinically relevant chelator concentrations. All combinations showed synergistic iron mobilization at 8 h with clinically achievable concentrations of sink and shuttle chelators. Greatest synergism was achieved by combining DFP with DFX, where about 60% of mobilized iron was attributable to synergistic interaction. These findings predict that the DFX dose required for a half‐maximum effect can be reduced by 3·8‐fold when only 1 μmol/l DFP is added. Mechanisms for the synergy are suggested by consideration of the iron‐chelate speciation plots together with the size, charge and lipid solubilities for each chelator. Hydroxypyridinones with low lipid solubilities but otherwise similar properties to DFP were used to interrogate the mechanistic interactions of chelator pairs. These studies confirm that synergistic cellular iron mobilization requires one chelator to have the physicochemical properties to enter cells, chelate intracellular iron and subsequently donate iron to a second ‘sink’ chelator.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Ovarian torsion in the third trimester of pregnancy leading to a midline laparotomy and caesarean section for the delivery of a preterm baby is an uncommon event. As the woman is likely to present ...with nonspecific symptoms of lower abdominal pain, nausea, and vomiting, ovarian torsion can often be misdiagnosed as appendicitis or preterm labour. Treatment and the opportunity to preserve the tube and ovary may consequently be delayed. We report the case of a multiparous woman who had undergone two previous caesarean sections at term, presenting at 35 weeks of gestation with a presumptive diagnosis of acute appendicitis. Ultrasonography described a cystic lesion 6 × 3 cm in the right adnexa, potentially a degenerating fibroid or a torted right ovary. MRI of the pelvis was unable to provide further clarity. The patient was managed by midline laparotomy and simultaneous detorsion of the ovarian pedicle and ovarian cystectomy together with caesarean section of a preterm infant. This report describes that prompt recognition and ensuring intraoperative access can achieve a successful maternal and fetal outcome in this rare and difficult scenario. Furthermore, we would like to emphasise that the risk for a pregnant woman and her newborn could be reduced by earlier diagnosis and management of ovarian masses (Krishnan et al., 2011).
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FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK
We have investigated the efficacy of mono- and combined therapy with green tea extract (GTE) in mobilizing redox iron, scavenging reactive oxygen species (ROS), and improving insulin production in ...iron-loaded pancreatic cells.
Rat insulinoma pancreatic β-cells were iron-loaded using culture medium supplemented with either fetal bovine serum or ferric ammonium citrate and treated with various doses of GTE for epigallocatechin-3-gallate (EGCG) equivalence and in combination with iron chelators. Cellular iron, ROS, and secretory insulin were measured.
The rat insulinoma pancreatic cells took up iron from fetal bovine serum more rapidly than ferric ammonium citrate. After treatment with GTE (0.23-2.29 μg EGCG equivalent), cellular levels of iron and ROS were dose dependently decreased. Importantly, secretory insulin levels were increased nearly 2.5-fold with 2.29 μg of EGCG equivalent GTE, indicating a recovery in insulin production.
Green tea EGCG ameliorated oxidative damage of iron-loaded β-cells by removing redox iron and free radicals and attenuating insulin production. The impact can result in the restoration of pancreatic functions and an increase in insulin production. Green tea extract exerts iron-chelating, free-radical scavenging, and pancreato-protective effects in the restoration of β-cell functions, all of which we believe can increase insulin production in diabetic β-thalassemia patients.
Introduction: Eltrombopag (ELT) is an orally active, nonpeptide, small-molecule thrombopoietin receptor agonist (TPO-R), used to treat chronic immune thrombocytopenic purpura (ITP). We have recently ...reported its ability to mobilise cellular iron, and act as an iron shuttle when combined with currently licensed chelation therapies (Vlachodimitropoulou et al, Blood 2014, Volume 124, 21). Tissue damage induced by ROS production in iron overload conditions includes endocrine dysfunction including type I diabetes. We have developed a model where iron overload of the pancreatic cell line (RINm5F) inhibits insulin secretion. We investigated the ability of ELT, compared with clinically licensed iron chelators, to reverse ROS production and concomitant suppression of insulin production by iron loading of these cells.
Methods:
Cell line: RINm5F is a clonal rat pancreatic b cell line (LGC ATCC Sales, UK). These cells secrete insulin following a glucose challenge (Praz et al., 1983, Biochemistry J).
Intracellular Iron: Cellular iron loading and mobilisation were measured as a decrease in cellular iron content using the ferrozine assay (Vlachodimitropoulou et al. 2015, British Journal of Haematology). A four-fold increase in intracellular iron compared to control was obtained by serially treating cells with 10% Fetal Bovine Serum (FBS) RPMI media in pancreatic cells over two ten hour periods (Figure 1A). The cells were then exposed to iron chelators/ELT, lysed and intracellular iron concentration determined, normalised against protein content.
Reactive oxygen species (ROS) estimation: A cell-permeable oxidation-sensitive fluorescent probe 5,6-carboxy-2’,7’- dichlorofluorescein diacetate (DCFH-DA); (Molecular Probes, Leiden, Netherlands) was used to measure intracellular ROS. Following iron loading, the cells were pre-incubated with 6 mM H2DCF-DA for 30 minutes at 37°C. Chelators were added and the fluorescence of control and treated cells was read throughout the treatment period in the plate reader (excitation 504 nm, emission 526 nm).
Insulin quantification: Following iron loading and chelator treatment, the cells were challenged with Kreb's Ringer Buffer twice, for one hour at a time, containing 2.8mM and 16.7mM glucose (Lu et al. 2010, Toxicology letter). The supernatant was then collected and insulin concentration determined using a standard rat insulin ELISA kit (Life Technologies Limited, UK).
Viability: The Sulforhodamide B (SRB) viability assay was used to ensure viability >98% and assess the toxicity on the pancreatic cell line. It is commonly used to measure drug-induced cytotoxicity and is a colorimetric assay dependent on healthy adherent cells.
Results: Pancreatic cell iron loading was achieved with serial changes of media containing 10% FBS. This loading method was comparable to treating cells with ferric ammonium citrate (FAC) for 24 hours, which was not adopted as FAC adheres to the extracellular surface and produces bias to our intracellular iron quantification system when using iron chelators (Figure 1A). When cells were then treated with increasing ELT concentrations, a dose-dependent cellular iron removal were demonstrated so that at 10μΜ for 8hours, approximately 40% of total cellular iron was mobilised (Figure 3A). Iron mobilisation by ELT was further enhanced by combination with DFO, DFX or DFP (Figure 3). For example, when 10μΜ DFP is combined with 3μΜ ELT, iron mobilisation increases by a further 17% when compared to DFP treatment alone (Figure 3C). ROS production was also decreased in iron-loaded cells in a concentration-dependent manner by increasing ELT concentrations (Figure 2). These reductions in ROS and cellular iron were associated with restoration of insulin secretion, which was reduced by 2.6 fold following iron loading (Figure 1B). The levels of insulin secretion returned back to higher than baseline levels (better than with DFX 1μΜ) (Figure 1C).
Conclusions: This is the first demonstration of a link between cellular iron overload and reduced insulin secretion using pancreatic b-cell line. This is also the first demonstration of improved pancreatic b-cell function, evidenced by restoration of insulin secretion, when iron is chelated and ROS decreased by ELT and other iron chelators. ELT may be useful alone or in combination with other chelators for decreasing iron-mediated ROS induced damage to pancreatic b-cells.
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Porter:Novartis: Consultancy, Honoraria, Research Funding; Bluebird Bio: Consultancy; Agios Pharmaceuticals: Consultancy, Honoraria; Celegene: Consultancy.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
INTRODUCTION
Eltrombopag (ELT) is an orally available, non-peptide, small-molecule thrombopoietin receptor (TPO-R) agonist approved for the treatment of chronic immune thrombocytopenic purpura (ITP). ...Additionally ELT appears to bind intracellular iron (Roth et al, 2012, Blood) and our group has previously demonstrated its ability to progressively mobilize iron from cardiomyocytes in vitro. (Vlachodimitropoulou et al, Blood 2014, Volume 124, 21). The ELT concentrations at which iron was mobilized were substantially less (1µM) than with the clinically available iron chelators Desferrioxamine (DFO), Deferiprone (DFP) and Deferasirox (DFX), where 30µM iron binding equivalents (ibe) were required to achieve similar effects (Vlachodimitropoulou et al, 2014. Blood, Volume 124, 21). Importantly , the 1µM effective concentration of ELT for mobilizing cellular iron is nearly twenty-fold less than peak plasma concentrations reported clinically, even with low doses (30mg) of ELT (Gabianski, Journal of Clinical Pharmacology, 2011;51:842-856). At this low dose, increments in platelet counts do not typically exceed 1.2 x the baseline values in healthy volunteers with repeat dosing (Jenkins et al 2007, Blood, 109; 11 ). Hence it is predicted that effective chelating doses of ELT could be given without promoting unacceptable thombocytosis. In principle, still lower concentrations could be used for iron chelation if combined with another iron chelator. Here we explore and compare the concentrations at which effective cellular chelation is achieved with ELT alone or in combination with another chelator.
METHODS
As cardiomyocytes are a target tissue for transfusional iron overload and provide a particular therapeutic challenge once iron has accumulated in them, the cardiomyocyte cell line H9C2, derived from embryonic rat ventricle, was chosen for investigation. As hepatocytes represent the cell type with the largest quantity of iron deposition, a human hepatocarcinoma HuH7 cell line was also evaluated. Cellular iron loading and iron mobilization were measured as a decrease in cellular iron content using the ferrozine assay (Vlachodimitropoulou et al 2015, British Journal of Haematology). The cells loaded with iron using 10% FBS containing media and then exposed to iron chelators/ELT. Cells were then lysed and intracellular iron concentration determined via the ferrozine assay, normalized against protein content. Acridine Orange/Propidium Iodide staining was used to ensure viability was consistently >98% during experiments, and to assess the toxicity of ELT on the cardiomyocyte and hepatocyte cell lines.
RESULTS
Monotherapy with 1µM ELT removed 42% of total cardiomyocyte iron following 8 hours of treatment. This was notably more efficient than in hepatocytes, where only 7% of cellular iron was removed with 1µM ELT monotherapy (Table 1). In Table 1 we can see the difference in iron removal between ELT monotherapy and combination with chelators after 8 hours. The effect in combination with all chelators was substantial. Viability was unaffected by combinations of 1µM ELT with other chelators. The hydrophilic hydroxypridinone iron chelator CP40, which has no iron mobilizing effects when used alone, enhanced iron mobilization by ELT, indicating that ELT can shuttle iron from cells onto a second chelator.
CONCLUSION
Remarkably low concentrations of ELT monotherapy mobilize cellular iron from cardiomyocytes compared with conventional iron chelators. Furthermore, when used at as little as 1μΜ, in combination with standard therapeutic concentrations of DFO, DFP and DFX, the percentage of iron mobilized from cardiomyocytes more than doubled. Experiments with CP40 indicate that ELT acts as a shuttle molecule for chelated iron onto a second 'sink chelator' and that this is the likely mechanism for the enhanced iron mobilization with other iron chelators. While the action of ELT on the TPO-R is highly species-specific and occurs only in humans and primates, we found effective iron mobilization from both rat cardiomyocytes and human hepatocyte cell lines. This is consistent with an iron chelating mechanismdistinct from the TPO-R downstream signaling mechanism of ELT. The concentrations of ELT used to achieve iron mobilization in combination are clinically achievable and are unlikely to increase platelet counts in patients without thrombocytopaenia.
Porter:Celgene: Consultancy; Shire: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Advancements in orally bioavailable iron chelators and MRI methods have improved life expectancy and reproductive potential in thalassemia major (TM) and thalassemia intermedia (TI). Pregnancy is ...associated with adverse maternal and neonatal outcomes, frequency of which has not been well delineated. This systematic review aims to provide risk estimates of maternal and fetal outcomes in TM and TI and explore pregnancy's impact on iron homeostasis. Fifteen studies (429 participants, 684 pregnancies) were included. Meta-analysis revealed a higher thrombosis risk in TI (3.7%) compared to TM (0.92%), unchanged from prepregnancy. Heart failure risks in the earlier years appeared similar (TM 1.6% vs TI 1.1%), and maternal mortality in TM was 3.7%, but with current management, these risks are rare. Gestational diabetes and pre-eclampsia occurred in 3.9% and 11.3% of TM pregnancies, respectively. Caesarean section rates were 83.9% in TM and 67% in TI. No significant difference in stillbirth, small for gestational age neonates, or preterm birth incidence between TM and TI was observed. In TM pregnancies, red cell requirements significantly increased (from 102 to 139 ml/kg/year, P = 0.001), and 70% of TI pregnancies required blood transfusions. As expected, increased transfusion alongside chelation cessation led to a significant increase in serum ferritin during pregnancy (TM by 1005 ng/mL; TI by 332 ng/mL, P < 0.0001). Deterioration in iron status was further reflected by an increase in liver iron concentration (from 4.6 to 11.9 mg/g dry weight, P < 0.0001), and myocardial T2-star (T2∗) magnetic resonance imaging decreased (from 36.2 ± 2.5 ms to 31.1 ms) during pregnancy. These findings emphasize the elevated maternal risk of iron-related cardiomyopathy during pregnancy and labor, stressing the importance of cardiac monitoring and postpartum chelation therapy resumption.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
INTRODUCTION
Eltrombopag (EP) is an orally bioavailable thrombopoietin receptor agonist developed to increase platelet production in a range of conditions associated with thrombocytopaenia. The ...finding that EP, in addition to increasing platelet counts in myelodysplastic syndromes (MDS), also slowed progression to acute myeloid leukemia has been linked to antiproliferative effects potentially mediated by chelation of labile intracellular iron pools in leukaemia cell lines (Roth et al, 2012, Blood). However, the ability of EP to progressively decrease total cellular iron and its relative efficacy in this regard compared with clinically available iron chelators such as Desferrioxamine (DFO), Deferiprone (DFP) and Deferasirox (DFX) have not been reported. Using a cell based assay system for total cellular iron we therefore compared cellular iron mobilization with EP to that achieved with clinically established iron chelators.
METHODS
The permanent cardiomyocyte cell line H9C2 derived from embryonic rat ventricle was chosen to model iron mobilization, as heart failure secondary to iron overload is the most common cause of death among patients with transfusion-dependent anaemias. Iron concentration was determined using the ferrozine assay (Riemer et al. Anal Biochem. 2004). A two fold increase of intracellular iron compared to control was obtained by serially treating cells with 10% FBS DMEM media. The cells were then exposed to iron chelators/EP, lysed, and intracellular iron concentration determined via the ferrozine assay, normalized against protein content. The LDH enzymatic viability assay was used to ensure viability was consistently >98% during experiments, and to assess the toxicity of EP on the cardiomyocyte cell line.
RESULTS
EP induced both dose and time dependent cellular iron removal from cells at 1, 2, 4 and 8 hours. At 1µM, EP was able to remove 42% of total cellular iron following 8 hours of treatment, and 60.1% and 65.62% at 10µM and 30µM respectively (Figure 1). Figure 2 shows that cell viability is compromised at concentrations of 10µM and 30µM to 96% and 92% respectively with eltrombopag, but not with the commercially used iron chelators, DFO, DFP and DFX. However, at 1µM EP the viability of the monolayer is maintained at >98%. The high effects of iron release noted in figure 1 by EP at 10µM and 30µM could be partially attributed to toxicity of the drug on the monolayer. In table 1 the difference in iron removal between EP and commercially used iron chelators after 8 hours of treatment is shown. Interestingly, with EP at only 1µM, 42.9% of cellular iron was removed, compared to 22.7 %, 34.9% and 19.3% in the case of DFO, DFX and DFP respectively, all at higher concentrations of 30µM iron binding equivalents (IBE).
DISCUSSION AND CONCLUSION
Remarkably low concentrations of EP (1µM) are required to mobilize cellular iron in our cell system while maintaining cell viability at this concentration. This concentration is achievable clinically (Cmax 2-3 µM two to six hours post administration of 75mg orally) (Neito et al, Haematologica, 2011; Deng et al, Drug Metabolism and Disposition, 2011), and when considered alongside with the long plasma half life and elimination in urine and feces, could render it an effective iron chelator for other indications, particularly if combined with existing iron chelation regimes. It would be of interest to explore how EP performed in an animal iron overloaded/MDS model in isolation or as an adjunct to established chelation therapies.
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Table 1:Comparison of iron mobilization by Eltrombopag and commercially used iron chelators following 8 hours of treatment.ChelatorIron/protein (nmol/mg)SD% iron removalcontrol26.782.5-DFO 30μM ibe20.702.222.70DFP 30μM ibe17.431.434.89DFX 30μM ibe21.611.019.28Eltrombopag 1μM15.531.942.94Eltrombopag 10μM10.701.260.06Eltrombopag 30μM9.210.365.62
No relevant conflicts of interest to declare.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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