Heparin Inhibition Research Articles

Using a Modified CPD Blood Bag to Store Blood from either ECMO or RRT Circuit Blood in Pediatric Patients.

By adapting a citrate phosphate dextrose (CPD) whole blood storage bag, residual blood from a renal replacement therapy (RRT) circuit can be saved in pediatric patients, decreasing in donor exposure later. The techniques used for autologous preoperative blood storage are the basis of storing the RRT circuit blood. The CPD anticoagulant has a benefit of having a commonly used reversal agent for its anticoagulant properties, i.e., calcium. Also, unlike the traditional anticoagulants used in extracorporeal membrane oxygenation (ECMO), i.e., heparin, and direct thrombin inhibitors, i.e., bivalirudin, there is no increase in anticoagulation laboratory parameters after administration. The CPD volume in the bag is reduced but keeps the original ratio the same between CPD and blood. This is accomplished by removing all CPD from the bag, adding back only the exact amount of CPD needed for the smaller amount of blood being transferred from the circuit. The RRT circuit managed at our institution uses 23 mL of CPD for 165 mL of circuit blood when stored with this technique. This calculation assumes a normal patient calcium level. This technique has been used successfully multiple times in more than 30 pediatric patients without incident for 7 years at our center. The CPD bag can also be used to store the residual blood from ECMO circuits after removal of ECMO to allow the blood to be given back to the patient at a later time by keeping the same citrate-to-blood ratio.

Host-Guest Complex for Heparin Binding and Sensing

Heparin is a naturally occurring highly charged polyanion and a well-known anticoagulant widely used in surgical practice.[1] More specifically, it is a negatively charged sulfated polysaccharide belonging to the group of glycosaminoglycans (Scheme 1). The anticoagulant effect of heparin is based on its ability to activate antithrombin-III, which, subsequently, inactivates vital coagulation cascade substance such as thrombin and factor Xa consequently preventing fibrin and clot formation.[2] To balance and neutralize heparin dosing, a heparin inhibitor is needed, and commonly, arginine-rich protein protamine sulfate (PS) is used for this purpose.[3] However, in recent years there has been an interest to investigate alternatives for protamine due to its adverse effects like hypotension and serious allergic reactions.[4]In this study, a host-guest complex capable in selective heparin binding and sensing is presented.[5] Heparin binding affinity was studied in solution with a variety of polycationic macrocyclic hosts, a pillar[5]arene and multiple resorcin[4]arenes, by dynamic light scattering, dye displacement assay, isothermal titration calorimetry, and anti-Xa assay. The measurements reveal the significant importance of multivalency in the electrostatic host-heparin binding in competitive, application-relevant media. Additionally, to monitor heparin concentration, a host-guest indicator displacement assay was performed by following the free and bound state of methyl orange dye in UV-Vis spectroscopic experiments. Furthermore, this colorimetric sensing based on the tertiary host-guest-heparin supramolecular assembly was utilized in the construction of a calibration curve in a range of blood plasma concentrations.

Monitoring the hemostasis process through the electrical characteristics of a graphene-based field-effect transistor

Graphene-based transistors are promising devices in the evaluation of carrier density in biological analytes. We report on the design and fabrication of a graphene-based field-effect transistor for monitoring and assessing the interaction between the coagulation factors based on the charge carrier density in a blood sample. When biochemical reactions occurred during the coagulation cascade process, a dopant effect was noticed on the graphene surface by the change in Dirac point voltage values. Additional experiments were performed using blood samples treated with activators (vitamin K, calcium chloride, and thromboplastin reagent) and inhibitors (heparin drugs) to evaluate the selectivity of the graphene field-effect transistor devices. Since the transfer characteristic curves presented divergent behaviours for different levels of procoagulants and anticoagulants, the measurements showed that the devices can assess changes in the concentrations of factors that inhibit or accelerate the cascade process when using untreated and treated samples. Reproducibility was verified by testing samples from different sources. To the best of our knowledge, this study is the first to demonstrate the potential of graphene in monitoring the hemostasis process through the analysis of the electrical properties of human whole blood.

Профилактика массивных интраоперационных кровотечений, ассоциированных с гепарином, при системном применении фибрин-мономера в эксперименте

Цель исследования - изучение способности фибрин-мономера предупреждать тяжелую интраоперационную кровопотерю, ассоциированную с введением нефракционированного гепарина, при дозированной травме печени. Методика. На кроликах «Шиншилла» индуцировали гипокоагуляцию нефракционированным гепарином (150 ед/кг). Профилактику интраоперационных кровотечений осуществляли внутривенным введением фибрин-мономера (0,25 мг/кг) за 1 ч до травмы или протамина сульфата (1,5 мг/кг) за 10 мин до травмы. После нанесения стандартной травмы печени оценивали объем (в % ОЦК) и темп (мг/с) кровопотери. Анализировали число тромбоцитов, активированное парциальное тромбопластиновое время, протромбиновое и тромбиновое время свертывания, уровень фибриногена и активность антитромбина III, параметры ротационной тромбоэластометрии крови. Результаты. Объем кровопотери в группах животных после в/в введения фибрин-мономера и протамина сульфата на фоне гепаринизации был, соответственно, в 5,1 и 4,0 раза меньше по сравнению с группой плацебо, получавшей тот же антикоагулянт. Вместе с тем, фибрин-мономер не влиял на параметры коагулограммы (отсутствие видимого гемостазиологического эффекта) и тромбоэластограммы, тогда как применение протамина сульфата в качестве антидота гепарина сопровождалось нормализацией данных тромбоэластометрии и коррекцией гипокоагуляционного сдвига по активированному парциальному тромбопластиновому времени, протромбиновому и тромбиновому времени. Заключение. Установлено, что фибрин-мономер (0,25 мг/кг) снижает посттравматическое кровотечение в условиях блокады свертывания крови гепарином без видимых признаков восстановления гемостатического равновесия. The research objective was to study the ability of fibrin monomer to prevent severe intraoperative blood loss associated with administration of unfractionated heparin in controlled liver injury. Methods. Hypocoagulation was induced in chinchilla rabbits with unfractionated heparin (150 U/kg). Intraoperative bleeding was prevented by administration of fibrin monomer (FM, 0.25 mg/kg, i.v.) one hour prior to the injury and of protamine sulfate (PS, 1.5 mg/kg, i.v.) 10 min prior to the injury. Following the liver injury, blood loss was assessed as percentage of circulating blood volume and the blood loss rate (mg/s). Platelet counts, aPTT, PT, TT, fibrinogen level, antithrombin III activity, and parameters of blood rotation thromboelastometry were analyzed. Results. The volume of blood loss was 5.1 times and 4.0 times less, respectively, after the FM and PS administration during heparinization compared to the placebo group treated with the same anticoagulant. However, FM affected neither coagulogram indexes (no visible hemostasiological effect) nor thromboelastogram while the use of PS as an antidote for heparin was associated with normalization of thromboelastometric data and correction of hypercoagulative changes in aPTT, PT, TT. Conclusion. FM at a dose of 0.25 mg/kg reduced severity of posttraumatic bleeding induced by heparin inhibition of coagulation with no visible signs of hemostatic balance recovery.

Comparison of Unfractionated Heparin and Bivalirudin for Treatment of Suspected Device Thrombosis during Heart Mate II Support

Purpose Although intravenous anticoagulation is the mainstay medical therapy employed during CF LVAD thrombosis, the comparative impact of indirect thrombin inhibition with unfractionated heparin (UH) and direct thrombin inhibition with bivalirudin (BV) is unknown. Methods We conducted a single center review of all patients with a Heart Mate(HM) II who were admitted for suspected device thrombosis (SDT) from September 2011 to September 2018. Device thrombosis was suspected due to elevated outpatient lactate dehydrogenase (LDH), evidence of systemic emboli, or device alarms. After admission, patients were categorized into those receiving UH or BV. Crossovers were excluded. Freedom from device exchange was calculated with Kaplan Meier analysis. Results Twenty-three patients were admitted for SDT, of whom 13 received UH and 10 received BV. There were no differences in age (UH: 49.2 vs. BV: 46.9 years, p=0.60) and time from implant to SDT (UH: 10.3 vs. BV: 10.4 months, p=0.98). LDH remained elevated after UH (879, IQR: 755-1049 to 1028, IQR: 550-1438 U/L, p=0.34) but dropped with BV (839, IQR: 733-914 to 453, IQR: 352-465 U/L, p=0.002). During the follow up period, there was a lower likelihood of a device exchange in patients treated with BV (HR: 0.28, 95% CI: 0.09-0.89, p=0.04, figure 1). Conclusion In this sample of HM II patients, intravenous direct thrombin inhibition was more effective in treating device thrombosis.

Environmental Cell-Matrix Adhesion Modulates Pluripotent Stem Cell Fate Toward Definitive Hemogenic Endothelium and Hematopoietic Progenitor Cells

[Background] Human pluripotent stem cell (hPSC) technology enables regenerative transplantation therapy and establishment of in vitro disease models. hPSCs can be maintained to proliferate limitlessly and also differentiated to specific lineage cells, but hematopoietic cell differentiation efficiency varies among each hPSC lines. It has been reported the intrinsic expression level of transcription factors in hPSCs determine the commitment capacity toward hematopoietic cells (i.e. c-MYC [J Exp Med. 2010; 207: 2817-2830], IGF2 [Cell Stem Cell. 2016; 19: 341-354]). However, whether extracellular scaffold factors that contribute to maintain hPSCs influence hematopoietic differentiation potential remain elusive.[Aim] In this study, we hypothesized that the extracellular environment such as cell-matrix adhesion signal regulates the commitment capacity toward hematopoietic cells in hPSCs. Moreover, we sought to improve the conventional hematopoietic differentiation PSC-sac method, which is simple but inefficient.[Results] The recombinant fragment of a laminin isoform, LM511-E8, [Neurosci Res. 2018; 96: 863-874, Nature. 2016; 531: 376-380, Nat commun. 2012; 3: 1236] is used in culture to maintain pluripotency of cGMP grade hPSCs instead of Matrigel in research grade hPSCs. However, cGMP grade hPSCs maintained on LM511-E8 showed decreased differentiation towards CD34+CD43+ Hematopoietic progenitor cells (HPCs) than hPSCs maintained on Matrigel or mouse embryonic fibroblasts as feeder cells. Meanwhile, hPSCs maintained on human recombinant laminin fragments LM121-E8 or LM421-E8 yielded significantly more HPCs than hPSCs maintained on LM511-E8.Microarray analysis revealed that hPSCs maintained on those “hematopoietic laminins (HLs)” showed upregulated activity of canonical Wnt/β-catenin signaling pathway. Activation of canonical Wnt signaling pathway by GSK3β inhibitor significantly improved the yield of HPC from hPSCs on LM511-E8. We then found using flow cytometry that hPSCs on HLs had higher cell surface expression of integrin β1 subunit (ITGB1), which is a part of integrin a6b1 complex, the laminin receptor, and also found the higher expression of ILK, pGSK3β (Ser9) and β-catenin by Western blot analysis. These results accordingly suggest that the ITGB1-ILK-pGSK3β-β-catenin axis modulate the commitment of hPSCs towards hematopoietic lineage. We also found lower concentrations of LM511-E8 increased ITGB1 surface expression and HPC differentiation on day14, without influencing the activity of pluripotency of hPSCs. These results indicate the modulation of hematopoietic capacity by extrinsic laminin at pluripotent state is determined by the affinity of laminin-integrin, not by the specificity of the laminin subunits, and this extrinsic modulation might be regulated through the expression level of ITGB1.Next, we revised hPSC-sac method, a conventional hematopoietic differentiation method which we originally proposed [Cell Stem Cell. 2017; 20(3) : 329-344.e7. Stem Cells. 2016; 34: 1541-1552. Blood. 2011; 111(11): 5298-5306.] By adding basic fibroblast growth factor (bFGF), TGFβ inhibitor SB431542 and heparin during differentiation period, the number of HPC increased dramatically on differentiation day14. The number of definitive hemogenic endothelium (CD34+ CD31+ CD73- CD144+ CD117+ KDR+ CD41- CD43- CD45- [Cell Reports, 2012; 2: 553-567][Blood, 2013; 121(5): 770-780]) on differentiation day 9 was also increased. Simultaneously, transcription factors specific for hematopoietic lineage, ERG, SPI1, Runx1c, GFI1b, TAL1, and TEK were upregulated in hPSCs derived cells on day 9.[Conclusion] This study revealed a mechanism in which the signals from cell-matrix adhesion by cell surface integrins and extracellular laminins modulate hematopoietic differentiation potential in hPSCs. Moreover, the revised hPSC-Sac differentiation method enables a simple, efficient and economical differentiation of hematopoietic cells. These findings thus should contribute to development of the fundamental research on hematopoietic differentiation from hPSCs towards further application. DisclosuresSekiguchi:Matrixome, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Nippi, Inc: Research Funding. Eto:Megakaryon co. Ltd.: Research Funding.

Safety and efficacy of switching from unfractionated heparin to bivalirudin during primary percutaneous coronary intervention.

To evaluate the safety and efficacy of switching to bivalirudin during primary percutaneous coronary intervention (PCI) for patients who received preprocedure unfractionated heparin (UFH). Current guidelines favor bivalirudin for primary PCI in patients at high risk of bleeding, particularly when femoral access is used. However, patients with ST-segment elevation myocardial infarction frequently receive UFH before arrival in the catheterization laboratory. Scientific databases and websites were searched for randomized controlled trials. Patients were divided into those who received heparin with or without glycoprotein IIb/IIIa inhibitors (heparin group); those switched to bivalirudin during primary PCI from preprocedure UFH (switch group); and those who received bivalirudin without preprocedure UFH (Biv-alone group). Both traditional pairwise meta-analyses using moderator analyses and network meta-analyses using mixed-treatment comparison models were performed. Data from five trials including13,547 patients were analyzed. In mixed-comparison models, switching to bivalirudin during primary PCI was associated with lower rates for all-cause mortality and major adverse cardiovascular events (MACEs) compared to the other strategies. Rates for all-cause mortality, MACEs, and net adverse clinical events (NACEs) were similar for the heparin and Biv-alone groups. Switching strategies was also associated with lower major bleeding rates compared to heparin alone. Similarly, in a standard pairwise model, both the switch and Biv-alone groups were associated with decreased bleeding risk compared to the heparin group. However, only the switch strategy was associated with decreased all-cause mortality (RR, 0.47; 95% CI, 0.30-0.75; P = 0.001), MACE (RR, 0.67; 95% CI, 0.49-0.91; P = 0.012), and NACE (RR, 0.61; 95% CI, 0.41-0.92; P = 0.019) compared with heparin alone. During primary PCI, use of bivalirudin for those receiving preprocedure UFH was associated decreased rates for major bleeding, NACEs, MACEs, and all-cause mortality compared to heparin +/- GPI. This strategy was also associated with decreased rates for MACEs and all-cause mortality compared to bivalirudin alone without preprocedure UFH.

An in-silico glimpse into the pH dependent structural changes of T7 RNA polymerase: a protein with simplicity

The capability of performing an array of functions with its single subunit structure makes T7 RNA polymerase (T7RNAP) as one of the simplest yet attractive target for various investigations ranging from structure determinations to several biological tests. In this study, with the help of molecular dynamics (MD) calculations and molecular docking, we investigated the effect of varying pH conditions on conformational flexibility of T7RNAP. We also studied its effect on the interactions with a well established inhibitor (heparin), substrate GTP and T7 promoter of T7RNAP. The simulation studies were validated with the help of three dimensional reconstructions of the polymerase at different pH environments using transmission electron microscopy and single particle analysis. On comparing the simulated structures, it was observed that the structure of T7RNAP changes considerably and interactions with its binding partners also changes as the pH shifts from basic to acidic. Further, it was observed that the C-terminal end plays a vital role in the inefficiency of the polymerase at low pH. Thus, this in-silico study may provide a significant insight into the structural investigations on T7RNAP as well as in designing potent inhibitors against it in varying pH environments.

Heparinase enables reliable quantification of circulating tumor DNA from heparinized plasma samples by droplet digital PCR

BackgroundHeparin is often used as a blood anticoagulant for tumor marker analysis but results in the inhibition of PCR detection of circulating tumor DNA (ctDNA), which has been deemed a potential “liquid biopsy”. We aimed to evaluate the impact of heparinase addition on heparinized plasma samples to allow ctDNA analysis. MethodsPlasma samples were collected in heparinized (n=194) and EDTA (n=8) tubes from hormone receptor-positive metastatic breast cancer (HR+MBC) (n=144) and pancreatic adenocarcinoma (PA) patients (n=50). Circulating ESR1 and KRAS mutations were detected with or without heparinase by digital PCR in HR+MBC and PA patients, respectively. Patients were classified into 2 subgroups i) inhibition, I+ and ii) no inhibition, I− based on a threshold of 200copies/μL for PCR inhibition by heparin. ResultsIn the I+ subgroup (91/144 HR+MBC and 26/50 PA), heparinase treatment significantly improved PCR efficacy, enabling ctDNA detection in 22/91 and 13/26 patients. Moreover, comparable results for ctDNA detection (4/8) were obtained with heparinized and EDTA PA samples. In the I− subgroup, heparinase addition did not quantitatively and qualitatively alter ctDNA detection. ConclusionHeparinase addition removes the heparin inhibition and allows accurate ctDNA detection in heparinized samples. These findings could make the samples from heparinized blood suitable for ctDNA analysis.

Morphological and growth responses of vascular smooth muscle and endothelial cells cultured on immobilized heparin and dextran sulfate surfaces.

Heparin has shown promise as a component of various biomaterial formulations, but its variable properties and inhibitory effects on some cell types have raised interest in use of dextran sulfate as an alternative. In this study, we characterized the interactions of vascular smooth muscle (SMC) and endothelial cells (EC) with heparin and dextran sulfate immobilized onto chitosan-based films. Films were modified by blending chitosan with type I collagen and covalently attaching heparin or dextran sulfate at various levels. Cell-material interactions were evaluated by quantifying cell spreading, shape and proliferation rate. ECs proliferated well on chitosan, but the polymer was a mediocre substrate for SMC growth. Immobilizing heparin on chitosan further inhibited SMC proliferation. However, blending collagen reversed the heparin inhibition of SMC growth, resulting in a pro-proliferative effect of heparin immobilized on chitosan-collagen films. Dextran sulfate surfaces supported both SMC and EC proliferation with or without the presence of collagen. The results indicate that inhibitory effects of heparin on SMC are reversed by immobilization in the presence of collagen, and that dextran sulfate may be superior to heparin as a biomaterial additive for promoting vascular cell growth in chitosan-based scaffolds. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1725-1735, 2017.

Label-free fluorescent assay of T4 polynucleotide kinase phosphatase activity based on G-quadruplexe−thioflavin T complex

T4 polynucleotide kinase phosphatase (T4 PNKP) is a bifunctional tool enzyme with 5′-kinase and 3′-phosphatase activities. Considering its important roles in the repair of strand breaks, assay of T4 PNKP activity is of great importance. In this work, we proposed a novel label-free sensing strategy for T4 PNKP activity based on G-quadruplexe−thioflavin T fluorescent indicator. In the assay, we used a single stranded oligonucleotide with 3′-phosphoryl and 5′-phosphoryl ends as enzyme substrate. Upon the addition of T4 PNKP, the 3′-PO3 of the substrate was changed to 3′-OH which initiated the polymerization in the presence of terminal deoxynucleotidyl transferase and G-rich dNTP substrates. The resultant elongated DNA can form G-quadruplex in the inducement of K+, resulting in strong fluorescence signal when using thioflavin T as a G-quadruplex−specific light-up fluorescent probe. The detection limit of this method is as low as 0.2U/mL. Additionally, the inhibition of T4 PNKP activity by the inhibitor heparin is demonstrated. This method is easy and convenient to operate in homogeneous solution, and the whole assay process can be completed in a single tube.

Circulating Histone Is Elevated in Acute Leukemia and Protects Leukemic Cell Death through Enhancement of Leukemic Cell Adhesion to Endothelium

Background: Leukocytes release nuclear contents into extracellular milieu called by extracellular traps (ET) when they are stimulated by reactive oxygen species (ROS). The nuclear contents mainly compose of histone-DNA complex and neutrophil elastase. This study investigated whether the leukemic cells could release ET and the released histone could induce endothelial activation, finally resulting in leukemic progression. Methods: The circulating ET biomarkers (histone-DNA complex, cell free DNA, neutrophil elastase) were measured in 80 patients with hematologic diseases and 40 healthy controls by ELISA. ET formation and ROS level were investigated during leukemic cell proliferation in vitro. Histone-induced endothelial adhesion molecule expression and survival were measured by flow cytometry. Results: Acute leukemia showed high ET biomarkers, which correlated with peripheral blast count. Leukemic cells produced high ROS and release extracellular histone, which was significantly blocked by antioxidants. Histone significantly induced 3 endothelial adhesion molecules expression, promoting leukemic cell adhesion to endothelial cells, which was inhibited by histone inhibitors (heparin, polysialic acid, activated protein C), 3 neutralizing antibodies against adhesion molecules, and Toll like receptor(TLR)9 antagonist. When the leukemic cell co-cultured with endothelial cells, the adherent leukemic cells showed better survival than the non-adherent, demonstrating that histone-treated endothelial cells protected leukemic cells from both spontaneous and chemotherapy-induced death. Conclusion: Our data for the first time supports that extracellular histone could be released from leukemic cells through ROS dependent mechanism. The released histone could promote leukemic cell adhesion through endothelial adhesion molecule induction and finally protect from leukemic cell death. Disclosures No relevant conflicts of interest to declare.

VP1 residues around the five-fold axis of enterovirus A71 mediate heparan sulfate interaction

Enterovirus A71 (EV-A71) is a neurotropic enterovirus that uses heparan sulfate as an attachment receptor. The molecular determinants of EV-A71-heparan sulfate interaction are unknown. With In silico heparin docking and mutagenesis of all possible lysine residues in VP1, we identified that K162, K242 and K244 are responsible for heparin interaction and inhibition. EV-A71 mutants with K242A and K244A rapidly acquired compensatory mutations, T100K or E98A, and Q145R-T237N respectively, which restored the heparin-binding phenotype. Both VP1-98 and VP1-145 modulates heparin binding. Heparin-binding phenotype was completely abolished with VP1-E98-E145, but was restored by an E98K or E145Q substitution. During cell culture adaptation, EV-A71 rapidly acquired K98 or Q/G145 to restore the heparin-binding phenotype. Together with next-generation sequencing analysis, our results implied that EV-A71 has high genetic plasticity by modulating positively-charged residues at the five-fold axis during in vitro heparin adaptation. Our finding has impact on EV-A71 vaccine production, evolutionary studies and pathogenesis.

Extracellular Histone Released from Leukemic Cells Increases Their Adhesion to Endothelium and Protects them from Spontaneous and Chemotherapy-Induced Leukemic Cell Death.

IntroductionWhen leukocytes are stimulated by reactive oxygen species (ROS), they release nuclear contents into the extracellular milieu, called by extracellular traps (ET). The nuclear contents are mainly composed of the histone–DNA complex and neutrophil elastase. This study investigated whether leukemic cells could release ET and the released histone could induce endothelial activation, eventually resulting in leukemic progression.MethodsThe circulating ET were measured in 80 patients with hematologic diseases and 40 healthy controls. ET formation and ROS levels were investigated during leukemic cell proliferation in vitro. Histone-induced endothelial adhesion molecules expression and cell survival were measured by flow cytometry.ResultsAcute leukemia patients had high levels of ET, which correlated with peripheral blast count. Leukemic cells produced high ROS levels and released extracellular histone, which was significantly blocked by antioxidants. Histone significantly induced 3 endothelial adhesion molecules expression, and promoted leukemic cell adhesion to endothelial cells, which was inhibited by histone inhibitors (heparin, polysialic acid, and activated protein C), neutralizing antibodies against these adhesion molecules, and a Toll like receptor(TLR)9 antagonist. When leukemic cells were co-cultured with endothelial cells, adherent leukemic cells showed better survival than the non-adherent ones, demonstrating that histone-treated endothelial cells protected leukemic cells from both spontaneous and chemotherapy-induced death.ConclusionOur data demonstrate for the first time that extracellular histone can be released from leukemic cells through a ROS-dependent mechanism. The released histone promotes leukemic cell adhesion by inducting the surface expression of endothelial adhesion molecules and eventually protects leukemic cells from cell death.

Effectiveness and Safety of Bivalirudin During Percutaneous Coronary Intervention in Acute Coronary Syndrome in the Real World: CARTAGOMAX Study.

The CARTAGOMAX study assessed the safety and efficacy of bivalirudin during real-world cardiac intervention. This was a single-center prospective study. Patients with acute coronary syndrome undergoing percutaneous coronary intervention were anticoagulated with bivalirudin alone or unfractionated heparin plus a glycoprotein IIb/IIIa inhibitor. Propensity score matching was performed to control for baseline imbalances and yielded 1168 patients. There was lower incidence of the composite outcome of death from any cause or major bleeding at 30 days (P = 0.005), 6 months (P = 0.005), and 12 months (P = 0.001) of follow-up in the bivalirudin group, compared with the heparin plus glycoprotein inhibitor group. The administration of bivalirudin was associated with lower rate of all-cause mortality at 1 year of follow-up (P = 0.009). The incidence of major bleeding was lower in the bivalirudin group at 1, 6, and 12 months of follow-up (P = 0.002, P = 0.013 and P = 0.017, respectively). The incidence of stroke and reinfarction were similar between groups during follow-up. The rate of stent thrombosis were slightly higher in the bivalirudin group, without reaching statistical significance at 1 and 12 months of follow-up (P = 0.06, P = 0.04, P = 0.07 at 1, 6, and 12 months, respectively). The CARTAGOMAX study found that the use of bivalirudin during percutaneous coronary intervention was associated with lower incidence of the composite outcome of death from any cause or major bleeding during follow-up. The use of bivalirudin was associated with similar rates of stroke, reinfarction, and stent thrombosis compared with heparin plus glycoprotein inhibitor. Bivalirudin proved to be a safe and effective anticoagulant during percutaneous coronary intervention.

Suramin is a potent inhibitor of Chikungunya and Ebola virus cell entry.

BackgroundChikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes high fever, rash, and recurrent arthritis in humans. It has efficiently adapted to Aedes albopictus, which also inhabits temperate regions and currently causes large outbreaks in the Caribbean and Latin America. Ebola virus (EBOV) is a member of the filovirus family. It causes the Ebola virus disease (EDV), formerly known as Ebola hemorrhagic fever in humans and has a mortality rate of up to 70 %. The last outbreak in Western Africa was the largest in history and has caused approximately 25,000 cases and 10,000 deaths. For both viral infections no specific treatment or licensed vaccine is currently available. The bis-hexasulfonated naphthylurea, suramin, is used as a treatment for trypanosome-caused African river blindness. As a competitive inhibitor of heparin, suramin has been described to have anti-viral activity.MethodsWe tested the activity of suramin during CHIKV or Ebola virus infection, using CHIKV and Ebola envelope glycoprotein pseudotyped lentiviral vectors and wild-type CHIKV and Ebola virus.ResultsSuramin efficiently inhibited CHIKV and Ebola envelope-mediated gene transfer while vesicular stomatitis virus G protein pseudotyped vectors were only marginally affected. In addition, suramin was able to inhibit wild-type CHIKV and Ebola virus replication in vitro. Inhibition occurred at early time points during CHIKV infection.ConclusionSuramin, also known as Germanin or Bayer-205, is a market-authorized drug, however shows significant side effects, which probably prevents its use as a CHIKV drug, but due to the high lethality of Ebola virus infections, suramin might be valuable against Ebola infections.

Heparinase treatment of heparin-contaminated plasma from coronary artery bypass grafting patients enables reliable quantification of microRNAs

BackgroundmicroRNAs have recently been identified as powerful biomarkers of human disease. Reliable polymerase chain reaction (PCR)-based quantification of nucleic acids in clinical samples contaminated with polymerase inhibitor heparin requires deheparinization. However, the effects of deheparinization procedure on quantification of nucleic acids remain largely unknown. The aim of this study was to determine whether the deheparinization procedure completely eliminates the inhibition of amplification, while maintaining RNA integrity and technical variability of the measured microRNA levels. MethodsHeparinized plasma from 9 patients undergoing coronary artery bypass grafting (CABG) and the heparin-free plasma from 58 rats were spiked with a synthetic RNA oligonucleotide and total RNA was extracted. The RNA solutions were then treated with heparinase I to remove contaminating heparin prior to reverse transcription. Levels of synthetic spike-in RNA oligonucleotide, as well as endogenous hsa-miR-1-3p and hsa-miR-208a-3p, were measured using quantitative reverse transcription PCR (RT-qPCR). The amplification efficiency and presence of inhibitors in individual samples were directly determined using calibration curves. ResultsIn contrast to RNA samples from rat plasma, RNA samples derived from the CABG patient plasma contained inhibitors, which were completely eliminated by treatment with heparinase. The procedure caused a decrease in the amount of detected RNA; however, the technical variability of the measured targets did not change, allowing for the quantification of circulating endogenous hsa-miR-1-3p and hsa-miR-208a-3p in the plasma of CABG patients. ConclusionsThe heparinase treatment procedure enables utilization of RT-qPCR for reliable microRNA quantification in heparinized plasma.

Competition Between Tamm‐Horsfall Protein and Heparin in Ligand Interactions

Tamm‐Horsfall protein (THP), also called uromodulin, is an 86kD acidic, single‐chain, glycoprotein produced in the kidney and excreted into the urine. THP binds various molecules of the immune system including immunoglobulins (Ig), complement 1q (C1q), and factor H (fH). Heparin is a negatively‐charged, sulfated polysaccharide that binds to a wide range of proteins, including C1q, fH, antithrombin III (ATIII), basic fibroblast growth factor (FGF2), and interleukin‐10 (IL‐10). The purpose of this study was to determine if heparin competed with THP for binding to proteins that interact with both THP and heparin (i.e. C1q and fH), and to screen other heparin‐binding proteins for previously unidentified interactions with THP. Dot blots were performed by immobilizing 1 μg of C1q, fH, and bovine serum albumin (BSA) to 4 strips of nitrocellulose (NC). One NC strip was stained with amido black and the other 3 NC strips were incubated with either THP (10 μg/ml), THP (10 μg/ml)/heparin (20 μg/ml) (sodium salt from porcine intestine), or no THP/heparin, before incubation with sheep anti‐THP antiserum and then donkey anti‐sheep IgG ‐ HRP. These blots showed that THP bound strongly to C1q and fH, and that this binding was inhibited by heparin. Heparin also inhibited the background binding of the antisera to C1q. ELISAs were used to quantify the ability of heparin to inhibit C1q (5 μg/ml) binding to immobilized THP (40 μg/ml). A steep inhibition curve was obtained, with no inhibition of C1q binding to immobilized THP occurring with 0.02 units/ml heparin, 50% inhibition of binding occurring with between 0.04 and 0.05 units heparin/ml, and 90% inhibition occurring with 0.1 unit heparin/ml (170 units/mg heparin). Control wells showed that heparin's inhibition of C1q binding to THP was not due to inhibition by heparin of the antisera's binding to C1q. Additionally, dot blots were used to evaluate binding of soluble THP to ATIII, IL‐10, and FGF2, three of the many proteins that heparin has been shown to bind. These blots demonstrated that THP bound to FGF2 and IL‐10, and this interaction was blocked by the addition of heparin. THP did not bind ATIII. In summary, heparin inhibited THP's binding to NC‐immobilized C1q and fH, as well as inhibiting immobilized THP's binding to soluble C1q in ELISAs. Protein‐protein interactions between THP and FGF2 and IL‐10, that were sensitive to heparin inhibition, were discovered. Heparin and THP coexist in the urine, especially after intravenous heparin administration or when heparin is instilled into the bladder as a therapy for interstitial nephritis. Mast cells are prominent in the wall of the urinary bladder in interstitial cystitis, so competition of heparin and THP for binding to other proteins may be important in this disease. Further studies to assess the physiological significance in THP/heparin competition for binding to various proteins is warranted.Support or Funding InformationFunded by the PNWU Research Seed Program.

Scalable Production of a Multifunctional Protein (TSG-6) That Aggregates with Itself and the CHO Cells That Synthesize It.

TNF-α stimulated gene/protein 6 (TNFAIP6/TSG-6) is a multifunctional protein that has a number of potential therapeutic applications. Experiments and clinical trials with TSG-6, however, have been limited by the technical difficulties of producing the recombinant protein. We prepared stable clones of CHO cells that expressed recombinant human TSG-6 (rhTSG-6) as a secreted glycoprotein. Paradoxically, both cell number and protein production decreased dramatically when the clones were expanded. The decreases occurred because the protein aggregated the synthesizing CHO cells by binding to the brush border of hyaluronan that is found around many cultured cells. In addition, the rhTSG-6 readily self-aggregated. To address these problems, we added to the medium an inhibitor of hyaluronan synthesis and heparin to compete with the binding of TSG-6 to hyaluronan. Also, we optimized the composition of the culture medium, and transferred the CHO cells from a spinner culture system to a bioreactor that controlled pH and thereby decreased pH-dependent binding properties of the protein. With these and other improvements in the culture conditions, we obtained 57.0 mg ± 9.16 S.D. of rhTSG-6 in 5 or 6 liter of medium. The rhTSG-6 accounted for 18.0% ± 3.76 S.D. of the total protein in the medium. We then purified the protein with a Ni-chelate column that bound the His tag engineered into the C-terminus of the protein followed by an anion exchange column. The yield of the purified monomeric rhTSG-6 was 4.1 mg to 5.6 mg per liter of culture medium. After intravenous injection into mice, the protein had a longer plasma half-life than commercially available rhTSG-6 isolated from a mammalian cell lysate, apparently because it was recovered as a secreted glycoprotein. The bioactivity of the rhTSG-6 in suppressing inflammation was demonstrated in a murine model.

Role of Heparan Sulfate in Cellular Infection of Integrin-Binding Coxsackievirus A9 and Human Parechovirus 1 Isolates.

Heparan sulfate/heparin class of proteoglycans (HSPG) have been shown to function in cellular attachment and infection of numerous viruses including picornaviruses. Coxsackievirus A9 (CV-A9) and human parechovirus 1 (HPeV-1) are integrin-binding members in the family Picornaviridae. CV-A9 Griggs and HPeV-1 Harris (prototype) strains have been reported not to bind to heparin, but it was recently shown that some CV-A9 isolates interact with heparin in vitro via VP1 protein with a specific T132R/K mutation. We found that the infectivity of both CV-A9 Griggs and HPeV-1 Harris was reduced by sodium chlorate and heparinase suggestive of HSPG interactions. We analyzed the T132 site in fifty-four (54) CV-A9 clinical isolates and found that only one of them possessed T132/R mutation while the other nine (9) had T132K. We then treated CV-A9 Griggs and HPeV-1 Harris and eight CV-A9 and six HPeV-1 clinical isolates with heparin and protamine. Although infectivity of Griggs strain was slightly reduced (by 25%), heparin treatment did not affect the infectivity of the CV-A9 isolates that do not possess the T132R/K mutation, which is in line with the previous findings. Some of the HPeV-1 isolates were also affected by heparin treatment, which suggested that there may be a specific heparin binding site in HPeV-1. In contrast, protamine (a specific inhibitor of heparin) completely inhibited the infection of both prototypes and clinical CV-A9 and HPeV-1 isolates. We conclude that T132R/K mutation has a role in heparin binding of CV-A9, but we also show data, which suggest that there are other HSPG binding sites in CV-A9. In all, we suggest that HSPGs play a general role in both CV-A9 and HPeV-1 infections.