Removal of dabigatran using sorbent hemadsorption.

BACKGROUND: The Redual PCI trial has demonstrated the safety of dabigatran and ticagrelor or clopidogrel combination in preventing strokes in patients with atrial fibrillation. There was 15.4% risk of hemorrhage in the dabigatran/ticagrelor or clopidrogel arm, lower than that of triple therapy with warfarin, aspirin and ticagrelor or clopidogrel. While idarucizumab is an effective antidote for dabigatran, there is no good method for antagonizing both dabigatran and ticagrelor. We tested in this study a hemadsorbtion method for removing dabigatran that we had previously successfully applied in the removal of ticagrelor from human blood. METHODS: 100 mL 4% BSA solution pre-incubated with dabigatran was passed through 10, 20 and 40 mL sorbent columns and dabigatran concentration was measured from the affluent and effluent solution using LC-MS/MS. For testing the effect of dabigatran removal on the aPTT value one human volunteer was administered oral dabigatran etexilate mesilate 150 mg. Plasma was collected 4 h after dabigatran administration and then in three experiments 20 mL of collected plasma was circulated through three different 10 mL CytoSorb columns over a duration of 5 min. aPTT was measured from plasma at baseline prior to drug administration, then post blood collection (mixed plasma) and from the adsorbed plasma as well. RESULTS: Dabigatran concentration, as measured by LC-MS/MS, decreased from 1456 ±â€¯331 nM (greater than the therapeutic level of 743 nM) to 67 ±â€¯59 nM (P = 0.002) with the 10 mL CytoSorb column, while with the 40 mL column it dropped to undetectable levels. In one human volunteer experiment the aPTT was on average 29.2 ±â€¯0.4 in the 3 baseline samples, 34.7 ±â€¯1.8 s after oral dabigatran (mixed plasma), and 25 ±â€¯0.7 s after plasma was passed through CytoSorb (adsorbed plasma) (P = 0.000025 and 0.0000002 for comparison between baseline plasma and mixed plasma, as well as the dabigatran mixed plasma and post-adsorption values respectively). CONCLUSION: Dabigatran is robustly removed by a sorbent hemadsorption method already proven successful for the P2Y12 receptor antagonist ticagrelor. Dabigatran removal restores the aPTT to below baseline values, suggesting that sorbent hemadsorption could clinically reverse the anticoagulant effect of this drug.

Patch-Clamp Study of Hepatitis C p7 Channels Reveals Genotype-Specific Sensitivity to Inhibitors.

Hepatitis C is a major worldwide disease and health hazard, affecting ∼3% of the world population. The p7 protein of hepatitis C virus (HCV) is an intracellular ion channel and pH regulator that is involved in the viral replication cycle. It is targeted by various classical ion channel blockers. Here, we generated p7 constructs corresponding to HCV genotypes 1a, 2a, 3a, and 4a for recombinant expression in HEK293 cells, and studied p7 channels using patch-clamp recording techniques. The pH50 values for recombinant p7 channels were between 6.0 and 6.5, as expected for proton-activated channels, and current-voltage dependence did not show any differences between genotypes. Inhibition of p7-mediated currents by amantadine, however, exhibited significant, genotype-specific variation. The IC50 values of p7-1a and p7-4a were 0.7 ± 0.1 nM and 3.2 ± 1.2 nM, whereas p7-2a and p7-3a had 50- to 1000-fold lower sensitivity, with IC50 values of 2402 ± 334 nM and 344 ± 64 nM, respectively. The IC50 values for rimantadine were low across all genotypes, ranging from 0.7 ± 0.1 nM, 1.6 ± 0.6 nM, and 3.0 ± 0.8 nM for p7-1a, p7-3a, and p7-4a, respectively, to 24 ± 4 nM for p7-2a. Results from patch-clamp recordings agreed well with cellular assays of p7 activity, namely, measurements of intracellular pH and hemadsorption assays, which confirmed the much reduced amantadine sensitivity of genotypes 2a and 3a. Thus, our results establish patch-clamp studies of recombinant viroporins as a valid analytical tool that can provide quantitative information about viroporin channel properties, complementing established techniques.

Analysis of the desialidation process of the haemagglutinin protein of influenza B virus: the host-dependent desialidation step.

It was reported previously that haemadsorption by the haemagglutinin (HA) protein of influenza B virus required that the protein must undergo desialidation. When MDCK and COS cells were infected with influenza B/Kanagawa/73 virus in the presence of a neuraminidase (NA) inhibitor, Zanamivir, haemadsorption on MDCK cells was inhibited but that on COS cells was not. The activity of the NA protein of the two types of infected cells was similar and both were inhibited by Zanamivir in a dose-dependent manner. A comparison of the desialidation of the HA protein was made on MDCK and COS cells in the presence of bacterial NA and both cells were found to have similar sensitivity. On the accumulation of the HA and NA proteins in the trans-Golgi network of MDCK cells by means of low-temperature treatment, desialidation of the HA protein in the presence of Zanamivir was detected by two-dimensional gel electrophoresis. Because this agent was reported to be unable to penetrate cells, these data suggest that, in MDCK cells, desialidation of the HA protein occurs on the cell surface but, in COS cells, the HA and NA proteins might accumulate in the trans-Golgi network, thus allowing NA desialidation before their migration to the cell surface.

Antiproliferative action of retinoic acid in cultured human brain tumour cells Gl-As-14(S).

We have employed human Gl-As-14(S) brain tumour cell line to study antiproliferative action of retinoic acid (RA). RA (20 microM) caused a time-dependent, dose-dependent, cell seeding density-dependent reduction in cell proliferation in liquid medium and inhibited growth in agar. Growth inhibitory effects of RA were also affected by the concentration of fetal bovine serum (PBS) in the medium. All these effects could be reversed within 48 h after removal of RA from the growth medium. RA-treated cells also displayed reduced concanavalin A (Con A) binding ability by microhemadsorption technique. The results demonstrated that RA can suppress in this tumour cell line the expression of some properties frequently associated with transformed cells.

Alterations in cell surface properties induced by modified purines.

Primary Chinese hamster embryo cultures exposed chronically to 1-methylguanine or 7-methylguanine, modified purines derived from nucleic acid turnover, exhibit a number of properties characteristic of transformed cell lines. One of the earliest effects observed following exposure of cells to the methylated purines is an alteration in cell surface properties as measured by the interaction of the cells with the lectin concanavalin A. Within sixteen hours following inclusion of the compounds in the culture medium, the cells exhibit an increase in concanavalin A mediated hemadsorption. The increase in hemadsorption is accompanied by an alteration in distribution of receptors within the cell population as measured by flow microfluorometry using fluorescin conjugated concanavalin A, and by a decrease in the total number of receptors as measured by binding of radiolabelled concanavalin A. Possible mechanisms for these alterations and their significance for growth control are discussed.

1-Methylguanine and 7-methylguanine increase cell agglutinability.

1-Methylguanine and 7-methylguanine, both metabolic products of tRNA degradation, are known to induce transformation of Chinese hamster fibroblasts in culture. The effects of these compounds on the cell membrane have been studied by the method of Concanavalin A-mediated hemadsorption. 1-Methylguanine or 7-methylguanine induced a 50% increase of Con A-mediated hemadsorption within 20 hours of exposure of the cells to the agent at a concentration of 10(-5) M. This alteration was reversed within 13 days when the cells were grown in the control medium. Prolonged treatment with 1-methylguanine or 7-methylguanine resulted in changes which were only slowly reversed during growth of the cells in the control medium. The effect of the methylated purines on the cell membrane could be completely inhibited by simultaneous addition of dibutyryl-cAMP at a concentration of 10(-5) M. The possible mechanism of cell membrane alteration by methylated purines and its relevance to transformation in vitro are discussed.

Association of morphological differentiation with enhanced surface antigen expression and susceptibility to natural killer cell lysis in theophylline-treated human melanoma cells.

Human and animal melanomas undergo maturation spontaneously in vivo and in vitro, and as a result of experimental manipulation in vitro. To gain a better understanding of this phenomenon, we studied the effects of theophylline on a cultured human malignant melanoma cell line (CaCL 73-36). We observed a dose-dependent inhibition of cell growth with a reduction in plating efficiency of 16, 64, and 99% at concentrations of theophylline of 0.1, 1.0, and 2.0 mM, respectively. Theophylline-treated cells showed morphological changes consistent with a more differentiated state such as increased dendrite formation and contact inhibition. Expression of surface HLA-A,B,C antigens and beta-2-microglobulin was enhanced 15- and fivefold, respectively. Finally, cells treated with theophylline for 96 h showed a five- to eightfold increase in sensitivity to lysis by natural killer cells. These findings have obvious bearing on the potential use of theophylline in the treatment of malignant melanoma.

Tissue-specificity of reactivity to concanavalin-A of human cells in culture.

Concanavalin-A (Con-A) reactivity was studied to identify the tissue-specificity of cells established from various normal human tissues. Cells were treated with Con-A-labelled human red blood cells (C-RBC). C-RBC was not absorbed on the cells derived from the bone marrow, skin and liver. Lung-derived fibroblast cells showed weak C-RBC adsorption. Kidney-derived cells showed epithelial morphology and easily adsorbed C-RBC. These suggest that a large number of Con-A receptors exists on the membrane surface of kidney cells.

Effects of glutaraldehyde and other drugs on concanavalin A-mediated red blood cell adsorption to nonsenescent, senescent and transformed human fibroblasts.

Concanavalin A (Con A)-mediated red blood cell (RBC) adsorption with the RBC coating method (in which Con A-coated RBC's are adsorbed to fibroblasts) was greatly increased by glutaraldehyde fixation of RBCs before Con A-coating and decreased by the fixation of fibroblasts. On the other hand, RBC adsorption with the fibroblast coating method (in which RBCs are adsorbed to Con A-coated fibroblasts) was decreased by the fixation of RBCs and increased by the fixation of fibroblasts before Con A coating. The fixation of RBCs or fibroblasts after Con A coating did not have these effects. In addition, the fixation of both RBCs and fibroblasts nearly completely abolished RBC adsorption with either method. However, the amount of [3H] Con A binding was not affected by the fixation. RBC adsorption with the fibroblast coating method was also affected by cytochalasin B, colchicine, NaN3 and dibucane treatments of fibroblasts. These drug treatments of fibroblasts, however, did not affect RBC adsorption with the RBC coating method, except cytochalasin B. In addition, the effects of drug treatments of fibroblasts examined occurred nearly to the same extent for nonsenescent, senescent, and transformed cells. Our results suggest that secondary processes after Con A binding, receptor mobility and receptor association with cytoskeletals, play important roles in RBC adsorption, but that the roles do not change with aging or transformation.

Increased agglutinability and haemadsorption of Marek's disease HPRS line 1 lymphoblastoic cells after concanavalin A treatment.

The HPRS line 1 lymphoblastoid cells derived from an ovarian lymphoma induced by Marek's disease virus in chickens displayed increased agglutinability and haemadsorption when incubated with Con A. The increase was proportional to the concentration of Con A used. The same Con A-untreated cells and normal chicken lymphocytes incubated with Con A exhibited no increase in agglutinability and haemadsorption, however.

Properties of influenza C virus grown in cell culture.

Influenza C virus was propagated successfully in primary chicken embryo lung (CEL) and fibroblast cells and in Madin-Darby canine kidney (MDCK) cells. In other cell lines, either no virus or only noninfectious hemagglutinin (HA) was produced. In productively infected cells (CEL), HA and infectious virus appeared by 24 h and reached a maximum by 36 to 48 h, cell-associated virus remaining at a constant low level. Infected Vero cells produced noninfective HA by 24 h which also remained predominantly cell associated until 60 to 72 h, when the cells disintegrated. Viral antigen was demonstrable on membranes of both CEL- and Vero-infected cells at 24 h; Vero cells yielded membrane vesicles containing HA, but none of the spherical or filamentous viral particles synthesized in CEL cells. Influenza C virus produced in cell culture or in eggs differed in several important respects from A and B viruses and from Newcastle diseases virus. All influenza C preparations, regardless of infectivity or source, lacked detectable neuraminidase activity, yet retained the ability specifically to inactivate receptors only for influenza C. Influenza C HA was not inhibited by soluble glycoproteins highly active against HA of A virus. A rat serum glycoprotein uniquely inhibited influenza C by binding to the surface components of virious.

Infectivity of DNA recovered from cells persistently infected with SV5 paramyxovirus.

Infectivity of DNA isolated from L cells chronically infected with SV5 paramyxovirus was demonstrated by inoculation of continuous RH and HEp-2 cells. Infectivity of the DNA was completely abolished by treatment with deoxyribonuclease or by alkaline hydrolysis but did not change after treatment with ribonuclease and specific anti SV5 serum. The virus obtained as a result of transfection caused haemadsorption in susceptible cells and was neutralized by specific antiserum like the prototype SV5 strain.

Some characteristics of salt-dependent haemagglutinating measles viruses.

Several strains of measles virus which did not agglutinate monkey erythrocytes in phosphate-buffered saline did so in buffer containing 0-8 M-ammonium sulphate. Haemadsorption to cells infected with these viruses was also salt-dependent. In a series of tests salt-dependent agglutinin was shown to be a stable structural component of the infectious virion. The relevance of these findings is discussed in the light of previous reports that many measles virus preparations do not agglutinate erythrocytes.

Markers to distinguish normal and neoplastic mammary epithelial cells in vitro: comparison of saturation density, morphology and concanavalin A reactivity.

Normal and premalignant mouse mammary epithelial cells can be prepared in high yields by collagenase dissociation of minced glands followed by a brief, differential centrifugation to remove contaminating fibroblasts and fat cells. The major difficulties in preparing pure cultures in quantity are 1) incomplete dissociation of gland material, and 2) cell death during enzymatic digestion. These problems are eliminated by careful selection of collagenases for dissociation. Normal and premalignant mammary epithelial cells are morphologically indistinguishable from malignant mouse mammary epithelial cells in primary monolayer cultures. In addition, the growth rates and saturation densities achieved by normal mammary epithelial cells are indistinguishable from those of malignant mammary epithelial cells in primary culture. In both cases, a monolayer of cells is preserved with no evidence of focal overgrowth. Malignant adenocarcinoma mammary cells can however be distinguished from normal mammary epithelial cells by virtue of differences in their surface interactions with concanavalin A. A hemadsorption assay using Con-A-coated erythrocytes was the most sensitive indicator for these differences. In hemadsorption assays malignant mammary epithelial cells were half-maximally reactive with 2.5 mug/ml concanavalin A, while normal cells were completely unreactive even at concanavalin A concentrations five-times higher. Premalignant mammary epithelial cells were as reactive as malignant mammary epithelial cells in the hemadsorption assays. Hemadsorption of malignant cells was observed in primary and secondary cultures of epithelium as well as in cell lines. Malignant cells forming mammary adenocarcinomas were as highly reactive as malignant cells forming scirrhous carcinomas. Malignant cells not releasing mammary tumor virus (MuMTV) were as reactive as cells releasing that virus. Adsorption of concanavalin-A-coated erythrocytes to normal mammary epithelial cells could be induced by brief treatment of cell monolayers with hyaluronidase. Exposure of active sites was not affected with either trypsin or collagenase. Our results show that while the growth of malignant cells does not serve to distinguish them from normal cells in monolayer culture, surface changes do exist which can be identified by differences in concanavalin A reactivity. Since the earliest transformants identifiable in vivo (premalignant) have undergone conversion of the surface marker, concanavalin-A-mediated hemadsorption provides a sensitive measure for mammary epithelial cell transformants in vitro.