Comparison of the effect of the anti-Xa direct oral anticoagulants apixaban, edoxaban, and rivaroxaban on coagulation assays.

INTRODUCTION: The purpose of this study is to compare the effect of increasing concentrations of direct anti-Xa oral anticoagulants (DOAC) apixaban-, edoxaban-, and rivaroxaban-enriched plasma samples on various prothrombin time (PT), activated partial thromboplastin time (APTT), heparin calibrated anti-Xa methods, and other coagulation assays. METHODS: Apixaban, edoxaban, or rivaroxaban was dissolved in dimethylsulfoxide and added to pooled normal plasma to obtain concentrations ranging from 0 ng/mL to approximately 600 ng/mL. The samples were tested using Innovin(®) , Neoplastine(®) CI Plus, Recombiplastin 2G, and Thromborel(®) S for PT testing and Actin, Actin(®) FS, Actin(®) FSL, APTT-Automate, and SynthaSIL for APTT. Samples were also tested using four different anti-Xa methods calibrated for unfractionated heparin or low molecular weight heparin. Special coagulation assays included antithrombin activity, lupus anticoagulant assays, and others. For special coagulation assays, the concentration of DOAC that resulted in a >15% change from baseline value was determined. DOAC quantification was performed using liquid chromatography-tandem mass spectrometry. RESULTS: All PT and APTT reagents demonstrated a higher sensitivity for edoxaban and rivaroxaban than for apixaban. Anti-Xa methods were able to detect low concentrations of DOAC. DOACs effected special coagulation assays to differing degrees, with lupus anticoagulant testing using dilute viper venom, the most sensitive test to the presence of anti-Xa DOAC. CONCLUSION: No PT or APTT reagent system effectively detected apixaban. All anti-Xa methods demonstrated sensitivity to low concentrations of DOAC. Dilute viper venom methods are exquisitely sensitive to anti-Xa DOAC, suggesting potential use of this assay for screening or measuring these drugs.

Evaluating the use of commercial drug-specific calibrators for determining PT and APTT reagent sensitivity to dabigatran and rivaroxaban.

Suitable laboratory methodologies for quantifying the non-vitamin K oral anticoagulants (NOAC) include liquid chromatography-tandem mass spectrometry (LC-MS/MS) or drug-calibrated assays such as the dilute thrombin time for dabigatran or anti-Xa measurements for rivaroxaban. In situations when these tests are unavailable, it has been suggested that using commercial drug calibrators on APTT and PT assays would theoretically provide reagent sensitivity to these drugs. The purpose of this study was to determine whether commercial drug calibrators deliver similar reagent sensitivity information as samples from patients receiving dabigatran or rivaroxaban as part of their routine care. Two laboratory sites tested commercial calibrator material for dabigatran and rivaroxaban (Hyphen Biomedical) using PT and APTT reagents and data was compared to samples collected from patients taking NOACs that were quantified by LC-MS/MS. Correlation statistics and calculating the amount of drug required to double the clotting time of normal plasma were performed. All drug calibrator material correlated more strongly (R²> 0.95) for any reagent/drug combination than patient samples (R² ranged from 0.29-0.86). Dabigatran calibrator results and patient data were equivalent for SynthASil and PTT-A APTT reagents. The dabigatran and rivaroxaban calibrator material over-estimated drug sensitivity for all PT reagents when compared to sensitivity data calculated based on drug levels obtained by LC-MS/MS from patient samples. In conclusion, drug-specific calibrators overestimated reagent sensitivity which may underestimate in vivo drug concentration in a given patient. Further studies are required to assess whether this method of determining relative sensitivity of NOAC on routine coagulation assays should be recommended.

Molecular mechanism of translocation through nuclear pore complexes during nuclear protein import.

The trafficking of macromolecules between cytoplasm and nucleus through nuclear pore complexes is mediated by specific carrier molecules such as members of the importin-beta family. Nuclear pore proteins (nucleoporins) frequently contain sequence repeats based on FG cores and carriers appear to move their cargo through the pores by hopping between successive FG cores. A major question is why some macromolecules are transported while others are not. This selectivity may be generated by the ability to bind FG repeats, a local concentration of carrier-cargo complexes near the entrance to the pore channel, and steric hindrance produced by high concentrations of nucleoporins in the channel.

A novel intubation technique for tracheoesophageal fistula in adults.

PURPOSE: To describe a novel technique of tracheal intubation and ventilation in an adult patient with a large tracheoesophageal fistula at the level of the carina. CLINICAL FEATURES: A 59 yr old woman with squamous cell carcinoma of the esophagus developed a large (2 cm diameter) tracheoesophageal fistula after radiotherapy. The level of her fistula precluded traditional use of a double-lumen endobronchial tube. Intubation and ventilation were managed with two endobronchial tubes. The ability to ventilate or collapse each lung individually was preserved and anesthesia and surgery proceeded uneventfully. CONCLUSION: Double endobronchial intubation is described to manage anesthesia in an adult patient with a tracheoesophageal fistula at the level of the carina.

Structural requirements for biological activity of the ninth and tenth FIII domains of human fibronectin.

The ninth and tenth type III domains of fibronectin each contain specific cell binding sequences, RGD in FIII10 and PHSRN in FIII9, that act synergistically in mediating cell adhesion. We investigated the relationship between domain-domain orientation and synergistic adhesive activity of the FIII9 and FIII10 pair of domains. The interdomain interaction of the FIII9-10 pair was perturbed by introduction of short flexible linkers between the FIII9 and FIII10 domains. Incremental extensions of the interdomain link between FIII9 and FIII10 reduced the initial cell attachment, but had a much more pronounced effect on the downstream cell adhesion events of spreading and phosphorylation of focal adhesion kinase. The extent of disruption of cell adhesion depended upon the length of the interdomain linker. Nuclear magnetic resonance spectroscopy of the wild type and mutant FIII9-10 proteins demonstrated that the structure of the RGD-containing loop is unaffected by domain-domain interactions. We conclude that integrin-mediated cell adhesion to the central cell binding domain of fibronectin depends not only upon specific interaction sites, but also on the relative orientation of these sites. These data have implications for the molecular mechanisms by which integrin-ligand interactions are achieved.

Module-module interactions in the cell binding region of fibronectin: stability, flexibility and specificity.

The structure of mosaic proteins depends on the nature and strength of interactions between individual modules. Here we investigated the structural significance of module-module interactions in the RGD-dependent cell binding region of human fibronectin, comprising the ninth and tenth fibronectin type III. A combination of protein engineering, thermodynamics and nuclear magnetic resonance methods was employed to establish a relationship between intermodular protein-protein interactions and the structural properties of the module pair. A poly(glycine) peptide link connecting the C terminus of the ninth and the N terminus of the tenth module was introduced to probe the range of the interaction. NMR studies (Chemical shifts and 15N relaxation) together with equilibrium and kinetic unfolding experiments were carried out on five different single and double module constructs. The results show that non-specific protein-protein interactions provide the bulk of the thermodynamic stabilization and the motional constraint of the two modules. Specific interactions between the two modules are restricted to the wild-type module pair and decline very rapidly with the insertion of additional linker residues. This low level of specificity is nonetheless sufficient to fine-tune the precise module-module orientation and to provide the full biological activity of the wild-type pair. This suggests that individual modules in mosaic proteins can achieve a high degree of motional constraint and mutual stabilization without the requirement for intricate and specific interactions in the module-module interfaces.

Fibronectin splice variants are differentially incorporated into the extracellular matrix of tumorigenic and non-tumorigenic hybrids between normal fibroblasts and sarcoma cells.

Recent reports have described transformation- and tumour-specific expression of fibronectin isoforms generated by alternative splicing of the fibronectin pre-mRNA. We have investigated the expression and distribution of EDIIIA+ and EDIIIB+ fibronectin splice variants in tumorigenic and non-tumorigenic somatic cell hybrids made by fusing fibrosarcoma-derived cells (HT1080) and normal fibroblasts (GM00097). Alternative splicing of EDIIIA and EDIIIB was assessed quantitatively by S1 nuclease analyses. The levels of EDIIIA+ and EDIIIB+ fibronectin mRNAs were similar in the parental and hybrid cells. Domain-specific monoclonal antibodies were used in immunohistochemical studies to identify EDIIIA+ and EDIIIB+ fibronectins in fixed cells. GM00097 and the non-tumorigenic hybrid (clone G3) showed high levels of both EDIIIA+ and EDIIIB+ fibronectin staining. The tumorigenic hybrid (clone C1) showed reduced amounts of EDIIIA+ fibronectin, but no detectable EDIIIB+ fibronectin. No fibronectin was detected on the surface of HT1080 cells. Western blots of protein extracted from culture supernatants and extracellular matrices revealed that GM00097 and G3 cells incorporated most of the EDIIIA+ and EDIIIB+ fibronectin into the extracellular matrix whereas C1 cells released a large proportion of the EDIIIA+ fibronectin, and almost all of the EDIIIB+ fibronectin, into the supernatant. We conclude that there are differences in the presence of EDIIIA+ and EDIIIB+ FNs on the surface of tumorigenic and non-tumorigenic cells and that these differences are due to differential incorporation of FN variants into the ECM.

Patient-controlled lumbar epidural fentanyl compared with patient-controlled intravenous fentanyl for post-thoracotomy pain.

Thirty-four patients undergoing thoracotomy were entered into a randomized, double-blind, placebo-controlled study to compare the effects of patient-controlled, lumbar epidural (PCA-E) fentanyl with patient-controlled intravenous (PCA-i.v.) fentanyl with respect to drug requirements, analgesic efficacy and respiratory function. Prior to chest closure patients received fentanyl 2 micrograms.kg-1 by the epidural or i.v. route. In the recovery room further doses of epidural or i.v. fentanyl, 50 micrograms, were administered by the patients who controlled two PCA pumps. Background fentanyl infusion rates were increased by 10 micrograms.hr-1 each time the patient administered a drug bolus and were decreased by 10 micrograms.hr-1 whenever visual analogue scale (VAS) pain scores were less than 2 on a maximum 10 scale. Twenty-nine patients completed the study. Patients in the PCA-E group (n = 14) required less total fentanyl than those in the PCA-i.v. (n = 15) group (1857 +/- 693 micrograms vs 2573 +/- 890 micrograms respectively, P less than 0.05). Fentanyl infusion rates were lower in the PCA-E group at most measurement times. There were no differences between groups in respiratory rates, PaCO2, VAS pain scores or changes in pulmonary function as measured by FVC and FEV1. It is concluded that satisfactory patient-controlled analgesia can be achieved with both epidural and i.v. fentanyl after thoracotomy but that fentanyl requirements are less when given via the epidural route. This supports a direct spinal cord site of action for lumbar epidural fentanyl.

Dipyridamole-thallium myocardial scanning in the preoperative assessment of patients undergoing abdominal aortic aneurysmectomy.

Dipyridamole thallium scanning (DTS) is an imaging technique with good sensitivity for coronary artery disease (CAD). The purpose of this study was to compare the haemodynamic courses and the correlation between pulmonary capillary wedge pressure (PCWP) and central venous pressure (CVP) in patients with normal DTS (Group 1: n = 12) with those whose scans demonstrated CAD (Group 2: n = 11). Haemodynamic profiles were obtained prior to anaesthesia and at several times during surgery. The haemodynamic courses in both groups were similar with significant decreases in cardiac index, stroke index, and left ventricular stroke work index during aortic cross-clamping compared with values prior to anaesthesia. There were no significant changes in PCWP and CVP throughout the study. The correlations between PCWP and CVP were significant in both groups as were the correlations between the changes in PCWP and the changes in CVP observed at the time of cross-clamping. These correlations all had large standard errors of the estimate, however, making it impossible to predict the PCWP from the CVP with precision. It is concluded that, in a limited study population, an abnormal DTS did not identify patients in whom the PCWP and CVP correlated poorly during abdominal aortic aneurysmectomy.

Perioperative transcutaneous oxygen monitoring in thoracic anaesthesia.

Transcutaneous oxygen tension (PtcO2) was measured in 30 patients scheduled for elective pulmonary resection requiring one-lung ventilation during anaesthesia. Simultaneous PtcO2 and arterial oxygen tension (PaO2) measurements were taken preoperatively (preop), intraoperatively during two-lung endotracheal (ET) and one-lung endobronchial ventilation (EB), and postoperatively (postop). There was a significant correlation (r) between PtcO2 and PaO2 at all time periods: 0.97 (preop); 0.91 (ET); 0.83 (EB); 0.81 (postop). There were no significant differences among the transcutaneous oxygen indices (tcO2 index = PtcO2/PaO2) in the preop (0.69 +/- 0.09), ET (0.68 +/- 0.10) and postop (0.71 +/- 0.12) time period. The tcO2 index was significantly lower during one-lung anaesthesia (0.61 +/- 0.14). The PtcO2 was consistently lower than the corresponding PaO2 measurement, thus providing a continuous estimation of the "minimum" PaO2 level throughout anaesthesia and recovery. In four patients a marked drop in PtcO2 occurred just after the initiation of one-lung ventilation. In three, this was associated with arterial hypoxaemia and in one, haemodynamic compromise. In all four cases the PtcO2 was the first monitored parameter to change. As there is a substantial risk of developing hypoxaemia during thoracic anaesthesia, PtcO2 monitoring provides valuable early warning of impending hypoxaemia or haemodynamic compromise, thereby facilitating early therapeutic intervention.

Modification by preoperative beta-blockade of the renin response to infrarenal aortic cross-clamping.

The activity of the renin-angiotensin system was measured before, during, and after infrarenal aortic cross-clamping in 13 patients. Five of the patients studied were taking propranolol preoperatively and formed a subgroup. Intraoperative blood loss, volume of crystalloid and colloid infused, haemodynamic parameters and urine output were similar for the two groups. In eight patients who were not taking propranolol mean plasma renin activity was 2.24 ng . ml-1 . hr-1 prior to induction, 3.78 ng . ml-1 . hr-1 during surgery prior to cross-clamping and 4.42 ng X ml-1 X hr-1 15 minutes after the aorta was cross-clamped (increases not statistically significant). Mean plasma renin activity measured ten minutes prior to release of the cross-clamp (5.02 ng X ml-1 . hr-1), 15 minutes after clamp release (5.47 ng X ml-1 X hr-1), and 30 minutes after reaching the recovery room (5.84 ng X ml-1 X hr-1) were significantly greater than preinduction levels. Four patients developed postoperative hypertension (mean blood pressure greater than 120 mmHg); there was not a correlation between the elevated plasma renin activity observed postoperatively and the occurrence of postoperative hypertension. The five patients taking propranolol had a markedly attenuated renin activity response during and after surgery; the mean plasma renin activity was less than 1.5 ng X ml-1 X hr-1 at all sampling times. Two of these five patients did develop postoperative hypertension. It is concluded that surgery involving infrarenal aortic cross-clamping is associated with increased plasma renin activity with peak levels occurring postoperatively.(ABSTRACT TRUNCATED AT 250 WORDS)

Prediction of the need for postoperative mechanical ventilation in myasthenia gravis: thymectomy compared to other surgical procedures.

In a recent report Leventhal, Orkin, and Hirsh described a scoring system felt to be of value in predicting the need for postoperative mechanical ventilation in patients with myasthenia gravis undergoing thymectomy. Leventhal, et al. identified four risk factors felt to have predictive value, namely: (1) duration of myasthenia gravis greater than or equal to 6 years, (2) chronic respiratory disease, (3) dose of pyridostigmine greater than or equal to 750 mg per day, and (4) vital capacity less than or equal to 2.9 litres. Forty-six patients with myasthenia gravis who received 68 general anaesthetics were studied retrospectively. They represented the past 10 years' anaesthetic experience with myasthenia gravis at the Vancouver General Hospital. The patients were divided into two groups: (1) those who underwent thymectomy, and (2) those who underwent procedures other than thymectomy. Using the risk factors of Leventhal, et al., a predictive score was assessed for each patient; the time of postoperative tracheal extubation was also noted for each patient. From this study it was concluded that the scoring system proposed by Leventhal, et al. may have been of some value in predicting whether or not a particular patient undergoing thymectomy was likely to need ventilation postoperatively. In 41 myasthenics who had procedures other than thymectomy, however, this scoring system was found to be of no value.