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Zinc protoporphyrin (ZnPP) has been found to have anticancer activity both in vitro and in vivo. We have recently demonstrated that ZnPP diminishes β-catenin protein expression in cancer cells. The present study examined the cellular mechanisms that mediate ZnPP's suppression of β-catenin expression. We demonstrate that ZnPP induces a rapid degradation of the β-catenin protein in cancer cells, which is accompanied by a significant inhibition of proteasome activity, suggesting that proteasome degradation does not directly account for the suppression. The possibility that ZnPP induces β-catenin exportation was rejected by the observation that there was no detectable β-catenin protein in the conditioned medium after ZnPP treatment of cancer cells. Further experimentation demonstrated that ZnPP induces lysosome membrane permeabilization, which was reversed by pretreatment with a protein transportation inhibitor cocktail containing Brefeldin A (BFA) and Monensin. More significantly, pretreatment of cancer cells with BFA and Monensin attenuated the ZnPP-induced suppression of β-catenin expression in a concentration- and time-dependent manner, indicating that the lysosome protein degradation pathway is likely involved in the ZnPP-induced suppression of β-catenin expression. Whether there is cross-talk between the ubiquitin-proteasome system and the lysosome pathway that may account for ZnPP-induced β-catenin protein degradation is currently unknown. These findings provide a novel mechanism of ZnPP's anticancer action and reveal a potential new strategy for targeting the β-catenin Wnt signaling pathway for cancer therapy.

The introduction of oral direct anti-Xa anticoagulants apixaban and rivaroxaban has significantly impacted the treatment and prevention of thromboembolic disease. Clinical scenarios exist in which a quantitative assessment for degree of anticoagulation due to these agents would aid management. The purpose of this work was to evaluate the chromogenic antifactor Xa assay calibrated with heparin standards at our institution for assessment of intensity of anticoagulation with rivaroxaban or apixaban in addition to its current use for unfractionated heparin or low-molecular-weight heparin. We also aimed to propose expected steady state peak and trough antifactor Xa activities for these agents based upon dosing regimens approved for nonvalvular atrial fibrillation. Antifactor Xa activity correlated very strongly with apixaban and rivaroxaban concentration in both spiked samples and treated patient plasma samples (r 2 = .99, P < .001). This correlation was observed over a broad range (20-500 ng/mL) of drug concentrations, as sample dilution with pooled normal plasma significantly extended the range of quantitative assessment. Based on drug concentrations previously published in pharmacokinetic studies, the expected steady state peak and trough antifactor Xa activity ranges for apixaban are 1.80 to 2.20 IU/mL and 0.70 to 1.10 IU/mL, respectively. For rivaroxaban, these ranges are 3.80 to 6.20 IU/mL and 0.60 to 1.00 IU/mL, respectively. In conclusion, our findings demonstrate that heparin-calibrated antifactor Xa activity correlates strongly with apixaban and rivaroxaban concentration. The dilution of samples allowed for this correlation to be extended over the majority of on-therapy drug concentrations.

The parenterally administered direct thrombin inhibitors (DTIs) argatroban and bivalirudin are effective anticoagulants for acute heparin-induced thrombocytopenia (HIT) treatment. The activated partial thromboplastin time (aPTT) has classically been used as the monitoring test to assess degree of anticoagulation, however concerns exist with using aPTT to monitor DTI therapy. In this observational study plasma samples from DTI treated patients were analyzed by aPTT, dilute thrombin time (dTT) and ecarin chromogenic assay (ECA) to delineate results into concordant and discordant groups. Discordant samples were further analyzed via liquid chromatography with tandem mass spectrometry (LC MS/MS). In total 101 patients with 198 samples were evaluated. Discordance between tests were frequent (59% of DTI treated patients). Bivalirudin aPTT vs dTT discordance was observed in 45% (57/126) of samples. Amongst bivalirudin samples with test discordance dTT results were more likely to be concordant with LC MS/MS than the aPTT (77% vs 9%, p < 0.0001). Argatroban aPTT vs dTT discordance was observed in 43% (31/72) and aPTT vs ECA discordance was observed in 40% (29/72) of samples. Amongst argatroban samples with test discordance both the dTT and ECA tests were more likely to have concordant results with LC MS/MS than the aPTT (88% vs 9%, p < 0.0001 for both dTT and ECA tests). There were no differences between discordant and concordant patient groups in a composite outcome of bleeding/thrombosis rate (23% vs 27%, p = 0.689). Further investigation is warranted to elucidate the effect of suitable monitoring assays on patient outcomes in the setting of DTI therapy.

The activated partial thromboplastin time (aPTT) test has been used for years to monitor parenteral direct thrombin inhibitors (DTIs) and unfractionated heparin. Because the aPTT correlates poorly with unfractionated heparin levels, we hypothesized that the aPTT may not be the best test for monitoring parenteral DTIs. Using 235 excess plasma specimens from 82 adult patients receiving treatment with DTIs (argatroban, bivalirudin, or dabigatran), we compared the aPTT with the ecarin chromogenic assay (ECA), the dilute thrombin time (dTT) test, and the prothrombinase-induced clotting time (PiCT) test. The aPTT correlated poorly with each of the other tests in both bivalirudin- and argatroban-containing samples (r(2) = 0.04-0.23). The ECA and dTT exhibited the best correlations (r(2) = 0.66-0.93). Intermediate correlations were seen when the results of the PiCT were plotted against the dTT or ECA (r(2) = 0.46-0.58). Nineteen specimens obtained from six patients receiving dabigatran showed a good correlation between the dTT and the ECA (r(2) = 0.92). The aPTT does not correlate well with other tests that might be used to monitor parental DTI administration. Further studies are needed to evaluate the clinical usefulness of alternative tests and their correlation with clinical outcomes.

Abstract Objectives We implemented a policy of reflex serotonin-release assay (SRA) testing for all patients with a positive heparin-induced thrombocytopenia (HIT) immunoassay. Methods We identified all patients who had SRA testing sent as a consequence of a positive HIT immunoassay test. We reviewed charts of patients to calculate the 4Ts clinical score, determined the effect of testing on clinical management, and documented the change in utilization of direct thrombin inhibitors (DTIs). Results The likelihood of a positive SRA varied with the optical density (OD) of the immunoassay. The performance of the immunoglobulin G (IgG)–specific and polytypic enzyme-linked immunosorbent assay was not statistically different. Both OD and 4Ts score correlated with the likelihood of a positive SRA but demonstrated poor specificity. Discontinuation of DTIs in patients with negative SRAs resulted in decreased drug utilization. Conclusions The IgG-specific HIT immunoassay OD correlates with the likelihood of a positive SRA but does not achieve high specificity. The reflex testing algorithm allows for definitive classification of patients, and the cost of such a reflex testing program may be offset by decreased utilization of DTIs.

Anticoagulation therapy is administered to patients to prevent or stop thrombin generation in vivo. Although plasma tests of in vivo thrombin generation have been available for more than 2 decades, they are not routinely used in clinical trials or practice to monitor anticoagulation therapy. We observed a fall in one such marker, the D-dimer antigen, in patients receiving anticoagulation therapy. We therefore conducted a systematic review of the medical literature to document the change in serum biomarkers of thrombin generation following the initiation of anticoagulation therapy. Using a defined search strategy, we screened PubMed and Embase citations and identified full-length articles published in English. Eighteen articles containing serial changes in 1 of 3 markers of thrombin generation (D-dimer antigen, thrombin–antithrombin complexes, and prothrombin fragment 1+2 antigen levels) in the 14 days following the initiation of anticoagulation were identified. Even though the assays used varied considerably, each of the 3 markers of thrombin generation declined in the initial period of anticoagulation therapy, with changes evident as early as 1 day after beginning therapy. These observations provide a rationale for further exploration of these markers as measures of the adequacy of anticoagulation using classic as well as novel anticoagulants. Particular patient groups that would benefit from additional means of monitoring anticoagulation therapy are discussed.

Zinc protoporphyrin (ZnPP) has been found to have anticancer activity both in vitro and in vivo. We have recently demonstrated that ZnPP diminishes [beta]-catenin protein expression in cancer cells. The present study examined the cellular mechanisms that mediate ZnPP's suppression of [beta]-catenin expression. We demonstrate that ZnPP induces a rapid degradation of the [beta]-catenin protein in cancer cells, which is accompanied by a significant inhibition of proteasome activity, suggesting that proteasome degradation does not directly account for the suppression. The possibility that ZnPP induces [beta]-catenin exportation was rejected by the observation that there was no detectable [beta]-catenin protein in the conditioned medium after ZnPP treatment of cancer cells. Further experimentation demonstrated that ZnPP induces lysosome membrane permeabilization, which was reversed by pretreatment with a protein transportation inhibitor cocktail containing Brefeldin A (BFA) and Monensin. More significantly, pretreatment of cancer cells with BFA and Monensin attenuated the ZnPP-induced suppression of [beta]-catenin expression in a concentration- and time-dependent manner, indicating that the lysosome protein degradation pathway is likely involved in the ZnPP-induced suppression of [beta]-catenin expression. Whether there is cross-talk between the ubiquitin-proteasome system and the lysosome pathway that may account for ZnPP-induced [beta]-catenin protein degradation is currently unknown. These findings provide a novel mechanism of ZnPP's anticancer action and reveal a potential new strategy for targeting the [beta]-catenin Wnt signaling pathway for cancer therapy.

At a specialty conference, a postdoctoral fellow describing a new study being conducted at a neighboring institution showed a recruiting letter from the investigator, which outlined the study and noted that a $350 finder's fee would be paid to resident physicians who referred patients who subsequently enrolled in the study. This provoked a few laughs and comments from the audience but seemed an isolated instance to many of us. A few weeks later I learned that investigators at two other local hospitals had proposed offering cash finder's fees to resident physicians in the emergency room for referring patients with specified . . .