Elevated TAT in COVID-19 Patients with Normal D-Dimer as a Predictor of Severe Respiratory Failure: A Retrospective Analysis of 797 Patients

Although previous studies have revealed that elevated D-dimer in the early stage of coronavirus 2019 (COVID-19) indicates pulmonary intravascular coagulation, the state of coagulation/fibrinolysis disorder with normal D-dimer is unknown. The study aimed to investigate how coagulation/fibrinolysis markers affect severe respiratory failure in the early stage of COVID-19. Among 1043 patients with COVID-19, 797 patients were included in our single-center retrospective study. These 797 patients were divided into two groups, the normal D-dimer and elevated D-dimer groups and analyzed for each group. A logistic regression model was fitted for age, sex, body mass index (BMI) ≥ 30 kg/m2, fibrinogen ≥ 617 mg/dL, thrombin-antithrombin complex (TAT) ≥ 4.0 ng/mL, and plasmin-alpha2-plasmin inhibitor-complex (PIC) > 0.8 µg/mL. A multivariate analysis of the normal D-dimer group demonstrated that being male and TAT ≥ 4.0 ng/mL significantly affected severe respiratory failure. In a multivariate analysis of the elevated D-dimer group, BMI ≥ 30 kg/m2 and fibrinogen ≥ 617 mg/dL significantly affected severe respiratory failure. The elevated PIC did not affect severe respiratory failure in any group. Our study demonstrated that hypercoagulation due to SARS-CoV-2 infection may occur even during a normal D-dimer level, causing severe respiratory failure in COVID-19.

Coagulation and Fibrinolysis Biomarkers as Potential Indicators for the Diagnosis and Classification of Ovarian Hyperstimulation Syndrome

Background: Accurate diagnosis and classification of ovarian hyperstimulation syndrome (OHSS) is important for its management. We employed a new high-sensitivity chemiluminescence immunoassay to detect the thrombin-antithrombin complex (TAT), plasmin alpha2-plasmin inhibitor complex (PIC), soluble thrombomodulin (sTM), and tissue plasminogen activator-inhibitor complex (TPAI-C), and evaluated their diagnostic and classification performance for OHSS.Methods: A total of 106 women were enrolled, including 51 patients with OHSS (25 mild or moderate OHSS, 26 severe OHSS), and 55 without OHSS (control group). TAT, PIC, sTM, and TPAI-C levels were measured using the Sysmex HISCL5000 automated analyzer.Results: Compared to the control group, TAT, PIC, and TPAI-C levels were significantly higher (P < 0.001, P < 0.001, P < 0.001, respectively), whereas the sTM level was significantly lower (P < 0.001) in the patients with OHSS. The receiver operating characteristic was used to evaluate the diagnostic efficiency. For the diagnosis of OHSS, the area under the curves (AUCs) for TAT, PIC, sTM, and TPAI-C were 0.991, 0.973, 0.809, and 0.722, respectively. In particular, the sensitivity, specificity, positive predictive value, and negative predictive value for TAT and PIC were all above 90%. For the differential diagnosis of mild–moderate and severe OHSS, the AUCs for TAT, PIC, and TPAI-C were 0.736, 0.735, and 0.818, respectively. The cutoff values of TAT, PIC, and TPAI-C for the differential diagnosis of mild–moderate and severe OHSS were 11.5 ng/mL, 2.4 μg/mL, and 5.8 ng/mL, respectively. Based on these cutoff values, eight cases of mild–moderate OHSS exceeded the cutoff values, two of which developed to severe OHSS in the following days. However, of the remaining 17 cases of mild–moderate OHSS patients with negative biomarkers, none subsequently developed severe OHSS.Conclusions: TAT, PIC, sTM, and TPAI-C can be used as sensitive biomarkers in the diagnosis of OHSS. Meanwhile, TAT, PIC, and TPAI-C also displayed remarkable potential in the classification of OHSS. In addition, the levels of TAT, PIC, and TPAI-C above the cutoff values in patients with mild–moderate OHSS might predict a high risk of developing severe OHSS.

Tailoring the effect of antithrombin-targeting therapy in haemophilia A using in silico thrombin generation

Factor (F) VIII deficiency causes bleeding in haemophilia A patients because of the reduced formation of procoagulant enzyme thrombin, which is needed to make the blood clot. We measured the dynamics of coagulation in haemophilia A patients by measuring thrombin generation (TG). Additionally, we quantified the procoagulant process of prothrombin conversion and anticoagulant process of thrombin inhibitor complex formation. In haemophilia A, prothrombin conversion is severely reduced, causing TG to be low. Nevertheless, the thrombin inactivation capacity of these patients is comparable to that in healthy subjects, leading to a severe imbalance between procoagulant and anticoagulant processes and a subsequent increased bleeding risk. A novel therapy in haemophilia A is the targeting of anticoagulant pathway, e.g. thrombin inhibitor antithrombin (AT), to restore the haemostatic balance. We simulated the effect of AT reduction on TG in silico. Lowering AT levels restored TG dose-dependently and an AT reduction of 90–95% led to almost normal TG in most patients . However, the variation in response to AT reduction was large between patients, indicating that this approach should be tailored to each individual patients. Ideally, TG and thrombin dynamics simulation could in the future contribute to the management of patients undergoing AT targeting therapy.

Functionalized Homologues and Positional Isomers of Rabbit 15-Lipoxygenase RS75091 Inhibitor

Background: RS75091 is a cinnamic acid derivative that has been used for the crystallization of the rabbit ALOX15-inhibitor complex. The atomic coordinates of the resolved ALOX15-inhibitor complex were later used to define the binding sites of other mammalian lipoxygenase orthologs, for which no direct structural data with ligand has been reported so far. Introduction: The putative binding pocket of the human ALOX5 was reconstructed on the basis of its structural alignment with rabbit ALOX15-RS75091 inhibitor. However, considering the possible conformational changes the enzyme may undergo in solution, it remains unclear whether the existing models adequately mirror the architecture of the ALOX5 active site. Methods: In this study, we prepared a series of RS75091 derivatives using a Sonogashira coupling reaction of regioisomeric bromocinnamates with protected acetylenic alcohols and tested their inhibitory properties on rabbit ALOX15 Results: A bulky pentafluorophenyl moiety linked to either ortho- or metha-ethynylcinnamates via aliphatic spacer does not significantly impair the inhibitory properties of RS75091. Conclusion: Hydroxylated 2- and 3-alkynylcinnamates may be suitable candidates for incorporation of an aromatic linker group like tetrafluorophenylazides for photoaffinity labeling assays.

Design, Synthesis and Binding Affinity Evaluation of Cytochrome P450 1B1 Targeted Chelators

Background: Cytochrome P450 1B1 (CYP1B1) is specifically expressed in a variety of tumors which makes it a promise imaging target of tumor. Objective: We aimed to design and synthesize CYP1B1 targeted chelators for the potential application in positron emission tomography (PET) imaging of tumor. Methods: 1,4,7-triazacyclononane-1,4-diiacetic acid (NODA) was connected to the CYP1B1 selective inhibitor we developed before through polyethylene glycol (PEG) linkers with different lengths. The inhibitory activities of chelators 6a-c against CYP1 family were evaluated by 7-ethoxyresorufin o-deethylation (EROD) assay. The manual docking between the chelators and the CYP1B1 are conducted subsequently. To determine the binding affinities of 6a-c to CYP1B1 in cells, we further performed a competition study at the cell level. Results: Among three chelators, 6a with the shortest linker showed the best inhibitory activity against CYP1B1. In the following molecular simulation study, protein-inhibitor complex of 6a showed the nearest F-heme distance which is consistent with the results of enzymatic assay. Finally, the cell based competitive assay proved the binding affinity of 6a-c to CYP1B1 enzyme. Conclusion: We designed and synthesized a series of chelators which can bind to CYP1B1 enzyme in cancer cells.To our knowledge, this work is the first attempt to construct CYP1B1 targeted chelators for radiolabeling and we hope it will prompt the application of CYP1B1 imaging in tumor detection.

A Microscopic Description of SARS-CoV-2 Main Protease Inhibition with Michael Acceptors. Strategies for Improving Inhibitors Design

The irreversible inhibition of the main protease of SARS-CoV-2 by a Michael acceptor compound known as N3 has been investigated using multiscale simulation methods. The noncovalent enzyme-inhibitor complex was simulated using classical Molecular Dynamics techniques and the pose of the inhibitor in the active site was compared to that of the natural substrate, a peptide containing the Gln-Ser scissile bond. The formation of the covalent enzyme-inhibitor complex was then simulated using hybrid QM/MM free energy methods. After binding, the reaction mechanism was found to be composed of two steps: i) the activation of the catalytic dyad (Cys145 and His41) to form an ion pair and ii) a Michael addition where the attack of the Sg atom of Cys145 to the Cb atom of the inhibitor precedes the water-mediated proton transfer from His41 to the Ca atom. The microscopic description of protease inhibition by N3 obtained from our simulations is strongly supported by the excellent agreement between the estimated activation free energy and the value derived from kinetic experiments. Comparison with the acylation reaction of a peptide substrate suggest that that N3-based inhibitors could be improving adding chemical modifications that could facilitate the formation of the catalytic dyad ion pair.