Intrinsic coagulation pathway-mediated thrombin generation in mouse whole blood.

Calibrated Automated Thrombography (CAT) is a versatile and sensitive method for analyzing coagulation reactions culminating in thrombin generation (TG). Here, we present a CAT method for analyzing TG in murine whole blood by adapting the CAT assay used for measuring TG in human plasma. The diagnostically used artificial and physiologic factor XII (FXII) contact activators kaolin, ellagic acid and polyphosphate (polyP) stimulated TG in murine blood in a dose-dependent manner resulting in a gradual increase in endogenous thrombin potential and peak thrombin, with shortened lag times and times to peak. The activated FXII inhibitor rHA-Infestin-4 and direct oral anticoagulants (DOACs) interfered with TG triggered by kaolin, ellagic acid and polyP and TG was completely attenuated in blood of FXII- (F12 -/-) and FXI-deficient (F11 -/-) mice. Moreover, reconstitution of blood from F12 -/- mice with human FXII restored impaired contact-stimulated TG. HEK293 cell-purified polyP also initiated FXII-driven TG in mouse whole blood and addition of the selective inhibitor PPX_Δ12 ablated natural polyP-stimulated TG. In conclusion, the data provide a method for analysis of contact activation-mediated TG in murine whole blood. As the FXII-driven intrinsic pathway of coagulation has emerged as novel target for antithrombotic agents that are validated in mouse thrombosis and bleeding models, our novel assay could expedite therapeutic drug development.

Simultaneous voltammetric determination of guanine and adenine using MnO2 nanosheets and ionic liquid-functionalized graphene combined with a permeation-selective polydopamine membrane.

Guanine and adenine in blood samples can be detected by using an electrochemical sensor based on the use of manganese dioxide (MnO2) nanosheets and ionic liquid functionalized graphene (IL-GR) bound to a polydopamine (PDA) membrane. Both guanine and adenine undergo a redox reaction on the surface of the modified electrode. Cyclic voltammetry and differential pulse voltammetry were used to evaluate the electrochemical behavior of a glassy carbon electrode (GCE) modified with PDA/MnO2/IL-GR. The sensor allows for individual as well as simultaneous determination of guanine and adenine. The working voltage of differential pulse voltammetry at which data were acquired to establish the calibration plot: 0.6-1.2 V for guanine, 0.8-1.4 V for adenine, 0.4-1.4 V for mixture of guanine and adenine. A wide detection range (10-300 µM), low detection limits (guanine: 0.25 µM; adenine: 0.15 µM), selectivity and reproducibility are demonstrated. The modified GCE was successfully applied to the analysis of guanine and adenine in spiked fetal bovine serum and mouse whole blood samples. Graphical abstract An electrochemical sensor is presented for the determination of guanine (G) and adenine (A) based on MnO2 nanosheets, ionic liquid functionalized graphene (IL-graphene) and polydopamine membrane.