Intrinsic coagulation pathway-mediated thrombin generation in mouse whole blood
Konrath, Sandra; Mailer, Reiner K; Beerens, Manu; Englert, Hanna; Frye, Maike; Kuta, Piotr; Preston, Roger J S; Maas, Coen; Butler, Lynn M; Roest, Mark; de Laat, Bas; Renné, Thomas
(2022) Frontiers in cardiovascular medicine, volume 9
(Article)
Abstract
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
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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.
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Keywords: CAT assay, coagulation, diagnostics, factor XII, mouse whole blood, plasma contact system, polyphosphate, thrombin generation, Cardiology and Cardiovascular Medicine
ISSN: 2297-055X
Publisher: Frontiers Media S. A.
Note: Funding Information: This research was supported by the Rudolf-Marx-Research-Grant of the Society of Thrombosis and Haemostasis Research e.V. (GTH) and the German Research Foundation (DFG, project number 470698011 to RM). TR acknowledges the German Research Foundation (grants A11/SFB 877, P6/KFO 306, and B8/SFB 841) for funding. Publisher Copyright: Copyright © 2022 Konrath, Mailer, Beerens, Englert, Frye, Kuta, Preston, Maas, Butler, Roest, de Laat and Renné.
(Peer reviewed)