Fluorescent Peptide for Detecting Factor XIIIa Activity and Fibrin in Whole Blood Clots Forming Under Flow
Document Type
Article
Publication Date
12-7-2023
Abstract
Background
During clotting, thrombin generates fibrin monomers and activates plasma-derived transglutaminase factor (F) XIIIa; collagen and thrombin-activated platelets offer thrombin-independent cellular FXIIIa (cFXIIIa) for clotting. Detecting fibrin on collagen and tissue factor surfaces in whole blood clotting typically uses complex reagents like fluorescent fibrinogen or antifibrin antibody.
Objectives
We want to test whether the peptide using the α2- antiplasmin crosslinking mechanism by FXIIIa is a useful tool in both monitoring FXIIIa activity, and visualize and monitor fibrin formation, deposition, and extent of crosslinking within fibrin structures in whole blood clots formed under flow.
Methods
We tested a fluorescent peptide derived from α2-antiplasmin sequence (Ac-GNQEQVSPLTLLKWC-fluorescein) to monitor the location of transglutaminase activity and fibrin during whole blood clotting under microfluidic flow (wall shear rate, 100 s−1).
Results
The peptide rapidly colocated with accumulating fibrin due to transglutaminase activity, confirmed by Phe-Pro-Arg-chloromethylketone inhibiting fibrin and peptide labeling. The FXIIIa inhibitor T101 had no effect on fibrin generation but ablated the labeling of fibrin by the peptide. Similarly, Gly-Pro-Arg-Pro abated fibrin formation and thus strongly attenuated the peptide signal. At arterial wall shear rate (1000 s−1), less fibrin was formed, and consequently, less peptide labeling of fibrin was detected compared with venous conditions. The addition of tissue plasminogen activator caused a reduction of both fibrin and peptide signals. Also, the peptide strongly colocalized with fibrin (but not platelets) in clots from laser-injured mouse cremaster arterioles. For clotting under flow, FXIIIa activity was most likely plasma-derived since a RhoA inhibitor did not block α2-antiplasmin fragment cross-linking to fibrin.
Conclusion
Under flow, the majority of FXIIIa-dependent fibrin labeling with peptide during clotting was distal of thrombin activity. The synthetic peptide provided a strong and sustained labeling of fibrin as it formed under flow.
Recommended Citation
Liu, Yue; Crossen, Jennifer; Stalker, Timothy J.; and Diamond, Scott L., "Fluorescent Peptide for Detecting Factor XIIIa Activity and Fibrin in Whole Blood Clots Forming Under Flow" (2023). Cardeza Foundation for Hematologic Research. Paper 77.
https://jdc.jefferson.edu/cardeza_foundation/77
Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 License.
Supplementary material
Language
English
Comments
This article is the author's final published version in Research and Practice in Thrombosis and Haemostasis, Volume 8, Issue 1, January 2024, Article number 102291.
The published version is available at https://doi.org/10.1016/j.rpth.2023.102291. Copyright © 2023 The Author(s). Published by Elsevier Inc. on behalf of International Society on Thrombosis and Haemostasis.