Fluorescent peptide for detecting factor XIIIa activity and fibrin in whole blood clots forming under flow

Yue Liu; Jennifer Crossen; Timothy J Stalker; Scott L Diamond

doi:10.1016/j.rpth.2023.102291

Fluorescent peptide for detecting factor XIIIa activity and fibrin in whole blood clots forming under flow

Res Pract Thromb Haemost. 2023 Dec 7;8(1):102291. doi: 10.1016/j.rpth.2023.102291. eCollection 2024 Jan.

Authors

Yue Liu¹, Jennifer Crossen¹, Timothy J Stalker², Scott L Diamond¹

Affiliations

¹ Department of Chemical and Biomolecular Engineering, Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
² Department of Medicine, The Cardeza Foundation for Hematologic Research, Thomas Jefferson University, Philadelphia, Pennsylvania, USA.

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.

Keywords: alpha-2-antiplasmin; blood platelets; fibrin; thrombosis.