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A novel method to quantify fibrin–fibrin and fibrin–α2-antiplasmin cross-links in thrombi formed from human trauma patient plasma.

Morrow, Gael B.; Flannery, Sarah; Charles, Philip D.; Heilig, Raphael; Feller, Timea; McQuilten, Zoe; Wake, Elizabeth; Ariens, Robert A.S.; Winearls, James; Mutch, Nicola J.; Fischer, Roman; Laffan, Mike A.; Curry, Nicola


Sarah Flannery

Philip D. Charles

Raphael Heilig

Timea Feller

Zoe McQuilten

Elizabeth Wake

Robert A.S. Ariens

James Winearls

Nicola J. Mutch

Roman Fischer

Mike A. Laffan

Nicola Curry


The widespread use of the anti-fibrinolytic agent, tranexamic acid (TXA), interferes with the quantification of fibrinolysis by dynamic laboratory assays such as clot lysis, making it difficult to measure fibrinolysis in many trauma patients. At the final stage of coagulation, Factor XIIIa (FXIIIa) catalyses the formation of fibrin-fibrin and fibrin-α2-antiplasmin (α2AP) cross-links which increases clot mechanical strength and resistance to fibrinolysis. Here, we develop a method to quantify fibrin-fibrin and fibrin-α2AP cross-links that avoids the challenges posed by TXA in determining fibrinolytic resistance in conventional assays. Fibrinogen alpha chain (FGA-FGA), fibrinogen gamma chain (FGG-FGG) and FGA-α2AP cross-links were quantified using liquid-chromatography-mass spectrometry (LC-MS) and parallel reaction monitoring (PRM) in paired plasma samples from trauma patients pre- and post-fibrinogen replacement. Differences in the abundance of cross-links in trauma patients receiving cryoprecipitate (cryo) or fibrinogen concentrate (Fg-C) were analysed. The study found that the abundance of cross-links was significantly increased in trauma patients post-cryo, but not Fg-C, transfusion (p < 0.0001). The abundance of cross-links was positively correlated with the toughness of individual fibrin fibres, the peak thrombin concentration and FXIII antigen (p < 0.05). We have developed a novel method that allows us to quantify fibrin cross-links in trauma patients who have received TXA, providing an indirect measure of fibrinolytic resistance. Using this novel approach we have avoided the effect of TXA and shown that cryo increases fibrin-fibrin and fibrin-α2AP cross-linking when compared to Fg-C, highlighting the importance of FXIII in clot formation and stability in trauma patients.


MORROW, G.B., FLANNERY, S., CHARLES, P.D., HEILIG, R., FELLER, T., MCQUILTEN, Z., WAKE, E., ARIENS, R.A.S., WINEARLS, J., MUTCH, N.J., FISCHER, R., LAFFAN, M.A. and CURRY, N. 2024. A novel method to quantify fibrin–fibrin and fibrin–α2-antiplasmin cross-links in thrombi formed from human trauma patient plasma. Journal of thrombosis and haemostasis [online], 22(6), pages 1758-1771. Available from:

Journal Article Type Article
Acceptance Date Mar 4, 2024
Online Publication Date Mar 9, 2024
Publication Date Jun 30, 2024
Deposit Date Mar 11, 2024
Publicly Available Date Mar 11, 2024
Journal Journal of thrombosis and haemostasis
Print ISSN 1538-7933
Electronic ISSN 1538-7836
Publisher Wiley
Peer Reviewed Peer Reviewed
Volume 22
Issue 6
Pages 1758-1771
Keywords Fibrinogen; Fibrinogen cross-linking; Fibrinolysis; Anti-fibrinolytic agents; Trauma coagulopathy; Tranexamic acid
Public URL
Additional Information This article has been published with separate supporting information. This supporting information has been incorporated into a single file on this repository and can be found at the end of the file associated with this output.


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