Abstract Number: PB0538
Meeting: ISTH 2022 Congress
Background: It has been shown previously that thrombin becomes trapped within fibrin fibers during coagulation [Zhu, Chen, and Diamond, ATVB 2018], but not much is known about thrombin activity while bound, nor have there been direct measurements of clot-bound thrombin by its enzymatic activity.
Aims: In this study, we investigated the activity and stability of thrombin bound to whole blood and plasma clots formed under flow.
Methods: A fluorogenic thrombin sensitive substrate BOC-VPR-AMC (628 Da, Km = 21 μM, kcat = 109 s-1) was perfused over whole blood and plasma clots formed in microfluidic devices and washed with buffer to remove free thrombin. The flow was repeatedly stopped and started to allow for cleavage of the substrate by bound thrombin during stopped flow cycles and washed away during start flow cycles.
Results: It was observed that bound thrombin maintains its enzymatic activity over long periods of time under flow over repeated stop-start cycles. Three stop/start cycles were averaged to approximate the substrate conversion V0 = 0.78 ± 0.03 µM/s and apparent active clot bound thrombin (IIa) concentration within the reaction volume of 9.24 ± 0.35 nM. The signal from the substrate cleavage comes from a 60 x 250 x 250 µm volume in which the clot forms upon a collagen/tissue factor patterned surface. Clot bound thrombin is confined to a thin fibrin layer ~10-20 µM in height, therefore it is expected that the actual concentration of active clot bound thrombin is up to 10-fold higher. This was further expanded to investigate the effect of clot bound thrombin of heparinized plasma perfused with substrate over fully formed clots.
Conclusion(s): Overall, it is important to fully understand the extent that fibrin acts as an anticoagulant via thrombin retention. Thrombin appears to remain stably bound and active within fibrin fibers of clots for long periods of time.
To cite this abstract in AMA style:Crossen J, Diamond S. Enzymatic activity of bound thrombin within fibrin fibers [abstract]. https://abstracts.isth.org/abstract/enzymatic-activity-of-bound-thrombin-within-fibrin-fibers/. Accessed February 20, 2024.
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