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.

Image

Figure 1: Stop-start flow experiment using BOC-VPR-AMC to determine the activity of bound thrombin. -A- Experimental procedure. Whole blood HCTI clots were formed in microfluidic devices under venous flow for 3 minutes to allow for platelet deposition and fibrin formation. The clots were washed with buffer for 5 minutes to remove any free thrombin. 70 µM BOC-VPR-AMC was then flowed over the clots and subjected to repeated stop-start flow cycles in order to allow for substrate cleavage of thrombin during stopped cycles and washed away with non-cleaved substrate during stop cycles. -B- Image of BOC-VPR-AMC at t=150 s, 120 s into a stopped flow cycle. -C- Data of BOC-VPR-AMC cleavage over repeated stop-start flow cycles. Red arrow indicates where flow was stopped at 8.5, 12, and 15.5 minutes into the experiment. Green arrow indicates where flow was started to wash away cleaved substrate at 11.5 and 15 minutes. N = 1 donor, n = 8 clots. -D- Conversion of BOC-VPR-AMC with three stop/start cycles averaged. Data was used to calculate V0 of substrate across three cycles and the apparent concentration of active clot bound thrombin -IIa-.

« Back to ISTH 2022 Congress

ISTH Congress Abstracts - https://abstracts.isth.org/abstract/enzymatic-activity-of-bound-thrombin-within-fibrin-fibers/