PB0542 - Investigating thrombin-loaded fibrin in whole blood clot microfluidic assay via fluorogenic peptide.

Background: During clotting under flow, thrombin rapidly generates fibrin while fibrin potently sequesters thrombin. Previous work has studied thrombin incorporated into fibrin and plasma gels formed under isotropic conditions with exogeneous thrombin. However, little is known about thrombin activity while bound, nor have there been direct measurements of clot-bound thrombin by its enzymatic activity.

Aims: We investigated the enzymatic activity and capabilities of clot-bound thrombin made in clots of multiple morphologies, shear rates, and coagulation inhibition conditions. We compared the diffusional limitations of similarly sized molecules to thrombin and probed bound thrombin’s ability to polymerize fibrinogen from its immobilized state.

Methods: The co-regulation of fibrin and thrombin was studied using microfluidic whole blood clotting on collagen/tissue factor, followed by buffer wash, and a start/stop cycling flow assay using the thrombin fluorogenic substrate, boc-val-pro-arg-AMC. Thrombin activity was estimated via a 2-D-reaction-diffusion simulation.

Results: After 3-min of clotting and 5-min buffer wash, non-elutable thrombin activity was detected during pulse cycles. Non-elutable thrombin was similarly detected in plasma or arterial whole blood clots. Reaction-diffusion simulations predicted 108 nM thrombin within the clot. Heparin addition to the start/stop assay had little effect on fibrin-bound thrombin, while addition of heparin-antithrombin required over 6-min to inhibit thrombin, indicating a substantial diffusion limitation. Perfusion of AF647-fibrinogen over washed FITC-fibrin clots resulted in an intense red layer around, but not within, the original FITC-fibrin. Similarly, introduction of plasma/AF647-fibrinogen generated substantial red fibrin masses that did not penetrate the original green clots, demonstrating that fibrin cannot be re-clotted with fibrinogen.

Conclusion(s): Thrombin within fibrin is non-elutable, easily accessed by peptides, slowly accessed by average-sized proteins (heparin/AT), and not accessible to fresh fibrinogen. Although we demonstrated that bound thrombin is unable to significantly propagate coagulation from its bound states, this would be relevant in clinical settings where there is a risk of embolization and re-thrombosis