Dynamics of Thrombin Generation and Flux from Clots during Whole Human Blood Flow over Collagen/Tissue Factor Surfaces.

ABSTRACT

Coagulation kinetics are well established for purified blood proteases or human plasma clotting isotropically. However, less is known about thrombin generation kinetics and transport within blood clots formed under hemodynamic flow. Using microfluidic perfusion (wall shear rate, 200 s-1) of corn trypsin inhibitor-treated whole blood over a 250-µm long patch of type I fibrillar collagen/lipidated tissue factor (TF; ∼1 TF molecule/µm2), we measured thrombin released from clots using thrombin-antithrombin immunoassay. The majority (>85%) of generated thrombin was captured by intrathrombus fibrin as thrombin-antithrombin was largely undetectable in the effluent unless Gly-Pro-Arg-Pro (GPRP) was added to block fibrin polymerization. With GPRP present, the flux of thrombin increased to ∼0.5 × 10-12 nmol/µm2-s over the first 500 s of perfusion and then further increased by ∼2-3-fold over the next 300 s. The increased thrombin flux after 500 s was blocked by anti-FXIa antibody (O1A6), consistent with thrombin-feedback activation of FXI. Over the first 500 s, ∼92,000 molecules of thrombin were generated per surface TF molecule for the 250-µm-long coating. A single layer of platelets (obtained with αIIbß3 antagonism preventing continued platelet deposition) was largely sufficient for thrombin production. Also, the overall thrombin-generating potential of a 1000-µm-long coating became less efficient on a per µm2 basis, likely due to distal boundary layer depletion of platelets. Overall, thrombin is robustly generated within clots by the extrinsic pathway followed by late-stage FXIa contributions, with fibrin localizing thrombin via its antithrombin-I activity as a potentially self-limiting hemostatic mechanism.