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-m long patch of type I fibrillar collagen/lipidated tissue factor (TF; ϳ1 TF molecule/ m 2 ), 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 ϳ0.5 ؋ 10 ؊12 nmol/m 2 -s over the first 500 s of perfusion and then further increased by ϳ2-3-fold over the next 300 s. The increased thrombin flux after 500 s was blocked by antiFXIa antibody (O1A6), consistent with thrombin-feedback activation of FXI. Over the first 500 s, ϳ92,000 molecules of thrombin were generated per surface TF molecule for the 250-m-long coating. A single layer of platelets (obtained with ␣ IIb ␤ 3 antagonism preventing continued platelet deposition) was largely sufficient for thrombin production. Also, the overall thrombin-generating potential of a 1000-m-long coating became less efficient on a per m 2 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 selflimiting hemostatic mechanism.

Comparison between manufacturing sites shows differential adhesion, activation, and GPIbα expression of cryopreserved platelets

“…Under shear flow, factor availability depends on convective hydrodynamic forces rather than diffusion. 36 Our data confirm, however, that also under shear flow, coagulation is not affected by cryopreservation of PLTs. In contrast, contact-induced coagulation initiation in the absence of TF was even shorter than control.…”

“…In this case, the cascade can quickly assemble tenase and prothrombinase complexes on the abundant negatively charged phospholipid surface provided by damaged cryoplatelets and microparticles. Under shear flow, factor availability depends on convective hydrodynamic forces rather than diffusion . Our data confirm, however, that also under shear flow, coagulation is not affected by cryopreservation of PLTs.…”

Recombinant factor VIIa addition to haemophilic blood perfused over collagen/tissue factor can sufficiently bypass the factor IXa/VIIIa defect to rescue fibrin generation

“…In contrast, fibrin deposition with healthy blood is extremely responsive to thrombin production and can increase >10 to 20-fold with TF. Based on a separate study [29], we conclude fibrin formation is more dependent on TF (and thrombin) and somewhat less dependent on platelets during the first 500 sec of clotting, once the first layer of platelets have deposited on the surface. For healthy blood, the first layer of platelets is largely sufficient for thrombin production by surface TF during the first 500 sec.…”

“…In our earlier work [23], a PTT clotting time of > 40 sec was fully predictive of poor fibrin formation under flow over collagen (no TF). Yet, recent measurements of thrombin generation during whole blood flow (using TAT assay) indicate a different kinetics may be in place under flow where thrombin flux increases linearly with time to about 0.5 × 10 −12 nmole/μm 2 -sec by 500 sec while dense intraclot fibrin is a powerful inhibitor of thrombin [29]. Viewed from a “cell-based” perspective of rFVIIa function, the microfluidic data supports a mechanism for: (1) initiation of coagulation (TF or FXIIa), and (2) the ability to propagate FXa and thrombin generation on the platelet surface (via FIXa/FVIIIa or rFVIIa) to support platelets and especially fibrin deposition (Fig.…”

Microfluidic whole blood testing of platelet response to pharmacological agents

“…The collagen surface allows evaluation of GPVI pathways and α 2 β 1 -mediated adhesion to collagen. Inhibition of α 2b β 3 prevents continued platelet buildup on the first layer of collagen-adherent platelets [23]. Buildup of the deposit under flow is autocrinic and dependent on platelet release of ADP to activate P2Y 1 and P2Y 12 .…”