Thrombus Growth and Embolism On Tissue Factor-Bearing Collagen Surfaces Under Flow Role of Thrombin with and without Fibrin

 At sites of vascular injury thrombin is an important mediator in thrombus growth and stability. Using microfluidic flow devices as well as patterned surfaces of collagen and tissue factor we sought to determine the role that fibrin plays in clot stability without interfering with the production of thrombin. We deployed an 8-channel microfluidic device to study coagulation during corn trypsin inhibitor-treated (XIIa-inhibited) whole blood perfusion over lipidated tissue factor (TF) linked to a fibrillar collagen type 1 surface.  Clot growth and embolization were measured at initial inlet venous (200 s^-1) or arterial (1000 s^-1) wall shear rates under constant flow rate or pressure relief mode in the presence or absence of Gly-Pro-Arg-Pro (GPRP) to block fibrin polymerization.   Numerical calculations for each mode defined hemodynamic forces on the growing thrombi. In either mode at inlet venous flow, increasing amounts of TF on the surface led to a modest dose-dependent increase (up to 2-fold) in platelet deposition, but resulted in massive fibrin accumulation (> 50-fold) only when exceeding a critical TF threshold. At a venous inlet flow, GPRP led to a slight 20% increase in platelet accumulation (p < 0.01) in pressure relief mode with thrombi resisting ~1500 s^-1 prior to full channel occlusion. GPRP-treated thrombi were unstable under constant flow rate, where shear forces caused embolization at a maximum shear rate of ~2300 s^-1 (69 dynes/cm²).   In constant flow rate mode, the nonocclusive platelet-fibrin deposits (no GPRP) withstood maximum shear rates of ~29,000 s^-1 (870 dyne/cm²) or ~95% of full channel occlusion.   For arterial inlet shear rate, embolization was marked for either mode with GPRP present when shear forces reached 87 dynes/cm² (~2900 s^-1).  Under constant flow rate, platelet-fibrin deposits (no GPRP) withstood maximums of 2400 dynes/cm² (80,000 s^-1) or ~90% of full channel occlusion before embolization. Fibrin increased clot strength by 12 to 28-fold. Under pressure relief mode, ~2-fold more fibrin was produced under venous flow (p < 0.001). These studies define embolization criteria for clots formed with surface TF-triggered thrombin production (± fibrin) under venous and arterial flows.

                                                

Figure 1 Whole blood (CTI 40 μg/mL) in the presence or absence of 5 mM GPRP was perfused over a fibrillar collagen surface at an initial arterial wall shear rate of 1000 s^-1, with (left, n=4) or without (right, n=8) pressure relief. Platelet accumulation data was used to calculate the hemodynamic conditions at the thrombus boundary based on simulation data.