Fibrin, γ′-Fibrinogen, and Transclot Pressure Gradient Control Hemostatic Clot Growth During Human Blood Flow Over a Collagen/Tissue Factor Wound

Abstract

Objective— Biological and physical factors interact to modulate blood response in a wounded vessel, resulting in a hemostatic clot or an occlusive thrombus. Flow and pressure differential (Δ P ) across the wound from the lumen to the extravascular compartment may impact hemostasis and the observed core/shell architecture. We examined physical and biological factors responsible for regulating thrombin-mediated clot growth. Approach and Results— Using factor XIIa-inhibited human whole blood perfused in a microfluidic device over collagen/tissue factor at controlled wall shear rate and Δ P , we found thrombin to be highly localized in the P-selectin + core of hemostatic clots. Increasing Δ P from 9 to 29 mm Hg (wall shear rate=400 s −1 ) reduced P-selectin + core size and total clot size because of enhanced extravasation of thrombin. Blockade of fibrin polymerization with 5 mmol/L Gly-Pro-Arg-Pro dysregulated hemostasis by enhancing both P-selectin + core size and clot size at 400 s −1 (20 mm Hg). For whole-blood flow (no Gly-Pro-Arg-Pro), the thickness of the P-selectin-negative shell was reduced under arterial conditions (2000 s −1 , 20 mm Hg). Consistent with the antithrombin-1 activity of fibrin implicated with Gly-Pro-Arg-Pro, anti-γ′-fibrinogen antibody enhanced core-localized thrombin, core size, and overall clot size, especially at venous (100 s −1 ) but not arterial wall shear rates (2000 s −1 ). Pathological shear (15 000 s −1 ) and Gly-Pro-Arg-Pro synergized to exacerbate clot growth. Conclusions— Hemostatic clotting was dependent on core-localized thrombin that (1) triggered platelet P-selectin display and (2) was highly regulated by fibrin and the transclot ΔP. Also, γ′-fibrinogen had a role in venous but not arterial conditions.