Thrombin flux and wall shear rate regulate fibrin fiber deposition state during polymerization under flow

Abstract

Thrombin is released as a soluble enzyme from the surface of platelets and tissue-factor-bearing cells to trigger fibrin polymerization during thrombosis under flow conditions. Although isotropic fibrin polymerization under static conditions involves protofibril extension and lateral aggregation leading to a gel, factors regulating fiber growth are poorly quantified under hemodynamic flow due to the difficulty of setting thrombin fluxes. A membrane microfluidic device allowed combined control of both thrombin wall flux (10(-13) to 10(-11) nmol/mum(2) s) and the wall shear rate (10-100 s(-1)) of a flowing fibrinogen solution. At a thrombin flux of 10(-12) nmol/mum(2) s, both fibrin deposition and fiber thickness decreased as the wall shear rate increased from 10 to 100 s(-1). Direct measurement and transport-reaction simulations at 12 different thrombin flux-wall shear rate conditions demonstrated that two dimensionless numbers, the Peclet number (Pe) and the Damkohler number (Da), defined a state diagram to predict fibrin morphology. For Da < 10, we only observed thin films at all Pe. For 10 < Da < 900, we observed either mat fibers or gels, depending on the Pe. For Da > 900 and Pe < 100, we observed three-dimensional gels. These results indicate that increases in wall shear rate quench first lateral aggregation and then protofibril extension.

Figure 1

Membrane microfluidic device, operation, and data analysis. The device consists of two microfluidic channels oriented perpendicular to each other and separated by a polycarbonate membrane. Fibrinogen is perfused through the lower channel at a desired wall shear rate. Thrombin is introduced into the flowing fibrinogen at a controlled flux. (A and B) The schematic depicts the side and bottom views of the device. (C) FITC is used as a tracer molecule to measure flux through the membrane before introduction of fibrinogen and thrombin. (D–F) After the experiment, deposited fibrin is fixed and imaged by electron microscopy. The progression of the polymerization is observed as a function of distance from the channel intersection. Scale bar, 1 μm.

Figure 2

Thrombin and fibrinogen concentration profiles from simulations at wall shear rates of 10, 25, 50, and 100 s⁻¹ and a thrombin flux of 10⁻¹² nmol/μm² s. Thrombin is introduced at a constant flux from the top of the channel from the origin (x = 0 μm) to the dotted line (x = 250 μm). Direction of flow is from left to right.

Figure 3

Fibrin morphology as a function of wall shear rate. Electron micrographs of fibrin formed after 5 min at a thrombin flux of 10⁻¹² nmol/μm² s and wall shear rates of 10 s⁻¹ (A), 25 s⁻¹ (B), 50 s⁻¹ (C), and 100 s⁻¹ (D). Dark disklike objects in D are the membrane pores. Scale bar, 1 μm.

Figure 4

Fibrin morphology as a function of thrombin flux. Electron micrographs of fibrin formed at a wall shear rate of 50 s⁻¹ and thrombin flux of 10⁻¹³ (A), 10⁻¹² (B), and 10⁻¹¹ (C) nmol/μm² s. Dark disklike objects in A are the membrane pores. Scale bar, 1 μm.

Figure 5

Fibrin state diagram. Fibrin morphology can be described in terms of the Peclet number and the Damkohler number as calculated from simulations. Symbols represent observations from electron miscroscopy; circles = three-dimensional gels, squares = two-dimensional mat, and triangles = thin films. Dashed lines represent the experimentally observed transitions between different morphologies.