Fibrin Networks Regulate Protein Transport during Thrombus Development

<div><p>Thromboembolic disease is a leading cause of morbidity and mortality worldwide. In the last several years there have been a number of studies attempting to identify mechanisms that stop thrombus growth. This paper identifies a novel mechanism related to formation of a fibrin cap. In particular, protein transport through a fibrin network, an important component of a thrombus, was studied by integrating experiments with model simulations. The network permeability and the protein diffusivity were shown to be important factors determining the transport of proteins through the fibrin network. Our previous <i>in vivo</i> studies in mice have shown that stabilized non-occluding thrombi are covered by a fibrin network (‘fibrin cap’). Model simulations, calibrated using experiments in microfluidic devices and accounting for the permeable structure of the fibrin cap, demonstrated that thrombin generated inside the thrombus was washed downstream through the fibrin network, thus limiting exposure of platelets on the thrombus surface to thrombin. Moreover, by restricting the approach of resting platelets in the flowing blood to the thrombus core, the fibrin cap impaired platelets from reaching regions of high thrombin concentration necessary for platelet activation and limited thrombus growth. The formation of a fibrin cap prevents small thrombi that frequently develop in the absence of major injury in the 60000 km of vessels in the body from developing into life threatening events.</p></div

A: Confocal images of fibrin networks for different fibrinogen concentrations (left to right): 0.5 mg/mL, 2 mg/mL, 3 mg/mL, and 4 mg/mL.

<p>Scale bar is 30 . B,C: Diffusivity of different probes in fibrin gel as a function of (B) and fibrinogen concentration (C): circles - thrombin molecules, squares - Fab IgG fragments, diamonds - azobenzene-labeled BSA molecules <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003095#pcbi.1003095-Stewart1" target="_blank">[19]</a>, left triangles - 228 nm diameter PEG-coated particles <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003095#pcbi.1003095-Spero1" target="_blank">[20]</a>, right triangles - 526 nm diameter PEG-coated particles <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003095#pcbi.1003095-Spero1" target="_blank">[20]</a>, 1 - Johnson's model <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003095#pcbi.1003095-Johnson1" target="_blank">[38]</a> <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003095#pcbi.1003095.e106" target="_blank">Equation (3)</a> (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003095#pcbi.1003095.s004" target="_blank">Text S2</a>); 2 and 3 - thrombin and IgG fits obtained using <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003095#pcbi.1003095.e099" target="_blank">Equation (2)</a>. All data points refer to fibrin gels formed at 7.5 pH and thrombin concentration of 1 NIH U/mL.</p

Evolution of thrombin concentration for low () and high () permeable fibrin networks in time: 0 s (A, D), 13 ms (B, E), 38 ms (C, F).

<p>‘TC’ denotes a thrombus core. The arrow shows the direction of flow. The flow Reynolds number, . The color bar shows thrombin concentration relative to its initial value when thrombin is uniformly distributed in a 2 m thick layer near the thrombus core ( = 0 s).</p

Composition of developing thrombi obtained from vertical stacks of images collected by multiphoton microscopy of laser induced injuries in mesenteric veins of a mice.

<p>3D image reconstruction of a late stage thrombus is shown in (a) (luminal view) and (b) cross section in a vertical plane (wall is on the left, lumen is on the right). Regions composed mostly of platelets are red, mostly of fibrin - green, composed of platelets and fibrin -yellow; and regions excluding plasma, fibrin and platelets (other material, cells) - black. (c) Shows evolution of the thrombus composition as it stabilizes. Stabilization is associated with decreasing amounts of platelets and increasing amounts of fibrin on the surface.</p

A: Diffusion and advection layers identified in the fibrin cap for thrombin and Fab IgG.

<p>Diffusion prevails near the thrombus surface and depends on molecular size. Smaller than Fab IgG complexes thrombin molecules have higher mobility resulting in thicker diffusion layer. Diffusion layer profiles for thrombin and Fab IgG for low () and high () fibrin cap permeabilities are shown. Flow Reynolds number , , . B: Force acting on a thrombus surface as a function of the permeability of the fibrin cap for different radii values of the core, , and the thrombus, . For each permeability value, the force is non-dimensionalized by the force acting on a nonpermeable thrombus of the same size.</p

Mobile fraction of Fab IgG (A) and thrombin (B) in the fibrin gel at different fibrinogen concentrations.

<p>Mobile fraction of Fab IgG (A) and thrombin (B) in the fibrin gel at different fibrinogen concentrations.</p

A: FRAP microscopy of fluorescently labeled Fab fragments of IgG.

<p>For a typical FRAP experiment performed on a fibrin network, the regions of interest (ROIs, radius of 27 m) before, immediately after photobleaching, and after 98 s are shown (green circle). Normalized fluorescence recovery for the corresponding ROIs and their fits according to a model (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003095#pcbi.1003095.e163" target="_blank">Equation (7)</a>, ) are shown by symbols and a line, respectively. Fluorescence intensity values before () and after () photobleaching, and at the end of the experiment (I_∞), are shown. For each sample, FRAP measurements were repeated 3 times in 8 different locations over the sample and the arithmetic mean of the intensity curves was taken. B: Hydrodynamic diameter measurements by dynamic light scattering: circles denote -thrombin and squares denote Fab IgG.</p

A: Permeability as a function of fiber volume fraction in fibrin gels.

<p>Circles correspond to data collected for clots in plastic tubes and squares show the data for clots in glass capillaries. Line is a fit: , . B: Fiber radius versus fibrinogen concentration determined from permeability measurements and <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003095#pcbi.1003095.e021" target="_blank">Equation (1)</a> (circles) and from confocal microscopy images (squares).</p

A: Model representation of a thrombus consisting of a nonpermeable core of radius and a fibrin cap of thickness , where is the radius of the thrombus.

<p>Flow is uniform at infinity. B,C: Velocity field near the thrombus (, ) with low (B) () and high (C) () permeable fibrin cap, the flow Reynolds number, . The flow at infinity is along the horizontal axis. Color scale shows the relative absolute velocity .</p

Supplementary Information to the paper in J. R. Soc. Interface \Model Predictions of Deformation, Embolization, and Permeability of Partially Obstructive Blood Clots under Variable Shear Flow" from Model predictions of deformation, embolization and permeability of partially obstructive blood clots under variable shear flow

Supplementary_Information_Fina