(489b) Fibrin Polymerization and Deposition in a Shear Flow

Authors: 
Diamond, S. L., University of Pennsylvania

Background: The conversion of fibrinogen to fibrin by the enzyme thrombin is a central event in the growth and stability of a blood clot. However, there is little known about how fibrin fibers polymerize and deposit to a surface in shear flows that are representative of those found in blood vessels. In this study we will test the hypothesis that there exists a critical thrombin flux necessary for fibrin deposition for a given wall shear rate. The significance of this work is that many bleeding disorders, such as hemophilia, result in a diminished ability to generate thrombin and, as a result, fibrin.

Methods: Experiments were conducted in a previously developed membrane microfluidic device. The device consisted of a track-etched polycarbonate membrane reversibly sealed between two microfluidic channels; one channel contained fibrinogen (3 mg/mL) flowing at a physiological wall shear rate (25-100 s-1), and the other channel contained thrombin (10-500 nM). These two channels are oriented perpendicular to each other, at their intersection the thrombin flux is dictated by the user-defined transmembrane pressure. Thrombin was introduced into flowing fibrinogen to determine the critical thrombin flux necessary for fibrin deposition at a given wall shear rate. Fibrin deposition and was monitored by epifluorescence during the experiment and morphology was measured by post hoc confocal and electron microscopy.

Results: At each wall shear rate, we observed three distinct morphologies of fibrin. For example, at 25 s-1 and at a low thrombin flux (1 x 10-8 mol/m2 s) we observed a thin film of fibrin with little or no measurable fibers. Above a certain critical flux (~1 x 10-9 mol/m2 s), we measured protofibrils (~10 nm) and fibers (50-100 nm). Finally, we observed a second critical flux (5 x 10-9 mol/m2 s) that resulted in large aggregates (200-1000 nm) of fibers and occlusion of microfluidic channel. Similar results were found at wall shear rates of 50 s-1 and 100 s-1.

Conclusions: This is the first demonstration of the threshold thrombin flux required to induce fibrin deposition and polymerization in a shear flow. The differences in fibrin morphology at subthreshold thrombin fluxes could lead to diminished platelet adhesion and aggregation. This is one possible mechanism for why individuals with bleeding disorders for unstable clots.