Microfluidic Tools To Probe the Spatial Dynamics of Coagulation.

To investigate the biophysical mechanisms that regulate the spatial dynamics of blood coagulation, we have developed a set of microfluidic tools that allow analysis and perturbation of blood coagulation on the micrometer scale with precise control of fluid flow, geometry, and surface chemistry. Physiological coagulation occurs in a localized manner; specifically, coagulation is believed to occur exclusively at regions of substantial vascular damage and does not spread throughout the entire vascular system. In vitro analysis and characterization of these spatial dynamics requires the ability to reproduce and perturb this system, an ability that is not provided by the mixed reactor systems commonly used for in vitro studies of blood coagulation. We developed microfluidic devices with micrometer-scale channels and methods to coat these channels with various phospholipids, including components of the blood coagulation network such as thrombomodulin and tissue factor, to reproduce in vitro the geometry and surface chemistry of blood vessels in vitro. In a microfluidic device with channels coated with phospholipids and thrombomodulin, we demonstrated that clots propagate in a wave-like fashion with a constant velocity in the absence of flow. We also showed that propagation of coagulation from an occluded channel to a channel with flowing blood plasma can be regulated by the geometry of the junction and the shear rate in the channel with flowing plasma. We also developed microfluidic tools to probe the spatial dynamics of initiation of clotting by patterning surfaces with tissue factor reconstituted into phospholipids bilayers. When human plasma or whole blood was exposed to these surfaces in a microfluidic device, clotting occurred only on patches of tissue factor larger than a threshold size. This threshold patch size is controlled by the rate of activation of clotting factors at the patch and the rate of transport of activated factors off the patch. These results suggest a mechanism for how tissue factor can circulate in blood without causing clotting, and how small regions of vascular damage can exist without causing clotting. These results also suggest new biophysical mechanisms that may control interactions between the coagulation cascade and bacterial surfaces.