Microfluidics and fluorescent imaging combined to study platelet activation

Platelets are cell fragments that play a central role in hemostasis and thrombosis. Typically platelets circulate as thin flat disks; however upon encountering a damaged vessel wall they are recruited to the exposed subendothelial matrix where they adhere and activate. The platelets interact with the exposed matrix proteins via specific adhesive glycoproteins found on the surface of the platelet. Activated platelets have extensive pseudopodia due to a change in the assembly of cytoskeletal proteins such as actin and tubulin. The rearrangement of the cytoskeleton also corresponds with the secretion of granule content leading to the amplification of the platelet stimulation and the formation of a stable platelet-fibrin plug. A popular model assumes that the granules are drawn to the center of the platelet before release occurs, indicating the importance of the cytoskeletal components that drive this centralization leading to the eventual clot formation.

The goal of our research is to examine cytoskeletal protein arrangements in granule release. To accomplish this we used fluorescent imaging to study the activated platelets that aggregate on the protein coatings along a microfluidic channel. These protein coatings will include proteins found in damaged vessel walls such as collagen and fibrinogen. Here we will describe the microfluidic channels we used to model blood vessels. We will explain the process of photolithography that we use to make these devices, and explain the design and fabrication process. We will also discuss methods that were used to stain cytoskeletal proteins to reduce the premature activation of the platelets. A better understanding of these models will lead to a greater understanding clot formation.