Just a Drop of Blood: Exploration of Shear-Dependent Platelet Adhesion and Activation in Microfluidic Devices.

Abstract

Interactions of platelets and their adhesion receptors with extracellular matrices are essential for hemostasis. Platelets experience different shear rates as they circulate through the vasculature. Conventional studies of platelets in shear flow are performed in simple flow chambers with relatively large volumes of cells and reagents, limiting testing when these are in short supply. Microfluidics technology should enable the concurrent study of multiple small volume samples across a wide range of shear rates, thereby allowing trends to emerge that might be difficult to detect otherwise. To achieve this goal, we fabricated PDMS microfluidic devices that permit testing of dynamic platelet adhesion over a 100-fold span of shear rates from a single 200μl blood sample. Alternate device design permits synchronous monitoring of platelet adhesion from two genetically distinct blood samples or treatment groups. We have used this technology to gain new insights into integrin αIIbβ3 function in mepacrine-labeled platelets under shear flow. In whole blood, the adhesion of wild-type mouse platelets to a fibrinogen-coated substrate was shear rate-dependent, similarly to human platelets. In contrast, adhesion of αIIbβ3-deficient (β3−/−) platelets was virtually absent above a shear rate of 100 s−1. To distinguish between requirements for the presence of an intact extracellular αIIbβ3 ligand binding domain versus an intact integrin activation process, we used mice with a Y/A mutation at residue 747 in the β3 cytoplasmic tail (β3Y747A), that selectively blocks talin interaction with β3, agonist-induced αIIbβ3 activation, and platelet thrombus formation in vivo. When compared to wild-type and β3−/− platelets, a normal αIIbβ3 extracellular domain on β3Y747A platelets partially rescued dynamic adhesion to fibrinogen by 50–80% at ≤130 s−1, but by only 25% at 250 s−1. Treatment of wild-type platelets with PGE1 to inhibit platelet activation similarly reduced adhesion to fibrinogen at higher shear rates. On fibrillar type I collagen, wild-type platelets formed an initial monolayer and progressively larger thrombi over time. In addition, platelets supported rolling and firm adhesion of granulocytes in a manner dependent on shear rate, platelet P-selectin and granulocyte PSGL1. In contrast, no platelet thrombus growth on collagen was observed with αIIbβ3-deficient or β3Y747A platelets, or with wild-type platelets treated with PGE1. Furthermore, even the initial adhesion of αIIbβ3-deficient and β3Y747A platelets to collagen rapidly declined at increasing shear rates (120%, 30% and 7% of wild-type platelets at 70, 1000 and 4000 s−1, respectively (p < 0.01)). Taken together, these studies establish that microfluidics provides an efficient and high-throughput platform to study mechanisms of dynamic platelet adhesion, activation and thrombus formation on extracellular matrices. Furthermore, they demonstrate a role for talin-dependent αIIbβ3 activation in all of these processes. This platform will be particularly useful under conditions where blood sample volumes or reagents are limiting, such as in neonates, and genetically-modified model organisms.