Microfluidic devices for studies of shear-dependent platelet adhesion

Adhesion of platelets to blood vessel walls is a shear stress dependent process that promotes arrest of bleeding and is mediated by the interaction of receptors expressed on platelets with various extracellular matrix (ECM) proteins that may become exposed upon vascular injury. Studies of dynamic platelet adhesion to ECM-coated substrates in conventional flow chambers require substantial fluid volumes and are difficult to perform with blood samples from a single laboratory mouse. Here we report dynamic platelet adhesion assays in two new microfluidic devices made of PDMS. Small cross-sections of the flow chambers in the devices reduce the blood volume requirements to < 100 mu l per assay, making the assays compatible with samples of whole blood obtained from a single mouse. One device has an array of 8 flow chambers with shear stress varying by a factor of 1.93 between adjacent chambers, covering a 100-fold range from low venous to arterial. The other device allows simultaneous high-resolution fluorescence imaging of dynamic adhesion of platelets from two different blood samples. Adhesion of platelets in the devices to three common ECM substrate coatings was verified to conform with published results. The devices were subsequently used to study the roles of extracellular and intracellular domains of integrin alpha IIb beta 3, a platelet receptor that is a central mediator of platelet aggregation and thrombus formation. The study involved wild-type mice and two genetically modified mouse strains and showed that the absence of the integrin impaired adhesion at all shear stresses, whereas a mutation in its intracellular domain reduced the adhesion only at moderate and high stresses. Because of small sample volumes required, the devices could be employed in research with genetically-modified model organisms and for adhesion tests in clinical settings with blood from neonates.