Microfluidic focal thrombosis model for measuring murine platelet deposition and stability: PAR4 signaling enhances shear-resistance of platelet aggregates.

BACKGROUND Flow chambers allow the ex vivo study of platelet response to defined surfaces at controlled wall shear stresses. However, most assays require 1-10 mL of blood and are poorly suited for murine whole blood experiments. OBJECTIVE To measure murine platelet deposition and stability in response to focal zones of prothrombotic stimuli using 100 microL of whole blood and controlled flow exposure. METHODS Microfluidic methods were used for patterning acid-soluble collagen in 100 microm x 100 microm patches and creating flow channels with a volume of 150 nL. Within 1 min of collection into PPACK and fluorescent anti-mouse CD41 mAb, whole blood from normal mice or from mice deficient in the integrin alpha(2) subunit was perfused for 5 min over the patterned collagen. Platelet accumulation was measured at venous and arterial wall shear rates. After 5 min, thrombus stability was measured with a 'shear step-up' to 8000 s(-1). RESULTS Wild-type murine platelets adhered and aggregated on collagen in a biphasic shear-dependent manner with increased deposition from 100 to 400 s(-1), but decreased deposition at 1000 s(-1). Adhesion to patterned collagen was severely diminished for platelets lacking a functional alpha(2)beta(1) integrin. Those integrin alpha(2)-deficient platelets that did adhere were removed from the surface when challenged to shear step-up. PAR4 agonist (AYPGKF) treatment of the thrombus at 5 min enhanced aggregate stability during the shear step-up. CONCLUSIONS PAR4 signaling enhances aggregate stability by mechanisms independent of other thrombin-dependent pathways such as fibrin formation.