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

Abstract

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.