Background: The aim of this research is to develop an in vitro system with spatially controlled presentation of collagen and tissue factor. A technical challenge is to vary the tissue factor concentration in a controlled manner. We have developed a bead-based protocol for presenting tissue factor in conjunction with micropatterned collagen into a microfluidic thrombosis model.

Methods: Biotin-streptavidin chemistry is used to link relipidated recombinant human tissue factor to 1mm-diameter latex beads. Beads are delivered by acoustic dispensing to the surface of glass slides with high spatial resolution. Standard soft-lithography methods are utilized to cast PDMS devices which are bonded reversibly to glass for protein deposition and blood presentation. The accumulation of platelets and fibrin is monitored in real time by epifluorescence.

Results: The activity of tissue factor was measured and confirmed at each level of synthesis, and two modes of modulation allows for precise presentation of active tissue factor at a specific area: the tissue factor concentration in the liposomes (5 to 20x10³ lipid molecules per tissue factor molecule) and the number of particles on the surface (0.3 to 2.6 particles/mm²). Microfluidic devices allow for perfusion of less than 100mL of human or murine blood over tissue factor and collagen, which was performed to investigate the relative roles of the two main receptors (a2b1 and GPVI) in knock-out mice. The data imply independent roles of firm adhesion and activation of murine platelets to collagen at an average wall shear rate of 400s^-1.