Matrix Protein Microarrays for Spatially and Compositionally Controlled Microspot Thrombosis under Laminar Flow

Abstract

Microarraying allows the spatial and compositional control of surfaces, typically for the purpose of binding reactions. Collagen and/or von Willebrand Factor (vWF) in 5% glycerol was contact printed onto glass slides to create defined microspots (176- μm diameter) of adsorbed protein without sample dehydration. The arrays were mounted on flow chambers allowing video microscopy during perfusion (wall shear rate of 100–500 s −1) of recalcified corn trypsin inhibitor-treated whole blood or platelet rich plasma and subsequent array scanning via anti-GPIb α and anti-fibrin(ogen) immunofluorescence. To mimic the subendothelial matrix, vWF was microarrayed over sonicated type I collagen microspots. For whole blood perfusion (500 s −1, 10 min) over collagen, vWF, and collagen/vWF microspots, the amount of platelet deposition on the collagen/vWF spots was ∼2 times greater in comparison to the collagen spots and ∼18 times greater in comparison to the vWF spots. The amount of fibrin(ogen) deposition on the collagen/vWF spots was ∼2 times greater in comparison to the collagen spots and ∼4 times greater in comparison to the vWF spots. This protocol allowed for highly uniform (CV = 18%) and precisely located thrombus formation at a density of ≥400 spots/cm 2. Microarrays are ideal for the combinatorial assembly of adhesive and procoagulant proteins to study thrombosis as well as to study axial and lateral transport effects between discrete microspots of distinct composition.