Human blood coagulation on matrix protein microarrays under venous and arterial flow
Microarraying allows the spatial and compositional control of surfaces, typically for the purpose of binding reactions. We have developed a matrix protein microarray to study blood coagulation with spatially controlled surface biochemistry, thus allowing numerous conditions to be studied in a single flow experiment. Fibrillar collagen, von Willebrand Factor (vWF) and/or Tissue Factor (TF) 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 of recalcified corn trypsin inhibitor-treated whole blood or platelet rich plasma and subsequent array scanning via anti-GPIbα and anti-fibrin(ogen) immunofluorescence. Another microarray used collagen and increasing levels of lipidated tissue factor (TF) to assay simultaneous platelet deposition and fibrin formation. TF displayed a switch-like regulatory function over a very narrow concentration range allowing a determination of the surface EC50 of 3.63, 8.40, and 10.2 TF-molecules/μm2 at 100, 500, 1000 s-1, respectively. As expected, higher shear rates helped suppress fibrin formation. To further investigate the role of very low sub-picomolar soluble levels of TF (i.e. blood-borne TF), whole blood IX (with and without exogenously supplemented 100 fM TF) was used in this system under static and flow conditions. The addition of TF had no significant effect under static conditions. However under flow, the addition of this small amount of TF caused and significant increase in the coagulation response. We conclude that sub-pM TF in the blood may synergize with the TF on the array surface to evoke this response under flow. 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. This is one of the first in vitro systems that can incorporate the major components of the in vivo hemostasis system: flow, focal surface chemistry, and combinations of matrix proteins.
Molecular biology|Biomedical research|Chemical engineering
Okorie, Uzoma Michelle, "Human blood coagulation on matrix protein microarrays under venous and arterial flow" (2007). Dissertations available from ProQuest. AAI3294690.