Relipidated Tissue Factor Linked to Collagen Surfaces Potentiates Platelet Adhesion and Fibrin Formation in a Microfluidic Model of Vessel Injury

Microfluidic devices allow for the controlled perfusion of human or mouse blood over defined prothrombotic surfaces at venous and arterial shear rates. To mimic in vivo injuries such a plaque rupture, the need exists to link lipidated tissue factor (TF) to surface bound collagen fibers. Recombinant TF was relipidated in liposomes of phosphatidylserine/phosphatidylcholine/biotin-linked phosphatidylethanolamine (20:79:1 PS/PC/bPE molar ratio). Collagen was patterned in a 250 mu m-wide stripe and labeled with biotinylated anticollagen antibody which was then bound with streptavidin, allowing the subsequent capture of the TF liposomes. To verify and detect the TF liposome collagen assembly, individual molecular complexes of TF-factor Vila on collagen were visualized using the proximity ligation assay (PLA) to produce discretely localized fluorescent events that were strictly dependent on the presence of factor Vila and primary antibodies against TF or factor VIM. Perfusion for 450 s (wall shear rate, 200 s(-1)) of corn trypsin inhibitor (CTI, a factor XIIa inhibitor) treated whole blood over the stripe of TF-collagen enhanced platelet adhesion by 30 +/- 8% (p < 0.001) and produced measurable fibrin (>50-fold increase) as compared to surfaces lacking:IT. PS/PC/bPE liposomes lacking TF resulted in no enhancement of platelet deposition. Essentially no fibrin was formed ding perfusion over collagen surfaces or collagen surfaces with liposomes lacking TF despite the robust platelet deposition, indicating a lack of kinetically significant platelet-borne tissue factor in healthy donor blood. This study demonstrates a reliable approach to link functionally active TF to collagen for microfluidic thrombosis studies.