Flow-Dependent Functions Of Soluble Or Immobilized Tissue Factor In Mural Thrombus Formation Mediated By Von Willebrand Factor

Mural thrombus formation at sites of damaged vessel wall, essential for both physiologic hemostasis and pathological thrombosis, is established by platelet adhesion/aggregation and blood coagulation mechanisms. Although tissue factor (TF) is up-regulated upon vessel wall damage and plays a pivotal role in the latter process, its functional relevance under physiologic blood flow conditions is poorly understood. Using an in vitro perfusion chamber system, we have therefore studied the relevant role of TF in thrombus formation mediated by von Willebrand factor (VWF), a distinctive flow-dependent thrombogenic surface, under whole blood flow conditions with varying shear rates. Human recombinant TF (Innobin) were co-coated with purified VWF (100 ug/ml) onto a glass plate to prepare ‘surface-immobilized TF/VWF complex’. Surface density of immobilized TF, evaluated by the ELISA-based assay using an anti-TF monoclonal antibody, was increased in a concentration-dependent and saturated manner by soluble TF (1-100 pM) added on a plate. Citrated whole blood, recalcified with 8 mM CaCl₂ prior to perfusion, was perfused over a VWF-surface in the presence or absence of surface-immobilized TF. Platelet adhesion and aggregation was evaluated by the surface coverage of generated thrombi in a defined area after 5-min perfusion. Mural thrombi formed on VWF-surface were also double-stained with fluorescently labeled anti-fibrin and anti-fibrinogen antibodies. Fibrin generation was evaluated by confocal laser scanning microscopy as a ratio of fibrin relative to fibrinogen fluorescence within mural thrombi. As a result, surface-immobilized TF significantly augmented flow-dependent fibrin generation as a function of increasing surface density of TF under both low (250 s^-1) and high (1500 s^-1) shear rate conditions. In this regard, soluble TF, when added to sample blood, similarly increased intra-thrombus fibrin generation in a dose-dependent manner in the absence of immobilized TF. However, coagula formation in sample blood was enormously amplified by soluble TF during perfusion, as judged by the flow-path occlusion time. In addition to the enhancing effects on fibrin generation, immobilized TF significantly up-regulated VWF-dependent platelet adhesion and aggregation under high shear rate conditions, albeit with no appreciable effects under low shear rate conditions. These results suggest a synergistic functional link between immobilized TF and VWF in mural thrombus formation under high shear rate conditions. Our results clearly illustrate the thrombogenic potentials of two distinct forms (soluble or surface-immobilized) of TF, in which surface-immobilized TF plays a concerted role on VWF-dependent thrombus formation with lesser risk of systemic hypercoagulability which may be induced by circulating soluble TF under high shear rate conditions.