Mural thrombus generation at sites of damaged vessel walls is essential for both physiological haemostasis and pathological intravascular thrombosis. While thrombi are established by the concerted action of platelet aggregation and blood coagulation, most previous in vitro coagulation assays have evaluated fibrin clot formation in a closed stirring situation that lacks blood cells including platelets. We describe here a modified flow chamber system, established originally for platelet functional studies, that enables real-time observation of intra-thrombus fibrin accumulation during platelet thrombogenesis under flow conditions. Analysis by confocal laser scanning microscopy during perfusion of whole blood anticoagulated to various extents revealed that the size and shape of mural thrombi can depend on the intra-thrombus fibrin development under high shear rate conditions. These observations were confirmed by perfusion of heparinized blood or blood from haemophilia patients with or without addition of activated factor VII. Thus, our experimental system provides visual evidence supporting the concept of "cell-based coagulation under whole blood flow", which might be the most physiologically relevant model of comprehensive thrombogenicity in vivo to date. This system promises to help formulate strategies for haemostatic management of congenital coagulation disorders as well as for antithrombotic therapy targeting fatal arterial thrombosis.
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