Flow profoundly influences fibrin network structure: Implications for fibrin formation and clot stability in haemostasis

Haemostasis requires fibrinogen conversion to fibrin and formation of a stable fibrin network.Fibrin network properties, including fibre thickness, branchpoint density, fibre density, mechanical stability, porosity, and resistance to lysis can differentiate plasma clots of healthy individuals from those with haemostatic or thrombotic disorders.Plasma from patients with a bleeding history produces thick, minimally-branched fibres in coarse, deformable networks that are highly susceptible to lysis, whereas plasma from patients with a personal or family history of thrombosis produces thin, highly-branched fibres in impermeable, rigid networks that are relatively resistant to fibrinolysis (1).Most previous studies have examined fibrin networks produced in the absence of flow or cells.Networks formed under these conditions show an isotropic distribution of fibres with relatively uniform diameters.However, although reduced flow promotes fibrin deposition (2), it is unlikely that blood flow ever fully stops in vivo, even under "stasis".Fibrin formation studied in systems modelling venous (10-100 s -1 ) or arterial (500-1,500 s -1 ) shear shows orientation of fibres along flow vectors (3).Few studies, however, have examined fibrin formation under very low flow rates expected during extravasation of blood into extravascular tissue factor (TF)-rich tissues.The impact of flow on fibrin morphology under these conditions is not known.To mimic extravascular TF-bearing cells, immortalised human dermal fibroblasts (NHF 1 -hTert) were cultured on sterile Thermanox cover slips (Miles Laboratories, Elkhart, IN, USA) to ~90% confluence and washed in 10 mM sodium phosphate pH 7.4, 150 mM NaCl."Stasis clots" were formed on these cells by incubating 150 µl re-calcified (10 mM, final) contact-inhibited, normal-pooled, platelet-free plasma (PFP) (4) with fibroblasts for 10 minutes (min)."Flow clots" were formed by pumping (0.1 ml/min) re-calcified PFP over fibroblasts for 10 min using a Genie Syringe Pump (Kent Scientific Corporation, Torrington, CT, USA) with the cover slip at a ~30° decline to allow plasma to slowly flow over fibroblasts and model haemostatic injury.For both conditions, clots were left still for 10