RESUMO
BACKGROUND: In healthy subjects fibrinogen γ/γ' circulates at 8-15% of the total plasma fibrinogen concentration. Elevated levels of this variant have been associated with arterial thrombosis, and its diminution with venous thrombosis. The aims of the present work were to analyze the structure of the fibrin network formed on the top of human dermal microvascular endothelial cells (HMEC-1) at different fibrinogen γ/γ' concentrations, as well as its influence on the secretion of fibrinolytic components. The kinetics of fibrin polymerization on top of HMEC-1 cells with 3, 10, and 30% fibrinogen γ/γ' was followed at 350 nm. The secretion of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI 1) by HMEC-1 were measured in the supernatant and cell lysates, after incubation with 1 nM thrombin, fibrin with 3, and 30% fibrinogen γ/γ', using commercial kits. The influence of fibrinogen γ/γ' on fibrin structure on the surface of the HMEC-1 was followed with laser scanning confocal microscopy (LSCM). RESULTS: The kinetics of fibrin formation on HMEC-1 with 3 and 10% fibrinogen γ/γ' were similar. However, with 30% fibrinogen γ/γ' both the slope and final turbity were approximately 50% less. The LSCM images showed the dramatic effects of increasing fibrinogen γ/γ' from 3 to 30%. The uPA and PAI 1 concentrations in culture supernatants HMEC-1 cells treated with thrombin or 30% γ/γ' fibrin were two-fold increased as compared to basal culture supernatants and 3% γ/γ' fibrin-treated HMEC-1. In all stimulatory conditions the intracellular concentration of uPA was higher than in supernatants. In contrast, the intracellular PAI 1 concentration was decreased as compared to that measured in the supernatant, including the basal condition. CONCLUSION: A concentration of 30% fibrin γ/γ' alter drastically fibrin structure on the cell surface and affects the secretion of uPA and PAI 1 through its capacity to bind thrombin.
