Endothelial Cell Influence on Fibrinolysis

Endothelial cells (EC) actively participate in many physiologic processes like inflammation, coagulation and fibrinolysis. EC influence fibrinolysis in both directions, pro- and antifibrinolytic, by secretion of tissue Plasminogen Activator (t-PA) and its antagonist Plasminogen Activator Inhibitor 1 (PAI-1). To understand the role of EC in the situation of a fresh occlusive thrombus, which is i.e. the reason for a myocardial infarction, we developed an in-vitro model with a glass tube, coated with a confluent monolayer of HUVEC (human umbilical venous endothelial cells). In the presence of differently stimulated EC (TNF-α, TGF-β, PMA and Forskolin) a fibrin clot was formed by adding thrombin to a mixture of fibrinogen and plasminogen in a HEPES buffered Geyʹs salt solution. After intervals of 2 and 4 hours the clot was slowly perfused with a buffer solution additionally containing t-PA to induce lysis. Thrombus growth and lysis were measured by laser light scattering and lysis times were determined. In parallel the clots were stained for PAI-1 and analyzed by intermediate voltage electronmicroscopy (IVEM). We found that cytokine-activated EC strongly delay t-PA induced lysis of a fibrin clot by secreting active PAI-1, which is bound to the fibrin strands. Therefore EC seem to be a major reason for “time dependent thrombolytic resistance”, a term used to describe the reduced sufficiency of thrombolytic therapy starting more than 6 hours after the onset of the symptoms of a myocardial infarction. Additionally we present data about the behaviour of EC from different vascular beds and show EC morphology and behaviour under the fibrin clot.