Abstract 483: Clot Degradation Assay for the Analysis of Vascular Bed Fibrinolytic Reactivity

Abstract

Introduction Pulmonary emboli cause significant morbidities and risk of mortality to affected patients, yet the mechanism behind their formation is still elusive. A greater understanding of clot formation and fibrinolysis in the pulmonary artery (PA) and iliac vein (IV) vascular beds has become an important area of research in order to find better therapeutics for the treatment of deep vein thrombi and pulmonary emboli. The aim of this study was to develop a novel in vitro clot degradation assay for the comparison of fibrinolytic reactivity of different vascular beds. Methods Thrombin was added to fibrinogen at 37 ° C for 24 hours and the resulting fibrin clot was centrifuged, dried and weighed. Human PA and IV endothelial cells were incubated for 24 hours and cellular extract and media samples were collected and evaluated for their ability to degrade fibrin clots in the presence of plasminogen. Fibrinolysis, stimulated by plasmin formed by endothelial cell derived plasminogen activator, was measured as percent clot degradation by weight. Results Fibrinogen and thrombin dose responses were completed to find the appropriate concentrations of each to use for clot formation: fibrinogen (6.04x10 -6 M) and thrombin (0.0005 NIH units). To validate the clot degradation assay, a uPA dose response was conducted from 150fM to 15nM uPA and demonstrated that each ten-fold increase in uPA concentration corresponded to an average of 14% degradation (p<0.05, n=3) up to 72.5% degradation in the presence of 15 nM uPA. While PA cell extracts showed no effect on clot degradation, IV cell extracts increased degradation by 30%. However, when clots were exposed to PA media there was a 14% increase in clot degradation (p<0.05, n=3), whereas the corresponding IV media samples had no effect on clot weight. Conclusions This study demonstrates the development of a novel and reproducible methodology to examine fibrinolytic reactivity in cell culture samples. Endothelial cells from the pulmonary artery circulation exhibit greater ability to extrinsically degrade fibrin clots when compared to endothelial cells from the iliac venous bed which may explain, in part, why more clots are found in the deep veins and not the pulmonary vasculature.