B. R. Huebner1,3, C. C. Silliman1,2,4, E. Gonzalez1,3, S. Mitra1, A. Banerjee1, E. E. Moore1,3 1University Of Colorado Denver,Department Of Surgery,Aurora, CO, USA 2University Of Colorado Denver,Department Of Pediatrics,Aurora, CO, USA 3Denver Health Medical Center,Aurora, CO, USA 4Bonfils Blood Center,Denver, CO, USA
Introduction:
Recently, there’s evidence that injured patients exhibit diverse phenotypes ranging from hyperfibrinolysis coagulopathy to fibrinolysis-shutdown resulting in microvascular occlusion and multi-organ failure. The primary mediators of these phenotypes are tissue plasminogen activator (tPA), a driving force behind hyperfibrinolysis, and plasminogen activator inhibitor-1 (PAI-1), a serine protease inhibitor that couples tPA nullifying its physiologic effect. Endothelial cells activate during stress and are capable of releasing tPA and PAI-1, but their role in critically injured patients remains unclear. Thrombin is a key agent which can stimulate shutdown or enhancement of fibrinolysis. While it is known that tPA is stored in Weibel-Palade bodies in endothelium, the timing and source of PAI-1 remains unclear. We hypothesize that thrombin causes PAI-1 release from preformed vesicles in endothelium thus serving as a rapid counter-regulation of tPA release.
Methods:
Human liver sinusoidal endothelial cells (HLSECs) were grown to an 80-90% confluence in wells after passage 3. HLSECs were treated with thrombin at a concentration of 5units/ml known to be present in the plasma of trauma patients. Supernatant samples were collected at 15 and 30 minute timepoints. Each timepoint was run in duplicate. The supernatants were analyzed with ELISAs for total (free) tPA, total (free) PAI-1, and tPA-PAI-1 complex.
Results:
Thrombin-stimulated endothelial cells released more PAI-1 than the control (>600ng/ml for thrombin 15 min and 30 min vs 441ng/ml control). Free tPA was lower in the thrombin treated cells (0.781ng/ml at 15m, 0.617ng/ml at 30m, 0.977ng/ml control). tPA-PAI-1 complex values were slightly higher from the control at 15min and lower at 30min for the thrombin-treated cells (10.95ng/ml for 15m, 8.56ng/ml for 30m, and 10.2ng/ml control).
Conclusion:
Thrombin provokes the rapid release of PAI-1 from hepatic endothelium with the subsequent formation of tPA-PAI-1 complexes causing a decrease in free tPA and an increase in PAI-1 resulting in fibrinolysis shutdown. Endothelial cells are important in contributing to the fibrinolysis phenotype seen in severely injured patients.
