Reduced Fibrinolysis in the Hepatic Venous Occlusive Disease: Effect of Defibrotide on Plasmin Activity.

Introduction: The fibrinolytic activity has been shown to be reduced in many vascular diseases, including hepatic veno-occlusive disease (VOD) after stem cells transplantation (SCT). Defibrotide (DF) is a polydisperse oligonucleotide with antithrombotic, profibrinolytic, anti-ischemic, and anti-adhesive properties. Numerous clinical studies have shown promising results with DF in the treatment of VOD, with minimal toxicity. In laboratory studies, DF has been shown to decrease plasminogen activator inhibitor-1 (PAI-1), increase tissue plasminogen activator (t-PA) levels and increase the overall plasma fibrinolytic activity in patients with VOD. Similar results have been observed in pre-clinical studies with endothelial cells stimulated by lipopolysaccharide. Plasmin is a potent and nonspecific serine protease, generated from the proenzyme plasminogen by plasminogen activators. Plasmin plays a pivotal role in fibrinolysis by virtue of its ability to effectively degrade fibrin clots. The purpose of this study was to investigate whether DF is capable of modulating the activity of plasmin in different in vitro studies.

Method: Plasmin activity was measured in the presence and absence of DF, at different concentrations, by following the cleavage of the chromogenic substrate S-2251. S-2251 contains the preferential cleavage site for plasmin and mimics the fibrin polymer substrate present in fibrin clots. In addition, we evaluated the activity of plasmin generated by t-PA and urokinase-plasminogen activation as well as the plasmin activity in plasma in the presence and absence of DF. Further, plasmin activity generated in fibrin clot plate was measured in the presence and absence of DF. Finally, the inhibition of plasmin by aminocaproic acid was tested in these systems.

Results: DF increased the activity of plasmin, to hydrolyze its substrate, in a dose dependent manner. Similar concentration-dependent effects of DF were observed when plasmin was generated by t-PA or urokinase activation of plasminogen and on plasmin in plasma samples (p<0.001). In contrast, DF had no direct effect in activation of plasminogen to plasmin. We also showed that DF was able to enhance the activity of plasmin to degrade the fibrin clot formed from fibrinogen, plasminogen and thrombin (p<0.001). This effect was concentration dependent and directly correlated with the enzymatic activity of plasmin.

Conclusion: The present study demonstrates that DF is capable of enhancing the activity of plasmin. The stimulation of plasmin by DF was concentration-dependent. These findings can, in part, explain the fibrinolytic activity of DF and support its effect in restoring the fibrinolytic vascular phenotype in conditions such as VOD.