Abstract

Introduction: Patients with Multiple myeloma are at relatively high risk of developing thromboembolic events, usually deep vein thromboses (DVT). There are numerous contributing factors, including therapy, such as thalidomide, where DVT has been identified as a major toxicity, especially when thalidomide is used in combination with other treatments such as dexamethasone. The mechanisms by which thalidomide predisposes to thrombosis are not well understood. Defibrotide (DF) is an orally biovailable polydisperse oligonucleotide with anti-thrombotic, pro-fibrinolytic and anti-adhesive properties. Previously, DF has been shown to dose-dependently counteracted the increase in Plasminogen Activator Inhibitor-1 (PAI-1) expression and decrease on tissue plasminogen activator (t-PA) activity after lipopolysaccharide (LPS) stimulation of endothelial cells in vitro.

Methods and Results: We have conducted in vitro studies using human microvascular endothelial cells (HMEC) in order to investigate the effect of different doses of thalidomide on various fibrinolytic factors. In addition, we evaluated whether DF modulates changes of fibrinolysis induced by thalidomide. HMEC were treated with 50 and 100μg/ml of thalidomide for 24 hours in presence and absence of DF (at a dose of 150μg/ml). t-PA and PAI-1 gene expression were evaluated through real time polymerase chain reaction (RT-PCR) of cDNA prepared from HMEC. Release of t-PA and PAI-1 were evaluated by imunoenzymatic assay (ELISA). Furthermore, we evaluated the fibrinolytic activity of cell surpernatant using a fibrin clot plate assay. In this method the fibrin clot was formed by mixing fibrinogen, plasminogen and thrombin. The plasmin generated by the cell surpernatant was able to digest fibrin and also hydrolyzed the chromogenic substrate S-2251. The RT-PCR results showed that thalidomide reduces t-PA (2.2 fold) and increases PAI-1 gene expression (4.0 fold) in HMEC cells, whereas DF was able to counteract this effect by up-regulating the t-PA and down-regulating PAI-1 gene expression induced by thalidomide (8.8 and 2.0 fold, respectivielly). Similar results was observed analyzing t-PA release by HMEC cells treated with different concentrations of thalidomide with and without DF. Thalidomide significantly reduces the t-PA released in both concentrations (p<0.001) and DF significantly increase the release of t-PA reduced by thalidomide (p<0.01). The changes of fibrinolytic activity in HMEC by thalidomide and the capacity of DF to restore the fibrinolysis was confirmed by analyzing the lyses of fibrin clots with endothelial cell surpernatant (p<0.01).

Conclusions: These results show that DF is able to counteract the alterations of fibrinolytic factors in HMEC treated with thalidomide. Whilst further studies in preclinical MM models are underway, these data suggest a potential role for DF in the prevention of DVT induced by thalidomide and support ongoing clinical trials of DF in combination with thalidomide-based treatment.

Author notes

Disclosure:Employment: Cinara Echart; Barbara Graziadio; Cinzia Repice and Massimo Iacobelli are full time employees of Gentium. Membership Information: Kenneth Anderson is member of the Board of Directors of Gentium.