Immunomodulatory Drugs (Thalidomide and Lenalidomide) Up-Regulate Endothelial Tissue Factor in Vitro Resulting in An Acquired Hypercoagulable State.
Authors: Valsami Serena , Leikauf Maria Sybille , Madon Jerzy , Ruf Wolfram , Asmis Lars M 
Published: April 2020

Abstract
Background: The immunomodulatory drugs (IMIDs), thalidomide and lenalidomide, are considered first line treatments for multiple myeloma and are being investigated for an expanding range of indications including myelodysplastic syndrome, chronic lymphocytic leukaemia, and other B-Cell malignancies. One of the adverse effects of IMIDs are venous thromboembolic events (VTE). Tissue factor (TF) is the cellular receptor for clotting factor VII/VIIa and plays a central role in the initiation of coagulation in vivo. Presently there is no consensus regarding the optimal thromboprophylaxis strategy in IMID treated patients. We hypothesize that in the presence of an inflammatory stimulus IMIDs induce a hypercoagulable state through increased endothelial TF expression and activity.
Materials and Methods: Commercially available endothelial cells (HUVEC) were cultured to subconfluent conditions and incubated with thalidomide, lenalidomide or vector control (DMSO) under inflammatory conditions induced by TNFα (10ng/ml). Drug concentrations used were similar to therapeutic human plasma levels. The following parameters were evaluated: TF procoagulant surface activity (colorimetric assay), TF antigen in whole cell lysates (ELISA), and TF mRNA (real time-PCR). Each experiment was repeated at least 3 times. Neutralizing anti TF antibodies were used as a quality control for the functional TF assay. To investigate endothelial cell derived “micro”particles culture supernatants were first centrifuged for 10 minutes at 300g to remove detached whole cells. The resulting supernatant was centrifuged twice at 17000 g for 30min to precipitate potential “micro”particles. Size and surface markers of resuspended particles were investigated by flow cytometry.
Results: In the presence of inflammatory conditions (TNFα 10 ng/ml) IMIDs, thalidomide and lenalidomide (both tested at 0.5μM), increase surface related TF activity by 31% and 16% respectively (p&lt;0.01, Wilcoxon test) compared to inflammatory baseline levels. IMID induced increased TF activity could be specifically inhibited by a cocktail of two monoclonal anti TF antibodies. These findings suggest that TF protein accounts for &gt;95% of the TF activity we observed. TF antigen estimated in whole cell lysates was upregulated by 26% by thalidomide and 24% by lenalidomide (vs TNFα treated cells). To evaluate whether the observed TF surface activity and antigen up-regulation was due to a possible effect of IMIDs on TF gene transcription, TF mRNA was measured by RT-PCR after one and six hours. Compared to TNFα treated cells thalidomide and lenalidomide increased TF mRNA by 53 and 33% at one hour while at six hours the corresponding values were 17 and 18% respectively. Cell culture media from TNF, thalidomide and lenalidomide treated cells contained precipitable particles that when resuspended showed TF activity while media from control cells lacked such activity.
Conclusions: Under inflammatory conditions thalidomide and lenalidomide lead to increased endothelial cell TF synthesis, increased TF antigen and increased activity of surface bound TF. We furthermore detected increased endothelial derived “micro”particles with TF activity indicating an overall increased turnover and shedding of endothelial TF. Our findings need to be reproduced in vivo but may provide insights into the hypercoaglubale state observed in patients treated with IMIDs und thus may be utile in designing efficient thromboprophylaxis strategies.