PB1351 - Altered haemostatic behaviour of endothelial colony-forming cells from patients with the truncating thrombomodulin variant Pro496Argfs*10 and a bleeding phenotype

Background: Thrombomodulin (TM; encoded by THBD) is an anticoagulant, transmembrane glycoprotein, predominantly expressed on the surface of vascular endothelial cells (ECs). We previously identified a frameshift mutation in the THBD gene in two related patients with elevated plasma soluble TM (sTM) levels and an unexplained bleeding disorder.

Aims: To investigate the impact of endothelial colony-forming cells (ECFCs) cultured from the two patients harbouring the TM mutation (MT-TM) on clot lysis and thrombin generation.

Methods: ECFCs were isolated and cultured from five wildtype TM (WT-TM) donors and two MT-TM patients. Turbidometric clot lysis assays were performed in the absence and presence of WT-TM and MT-MT ECFCs using 30% pooled normal plasma (PNP), 16μM phospholipids, 50pM tPA, 0.1U/ml thrombin and 10.8mM Ca2+ in TBS. Fluorometric thrombin generation (TG) assays were performed using PNP and 1pM tissue factor. ELISA was used to determine the levels of soluble TM (sTM) in cell culture supernatants.

Results: WT-TM ECFCs prolonged clot lysis time by 30min (p < 0.0001) compared to wells devoid of ECFCs (nude wells). This increase was partially reversed by an anti-TM antibody that binds the thrombin-interacting EGF5 domain of TM. Clot lysis on MT-TM ECFCs was prolonged albeit to a lesser extent (17min increase; p< 0.0001) than on WT-TM ECFCs and fully reversed by anti-TM. TG was significantly diminished to a similar degree in the presence of WT-TM and MT-TM ECFCs compared to nude wells. This decrease in TG was attenuated by anti-TM and protein C depletion. The levels of sTM in the supernatants of MT-TM ECFCs was markedly higher than in those of WT-TM ECFCs (343pg/ml vs 111pg/ml; p< 0.0001).

Conclusion(s): Collectively, the data suggest that the reduced capacity of ECs to prolong clot lysis due to membrane TM shedding may underlie the bleeding abnormality observed in the patients with this TM variant.