Administration Of rTM Research Articles

Recombinant human soluble thrombomodulin safely and effectively rescues acute promyelocytic leukemia patients from disseminated intravascular coagulation

We treated individuals for disseminated intravascular coagulation (DIC) caused by acute promyelocytic leukemia (APL) (n=9) using human soluble thrombomodulin (rTM) in combination with all-trans retinoic acid (ATRA) and chemotherapy, and compared the clinical outcomes with historical control patients (n=8) treated with ATRA and/or chemotherapy. Two control patients developed intracranial vascular incidents. On the other hand, no bleeding related mortality was noted in rTM-treated patients. Notably, treatment with rTM rescued patients from DIC earlier than historical controls (log rank test, p=0.019). These results suggest that administration of rTM should be considered for the treatment of individuals with DIC associated with APL.

Thrombomodulin enhances the antifibrinolytic and antileukemic effects of all–trans retinoic acid in acute promyelocytic leukemia cells

This study found that levels of thrombomodulin (TM) were downregulated in freshly isolated leukemia cells from patients with acute promyelocytic leukemia (APL, n = 7) and acute myelogenous leukemia (n = 14), as compared with CD34(+)/CD38(-) hematopoietic stem/progenitor cells and CD34(-)/CD33(+)/CD11b(-) promyelocytes isolated from healthy volunteers (n = 3). Exposure of APL NB4 cells to recombinant human soluble TM (rTM, 1500 ng/mL) inhibited clonogenic growth of these cells by approximately 30%, and induced expression of CD11b, a marker of myeloid differentiation, on their surfaces, in association with upregulation of nuclear levels of myeloid-specific transcription factor CCAAT/enhancer binding protein ε. These antileukemic effects of rTM were mediated by thrombin/activated protein C-dependent mechanisms, as hirudin, an inhibitor of thrombin and a blocking antibody against endothelial receptor for protein C to which activated protein C binds, hampered the ability of rTM to induce expression of CD11b in NB4 cells. This study also found that rTM downregulated expression of Annexin II, a receptor for both plasminogen and tissue plasminogen activator, and inhibited plasmin activity in APL cells. Interestingly, rTM significantly enhanced the ability of all-trans retinoic acid to induce growth arrest, differentiation and apoptosis, and inhibited plasmin activity in APL cells. Taken together, these results suggest that administration of rTM should be considered for treatment of individuals with disseminated intravascular coagulation associated with APL.

Recombinant Human Soluble Thrombomodulin Enhances the Anti-Fibrinolytic and Anti-Leukemia Effects of All-Trans Retinoic Acid In Acute Promyelocytic Leukemia Cells.

AbstractAbstract 1079Recombinant human soluble thrombomodulin (rTM) is composed of the active, extracellular domain of thrombomodulin, and inactivates coagulation by binding to thrombin. In addition, the thrombin-rTM complex activates protein C to produce activated protein C (APC), which in the presence of protein S, inactivates factors VIIIa and Va, thereby inhibiting further thrombin formation. Use of rTM for treatment of disseminated intravascular coagulation (DIC) associated with haematological malignancies or infections has been approved in Japan, although its effects on hyperfibrinolysis caused by acute promyelocytic leukemia (APL), as well as all-trans retinoic acid (ATRA)-mediated anti-fibrinolytic activity, remain to be fully elucidated. This study examined the effects of rTM on the plasmin activity in APL NB4 cells in vitro and found that rTM (100 nM, 48 h) alone inhibited the plasmin activity by approximately 35% when compared to the control diluent-treated NB4 cells. ATRA (100 nM, 48 h) also inhibited the plasmin activity by 40%, and when ATRA (100 nM, 48 h) was combined with rTM (100 nM, 48 h), the plasmin activity was inhibited by 60%. In further studies, neither anti-proliferative nor pro-differentiative activity was noted in NB4 cells after exposure to rTM (3-100 nM, 48 h). However, rTM significantly enhanced the ability of ATRA to induce growth arrest, differentiation and apoptosis in NB4 cells, as measured by colony forming assay, quantification of CD11b cell surface antigens and nitroblue tetrazolium reduction test, respectively. Western blot analyses showed that ATRA (30 or 100 nM, 48 h) increased nuclear levels of CCAAT/enhancer binding protein e in NB4 cells, a critical transcription factor for myeloid differentiation, which was further increased in the presence of rTM (3-100 nM, 48 h). Furthermore, we treated individuals with DIC caused by APL (n=4) with rTM (380 U/kg) in combination with ATRA (45 mg/m2) and chemotherapy (idarbicine and cytarabine), and compared their clinical outcomes with historical control patients (n=6) treated with ATRA and/or chemotherapy (idarbicine and cytarabine). Notably, treatment with rTM rescued patients from DIC earlier than historical controls (8.3 ± 4.5 days vs 12.5 ± 5.2 days in historical controls) and significantly reduced the transfusion of fresh frozen plasma to maintain plasma levels of fibrinogen at more that 150 mg/dl (0.13 ± 0.25 U/day vs 3.93 ± 1.18 U/day in historical control, p=0.0131). Taken together, the present results suggest that administration of rTM be considered in the treatment of individuals with DIC associated with APL. Disclosures:Honda:Asahi Kasei Pharma: Employment.

Prophylaxis Against Endothelial Damage Using Recombinant Human Soluble Thrombomodulin (rTM) In Allogeneic Hematopoietic Stem Cell Transplantation (allo-HSCT).

AbstractAbstract 4523Thrombomodulin (TM) is a vascular endothelial cell protein which controls coagulation. Recombinant human soluble thrombomodulin (rTM) is a new agent for treatment of disseminated intravascular coagulation. rTM also exhibited anti-inflammatory effects and ameliorated endothelial injuries in our pilot study where rTM was administered to a patient during cord blood transplantation (CBT) as a prophylaxis against veno-occulsive disease (VOD). Major complications following allogeneic hematopoietic stem cell transplantation (allo-HSCT) triggered by endothelial damage not only include VOD, but also thrombotic microangiopathy (TMA). Two case reports showed that rTM dramatically ameliorated VOD and TMA (Bone Marrow Transplant. 2010;45:783 and 803, respectively) following allo-peripheral blood stem cell transplantation (PBSCT). In the present study, we examined the effectiveness of rTM as a prophylaxis against endothelial damage in patients receiving allo-HSCT.We evaluated seven patients (age range 50–72 y; median 61 y) with hematological malignancies receiving allo-HSCT at our institute from July 2009 to February 2010. Each patient provided written informed consent prior to inclusion in the study. One patient received conventional SCT, and the others received reduced-intensity SCT using a fludarabine-based conditioning regimen. Two patients were transplanted from related PBSC and the others were transplanted from unrelated CB using rabbit anti-thymoglobulin (rATG) before transplantation as a prophylaxis against graft-versus-host disease (GVHD). Four patients received rTM as a prophylaxis against endothelial damage from day −6 to day 30 (long period) based on the day they received transplantation, and three patients received rTM from day 6 to day 19 (short period) and heparin on the other days. IL-1β, IL-6, IL-8, TNFα, HMGB1, RANTES, SDF-1, E-selectin, and VCAM-1 levels were measured using ELISA before and after rTM administration, and changes were evaluated using a two-tailed Wilcoxon test.IL-1β (p=0.018), IL-6 (p=0.018), IL-8 (p=0.018), TNFα (p=0.017), RANTES (p=0.017), HMGB1 (p=0.018) decreased significantly following rTM administration. No significant change in CRP level, considered to affect cytokine measurements, was observed (p=0.063), and neither E-selectin nor VCAM-1 level increased. No significant differences were observed between changes in these parameters among patients treated with rTM during the long and short periods. All patients achieved engraftment by the mean post-transplantation day as previously reported (range 16–29 days; median 22 days). Of the seven patients, only one developed VOD, and no patients developed TMA or acute GVHD. Pre-engraftment immune reaction was not observed in any patients who underwent CBT. Four patients were alive and three patients were deceased as of July 30, 2010, with main causes of death being cerebral hemorrhage, VOD and relapse of primary disease, respectively. Median event-free survival and overall survival after transplantation were 54 days (26–283 days) and 168 days (55–283 days), respectively.rTM showed potent inhibitory effects on inflammatory cytokines, including TNFα, IL-1β, IL-6, IL-8, and prevented the release of HMGB1 through absorption of alarmin. It has been reported that rATG contributes to an increase in inflammatory cytokines; however, rTM treatment prohibited these increases among patients receiving CBT. We previously observed increased levels of E-selectin and VCAM-1 after allo-HSCT (Biol Blood Marrow Transplant. 2008;14:766); however, rTM prohibited these increases in the present study, indicating that it prevents allo-HSCT-induced endothelial injuries. One patient died of cerebral hemorrhage 26 days after the end of prophylactic administration of rTM. An association between this major bleeding event and rTM administration is unlikely because the t1/2 of rTM is approximately 20 h. One patient developed fatal VOD, despite controlled cytokine levels. This patient underwent CBT on primary disease progression. We speculated that disease progression in this case contributed to the development of VOD in spite of prophylactic administration of rTM. Although this study involved a small number patients and median follow-up was relatively short, given the present findings, rTM may prevent VOD and TMA following allo-HSCT. Disclosures:No relevant conflicts of interest to declare.

Recombinant human thrombomodulin inhibits arterial neointimal hyperplasia after balloon injury

Clinical relevance Results of angioplasty/stenting and surgical bypass remain hindered by restenosis and thrombosis. Intravenous administration of thrombomodulin (TM) demonstrated a multifactorial effect resulting in significant inhibition of neointimal hyperplasia and thrombus formation following balloon injury in a small-caliber artery model. This, along with TM being an endogenous glycoprotein expressed on the luminal surface of the normal vessel wall, makes it an attractive target to exploit in modulating the pathologic arterial response to injury with less risk of toxicity. Administration of rTM intravenously has direct clinical applicability with additional efforts toward local delivery and modification of synthetic bioprostheses.