1. Potential of the Factor Xa Inhibitor Rivaroxaban for the Anticoagulation Management of Patients with Heparin-Induced Thrombocytopenia Jeanine M. Walenga, Walter P. Jeske, Margaret Prechel, Debra Hoppensteadt, Amanda Drenth, Jessica Swank, Meredith McDonald, Luke Sheen, Omer Iqbal, Brian Neville Presented at the XXIst Congress of International Society on Thrombosis and Haemostasis (ISTH) 2007 Meeting, July 6-12th in Geneva, Switzerland.

2. Potential of the Factor Xa Inhibitor Rivaroxaban for the Anticoagulation Management of Patients with Heparin-Induced Thrombocytopenia Approximately 1–5% of patients receiving heparins (unfractionated heparin [UFH] and the low molecular weight heparins [LWMHs]) develop heparin-induced thrombocytopenia (HIT), which is associated with a substantially increased risk of thrombosis HIT is caused by the production of antibodies to a complex of heparin and platelet factor 4 (PF4), and subsequent cross-reaction of these HIT antibodies with heparin–PF4 Background: Walenga JM, et al. Presented at ISTH 2007 in Geneva, Switzerland, abstract #P-M-648.

3. Potential of the Factor Xa Inhibitor Rivaroxaban for the Anticoagulation Management of Patients with Heparin-Induced Thrombocytopenia Current guidelines recommend the use of a direct thrombin inhibitor (DTI) in patients with HIT DTIs administered parenterally are associated with a high bleeding risk, as well as other drug- specific limitations Background: Walenga JM, et al. Presented at ISTH 2007 in Geneva, Switzerland, abstract #P-M-648.

4. Potential of the Factor Xa Inhibitor Rivaroxaban for the Anticoagulation Management of Patients with Heparin-Induced Thrombocytopenia Rivaroxaban is a novel, oral, direct Factor Xa (FXa) inhibitor in advanced clinical development for the prevention and treatment of thromboembolic disorders Rivaroxaban may be suitable for the prevention and treatment of thrombosis in patients with HIT, because it is structurally unrelated to UFH, LMWHs and the heparin-based indirect FXa inhibitor fondaparinux, and, therefore, would not be expected to generate or cross-react with HIT antibodies Background: Walenga JM, et al. Presented at ISTH 2007 in Geneva, Switzerland, abstract #P-M-648.

5. Potential of the Factor Xa Inhibitor Rivaroxaban for the Anticoagulation Management of Patients with Heparin-Induced Thrombocytopenia To evaluate, in vitro, the potential of rivaroxaban as an anticoagulant for the management of patients with HIT Objective: Walenga JM, et al. Presented at ISTH 2007 in Geneva, Switzerland, abstract #P-M-648.

6. Potential of the Factor Xa Inhibitor Rivaroxaban for the Anticoagulation Management of Patients with Heparin-Induced Thrombocytopenia Several drugs were compared to rivaroxaban in the treatment of HIT: –UFH –the LMWH, enoxaparin –fondaparinux –the DTI, argatroban –saline, as a control Methods: Walenga JM, et al. Presented at ISTH 2007 in Geneva, Switzerland, abstract #P-M-648.

7. Potential of the Factor Xa Inhibitor Rivaroxaban for the Anticoagulation Management of Patients with Heparin-Induced Thrombocytopenia Serum was collected from 89 patients with HIT Three functional assays of platelet activation or aggregation were used to determine any cross- reaction between rivaroxaban, or other drugs, and HIT antibodies – 14 C-serotonin release assay (in washed platelets) –Heparin-induced platelet aggregation assay (in platelet- rich plasma; platelet activation defined as >20% aggregation) –Flow cytometric analysis of platelet activation (platelet microparticle formation, platelet aggregation, and platelet P-selectin expression [% positive platelets and mean fluorescence intensity]) in whole blood Methods: Walenga JM, et al. Presented at ISTH 2007 in Geneva, Switzerland, abstract #P-M-648.

8. Potential of the Factor Xa Inhibitor Rivaroxaban for the Anticoagulation Management of Patients with Heparin-Induced Thrombocytopenia Several drugs were compared to rivaroxaban in the treatment of HIT: –UFH –The LMWH, enoxaparin –Fondaparinux –The DTI, argatroban –Saline, as a control Rivaroxaban was tested in a total of 152 different HIT antibody/donor platelet combinations using the three assays Methods: Walenga JM, et al. Presented at ISTH 2007 in Geneva, Switzerland, abstract #P-M-648.

9. Potential of the Factor Xa Inhibitor Rivaroxaban for the Anticoagulation Management of Patients with Heparin-Induced Thrombocytopenia PF4 release assay: –The release of PF4 from platelets was measured by ELISA –Whole blood or platelet-rich plasma was incubated at 37°C for 30 minutes with stirring –The assay was repeated after activation of platelets by tissue factor PF4 binding assay: –The activity of study drugs (anti-FXa activity of rivaroxaban and fondaparinux; anti-thrombin activity of heparins and argatroban) was measured before and after incubation with 10 μg/ml purified PF4 at 37°C for 30 minutes, with stirring Methods: Walenga JM, et al. Presented at ISTH 2007 in Geneva, Switzerland, abstract #P-M-648.

10. Potential of the Factor Xa Inhibitor Rivaroxaban for the Anticoagulation Management of Patients with Heparin-Induced Thrombocytopenia 14 C-serotonin release assay: –Rivaroxaban, fondaparinux and argatroban did not activate platelets in the presence of HIT antibodies i.e. no concentration-dependent increase in activity –As expected, strong, concentration-dependent platelet activation was observed with therapeutic concentrations of UFH and enoxaparin –The absence of platelet activation at supra-therapeutic concentrations is typical of a heparin-dependent, platelet antibody activation response Results: Walenga JM, et al. Presented at ISTH 2007 in Geneva, Switzerland, abstract #P-M-648.

11. 14 C-Serotonin Release Assay Values are shown as mean±SD. Walenga JM, et al. Presented at ISTH 2007 in Geneva, Switzerland, abstract #P-M-648. 100 80 60 40 20 0 00.01 0.11 10100 Concentration (  g/ml or U/ml) Serotonin Release (%) Rivaroxaban Fondaparinux Argatroban Enoxaparin Unfractionated heparin

12. Potential of the Factor Xa Inhibitor Rivaroxaban for the Anticoagulation Management of Patients with Heparin-Induced Thrombocytopenia Heparin-induced platelet aggregation assay: –Rivaroxaban did not activate platelets in the presence of HIT antibodies –Fondaparinux caused activation of platelets in only one of the 18 sera samples tested –UFH and enoxaparin strongly induced platelet activation in 100% and 70% of sera samples, respectively Results: Walenga JM, et al. Presented at ISTH 2007 in Geneva, Switzerland, abstract #P-M-648.

13. Values are shown as mean±SD. Walenga JM, et al. Presented at ISTH 2007 in Geneva, Switzerland, abstract #P-M-648. Heparin-Induced Platelet Aggregation Assay 100 80 60 40 20 0 Saline Aggregation (%) Rivaroxaban Fondaparinux Enoxaparin UFH

14. Potential of the Factor Xa Inhibitor Rivaroxaban for the Anticoagulation Management of Patients with Heparin-Induced Thrombocytopenia Flow cytometric analysis of platelet activation: –Rivaroxaban, fondaparinux and argatroban did not activate platelets in the presence of HIT antibodies (i.e. no concentration-dependent increase in parameters of platelet activation; data not shown) –UFH and enoxaparin induced strong, concentration-dependent platelet activation in the presence of HIT antibodies (data not shown) PF4 release assay: –Rivaroxaban and fondaparinux did not activate platelets, as measured by PF4 release –UFH and enoxaparin caused strong platelet activation Results: Walenga JM, et al. Presented at ISTH 2007 in Geneva, Switzerland, abstract #P-M-648.

15. PF4 Release Assay Values are shown as mean±SD. Walenga JM, et al. Presented at ISTH 2007 in Geneva, Switzerland, abstract #P-M-648. 100 80 60 40 20 0 Saline PF4 Release (ng/ml) Rivaroxaban (1  g/ml) Fondaparinux (1  g/ml) Enoxaparin (5  g/ml) UFH (5  g/ml) 140 120

16. Potential of the Factor Xa Inhibitor Rivaroxaban for the Anticoagulation Management of Patients with Heparin-Induced Thrombocytopenia PF4 binding assay: –The anti-FXa activity of rivaroxaban and fondaparinux was unchanged after incubation with PF4, as was the anti-thrombin activity of argatroban –The anti-thrombin activity of UFH and enoxaparin was decreased by 64% and 43%, respectively Results: Walenga JM, et al. Presented at ISTH 2007 in Geneva, Switzerland, abstract #P-M-648.

17. PF4 Binding Assay Values are shown as mean±SD. Walenga JM, et al. Presented at ISTH 2007 in Geneva, Switzerland, abstract #P-M-648. 70 60 50 40 30 20 10 0 Saline Rivaroxaban (1  g/ml) Fondaparinux (1  g/ml) Enoxaparin (5  g/ml) UFH (5  g/ml) Argatroban (1  g/ml) Anti-Xa or Anti-IIa Activity (% inhibition) Control PF4

18. Potential of the Factor Xa Inhibitor Rivaroxaban for the Anticoagulation Management of Patients with Heparin-Induced Thrombocytopenia Unlike UFH and enoxaparin, Rivaroxaban –Did not cross-react with HIT antibodies –Did not interact with PF4 –Did not mobilize PF4 from platelets Rivaroxaban may be considered an alternative anticoagulant for the management of patients with HIT Conclusions: Walenga JM, et al. Presented at ISTH 2007 in Geneva, Switzerland, abstract #P-M-648.

19. Potential of the Factor Xa Inhibitor Rivaroxaban for the Anticoagulation Management of Patients with Heparin-Induced Thrombocytopenia Rivaroxaban, if used as the initial anticoagulant in place of heparin, may not initiate HIT antibody production Rivaroxaban, administered orally, could be used for long-term secondary prevention after an acute episode of HIT Conclusions: Walenga JM, et al. Presented at ISTH 2007 in Geneva, Switzerland, abstract #P-M-648.

20. Potential of the Factor Xa Inhibitor Rivaroxaban for the Anticoagulation Management of Patients with Heparin-Induced Thrombocytopenia Further clinical investigation is required to confirm the potential of rivaroxaban as an alternative anticoagulant for the management of patients with HIT Rivaroxaban may offer certain advantages over currently approved DTIs in patients with HIT, due to its –Oral route of administration –Wide therapeutic window –And potentially improved bleeding profile Conclusions: Walenga JM, et al. Presented at ISTH 2007 in Geneva, Switzerland, abstract #P-M-648.