Influence of Edoxaban on Coagulability and Thrombin Generation: An in Vitro Study Focusing on Thrombelastography

Experimental protocol

1. On the day of experiment blood samples will be collected in 3.2% citrate tubes.

2. One citrated blood tube will be centrifuged to collect plasma for biomarker
measurements (C-reactive protein (CRP), fibrinogen, von Willebrand factor (vWF),
interleukin (IL)-6, p-selectin, plasminogen activator inhibitor (PAI)-1, matrix
metalloproteinase (MMP)-9).

3. Blood samples will be incubated with different concentrations of edoxaban (no edoxaban,
subtherapeutic range - 30 nM, therapeutic range -300 nM, and supratherapeutic range-
900 nM) (3).

A) Cora® Hemostasis Analyzer System (Cora®) will be used to assess qualitative and
quantitative assessment of the hemostatic properties of a blood sample in the presence or
absence of edoxaban. The CORA is an integrated computer module with Ethernet connection
capability and provides continuous resonance-frequency viscoelasticity measurements using a
disposable four-channel microfluidic cartridge to determine simultaneous maximal
platelet-fibrin clot strength, fibrin clot strength, and response to antiplatelet agents or
anticoagulants. The cartridge has four channels - citrated Kaolin (CK) channel that measures
platelet-fibrin clot strength, anti-Xa channel, DTI channel and FFC channel that measures
contribution of functional fibrinogen.

In addition, using the V-curve software, the following parameters of thrombin generation
kinetics will be evaluated from the CK channel- R - Period of time of latency from the time
that the blood was placed in the TEG® analyzer until the initial fibrin formation. This
represents the enzymatic portion of coagulation.

K - K time is a measure of the speed to reach a certain level of clot strength. This
represents clot kinetics.

alpha - measures the rapidity of fibrin build-up and cross-linking (clot strengthening).
This represents fibrinogen level.

MA - Maximum Amplitude is a direct function of the maximum dynamic properties of fibrin and
platelet bonding via GPIIb/IIIa and represents the ultimate strength of the fibrin clot.
This represents platelet function/aggregation.

TMRTG - Time to maximum rate of thrombus generation. MRTG - Maximum rate of thrombus
generation. TG - Total thrombus generated. TMRL - Time to maximum rate of lysis MRL -
Maximum rate of lysis L - Total lysis D - Delta is the difference between R time and the
time of initial split point (SP, mins) of the TEG tracing (R - SP), representing the time
interval of greatest clot growth secondary to peak thrombin generation.

B) Calibrated Automated Thrombogram® (CAT) System: Lag time, peak thrombin production, mean
velocity rate index and endogenous thrombin potential (ETP) will be assessed by calibrated
automated thrombogram in platelet poor plasma (Thrombinoscope by Stago).

Inclusion Criteria:

Patients with heart failure (class I-IV) are eligible for enrollment:

1. Patient must have documented symptomatic chronic HF for at least 3 months prior to
screening. Exacerbation of chronic HF is defined as symptoms of worsening dyspnea or
fatigue, objective signs of congestion such as peripheral edema or ascites, and/or
adjustment of pre-hospitalization HF medications.

2. Subject must have a documented LVEF of less than or equal to 40% within 3 months of
the study. If more than one LVEF is available, the most recent one should be used,
but it must be less than or equal to 40%. The ejection fraction will be determined by
one of the following methods: echocardiogram, nuclear multigated acquisition (MUGA)
scan, cardiac MRI, cardiac CT scan, or left ventriculography.

3. Patient must be receiving appropriate HF treatment at the appropriate dosing per
guidelines:

- Diuretic (required for study entry) Renin-angiotensin system (RAS) inhibitors
such as an ACE inhibitor, or ARB if intolerant of ACE inhibitor, or vasodilator
therapy such as hydralazine or nitrates if intolerant to ACE inhibitor and ARB

- Beta blocker therapy

- Aldosterone antagonist therapy.

4. Patient must have completed all prophylactic anticoagulation (such as enoxaparin,
warfarin, heparin, etc) for at least one week before study.

5. Each patient (or their legally acceptable representative) must sign an informed
consent form (ICF) indicating that he or she understands the purpose of and is
willing to participate in the study.

Exclusion Criteria:

For Healthy Subjects:

subjects currently on antiplatelet therapy or any other agents that are known to influence
platelet function and coagulation.

For Subjects with Heart Failure:

1. Hemodynamic instability, active bleeding and bleeding diatheses, oral anticoagulation
therapy, leukocyte count < 3,000/mm3, platelet count < 100,000/mm3, aspartate
aminotransferase or alanine aminotransferase levels ≥ 3 times upper normal, and
creatinine >2mg/dL.

2. Patient has a severe concomitant disease such as: Atrial fibrillation (AFib) or
another condition that requires chronic anticoagulation.