Stroke Risk Assessment and Markers of Blood Clotting in Patients With Newly Diagnosed Non-valvular Atrial Fibrillation (NVAF), Who Have Not Received Oral Anticoagulation Therapy (OAC-therapy) Prior to Inclusion

Background:

Non-valvular atrial fibrillation (NVAF) is the most prevalent cardiac arrhythmia worldwide, and it is associated with a five-fold increased risk of ischemic stroke compared to a healthy population. Patients with stroke due to atrial fibrillation (AF) have a higher mortality and greater disability compared to stroke patients without AF.

Oral anticoagulants (OACs), either vitamin K antagonists (VKAs) or direct oral anticoagulants (DOACs), are the cornerstones for the prevention of ischemic strokes in patients with AF. Different stroke risk stratification schemes have been developed to guide OAC therapy decision-making in AF patients. The most commonly used scheme is the CHA2DS2-VASc score, including the clinical risk factors congestive heart failure, hypertension, age, diabetes, prior stroke, vascular disease, and female gender. One point is assigned for every risk factor encompassed by the CHA2DS2-VASc score, except age ≥75 years and prior stroke, which account for two points each. The score ranges from 0-9 points, with higher scores representing a higher risk of stroke. The annual risk of ischemic stroke for CHA2DS2-VASc score of 0, 1, and 2 is approximately 0-0.68 %, 1.3-1.61 %, and 2.2-2.49 %, respectively. Novel stroke risk stratification schemes, also incorporating biomarkers, have been developed, e.g., the ABC-stroke score (Age, Biomarkers, and Clinical history). The components of the ABC-stroke score include age, plasma levels of N-terminal pro-B-type Natriuretic Peptide (NT-proBNP) and high-sensitivity troponin T (hs-TNT), and prior stroke/transient ischemic attack (TIA). ABC-stroke risk categories are defined as low-risk (<1 % predicted one-year risk of stroke), medium-risk (1-2 % predicted one-year risk of stroke), and high-risk (>2 % predicted one-year risk of stroke). Studies have shown that the ABC-stroke score outperforms the CHA2DS2-VASc score in terms of stroke risk prediction and stratification of AF patients. However, the CHA2DS2-VASc score is still recommended as the primary stroke risk stratification scheme for AF patients in current guidelines. Despite the broad use of stroke risk stratification schemes in clinical practice, stroke risk assessment remains particularly challenging in AF patients with intermediate stroke risk, i.e., men with a CHA2DS2-VASc score of 1 and women with a CHA2DS2-VASc score of 2, where current guidelines recommend considering OAC therapy. OAC therapy is generally recommended for AF patients at high risk of stroke, i.e., men with CHA2DS2-VASc score of ≥ 2 and women with CHA2DS2-VASc score of ≥ 3. OAC therapy is generally not recommended for AF patients at low risk of stroke, i.e., men with a CHA2DS2-VASc score of 0 and women with a CHA2DS2-VASc score of 1.

The CHA2DS2-VASc score is broadly used in clinical practice due to its simplicity and low cost, however, the score has limitations. It exclusively includes clinical stroke-related risk factors, but not AF burden and left atrial size/function, which are proven independent markers of a hypercoagulable state. Treatment and stroke risk assessment in AF patients is moving towards a more individualized patient care, in which biomarkers play an essential role in improving risk stratification and personalizing treatment, as proposed in the ABC-stroke score. In recent years, measurements of hemostatic biomarkers in AF patients have been investigated in several studies, illustrating a hypercoagulable state. Of note, the CHA2DS2-VASc score also predicts stroke risk in patients without AF. Hence, there are concerns about the specificity for predicting AF-related stroke versus non-AF related stroke. Furthermore, the CHA2DS2-VASc score does not consider whether the modifiable components are well-controlled or not, which could lead to OAC overtreatment. For these reasons, there is still a need for refining stroke risk prediction using more AF-sensitive factors (i.e., AF-burden and left atrial size/function), in addition to the clinical factors, and for more exhaustive assessment of the modifiable components, to optimize stroke risk stratification in the future. It remains to be established whether the abovementioned AF-sensitive factors can be used to improve OAC decision-making, particularly in NVAF patients with intermediate stroke risk, where the balance between benefit and harm is less clear. When doubt persists, the presence of one or more non-CHA2DS2-VASc stroke risk factors could possibly strengthen the decision to initiate OAC therapy.

The annual risk of ischemic stroke in NVAF patients depends on the overall CHA2DS2-VASc score. However, the annual risk of ischemic stroke in patients with NVAF may differ depending on the individual components of the CHA2DS2-VASc score that determine the score, despite the overall score being the same, as well as whether the modifiable components are well-controlled or not. Glowicki et al. conducted a study comparing the hemostatic profiles of AF patients with low stroke risk to those with intermediate stroke risk, as determined by the CHA2DS2-VASc score. As opposed to the study by Glowicki et al., this study will compare all three stroke risk groups, i.e., low, intermediate, and high risk, as determined by the CHA2DS2-VASc score and ABC-stroke score, and will incorporate a broader panel of hemostatic biomarkers, including the contact activation system. Furthermore, this study will investigate AF-burden and left atrial size/function, and their association with the hemostatic profile. Patients included in this study will be genuinely OAC-naïve, as opposed to the study by Glowicki et al. Comparison of hemostatic profiles between different stroke risk groups could be essential for the future improvement of OAC therapy decision-making, especially in NVAF patients with intermediate stroke risk.

Objectives:

• To evaluate how the individual components of the CHA2DS2-VASc score and ABC-stroke score are associated with hemostatic biomarkers, including an assessment of whether the modifiable components are well-controlled or not, and how the hemostatic profile differs among stroke risk groups in OAC-naïve NVAF patients.
• To compare the hemostatic profiles of the different CHA2DS2-VASc scores and ABC-stroke scores in OAC-naïve NVAF patients.
• To evaluate how AF-burden is associated with hemostatic biomarkers in OAC-naïve NVAF patients.
• To evaluate how left atrial size/function is associated with hemostatic biomarkers in OAC-naïve NVAF patients.
• To evaluate how underlying heart failure with preserved ejection fraction (HFpEF) is associated with hemostatic biomarkers in OAC-naïve NVAF patients.

Materials and methods:

Study Design: The study consists of five substudies. The first substudy will investigate the association of the different CHA2DS2-VASc score and ABC-stroke score components with the hemostatic profile in OAC-naïve NVAF patients. Furthermore, assessment of whether the modifiable components of the CHA2DS2-VASc score (i.e., diabetes and hypertension) are well-controlled or not, and its impact on the hemostatic profile will be investigated. Likewise, the hemostatic profile of OAC-naïve NVAF patients with intermediate stroke risk will be compared to that of OAC-naïve NVAF patients with low and high stroke risks, respectively, to determine which of the two groups most closely resembles patients with intermediate stroke risk in terms of their hemostatic profile. The second substudy will compare the hemostatic profile between the CHA2DS2-VASc score and ABC-stroke score in OAC-naïve NVAF patients. The third substudy will investigate how AF-burden is associated with hemostatic biomarkers in OAC-naïve NVAF patients. AF-burden will be determined as a percentage based on the total number of AF-events during seven days heart rhythm monitoring. In the fourth substudy, investigators will evaluate how left atrial size/function is associated with hemostatic biomarkers in OAC-naïve NVAF patients. In the fifth substudy, investigators will investigate how underlying HFpEF is associated with hemostatic biomarkers in OAC-naïve NVAF patients. HFpEF will be evaluated using the HFA-PEFF and H2FPEF scores. The study is a cross-sectional, single-center observational study and will take place at Esbjerg Hospital - University Hospital of Southern Denmark, involving collaboration between the Unit for Thrombosis Research, Department of Clinical Diagnostics and the Department of Cardiology.

Study population: The study population will consist of patients with newly diagnosed NVAF. Patients will have to be naïve to oral and parenteral anticoagulants prior to inclusion. Investigators will check for this upon inclusion by systematic screening of an online medication database, called FMK, which is widely used among health care personnel in Denmark. According to the annual report (2023) from the AF database in Denmark (AFDK), there are about 800 patients with newly diagnosed AF in the catchment area of the University Hospital of Southern Denmark, Esbjerg, why investigators are confident that it will be possible to include the necessary number of patients. Collaboration with the general practitioners will be essential for patient inclusion. Investigators will provide comprehensive information to general practitioners about this study and the importance of withholding OAC treatment before subacute referral to the Department of Cardiology. Patients with newly diagnosed NVAF who are willing to participate in this study and sign the patient consent form will be scheduled for fast track outpatient clinic visit within four days of their consultation with the general practitioner for blood sampling, transthoracic echocardiography (TTE), and heart rhythm monitoring. OAC treatment will be initiated immediately after blood sampling, based on current guidelines for the management of AF, and investigators will also do a tailored comprehensive work-up of the AF patients as necessary. Demographic data will be collected, as well. Likewise, symptoms attributable to AF will be quantified according to the modified EHRA-score (European Heart Rhythm Association). To exclude other causes of coagulopathy and characterize the patients at baseline, investigators will measure the following variables in each participant: Full blood count, Activated Partial Thromboplastin Time (APTT), International Normalized Ratio (INR), renal function, Glycated Hemoglobin (HbA1c), C-reactive Protein (CRP), liver function, and lipid profiles. Furthermore, plasma levels of NT-proBNP and hs-TNT will be determined with high-sensitivity immunoassays to estimate the ABC-stroke score. The risk of thromboembolic events will be estimated using the CHA2DS2-VASc score and ABC-stroke score for all patients in this study population, and the patients will be grouped according to their overall individual score. Moreover, each group will be subdivided according to the individual components of the CHA2DS2-VASc score and ABC-stroke score that constitute the overall score. Investigators will determine whether hypertension and diabetes are effectively managed by conducting home blood pressure (BP) measurements and assessing HbA1c levels. BP will be measured at home three times in the morning and evening over three consecutive days, and mean BP will be calculated based on the measurements from days two and three. For simplicity, well-controlled hypertension will be defined as a BP ≤ 135/85 mmHg, and a systolic BP ≤ 145 mmHg for age groups < 80 years and ≥ 80 years, respectively. The treatment goals for type 1 and 2 diabetes patients are HbA1c ≤ 53 and ≤ 48 mmol/mol, respectively, and will be used as thresholds for whether diabetes is well controlled or not.

Sample size estimation and statistical analyses: For sample size calculation, investigators utilized the median values and interquartile ranges of endogenous thrombin potential (ETP), as previously compared between AF patients with low and intermediate stroke risk by Glowicki et al. If the true difference of ETP between the low and intermediate stroke risk means is 120 nmol/L x min., and if investigators want to achieve power of 80 %, the sample size of the smallest group needs to contain at least 18 patients. According to the annual report (2023) from AFDK, approximately 12 % of incident AF patients in the Region of Southern Denmark were low-risk, 30 % were intermediate-risk, and 58 % were high-risk, as estimated by the CHA2DS2-VASc score. Given this distribution, investigators would need 150 AF patients in the overall study population to be able to answer whether ETP differs between the three stroke risk groups, i.e., low-risk, intermediate-risk, and high-risk groups. Investigators will use analysis of variance (Anova) to compare the three different stroke risk groups. Pearson's correlation or Spearman's rank correlation will be used to assess correlations between components of the CHA2DS2-VASc and ABC-stroke scores, AF-burden, left atrial size/function, and hemostatic biomarkers. A P-value <0.05 will be considered statistically significant.

Blood sampling: Fasting blood samples will be drawn from an antecubital vein with minimal stasis, using a 21 gauge needle. The first 2 mL of blood will be discarded, and the following 6 x 2.7 mL blood will be collected into 0.109 M sodium citrate tubes for APTT, INR, and hemostasis variables. Then, 2 x 3 mL of blood will be collected in Li-Heparin tubes for lipids, liver enzymes, renal function, CRP, NT-proBNP, and hs-TNT. Finally, 3 mL of blood will be collected in EDTA-tubes for HbA1c and full blood count. To generate platelet poor plasma (PPP), the tubes will be centrifuged at 2000 g for 20 min. at room temperature. PPP will be stored at -80°C until analysis.

Hemostatic biomarkers: Investigators will measure biomarkers related to the primary and secondary hemostasis, as listed below:

Primary hemostasis (platelet plug formation): To assess the influence of the primary hemostasis, investigators will measure the levels of von Willebrand factor (vWF) antigen using an in-house enzyme-linked immunosorbent assay (ELISA). If the concentrations of vWF are elevated compared with reference values, p-selectin and ADAMTS13 will be analyzed.

Secondary hemostasis (fibrin formation and resolution): Assessment of the secondary hemostasis will be done by analyzing thrombin turnover, and investigate whether thrombin generation is initiated though the contact activation system (CAS), i.e., the intrinsic pathway of the coagulation cascade, or the tissue factor (TF) pathway, i.e., the extrinsic pathway. In relation to CAS, measures of kallikrein generation (lag time, peak kallikrein concentration, time to peak, and endogenous kallikrein potential (EKP)) and concentrations of cleaved high-molecular weight kininogen (cHK), factor XII, prekallikrein, and HK will be measured. Concentrations of C1-inhibitor will be measured with commercial C1-inhibitor antibodies. Thrombin generation will be assessed through measures of lag time, peak thrombin concentration, time to peak, and endogenous thrombin potential (ETP). Endogenous activation of prothrombin to thrombin will be estimated from concentrations of prothrombin fragment 1 + 2 (F1+2), by a commercial ELISA. Furthermore, factor VII, factor X, factor II, protein C, protein S, tissue factor pathway inhibitor (TFPI), and antithrombin (AT) will be measured using commercial ELISAs to assess concentrations of activators and inhibitors of blood coagulation. Fibrin turnover will be assessed through fibrin clot lysis and concentrations of fibrinogen, which will be measured on a BN II analyzer. To assess fibrinolysis, D-dimer, a fibrin degradation product, will be measured by an immunoturbidimetric method. Other markers of fibrinolysis will be measured as well, i.e., tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor 1 (PAI-1), plasminogen, plasmin inhibitor (PI), factor XIII, and thrombin activatable fibrinolysis inhibitor (TAFI), using in house and commercial assays.

AF burden, left atrial function, and HFpEF: Participants will undergo transthoracic echocardiography (TTE) as well as seven days heart rhythm monitoring. Through TTE, a rhythm independent expression of left atrial function, known as left atrial function index (LAFI), will be calculated. LAFI is a ratio that incorporates analogues of cardiac output, atrial reservoir function, and left atrial (LA) size. It is calculated as LAFI = (LA Emptying Fraction (LAEF) x Left Ventricular Outflow Tract - Velocity Time Integral (LVOT-VTI))/LA End-Systolic Volume Index (LAESVI). LAEF is the difference between LA end-systolic volume (LAESV) and LA end-diastolic volume (LAEDV) divided by LAESV and multiplied by 100, which is illustrated as LAEF = ((LAESV-LAEDV)/LAESV) x 100. LVOT-VTI is calculated by placing the pulsed wave doppler sample volume in the LVOT below the aortic valve and trace the velocity curves. LVOT-VTI is expressed in cm and describes the distance blood travels in one heartbeat. LAESVI is the largest LA volume in end-systole in mL indexed to body surface area (BSA). Left atrial function has been proven to modify the risk of stroke in patients with NVAF. Left Ventricular Ejection Fraction (LVEF) will also be assessed as part of the TTE. Furthermore, echocardiographic assessment of underlying HFpEF will be made through the HFA-PEFF score and the H2FPEF score. AF-burden will be determined as a percentage based on the total number of AF-events during the seven days heart rhythm monitoring. This is of prognostic importance due to growing evidence indicating a substantial association between AF burden and stroke.

Primary and secondary endpoints: Studies have shown that activation of the coagulation cascade (secondary hemostasis) is the most significant cause of a prothrombotic state, for which thrombin plays an essential role. Therefore, primary endpoints will be related to thrombin generation, kallikrein generation, and F1 + 2. Secondary endpoints will be the remaining hemostatic biomarkers, AF-burden, LAFI, and underlying HFpEF.

Ethical considerations:

Participants will be meticulously informed about this study, and all questions paticipants might have will be answered. Participants will have to sign the patient consent form prior to enrollment. Data will be stored in a secure database, in accordance with Danish and European legislation. In general, this research will be conducted in accordance with the Declaration of Helsinki.

Clinical relevance:

OAC therapy plays an essential role in stroke risk reduction in patients with AF. The CHA2DS2-VASc score is used in everyday clinical practice to determine the potential need for OAC therapy in patients with AF. However, novel stroke risk stratification schemes incorporating biomarkers have been developed, e.g., the ABC-stroke score, which has been shown to outperform the CHA2DS2-VASc score, in terms of stroke risk prediction and stratification of AF patients. It remains unknown how the individual components of the CHA2DS2-VASc score and ABC-stroke score affect the hemostatic profile in patients with NVAF, and how the hemostatic profile differs among stroke risk groups. Furthermore, the CHA2DS2-VASc score does not consider whether the modifiable components are well-controlled or not, which could possibly lead to OAC overtreatment. Likewise, the CHA2DS2-VASc score and ABC-stroke score do not account for key factors (e.g., AF-burden and left atrial size/function) that are able to impact stroke event rates. The lack of abovementioned factors is believed to be a major limitation of the CHA2DS2-VASc score, in particular. Investigators believe that using AF-burden and left atrial size/function in addition to the CHA2DS2-VASc score and ABC-stroke score could improve OAC decision-making, especially in NVAF patients with intermediate stroke risk. The first step is to characterize the CHA2DS2-VASc score/ABC-stroke score, AF-burden, and left atrial size/function with respect to the hemostatic profile. In this way, this study lays the foundation for future prospective studies investigating whether adding non-clinical factors to the CHA2DS2-VASc score and ABC-stroke score can improve stroke risk stratification in OAC-naïve NVAF patients.

Impact paragraph:

AF accounts for 25 % of ischemic strokes. Stroke can cause long-term disability and remains the second leading cause of death worldwide. OAC therapy is important for stroke prevention in AF patients. The CHA2DS2-VASc score and ABC-stroke score are used for stroke risk stratification and OAC therapy decision-making in AF patients. However, the CHA2DS2-VASc score, in particular, is associated with limitations, i.e., lack of AF sensitive factors. This study will, through the use of AF-burden and left atrial size/function, in addition to the CHA2DS2-VASc score and ABC-stroke score, seek to lay the foundation for future improvement of OAC therapy decision-making, particularly in NVAF patients with intermediate stroke risk. Investigators believe that this project is in line with UN's sustainable development goal number three (i.e., good health and well-being) due to contributing to stroke risk stratification advances in OAC-naïve NVAF patients.