Coagulation factors and fibrinolytic activity in the left atrial appendage andother heart chambers i

Journal Pre-proofCoagulation factors and fibrinolytic activity in the left atrial appendage and other heartchambers in patients with atrial fibrillation is there a local intracardiac prothromboticstate? (HEART-CLOT study)Krzysztof Bartus, Radoslaw Litwinowicz, Joanna Natorska, Michal Zabczyk, AnettaUndas, Boguslaw Kapelak, Dhanunjaya Lakkireddy, Randall J. LeePII:S0167-5273(19)34017-3DOI:https://doi.org/10.1016/j.ijcard.2019.09.053Reference:IJCA 28035To appear in:International Journal of CardiologyReceived Date:7 August 2019Revised Date:19 September 2019Accepted Date:25 September 2019Please cite this article as: K. Bartus, R. Litwinowicz, J. Natorska, M. Zabczyk, A. Undas, B. Kapelak,D. Lakkireddy, R.J. Lee, Coagulation factors and fibrinolytic activity in the left atrial appendage andother heart chambers in patients with atrial fibrillation is there a local intracardiac prothromboticstate? (HEART-CLOT study), International Journal of Cardiology (2019), doi: https://doi.org/10.1016/j.ijcard.2019.09.053.This is a PDF file of an article that has undergone enhancements after acceptance, such as the additionof a cover page and metadata, and formatting for readability, but it is not yet the definitive version ofrecord. This version will undergo additional copyediting, typesetting and review before it is publishedin its final form, but we are providing this version to give early visibility of the article. Please note that,during the production process, errors may be discovered which could affect the content, and all legaldisclaimers that apply to the journal pertain.© 2019 The Authors. Published by Elsevier B.V.

Coagulation factors and fibrinolytic activity in the left atrial appendage and other heart chambers in patients with atrial fibrillation Is there a local intracardiac prothrombotic state? (HEART-CLOT study) Running Title: Differences in coagulation and fibrinolytic factor levels between various cardiac chambersKrzysztof Bartus1, Radoslaw Litwinowicz1, Joanna Natorska2, Michal Zabczyk2, Anetta Undas2, Boguslaw Kapelak1, Dhanunjaya Lakkireddy3, Randall J Lee41Department of Cardiovascular Surgery and Transplantology, Jagiellonian University Medical College, John Paul II Hospital, Krakow, Poland 2Institute of Cardiology, Jagiellonian University Medical College, Krakow, Poland 3The Kansas City Heart Rhythm Institute & Research Foundation, Overland Park, KS, USA 4Department of Medicine and Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA This study is the result of the research grant No. UMO-2014/13/D/NZ5/01351 funded by the National Science Centre Poland. Corresponding Author: Radoslaw Litwinowicz Department of Cardiovascular Surgery and Transplantology, Jagiellonian University, John Paul II Hospital, Pradnicka 80, 30-001 Kraków, Poland Phone: (12) 614 32 03 Fax: (12) 614-25-25 E-mail: [email protected] ABSTRACT:

Introduction: Atrial fibrillation (AF), a risk factor for strokes and systemic thromboembolisms, is associated with unfavorable fibrin clot properties and increased thrombus formation in peripheral blood. The left atrial appendage (LAA) is known to be the primary site of thrombus formation. Aim: We investigated the relative differences in plasma fibrin clot features including plasma fibrin clot permeability (Ks) and clot lysis time (CLT) between the right atrium (RA), right ventricle (RV), left atrium (LA), left ventricle (LV), LAA, and peripheral blood. Methods: Sixteen patients with nonvalvular AF who stopped oral anticoagulant therapy at least 2 days before a LARIAT procedure participated in a single-center prospective study. We measured fibrinogen and plasminogen levels along with Ks, CLT, and endogenous thrombin potential (ETP) during the LARIAT procedure in blood obtained from the right femoral vein, RA, RV, LA, LV and LAA. Results: LAA clot porosity was reduced by 16.2% in the LAA compared to peripheral blood (p=0.026), and also after adjustment for fibrinogen levels (p=0.038). Ks was similar for the RA, RV, LA, LV, and LAA (all p>0.05). We found 14.7% prolonged CLT for blood samples obtained from the LAA compared to peripheral blood, but no differences between the RA, RV, LA and LV (all p>0.05). There were no significant differences in other parameters, including ETP, among the five sites. Conclusions: Patients with AF have a local prothrombotic state as reflected by formation of compact fibrin clots in the LAA compared to peripheral blood, which may contribute to LAA thrombus formation and device-related thrombi. INTRODUCTION

Atrial fibrillation (AF) is the most common cardiac arrhythmia resulting in a prothrombotic state [1, 2]. Significant hemodynamic abnormalities in AF are caused by irregular contractions of the ventricles and a lack of effective atrial contraction with resulting turbulent blood flow [3, 4 ]. The structural and functional changes in the left atrium (LA) and left atrial appendage (LAA) impact their flow dynamics and coagulation status by significantly increasing the risk of thrombus formation and subsequent thromboembolization. AF patients have a significantly higher prothrombotic state as reflected by elevated thrombin generation markers (F1 + 2 prothrombin fragments and D-dimer [5-7]), and a prolonged clot lysis time (CLT). Higher levels of plasminogen activator inhibitor-1 (PAI-1) [8] and thrombin activatable fibrinolysis inhibitor (TAFI) [9] have also been described in AF patients [10]. Moreover, AF is associated with increased levels of plasma fibrinogen, von Willebrand Factor (vWF), soluble P-selectin, and α2-antiplasmin (α2AP) [11]. Vascular endothelial cell damage is also increased as measured by elevated soluble thrombomodulin (sTM) [9, 12, 13]. Tighter plasma fibrin clot structure, reflected by reduced clot permeability (Ks), along with decreased susceptibility to lysis as measured by prolonged clot lysis time (CLT) were found in patients with sinus rhythm experiencing a short-term AF episode [14] and in patients with permanent AF [15]. However, these altered levels parameters have mostly been studied in peripheral blood samples [9, 12, 13]. Although a systemic increase in prothrombotic state exists in AF patients, thrombosis is mostly observed in cardiac chambers, and evidence that the blood in certain cardiac chambers in AF patients is more thrombogenic than the peripheral blood [16] supports clinical observations that thrombi develop mostly in the LA and LAA. In conclusion, the regional differences in coagulation and fibrinolytic factor levels are poorly understood.

In the current study we evaluated the differences in plasma fibrin clot features between all heart chambers, including Ks and CLT, known to be associated with increased risk of thromboembolism in AF patients. METHODSPatient characteristics A prospective study was performed on 16 consecutive patients enrolled between September 2016 and November 2018. The study design was in accordance with the guiding principles of the Declaration of Helsinki and approved by the Jagiellonian University Ethical Committee. All patients signed a written informed consent form prior to inclusion. This study was funded by the Polish National Science Center (research grant UMO-2014/13/D/NZ5/01351). All patients met the following inclusion criteria: 1) age 18 years or older; 2) nonvalvular AF of > 6 months; 3) at least 1 risk factor of embolic stroke (CHADS21); 4) a poor or ineligible candidate for long term oral anticoagulation therapy. The exclusion criteria for Lariat procedure were: 1) previous cardiac surgery; 2) acute illness; 3) known cancer; 4) hepatic or renal dysfunction; 5) heart failure (New York Heart Association III or IV); 6) Left Ventricle Ejection Fraction (LVEF) < 35%; 7) myocardial infarction <3 months; 8) autoimmune disease; 9) steroid administration; or 10) any other cardiac disease required cardiac surgery procedure. Patients meeting enrollment criteria underwent a screening contrast cardiac computed tomography (CT) scan. Exclusion criteria based on LAA anatomy included: 1) LAA width 45 mm; 2) superiorly oriented LAA with the LAA apex directed behind the pulmonary trunk; 3) bilobed LAA or multilobed LAA in which lobes were oriented in different planes exceeding 45 mm; and 4) a posteriorly rotated heart. Left atrial appendage occlusion procedure and blood draw

Patients were hospitalized 1 or 2 days before the procedure. Patients receiving vitamin K antagonist (VKA) or direct oral anticoagulant (DOAC) on a long-term-basis were eligible if their anticoagulation was stable within the previous 3 months. Before the procedure, bridging therapy with low molecular weight heparin (LMWH) was performed. VKA was ceased five days and DOAC was ceased a minimum of two days before the LAAO procedure. In patients receiving aspirin or without oral anticoagulants, LMWH was started at least 2 days before the procedure. The last dose of LMWH was administered >12 hours before the procedure. All epicardial LAAO procedures were performed with the Lariat device (SentreHEART Inc, Redwood, CA). The LAAO procedure was described in detail in our previous studies [17-19]. Pericardial access was obtained using a telescoping-micropuncture technique at the beginning of the procedure followed by femoral venous access. Blood samples were quickly taken: (1) peripheral femoral venous (FV) sheath; (2) right atrium (RA); (3) right ventricle (RV); (4) left atrium (LA); (5) left atrial appendage (LAA); (6) left ventricle (LV) after gaining access to the right and left chambers of the heart. All blood samples were collected before the administration of unfractionated heparin. Briefly, punctures of the right femoral vein were performed using the Seldinger technique and a femoral cannula sheath was placed in the vein. Twenty ml of blood was drawn through the side arm of a short introducer immediately after access to the vein via 5F pigtail, from which the first 5 ml of blood was discarded in order to exclude intra-sheath hemostasis activation (FV sample). After femoral access, the 5F pigtail catheter (Medtronic, Kirkland, QC, Canada) was used again to draw all blood samples from all chambers with the first 5 ml discarded in order to exclude hemostasis activation. The blood was collected from the right atrium (RA sample). The 5F pigtail was then placed inside the right ventricle and the blood sample collected (RV sample). Next, transseptal catheterization was performed. A 5F pigtail catheter was inserted through the transseptal sheath to obtain blood samples from the left atrium, left

ventricle and LAA. The order of obtaining LA, LAA and LV blood samples was rotated between patients to eliminate temporal effects of collecting left-sided blood samples. Immediately after the collection of left heart blood samples, 150 IU/kg body weight intravenous heparin was administered and an epicardial LAAO procedure with suture-based LARIAT device was performed according to standard protocols [17, 20, 21]. Laboratory investigations Blood samples were collected in vacutainer tubes (tubes anticoagulated with K3-EDTA for complete blood count, tubes containing 0.109M sodium citrate and CTAD (buffered citrate, theophylline, adenosine, and dipyridamole) for hemostasis and fibrinolysis tests. In all cases, before the procedure, anti-Xa activity (IU/mL) was measured. International Normalized Ratio (INR), activated partial thromboplastin time (APTT), high sensitivity C-reactive protein (CRP), glycated hemoglobin (HbA1c), morphology including platelet count, comprehensive lipid profile including total cholesterol, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, and triglyceride levels were measured from antecubital vein blood samples of all patients upon hospital admission by routine methods. Tissue plasminogen activator (tPA) antigen, plasminogen activator inhibitor type 1 (PAI-1) antigen, thrombin activatable fibrinolysis inhibitor (TAFI) antigen, and plasminogen activity levels were determined by ELISA (Hyphen BioMed, Neuville-Sur-Oise, France). Fibrin clot analysisPlasma fibrin clot permeability was determined as described previously [22]. Briefly, 20 mM calcium chloride and 1 U/mL human thrombin (Merck, Darmstadt, Germany) were added to citrated plasma. Tubes containing the clots were connected to a reservoir of Tris-buffered saline. Its volume flowing through the gels was measured within 60 min. A permeation coefficient reflected by Ks indicating the average size of pores formed in the fibrin network