Analysis of Soluble Molecular Fibronectin-Fibrin Complexes and EDA-Fibronectin Concentration in Plasma of Patients with Atherosclerosis

Inflammation, Vol. 39, No. 3, June 2016 ( # 2016) DOI: 10.1007/s10753-016-0336-0 ORIGINAL ARTICLE Analysis of Soluble Molecular Fibronectin-Fibrin Complexes and EDA-Fibronectin Concentration in Plasma of Patients with Atherosclerosis Anna Lemańska-Perek,1,3 Dorota Krzyżanowska-Gołąb,1 Małgorzata Pupek,1 Piotr Klimeczek,2 Wojciech Witkiewicz,2 and Iwona Kątnik-Prastowska1 Abstract—Atherosclerosis, a chronic vascular disease, leads to molecular events bound with interplaying processes of inflammation and coagulation. In the present study, fibronectin (FN), FN containing extra domain A (EDA-FN), frequency of occurrence, and relative amounts of soluble plasma FN-fibrin complexes were analyzed in 80 plasma samples of patients suspected of coronary artery disease based on clinical evaluation and changes in arteries found by computed tomographic coronary angiography. The study showed that in the plasma of the patients’ group with high risk of coronary artery disease EDA-FN concentration was significantly higher (3.5 ± 2.5 mg/L; P < 0.025) and the molecular FN-fibrin complexes of 1000 kDa and higher occurred more often than in the groups of patients with mild risk of coronary artery disease and the normal age-matched. The increased level of EDA-FN and occurrence of FN-fibrin complexes could have a potential diagnostic value in the diagnosis and management of patients with coronary artery disease. KEY WORDS: fibronectin; fibronectin bearing EDA segment; FN-fibrin complexes; atherosclerosis. several adhesion molecules and their receptors [2–4]. The atheroprogression is manifested by the development of atheromatous plaque in the affected vessel wall, which leads to diverse vascular lesions and disturbs molecular vessel functions, vascular permeability, and flow of blood around the body [2, 3, 5]. A key event associated with plaque formation is expansion and remodeling of extracellular matrix components (ECM) in susceptible arterial areas [6]. During atherosclerotic lesions under diseasedependent conditions, the normal complex organization of ECM undergoes harmful structural and functional modifications which can lead to the occurrence of numerous diseases [6–8]. Among the major proteins of ECM responsible for the organization and regulation of ECMdependent molecular functions, a multifunctional and multidomain glycoprotein, fibronectin (FN), plays a crucial role [8–10]. FN, besides being an insoluble component of ECM and tissues (cFN), is also an abundant protein of plasma and other physiological fluids. In plasma and INTRODUCTION Development of atherosclerotic lesions, which increasingly affect the human population, causing high morbidity and mortality, are known to be associated with chronic inflammation and can lead to thrombosis and diverse cardiovascular diseases [1]. At the cellular and molecular levels, atherosclerosis is known to activate immune-inflammatory pathways, encompassing multiple complex interdependent interactions among inflammatory cells, vascular elements, extracellular matrix molecules, and plasma proteins through expression of cytokines, and 1 Department of Chemistry and Immunochemistry, Medical University of Wrocław, Bujwida 44a, 50-345 Wrocław, Poland 2 Provincial Specialist Hospital in Wrocław, Research and Development Center in Wrocław, Wrocław, Poland 3 To whom correspondence should be addressed at Department of Chemistry and Immunochemistry, Medical University of Wrocław, Bujwida 4 4 a , 5 0 - 3 4 5 Wr o c ł a w, P o l a n d . E - m a i l : a n n a . l e m a n s k a 1059 0360-3997/16/0300-1059/0 # 2016 The Author(s). This article is published with open access at Springerlink.com 1060 Lemańska-Perek, Krzyżanowska-Gołąb, Pupek, Klimeczek, Witkiewicz, and Kątnik-Prastowska tissues, FN can exist in diverse isoforms arising from posttranslational modifications (N- and O-glycosylation, phosphorylation), alternative splicing of FN premessenger RNA (i.e., inclusion of EDA, EDB, and IIICS segments), and moreover from variable conformations depending on environmental conditions (globular and fibrillar structures) [8, 9, 11]. FN is a large dimeric glycoprotein (450–500 kDa); each monomer of which consists of types I, II, and III of repeating amino acid units. The multiple copies of repeats are arranged into several domains able to bind fibrin, collagen, glycosaminoglycans, and cellular receptors [9, 10]. Two identical or nearly identical (depending on the included or excluded spliced extra domains) FN polypeptides are linked together by two disulfide bonds near their carboxyl termini. In contrast, their N-termini ends are unlinked, facilitating formation of many diverse conformational structures able to react with FN ligands [9, 11]. Its hepatic origin, plasma form of FN (pFN) lacking extra EDA and EDB segments, has a looped compact conformation which can be stretched to an unfolded conformation when FN is caught by cellular integrin receptors [9, 11]. The tissue cellular FN (cFN) is synthesized by many cell types (e.g., fibroblasts, endothelial cells, platelets, and monocytes) and bears variable proportions of EDA and EDB segments (EDA-FN and EDB-FN, respectively) [9, 12]. Both forms, pFN and cFN, are reported to be incorporated into the fibrillar network of ECM [9, 10, 13], where they play structural and functional roles regulating some cellular activities [9, 14]. The plasma-derived FN is reported to support hemostasis, regulate thrombosis [15, 16], and significantly accelerate healing, reducing the area of inflammation [17, 18]. Plasma FN is also a major component of the blood clot. With fibrin and its degraded forms, it readily forms a macromolecular complex which can be cross-linked covalently in the reaction catalyzed by transglutaminase XIIIa [16, 19]. Through binding with fibrin, pFN influences the rate of formation, stability, as well as the structure of the fibrin matrix [15, 20]. In spite of the fact that both EDA- and EDB-FN isoforms take part in vasculogenesis in embryos and angiogenesis in cancer and non-tumoral conditions, the specific integrin receptors have been identified exclusively for EDA-FN [12, 21, 22]. FN carrying the EDA segment recognized by integrin receptors is reported to be implicated in efficient adhesion, activation, and aggregation of platelets, promoting in that way inflammation and coagulation processes [23–25]. The initial step of platelet adhesion is immobilization of dimeric EDA-FN on platelets by integrins α5β1 and αIIbβ3 and stretching of a dimeric cFN to its fibrillar form. Maurer et al. [2015] provided experimental evidence that a fibrillar form of EDA-FN is a potent thrombogenic component of the subendothelium. The interaction of immobilized fibrillary EDA-FN to platelet integrins together with glycoprotein Ib-V-IX complex, a major signaling receptor for collagen, and Toll-like receptor 4 initiates molecular reactions which lead to activation of the coagulation cascade and promotion of thrombus formation (...truncated)