Prothrombotic Effects of Fibronectin Isoforms Containing the EDA Domain

Abstract

Objective-Fibronectin (FN) plays an important role in the formation of stable arterial thrombi at the site of vascular injury.FN containing Extra Domain A (EDA ϩ FN) is absent from normal plasma, but elevated plasma levels of EDA ϩ FN are found in several pathological conditions. We hypothesized that EDA ϩ FN plays a special role in thrombosis. Methods and Results-We used mouse strains constitutively including (EDA ϩ/ϩ ) or excluding (EDA Ϫ/Ϫ ) the EDA domain in all tissues and plasma. Using a flow chamber and the ferric-chloride injury model we found that EDA ϩ FN accelerates thrombosis both in vitro and in vivo at arterial shear rates. In EDA ϩ/ϩ mice thrombi (Ͼ30 m) grew faster when compared with EDA WT/WT (6.6Ϯ0.2 minutes versus 8.3Ϯ0.6 minutes, PϽ0.05) and the mean vessel occlusion time was shorter (9.9Ϯ0.4 minutes versus 14.6Ϯ1.7 minutes, PϽ0.05). However, the presence of EDA ϩ FN affected neither single platelet adhesion to subendothelium nor thrombosis in veins. In addition, the mortality rate of EDA ϩ/ϩ mice after collagen/epinephrine infusion was twice that of EDA WT/WT or EDA Ϫ/Ϫ mice. Conclusions-Our findings reveal that EDAϩ FN has prothrombotic activity, and its presence in plasma may worsen pathological conditions in which this form is elevated. (Arterioscler Thromb Vasc Biol. 2008;28:296-301) Key Words: plasma fibronectin Ⅲ fibronectin splice variants isoforms Ⅲ arterial thrombosis Ⅲ intravital microscopy Ⅲ thromboembolism Ⅲ arterial and venous injury T he formation of a thrombus in an injured vessel wall is a complex process that involves multiple adhesion molecules and their respective receptors on the platelet surface. von Willebrand factor (vWF) and fibrinogen are considered the major ligands mediating platelet adhesion and aggregation. However, in an experimental model 73% of the injured vessels of mice lacking both fibrinogen and vWF still formed occlusive thrombi either at the site of injury or downstream, 1 suggesting that other major adhesive proteins, such as fibronectin (FN), might contribute to the process. Incorporation of FN into a growing thrombus was shown both in vitro 2 and in vivo. 3 Additionally, it was documented in vivo that the depletion or lower levels (50%) of plasma FN result in serious defects in arterial thrombosis. 3,4 FN is a dimeric multidomain glycoprotein playing an important role in adhesion, migration, growth and differentiation of cells. 5,6 FN generates protein diversity as a consequence of alternative processing of a single primary transcript at 3 sites: the Extra Domain B (EDB, EDII, or EIII-B), Extra Domain A (EDA, EDI, or EIII-A), and the Type III Homologies Connecting Segment (IIICS) ( Figure 1A). 7-9 Two major forms of FN exist: soluble plasma FN (pFN), which lacks both the EDA and EDB domains ( Figure 1A); and cellular FN (cFN), which is deposited as insoluble fibrils in the extracellular matrix (ECM) and contains these domains at variable proportions.FN is a ligand for many members of the integrin receptor family and binds to thrombosis-related protein...

Thrombospondin 1 requires von Willebrand factor to modulate arterial thrombosis in mice

“…Platelets from adult donor mice were prepared as described 25,26 and briefly explained in the supplemental Methods on the Blood Web site. Intravital microscopy was performed as described.…”

“…Intravital microscopy was performed as described. 25,26 Platelets labeled with fluorescent calcein green, acetoxymethyl (AM) esters (1.25 3 10 9 platelets/kg) or fluorescent calcein red-orange, AM were infused through the retro-orbital plexus in mice anesthetized with 100 mg/kg ketamine and 10 mg/kg xylazine. An incision was made through the abdominal wall to expose the mesentery, and the arterioles of diameter (approximately 100-150 mm) were studied.…”

Cellular fibronectin containing extra domain A promotes arterial thrombosis in mice through platelet Toll-like receptor 4

“…9 In pathological disease states such as diabetes and ischemic stroke, plasma levels of cellular Fn containing extra domain A (Fn-EDA1) can become elevated. 13,14 Previously, it was shown that inclusion of the EDA segment gives Fn a prothrombotic potential in vivo 15 ; however, the underlying mechanisms of this effect still remain unknown. The EDA segment of cFn, but not other domains, has been shown to activate human Toll-like-receptor (TLR) 4 expressed in HEK293 cells.…”

Alternatively spliced fibronectin extra domain A is required for hemangiogenic recovery upon bone marrow chemotherapy

“…10 Although its function is not well understood, FN EIIIA has been previously linked to a variety of in vitro cellular events and in vivo molecular outcomes. Increased expression of EIIIA+ isoform modulates cell cycle progression, myofibroblast differentiation, 11 prothrombotic effects on platelets, 12 and protection of vessel walls in conditions of disturbed flow.…”