Regulation of fibrinolysis by S100A10 in vivo Running title: S100A10 and fibrinolysis

Endothelial cells form the inner lining of vascular networks and maintain blood fluidity by inhibiting blood coagulation and promoting blood clot dissolution (fibrinolysis). Plasmin, the primary fibrinolytic enzyme, is generated by the cleavage of the plasma protein, plasminogen, by its activator, tissue plasminogen activator (tPA). This reaction is regulated by plasminogen receptors at the surface of the vascular endothelial cells. Previous studies have identified the plasminogen receptor protein, S100A10 as a key regulator of plasmin generation by cancer cells and macrophages. Here we examine the role of S100A10 and its annexin A2 binding partner in endothelial cell function using a homozygous S100A10-null mouse. Compared to wild-type mice, S100A10-null mice displayed increased deposition of fibrin in the vasculature and reduced clearance of batroxobin-induced vascular thrombi, suggesting a role for S100A10 in fibrinolysis in vivo. Compared to WT cells, endothelial cells from S100A10-null mice demonstrated a 40% reduction in plasminogen binding and plasmin generation in vitro. Furthermore, S100A10-deficient endothelial cells demonstrated impaired neovascularization of Matrigel plugs in vivo suggesting a role for S100A10 in angiogenesis. These results establish an important role for S100A10 in the regulation of fibrinolysis and angiogenesis in vivo, suggesting S100A10 plays a critical role in endothelial cell function. Introduction Blood clots are continually forming in the vasculature due to activation of the coagulation process by sluggish blood flow, the presence of tissue debris in the blood, collagen or lipids or because of damage to small blood vessels and capillaries. In various diseases such as atherosclerosis, damage to the normally smooth vascular surface also results in the generation of blood clots. The endothelial cells that form the inner lining of the blood vessels are responsible for ensuring vascular patency by removing these potentially dangerous blood clots. To achieve this goal, the endothelial cells convert the plasma zymogen, plasminogen, to the active serine protease plasmin. The primary function of plasmin is to maintain vascular patency by degrading the fibrin-rich blood clots, a process called fibrinolysis. Fibrinolysis is a normal vascular process that occurs continuously and is required to prevent naturally occurring blood clots from growing and causing vascular occlusions which would result in heart attack and stroke. Fundamental to the process of fibrinolysis are the proteins that colocalize plasminogen with its activators to the endothelial cell surface, thereby stimulating the formation of plasmin in a tightly localized and highly regulated fashion. Among the protein and nonprotein plasminogen receptors that have been identified are a subset of plasminogen receptors that utilize their carboxylterminal lysine residue to interact with the kringle domains of plasminogen and tPA. Several of these plasminogen receptors, such as α-enolase, histone H2B, PLG-RTK and S100A10 have recently been the focus of detailed studies highlighting the importance of plasminogen receptors in the regulation of cellular plasmin generation. S100A10 is present on the surface of endothelial and other cells in a heterotetrameric complex with its binding partner, annexin A2. The complex, called the annexin A2 heterotetramer (AIIt), is composed of a dimer of S100A10 that links together two molecules of annexin A2. Our laboratory has demonstrated that the phospholipid-binding sites of annexin A2 anchor S100A10 to the cell surface whereas the carboxyl-terminal lysine of the S100A10 subunits provide the binding sites for tPA (Kd = 0.45 μM), plasminogen (Pg) (Kd = 1.81 μM) and plasmin (Pm) (Kd = 0.36 μM). The annexin A2 subunit may also play a role in Pg binding. However, the binding of Pg to annexin A2 is absolutely dependent on the cleavage of annexin A2 at Lys-307, an event which has not been demonstrated to occur on the cell surface in vivo. The role of S100A10 in Pg binding and Pm regulation has been verified by a series of studies which have examined S100A10 function in the absence of annexin A2 in vitro. For example, removal of the carboxyl-terminal lysine from S100A10 attenuates its binding to tPA and Pg, establishing this region of S100A10 as the tPA and Pg binding site. The binding of Pg to For personal use only. on August 31, 2017. by guest www.bloodjournal.org From