We have studied how fibrinolytic activity is expressed both in plasma and on vascular endothelial cells (VECs) under physiological and pathological conditions. The activation of coagulation cascade and the resulted fibrin formation is an important trigger to express high fibrinolytic activity in plasma. Binding of tissue plasminogen activator (tPA) and Glu-plasminogen (Glu-plg) on fibrin surface, and the resulted tri-molecular complex formation and the confomational change of Gluplg are the most important underlying mechanisms. We have analyzed those mechanisms and recently demonstrated using real-time imaging technique (Suzuki Y et al, Blood, 2011, Brzoska T et al, PLoSONE 2015). We have also proposed that the activated coagulation factors enhance tPA activity by neutralizing the activity of plasminogen activator inhibitor type 1 (PAI-1) (Urano T et al, JTH 2003;1:2615-2620, Iwaki T et al, JTH 2011;9:1200-1206). We recently focused on how fi brinolytic potential is highly maintained and its activity is effectively expressed on the surface of VECs.

Accelerated fi brinolysis and its propagation on vascular endothelial cells by secreted and retained tPA

tPA, the primary PA in the vasculature, is synthesized and released from VECs as an active form to initiate intravascular thrombolysis. We recently successfully visualized the secretory dynamics of tPA tagged by green fluorescent protein (tPA-GFP) from cultured VECs using total internal reflection fluorescence (TIRF) microscopy. tPA-GFP appeared to have unique secretory dynamics from VECs, showing that the release of tPA-GFP from the opened granule was very slow after the opening of its containing granule (Suzuki Y et al, Blood 2009;113:470-478). The retention of tPA on cell surfaces was heavy-chain dependent, and the release of tPA appeared to be facilitated by plasminogen activator inhibitor type 1 (PAI-1). The retained tPA on cell surface effi ciently expressed its activity to generate plasmin, which then proteolytically cleaved surface-associated proteins to expose lysine residues at their C-termini. This was demonstrated by both progressive accumulation of Alexa568 labeled Glu-plasminogen on the surface of active tPA-GFP expressing cells in a lysine binding sites (LBS) dependent manner and effective digestion of fibrin network formed over the cells (Suzuki Y et al, Blood 2011;118:3182-3185). Thus prolonged retention of tPA appeared to play an important role in initiating and amplifying plasmin generation on VECs. LBS-dependent binding of plasminogen was also observed as a narrow band at the lytic front of the fi brin mesh formed on active tPA-GFP expressing cells, which expanded outward as the lytic area increased. This binding was not observed on inactive mutant tPA-GFP expressing cells or in the presence of aprotinin. The binding of plasminogen to partially digested fi brin was directly proved indispensable for spontaneous fi brinolysis as was speculated for long time.

Time- and space-dependent control of both thrombus formation and its lysis revealed in in-vivo system

Since both thrombus formation and its lysis in vivo are finely regulated time- and space-dependently to repair vascular injury along with keeping vascular patency. Employing in-vivo real time confocal microscopy system, we have analyzed platelets activation, thrombus formation and its lysis in mesenteric vein in GFP expressing transgenic mice after laser-induced injury of vessel intima. Platelets adhesion and aggregation were clearly monitored and their activation was demonstrated by the specifi c binding of fluorescent-labeled annexin V to phosphatidylserine exposed on the outer leaflet of activated platelets. Surprisingly only platelets existing in the center of thrombus, in which sustained elevation of intracellular calcium ion concentration was observed, exposed PS, which was followed by fibrin formation (Hayashi T et al, Eur J Physiology 2008;456:1239-1251, Rybaltowski M et al, Pfl ugers Arch 2011;461:623-633, Kramkowski K et al, ATVB 2012;32:2149-2157). We also observed the accumulation of Glu-plasminogen at a later phase, but still early phase of thrombus formation, in the center of the thrombus in both LBS-dependent- and plasmin activity-dependent manner (Brzoska T, Tanaka A et al PLoSONE 2015). We are now analyzing physiological and pathological modification of the initiation of fibrinolysis.

Physiological function of PAI-1 both extra-cellular and intra-cellular space

In contrast to PAI-1 knock out mice, human PAI-1 deficient patients demonstrated life-threatening bleeding. Further, the patients showed impairment in the process of wound healing. To analyze the mechanism, we established induced pluripotent stem (iPS) cells from two distinct PAI-1 deficient patients, and started to analyze their characteristics in the process of differentiation to many kind of mature cells as well as possible difference in phenotypes of mature cells.

Mechanism to develop aneurysm: possible involvement of coagulation and fibrinolysis

Hiroki Tanaka, a newly joined laboratory member, revealed that the obstruction of vasa-vasorum and the resulted inflammation of the outer vascular wall are the trigger to develop aneurysm (Tanaka H et al PLoSONE 2013, Sano M, Tanaka H et al PloSONE 2014). We are analyzing how platelets function, coagulation- and fibrinolytic- system are involved in the development of aneurysm. We are also analyzing how intra-luminal thrombus formation affects inflammatory response in vascular wall.