Accelerated Fibrinolysis and Its Propagation on Vascular Endothelial Cells by Secreted and Retained tPA

Tetsumei Urano,Yuko Suzuki

doi:10.1155/2012/208108

Abstract

We successfully visualized the secretory dynamics of tissue-type plasminogen activator (tPA) tagged by green fluorescent protein (tPA-GFP) from cultured vascular endothelial cells (VECs) using total internal reflection fluorescence (TIRF) microscopy and demonstrated that tPA-GFP secreted from VECs was retained on cell surfaces in a heavy-chain-dependent manner. Progressive binding of Alexa568-labeled Glu-plasminogen was also observed on the surface of active tPA-GFP expressing cells via lysine binding sites (LBS), which was not observed on inactive mutant tPA-GFP expressing cells. These results suggest that retained tPA on VECs effectively activated plasminogen to plasmin, which then facilitated the binding of additional plasminogen on the cell surface by proteolytically cleaving surface-associated proteins and exposing their C-terminal lysine residues. 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 fibrin 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 fibrin appears to be indispensable for spontaneous fibrinolysis.

Highlights

Fibrinolysis takes place when undesirable fibrin is formed or when a hemostatic thrombus becomes unnecessary
The binding of plasminogen to fibrin through lysinebinding sites (LBS) existing in kringle domains is believed to play an essential role in its effective activation on the fibrin surface [1]
We introduce our recent findings on type plasminogen activators (PAs) (tPA) secretory dynamics from VECs, and on the subsequent process of secreted tPA catalyzing plasminogen activation and fibrinolysis on vascular endothelial cells

Summary

Introduction

Fibrinolysis takes place when undesirable fibrin is formed or when a hemostatic thrombus becomes unnecessary. That the accumulation of plasminogen increased during the time course of tPA secretion from VECs suggests that other proteins function as plasminogen binding molecules on the cell surface by providing newly exposed C-terminal lysine residues after plasmin-catalyzed cleavage (Figure 3). We believe that this augmentation of plasminogen binding by newly exposed C-terminal lysines as well as its effective activation plays essential roles in the degradation of fibrin or matrix proteins, and in proteasedependent signal transduction

Cell-Associated Fibrinolysis and Cell Function

Summary