Thrombin induces endothelial cell-surface exposure of the plasminogen receptor annexin 2.

Erica A Peterson,Michael E Nesheim,Michael R Sutherland,Edward L G Pryzdial

doi:10.1242/jcs.00434

Abstract

Cell-surface annexin 2 (A2) and its ligand p11 have been implicated in fibrinolysis because of their ability to accelerate tissue plasminogen activator (tPA)-mediated activation of plasminogen to plasmin. Because thrombin is a potent cell modulator obligately produced at the site of clot formation, we hypothesized that the amount of cell-surface A2 and p11 might be altered by thrombin with consequent effects on plasmin generation. In support of this hypothesis, immunofluorescence microscopy and hydrophilic biotinylation experiments showed that both A2 and p11 were significantly increased on the surface of human umbilical vein endothelial cells (HUVECs) treated with thrombin (0.8-8 nM) for 5 minutes followed by 1 hour at 37 degrees C. Intracellular immunofluorescence microscopy and immunoblot analyses of whole cell extracts revealed increased p11 but unchanged A2 in response to thrombin, suggesting that transbilayer trafficking of A2 might be controlled by p11. The thrombin receptor-activating peptide (TRAP) similarly affected cells, demonstrating that cell signaling at least involved the type-1 protease activated receptor (PAR-1). An effect on the fibrinolysis pathway after treatment of HUVECs with thrombin was shown by increased fluorescein-labeled plasminogen binding to cells, which was inhibited by an antibody specific for p11. This was confirmed by observing that thrombin pretreatment of HUVECs increased biotin-modified plasminogen binding. Utilizing a chromogenic assay, pretreatment of HUVECs by thrombin further enhanced activation of the Glu and Lys forms of plasminogen by tPA. These data suggest a novel mechanism that links the coagulation and fibrinolysis pathways by thrombin-mediated feedback.

Highlights

Annexin 2 (A2) is a Ca2+-dependent, anionic phospholipid-binding protein that belongs to the ubiquitous multigene annexin family
We addressed the hypothesis that an additional mode of communication could be facilitated by the cellmodulating capabilities of thrombin, which might affect activation of plasminogen on human umbilical vein endothelial cells (HUVECs) by altering the availability of cell-surface A2 and p11
HUVEC surface A2 and p11 are enhanced by thrombin or thrombin receptor-activating peptide (TRAP)

Summary

Introduction

Annexin 2 (A2) is a Ca2+-dependent, anionic phospholipid (aPL)-binding protein that belongs to the ubiquitous multigene annexin family (reviewed by Kim and Hajjar, 2002; Siever and Erickson, 1997; Waisman, 1995). The N-terminus of A2 can associate with p11, an 11 kDa member of the S100 protein family (Glenney et al, 1986; Johnsson et al, 1986; Johnsson et al, 1988) This property allows A2 to coexist as functionally distinct monomeric (A2m) and tetrameric (A2t) forms, the latter resulting from the noncovalent association of two A2m molecules bridged by a p11 dimer (Erikson et al, 1984; Gerke and Weber, 1984; Glenney, 1986). Both A2m and A2t can bind aPLcontaining membranes at millimolar Ca2+ concentrations, only A2t is able to bind at intracellular micromolar concentrations (Powell and Glenney, 1987). Both A2m and A2t can aggregate apposing aPL-containing membranes; only A2t can subsequently participate in membrane fusion through a complicated and incompletely understood mechanism (Ali et al, 1989; Drust and Creutz, 1988; Kang et al, 1997; Regnouf et al, 1995)

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