First published online 30 April 2003
doi: 10.1242/jcs.00434
Thrombin induces endothelial cell-surface exposure of the plasminogen receptor annexin 2
Erica A. Peterson1,2,
Michael R. Sutherland1,2,
Michael E. Nesheim3 and
Edward L. G. Pryzdial1,2,4,*
1 Canadian Blood Services, R&D Department, 1800 Alta Vista Drive, Ottawa, ON
K1G 4J5, Canada
2 Department of Biochemistry, Microbiology, and Immunology, University of
Ottawa, 451 Smyth Road, Ottawa, ON K1H 8M5, Canada
3 Department of Biochemistry, Queens University, Room A212 Botterell Hall,
Kingston, ON K7L 3N6, Canada
4 Department of Pathology and Laboratory Medicine, University of British
Columbia, 2211 Wesbrook Mall, Room GF-114, Vancouver, BC V6T 2B5, Canada
View larger version (46K):
[in a new window]
|
Fig. 1. Immunofluorescent detection of surface A2 on HUVECs. HUVECs grown on
gelatin-coated glass coverslips were treated for 5 minutes with either
thrombin or TRAP and allowed to recover for 1 hour. The cells were then
simultaneously stained under native conditions for annexin 2 (A2; red) and the
intracellular marker vimentin (Vmn; green) followed by fixation. The number
and position of individual cells were determined by detection of nuclei using
DAPI (blue).
|
|
View larger version (30K):
[in a new window]
|
Fig. 2. Detection of HUVEC surface A2 and p11 by biotinylation. Following treatment
with thrombin or TRAP, HUVECs were biotinylated using membrane-impermeable
sulfo-NHS-biotin. Surface-labeled proteins were affinity depleted using
avidin-Sepharose and probed by western blotting using anti-A2 mAb, while p11
was immuno-depleted and detected using HRP-avidin.
|
|
View larger version (45K):
[in a new window]
|
Fig. 3. Detection of A2 and p11 in permeabilized HUVECs. HUVECs were treated with
thrombin or TRAP for 5 minutes at 37°C, washed once with culture media
supplemented with 2 mM Ca2+, and allowed to recover for 1 hour at
37°C. After recovery, cells grown on glass coverslips (A) were detergent
permeabilized, fixed and stained for A2, p11 or vimentin (Vmn) and DAPI,
whereas those grown in multiwell plates (B) were subjected to western blot
analysis.
|
|
View larger version (77K):
[in a new window]
|
Fig. 4. Immunofluorescent detection of submembranous A2 in HUVECs. Following
treatment for 5 minutes with thrombin or TRAP and recovery for 1 hour, HUVECs
were fixed in the absence of detergent and stained simultaneously for A2,
vimentin (Vmn) and nuclei (DAPI).
|
|
View larger version (31K):
[in a new window]
|
Fig. 5. Effect of thrombin or TRAP on plasminogen binding to HUVECs. (A)
Fluorescein-plasminogen (F-Plg) binding to HUVECs was evaluated following
thrombin or TRAP treatment by incubating the cells with F-Plg for 30 minutes
at 37°C. The cells were subsequently fixed, and DNA was stained with DAPI.
Identical experiments were performed in which both media and F-Plg solutions
contained either polyclonal anti-p11 IgG or non-immune rabbit IgG (rIgG). (B)
Biotinylated plasminogen (B-Plg) binding to the surface of thrombin- or
TRAP-treated HUVECs was evaluated by incubating cells with B-Plg for 30
minutes at 37°C. After washing unbound ligand, the cells were lysed and
subjected to blot analysis using HRP-avidin. To ensure identical amounts of
cell lysate were loaded in each lane, the blots were reprobed with
anti-ß-actin antibody.
|
|
View larger version (15K):
[in a new window]
|
Fig. 6. Effect of thrombin on HUVEC-dependent plasmin generation. Following mock
( ) or 8 nM thrombin (•) treatment, HUVECs were incubated with 5 nM
recombinant tPA for 20 minutes at 37°C. Lys-plasminogen (A) or
Glu-plasminogen (B) were then added at a concentration of 150 nM, aliquots
were removed at the indicated times and assayed for plasmin activity by
chromogenic substrate (S2251) reactivity (n=3, ±s.d.).
|
|
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
© The Company of Biologists Ltd 2003
