THROMBOSIS RESEARCH 66; 385390,1992 0049-3848192 $5.00 + .OO Printed in the USA. Copyright (c) 1992 Pergamon Press Ltd. All rights reserved.
SUBCELLULAR
LOCALIZATION
DEFIBROTIDE
OF RADIOACTIVELY
IN CULTURED
LABELLED
ENDOTHELIAL
CELLS
BiLSEL, S., ERSAHiN, C., TAGA, Y., EMERK, K.
Department of Biochemistry, Faculty of Medicine, Marmara
(Received
University,
20.1.1992;
81326, Haydarpasa-jstanbui,
accepted
in revised form 54.1992
Turkiye
by Editor O.N. Ulutin)
PBSTFWT Defibrotide is a new antithrombotic trolled depolymerization
and fibrinolytic
drug which is obtained by con-
of mammalian DNA. In various models of arterial and ve-
nous thrombosis, it has been shown that it induces tissue plasminogen [tPA] and prostacyclin
activator
[PGl2] release from the vessel wall. We have previously
shown the presence of specific binding sites with a Kd of 4.2 ug/ml for radioactively labelled defibrotide. The present study was undertaken to identify the location of the binding site. Confluent
cultures of endothelial
were incubated with media containing
cells from
3H-acetyl-defibrotide
human umbilical for various
of time. Cells were then washed and harvested nonenzymatically. of 3H-defibrotide
was investigated ‘by fractionating
vein
intervals
Subcellular location
cells on discontinous
gradient and measuring the distribution of radioactivity. S-nucleotidase
sucrose
enzyme ac-
tivity was also measured to ensure the location of membrane fraction .Our results suggest that the major location of 3H-defibrotide
in endothelial
cells is the plasma
membrane. On the other hand, nuclei also contain a considerable amount of the drug which suggests a mechanism where binding to a membrane protein is followed by internalization.
Key words:
Endothelial cells, Defibrotide, Subcellular localization 385
386
Vol. 66, No. 4
DEFIBROTIDE IN ENDOTHELIAL CELLS
ROlWCTlQCj
Defibrotide ,a drug used in treatement of thrombotic processes, produces its action by modulating plasminogen
endothelium. activator
It significantly
production
stimulates endogenous
and inhibits plasminogen
prostacylin
and tissue
activator inhibitor
production
from the vascular endothelium in a dose dependent manner. In addition it inhibits platelet thromboxane
B2 synthesis
ischemia and reperfusion
(l-5).
It has also cardioprotective
effect in acute myocardial
as shown in cats and rabbits (6). Additional
clinical studies to
evaluate the efficacy of defibrotide in other vascular disorders are in progress at the time . Biochemical mechanism responsible for the pharmacological under investigation.
action of defibrotide is
In our previous studies, to investigate the interaction of the drug with
the endothelial cells, we had labelled defibrotide with 3H-acetic anhydride and we have established that 3H-defibrotide
binds to cultured human endothelial cells in a specific, satur-
able, reversible and time dependent manner with a Kd of 4.2ugIml (7). The purpose of the present study was to determine the subcellular binding location
and the possible biochemi-
cal mechanism responsible for the action of the drug.
n of et&Q&al
cell cultures
Endothelial cells were isolated from the veins of human umbilical cord according to the method of Jaffe with some modifications (8). The cell cultures were identified as endothelial cell cultures by their characteristics
such as the presence of von Willebrand factor.
Cells were cultured in 25 cm2 plastic flasks (Nunc, Denmark). Culture medium consisted of Medium199 units/ml),
(Sigma), 20% fetal calf serum (Sigma), and the anibiotics,
streptomycin
(80ug/ml)
and amphotercin
B (2 ug /ml). The cultures
refed after 24 hour and every other day until1 they are confluent cultures
were incubated
tain time intervals
(1.5,
with M 199 containing
penicillin
(8-10X104).
labelled defibrotide
3, 6 hours). At the end of incubation
(80 were
Confluent
(lOOug/ml) for cer-
the culture medium was
poured off and cells were washed with Hepes buffer solution (HBS) for three times. Cells were harvested by scraping them with rubber policemen rather than suspending with tyrpsin since the plasma membrane enzyme activities were investigated in the experiments.
Vol. 66, No. 4
DEFIBROTIDE
387
IN ENDOTHELIAL CELLS
Endothelial cells were isolated from the umbilical cord vein by 0.1% collagenase solution according to the method given above and suspended in M 199 containing the labelled defibrotide
(lOOug/ml) and incubated for given time intervals.
At the end of incubation,
cells were collected by centrifuging the medium at 180xg for 8 minutes. Cells which were attached to the flasks were harvested by scraping with a cell scraper.
Cells collected from 5 flasks of confuluent cultures (c) or directy isolated from 3-4 umbilical cords (s) were washed with HBS three times. Washing was done by resuspen%ion of cell pellets in HBS and recentrifugation at the force used previously. Cells were than suspended
re-
in ice cold 0.25 M sucrose solution and homogenized with a teflon homogenizer 3
times for 1 minute (approximately
15 strokes ). Microscopic examination of the homogen-
ate estimated 95% rupture of cells. Cell homogenate was than centrifuged for 5 minutes at 5009. The supernatent was decanted and the pellet (nuclear fraction)
was resuspended in
sucrose solution and centrifuged as described above. This procedure was repeated until the supernatant was clear. The post nuclear pellet was obtained by centrifuging
the combined
post nuclear supernatants for 45 minutes at lOO.OOOgat 4OC. Postnuclear pellet included plasma membrane, endoplasmic reticulum, Golgi membranes and all organelles except the nucleus. Post nuclear pellet was suspended in 200 ul of water and applied on top of a discontinous sucrose gradient consisting of 3.5 ml of 53% sucrose, 0.8 ml of 44% sucrose, 3 ml of 39% sucrose, 2.2 ml of 37% sucrose, 2.2 ml of 31% sucrose, 5.3 ml of 18% sucrose. The gradient was centrifuged
at 91.OOOg at 4OC for 45 minutes (9,lO). Gradients
were collected in 45 fractions of 0.4 ml each. Radioactivty of each fraction and of nuclear pellet was measured in Bray solution by a Pacard scintillation
counter. S-nucleotidase
ac-
tivity of fractions were assayed (by Biofvlerieux kit) to determine the location of the membrane fraction.
The destination investigated
of 3H-acetyl
by following
labelled defibrotide
the radioactivity
in subcellular
in cultured fractions.
endothelial Confluent
cells was cultures
of
the cells when incubated with media containing labelled drug for various times ranging’ from
388
DEFIBROTIDE
Vol. 66, No. 4
IN ENDOTHELIAL CELLS
1.56 hours, displayed an uptake of 0.8-2.3% in a time dependent manner. Nonezymatical harvesting and homogenization of cells in sucrose solution yielded a suspension for subcellular fractionation.
Post nuclear pellet which was obtained from the
centrifugation
of post
nuclear supernatants was fractionated on discontinous sucrose gradients as described in the methods.
The radioactivity
in the nuclear pellet and the post nuclear cell fractions were
measured. The amount of cell bound 3H-defibrotide was taken as the sum of these and the percentage of radioactivty in nudei and membranes were calculated and are shown in Table 1. The specificity quantitative
of defibrotide
distribution
binding
of radioactivity
fractions is shown. Approximately
was discussed
previously
and S-nucleotidase
(7). In Figurel,
the
activity between post nuclear
75 % of the total post nuclear radioactivity
was found to
be associated with the membrane fraction designated by 5’ nucleotidase activity. TABLE I
DISTRIBUTION OF RADlOLlGAND IN CELL FRACTIONS
Radioactivity (%) Incubation time
Type of assay
Nuclear
Membrane
90 min (r&)
Suspension
15f3
85 f 7
90 min (n=4)
culture
15k8
85f4
3 hours (r-M)
culture
33fl
67 *3
6 hours (n=3)
culture
17f
1
83 f3
Depending on the time of incubation, approximately 0.8-2.3 % of total radioactivity in culture medium was taken up by the cells. % radioactivities give the rough distribution of this fraction.
Our results indicate that the major location of 3H-defibrotide
in endothelial
cells is
the ptasma membrane. However, nuclei which also contain considerable amounts of the drug seem to be the final binding location. This suggests a mechanism in which the drug is internalized after binding to a specifc protein on the plasma membrane. Since defibrotide has a molecular weight of 30.000, and has negative charges,
such a carrier is needed to carry the
molecule across the plasma membrane to the nucleus.The possibility of this plasma membrane transporter being a receptor their pharmacological
should also be considered since drugs
usually
initiate
effects by binding to specific receptors. The specificity of this recep-
tor protein needs to be established.
Vol. 66, No. 4
DEFIBROTIDE
IN ENDOTHELIAL CELLS
389
12000 10000 8000 5’-NT
6000
cpmx5
0
20
10
30
4-o
50
Fraction no.
FIG. 1 The quantitative
distribution
of radioactivity
and S-nucleotidase
(V-NT)
activity
between post-nuclear fractions. Post nuclear sucrose crose,
gradient
pellet
consisting
was suspended
of 3.5 ml of 53% sucrose,
2.2 ml of 37% sucrose,
centrifuged
in 200 PI of water
2.2 ml of 31% sucrose,
at 91.OOOg at 4OC for 45 minutes.
Gradients
and applied
0.8 ml of 44%
to top of a discontinous
sucrose,
5.3 ml of 18% sucrose. were
collected
3 ml of 39% The gradient
in 45 fractions
suwas
of 0.4 ml
each.
Previous studies have shown that defibrotide increases the protein content of endothelial cells remarkably (11). The increase in protein concentration duction (increased
synthesis)
of profibrinolytic
may be due to the in-
and nonthrombogenic
mediators.
Ddfibro-
tide elevates t-PA (antigen and functions) and prostacyclin . These observations suggest that defibrotide exerts its effects by increacing or decreasing protein synthesis of some mediators via regulation of gene expression of these mediators. Nucleus’ being the final destination of defibrotide is in accordance with this suggested mechanism of action.
390
Vol. 66, No. 4
DEFIBROTIDE IN ENDOTHELIAL CELLS
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