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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Subcellular localization of radioactively labelled defibrotide in cultured endothelial cells.

Defibrotide is a new antithrombotic and fibrinolytic drug which is obtained by controlled depolymerization of mammalian DNA. In various models of arte...
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