Lipoproteins Inhibit the Secretion of Tissue Plasminogen Activator

438
Lipoproteins Inhibit the Secretion of Tissue
Plasminogen Activator From
Human Endothelial Cells
Eugene G. Levin, Lindsey A. Miles, Gunther M. Fless, Angelo M. Scanu, Patricia Baynham,
Linda K. Curtiss, Edward F. Plow
Downloaded from http://atvb.ahajournals.org/ by guest on October 19, 2016
Abstract We studied the effect of lipoprotein(a) [Lp(a)],
low-density lipoprotein (LDL), and high-density lipoprotein
(HDL) on tissue plasminogen activator (TPA) secretion from
human endothelial cells. At 1 /wnol/L, Lp(a) inhibited constitutive TPA secretion by 50% and phorbol myristate acetateand histamine-enhanced TPA secretion by 40%. LDL and
HDL also depressed TPA secretion by 45% and 35% (constitutive) and 40% to 60% (stimulated). TPA mRNA levels were
also examined and found to change in parallel with antigen
A
high levels, lipoprotein(a) [Lp(a)] is a major risk
factor for premature development of athero. sclerosis, myocardial infarction, and the severity of coronary artery disease. 14 Low-density lipoprotein (LDL) is also atherogenic, but Lp(a) and LDL are
independent risk factors for these diseases, suggesting a
difference in their pathogenic mechanisms. Lp(a) is
similar to LDL in composition and structural organization but contains an additional apoprotein, apo(a).5
Apo(a) is very similar in structure to plasminogen.6'7 It
may contain 36 repeats of the fourth kringle of plasminogen as well as a single kringle 5 and a protease-like
region.5 However, Lp(a) is not susceptible to cleavage
by plasminogen activators. This similarity in structure
between Lp(a) and plasminogen has led to the hypothesis that Lp(a) can interfere with the functions of
plasminogen.8 In vitro studies of such effects have
shown that Lp(a) inhibits plasminogen activation by
streptokinase, suppresses the fibrin and cell surfacedependent enhancement of plasminogen activation by
tissue plasminogen activator (TPA), and suppresses
plasminogen binding to U937 monocytoid and endothelial cells. 918 The LDL particle, which lacks apo(a), does
not compete for plasminogen binding sites.9'17 Thus,
Lp(a) interferes with plasminogen activation by inhibiting several molecular interactions of plasminogen.
secretion. In contrast to TPA, plasminogen activator inhibitor
type-1 secretion and mRNA levels were not affected by any of
the three lipoproteins. These results suggest that the interaction of lipoproteins with certain cell-surface binding sites may
interfere with the proper production and/or secretion of TPA.
(Arteriosder Thromb. 1994; 14:438-442.)
Key Words • Lp(a) • tissue plasminogen activator •
endothelial cells • plasminogen activator inhibitor type-1
In addition to the physical interference of Lp(a) with
plasminogen binding to cells and other proteins, a
prothrombotic state can be promoted by interference
with the normal production and/or secretion of the
fibrinolytic components associated with endothelial
cells, ie, TPA, the plasminogen activator primarily responsible for blood clot lysis,19 and plasminogen activator inhibitor-1 (PAI-1). For example, inhibition of TPA
release or elevation of PAI-1 production would create a
prothrombotic state in the endothelial cell environment.
It has been shown previously that addition of Lp(a) to
human endothelial cells in culture stimulates PAI-1
production up to twofold over a 16-hour period.20 In the
present study, we have examined the effects of Lp(a) on
endothelial cell expression of the fibrinolytic components PAI-1 and TPA. We report here that the addition
of Lp(a) to monolayers of human umbilical endothelial
cells suppresses TPA secretion while having a minimal
effect on PAI-1 secretion. However, similar results were
also observed with LDL and high-density lipoprotein
(HDL), suggesting a general mechanism by which lipoproteins might promote a prothrombotic state through
the downregulation of TPA secretion without significantly affecting PAI-1 expression.
Methods
Materials
Received February 17, 1993; revision accepted December 9,
1993.
From the Department of Molecular and Experimental Medicine
(E.G.L.), Committee on Vascular Biology (E.G.L., L.A.M., P.B.),
and Department of Immunology (L.K.C.), The Scripps Research
Institute, La Jolla, Calif; the Department of Medicine (G.M.F.,
A.M.S.) and Department of Biochemistry and Molecular Biology
(A.M.S.), University of Chicago (111); and the Center for Thrombosis and Vascular Biology (E.F.P.), The Cleveland Clinic (Ohio).
Correspondence to Dr Eugene G. Levin, Department of Molecular and Experimental Medicine, SBR13, The Scripps Research
Institute, 10666 N Torrey Pines Rd, La Jolla, CA 92037.
Phorbol myristate acetate (PMA) was from Calbiochem.
Monoclonal antibodies to PAI-1 (No. 1105) and TPA were
from Monozyme and Corvas, Inc, respectively.
Cell Culture
All tissue culture reagents were purchased from sources
previously described.21 Endothelial cells were isolated from
human umbilical vein cord veins21 and were cultured into
75-cm2 tissue culture flasks coated with 20 mg/mL calf skin
gelatin. Cells were grown to confluence in RPMI 1640 containing 10% fetal calf serum, 200 U/mL penicillin, 200 figJmL
streptomycin, 10 /ig/mL endothelial cell growth factor
Levin et al
Downloaded from http://atvb.ahajournals.org/ by guest on October 19, 2016
(ECGF), and 90 fig/mh heparin. Passaged cells were subcultured into 12-well dishes or 75-cm2 culture flasks and allowed
to grow to confluence under the same conditions as primary
cultures except that 50 jig/mL ECGF and 15% fetal calf serum
were used. All experiments used once-passaged cultures.
Average cell densities at confluence were 6X104 cells per
square centimeter.
Studies were performed by washing confluent cultures twice
with RPMI 1640 and incubating cultures at 37°C in 0.5 mL of
medium containing 5% NuSerum (Collaborative Research; a
final serum concentration of 1.25% newborn calf serum), 50
fig/mL ECGF, 90 /ig/mL heparin, and the indicated agent or
vehicle. Stock solutions of phorbol esters (Calbiochem) were
made in dimethyl sulfoxide (DMSO) (Calbiochem). In no case
was the final concentration of DMSO added to cells greater
than 0.1%. Vehicle concentration was kept constant in all
experimental samples regardless of differences in the concentration of the compound tested. The presence of ECGF,
heparin, or DMSO had no effect on TPA release in the
presence of any of the compounds used. When appropriate,
conditioned medium was centrifuged at 15 OOOg to remove cell
debris, adjusted to 0.1% Tween 80, and frozen at -70°C until
used.
Lipoprotein Isolation
LDL (<i=1.1019 to 1.063 g/mL) and HDL (<f=1.063 to 1.21
g/mL) were isolated from 600 to 800 mL of pooled normal
human plasma by ultracentrifugation in the presence of protease inhibitors as described.22 The isolated lipoproteins were
dialyzed against 0.15 mol/L NaCl containing 272 /i.mol/L
EDTA, pH 7.5, and their apoprotein composition was verified
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Endotoxin contamination of the lipoproteins during isolation
was reduced by the use of pyrogen-free sterile water, ovenbaked glassware (185°C for 4 to 18 hours), and E-Toxa
(Sigma)-cleaned supplies. Endotoxin was monitored by the
chromogenic Limulus amebocyte assay (Whittaker Bioproducts), which had a sensitivity of 0.1 endotoxin U/mL.
Lp(a) was isolated from fresh human plasma using flotation
centrifugation followed by affinity chromatography on lysine
Sepharose.6 The LDL preparation contained no apoA-1, and
the HDL contained no detectable apoB. Lipoprotein concentrations were determined by a modified Lowry assay.23 No
contamination of the Lp(a) preparations was detected by
immunoassay. These preparations were prepared from two
different donors and consisted of a single isoform with an
apoprotein M, of 280 000.
Quantitation of TPA and PAI-1
TPA and PAI-1 antigens were quantified by enzyme-linked
immunosorbent assay. Microtiter plates were coated with 10
Aig/mL rabbit antiserum to human TPA or PAI-1, in 50
mmol/L sodium borate, pH 9.0, at 4°C overnight and washed
with a wash buffer of 0.15 mol/L NaCl and 0.01 mol/L
phosphate, pH 7.3 (PBS), containing 0.05% Tween 20 and
0.1% bovine serum albumin (BSA). For the TPA assays,
samples or standards were diluted into culture medium, and
100 /xL was added to the wells. Immediately afterward, 20 fiL
anti-human TPA monoclonal antibody horseradish peroxidase
conjugate, diluted 1:50 into PBS/0.1% BSA, was added and
the plate incubated for 1.5 hours at room temperature with
rocking. The contents of the wells were removed, each plate
was washed with water three times, and 100 fih of substrate
was added. The substrate mixture was prepared by diluting
125 /xg/mL trimethoxybenzoic acid into 0.1 mol/L sodium
acetate, pH 4.5, containing 0.013% H2O2. The assays were
allowed to develop for 10 minutes in the dark, and the reaction
was stopped by the addition of 100 jtL of IN H^Q,. The plates
were read at 450 nm. For PAI-1 measurements, assay samples
and standards were diluted into PBS, 0.05% Tween 20, and
1% BSA, and 100-jiL aliquots were incubated in the coated
Lp(a) Inhibits TPA Production
439
wells for 2 hours at room temperature. The plates were washed
three times, and 0.25 figJmL of monoclonal antibody to PAI-1,
diluted into PBS/0.1% BSA/0.05% Tween 20 and 0.1% bovine
y-globulin, was added to the wells and incubated for 2 hours at
22°C. After washing, the wells were incubated with a 1:2500
dilution of horseradish peroxidase-conjugated rabbit antimouse IgG for 2 hours and then washed, and 1 mg/mL of
2,2'-azino-bis3-ethylbenzthiazoline-6-sulfonic acid in 0.0015%
H2O2 was added. Plates were read at 405 nm after 45 minutes
of incubation. Standard curves for both TPA and PAI-1 assays
were determined by log-logit transformation and linear regression analysis of logit B (absorbance) versus In TPA or PAI-1
concentration. Values in the range of 0.2 to 12.5 ng/mL TPA
and 1.6 to 25 ng/mL PAI-1 gave linear dose-response curves.
mRNA Quantitation
RNA was extracted by the method of Chomczynski and
Sacchi,24 and TPA mRNA levels were measured by slot blot
analysis as previously described.25 A full-length cDNA clone
encoding the mature TPA protein plus 200 bp of the 3'untranslated region and a PAI-1 cDNA clone containing the
complete coding region, 80 bp of the 5'-untranslated region,
and 140 bp of the 3'-untranslated region were used as probes
for hybridization. An actin probe was used as a control
(furnished by Dr L. Kedes, Stanford University).
Chromium-51 Labeling of Cells
Cells were labeled with 250 /xCi/mL 51Cr (Amersham International) for 4 to 6 hours in medium containing 10% fetal calf
serum. After the labeling period, cultures were washed twice
with RPMI 1640 and then treated with medium containing 5%
NuSerum and the indicated agonists in the presence or
absence of lipoproteins for 16 hours. Medium was collected
and cells were extracted with 0-5% Triton X-100. Both medium and cell extract were counted, and the chromium remaining was calculated as the percentage of cells incubated with
agonist in the absence of lipoprotein.
Results
The effect of three different classes of lipoproteins,
Lp(a), LDL, and HDL, on constitutive TPA secretion
(12.2±4.3 ng/mL in 16 hours; range, 6.5 to 22.0 ng,
n=17) was examined by addition of increasing concentrations of each to confluent monolayers of endothelial
cells. Lp(a) inhibited TPA secretion in a dose-dependent
manner, with 1 /imol/L Lp(a) reducing TPA levels to
49.1±8.1% of control after 16 hours (Fig 1, top). Lp(a)
was also effective against PMA-stimulated (100 nmol/L;
Fig 1, middle) and histamine-stimulated (10 ftg/mh; Fig
1, bottom) TPA secretion. The level of TPA measured in
the culture medium after 16 hours, a time when TPA
levels reached their maximum levels in both cases, was
reduced to 51.1 ±4.2% and 57±5.6%, respectively.
In contrast to the inhibitory effect of Lp(a) on
plasminogen binding to cell surfaces,9 other lipoproteins
were also effective at blocking both constitutive and
stimulated TPA secretion. LDL and HDL inhibited
constitutive release to 55.2±7.3% and 64.9±5% of
control, respectively (Fig 1, top), histamine-stimulated
TPA secretion to 49.9±7.9% and 67.9±1.5% (Fig 1,
bottom), and PMA-stimulated secretion to 30.6±4.8%
and 59.8±4.2% (Fig 1, middle), respectively. Addition
of plasminogen (1 jimol/L) to the cells under identical
conditions had no effect on TPA secretion (data not
shown). The effect of LDL on various concentrations of
histamine and PMA was also tested. At concentrations
ranging from 0.1 to 20 figJmL histamine, 1 /tmol/L LDL
inhibited the stimulated rate of TPA secretion by 30%
440
Arteriosclerosis and Thrombosis
Vol 14, No 3 March 1994
£ '
.01
M
0.1
Upoproteln cone.
0.5
.01
1.0
AS
0.1
OJ
Llpoprotatn cone. (
FIG 2. Une graph shows effect of lipoproteins on plasmlnogen activator inhibitor—1 (PAI-1) secretion. Increasing concentrations of lipoprotein(a) [Lp(a)] (o), low-density lipoprotein
(LDL) (•), or high-density lipoprotein (HDL) (A) were added to
confluent cultures of endotheliai cells for 16 hours. Culture
media were removed, prepared, and assayed for PAI-1 antigen
as described in "Methods." Data points represent mean±SD
of four experiments.
Downloaded from http://atvb.ahajournals.org/ by guest on October 19, 2016
.01
JOS
0.1
0J
1.0
Lipoprotein cone, {pit)
.01
.05
0.1
0.5
Upoproteln cone. GUI)
1X1
FIG 1. Une graphs show effect of lipoproteins on tissue plasminogen activator (tPA [TPA]) secretion from human endotheliai
cells. Increasing concentrations of lipoprotein(a) [Lp(a)] (o),
low-density lipoprotein (LDL) (•), or high-density lipoprotein
(HDL) (A) were added to confluent cultures of endotheliai cells
treated with nothing (top), 100 mmol/L phorbol myristate acetate
(middle), or 10 /ig/mL histamine (bottom) for 16 hours. Culture
media were removed, prepared, and assayed for TPA antigen as
described in "Methods." Data points represent mean±SD of at
least six determinations.
to 35% at each concentration. At concentrations of
lCT10 to l<r 7 mol/L PMA, 0.5 ^mol/L LDL inhibited
secretion by 50% at each point.
PAI-1 antigen levels were also examined in replicate
samples (Fig 2). Despite previous reports that Lp(a)
stimulates PAI-1 secretion from endotheliai cells,20 we
detected no significant change in PAI-1 levels after 16
hours of incubation either with this lipoprotein, LDL, or
HDL (96.6±16.0%, 98.5±5.9%, and 101.8+8.8%, respectively) (Fig 2).
To ensure that the decline in TPA secretion and the
absence of an effect on PAT-1 secretion were not due to
loss of cell viability, we performed 51Cr release assays in
cultures treated with each agonist in the presence or
absence of lipoproteins (Table). When compared with
the level of 5lCr associated with cells treated with
agonist alone, the addition of each lipoprotein for 16
hours did not decrease 51Cr content by more than 10%.
Thus, the effects observed in the presence of lipoproteins were not due to loss of cell viability.
To examine whether TPA mRNA levels are affected
in the same manner as antigen levels, we performed slot
blot analysis on cultures treated with vehicle, 1 /xmol/L
LDL, 100 nmol/L PMA/100 /xmol/L forskolin, and
PMA/forskolin and LDL. Forskolin, which elevates
cyclic AMP levels through the activation of adenylate
cyclase, potentiates the PMA effect fivefold, making the
quantitative measurement of TPA mRNA easier. Cyclic
AMP does not, however, change any of the characteristics of the TPA response to PMA.26 Slot blot analysis
of TPA mRNA showed a 67% decline in steady-state
levels after 8 hours of LDL treatment (n = 2) (Fig 3). In
the presence of PMA/forskolin, TPA mRNA increased
3.6-fold within the same time period but only 2.3 times
in the presence of LDL. Whether the same effect on
TPA mRNA is observed with HDL or Lp(a) is unknown. PAI-1 mRNA levels were unaffected by 1
/xmol/L LDL (data not shown).
Discussion
We have demonstrated that the treatment of human
umbilical vein endotheliai cells with either Lp(a), LDL,
or HDL results in a dose-dependent depression in both
constitutive and stimulated TPA secretion. This effect
on TPA secretion occurs with no significant change in
the level of PAI-1 antigen over the same period of time.
Chromlum-51 Release Assay
Counts per Minute, x10~ 3
Percent
Control
458±16
100
Buffer
412±15
90
LDL
428 ±3
93
Condition
HDL
427±15
93
Histamine
439±11
100
Histamine+LDL
390±8
89
Histamine+HDL
409±2
93
PMA
405±7
100
PMA+LDL
367 ±8
91
PMA+HDL
378±13
93
LDL indicates low-density lipoprotein; HDL, high-density lipoprotein; and PMA, phorbol myristate acetate.
Levin et al
Lp(a) Inhibits TPA Production
441
Control
PMA + fors
PMA + fors 1
Control
+ LDL
Actin tPA
Downloaded from http://atvb.ahajournals.org/ by guest on October 19, 2016
Fe 3. Blots show effect of low-density Iipoprotein (LDL) on tissue piasminogen activator (TPA) mRNA levels. Cultures were treated with
or without 100 mmol/L phorbol myiistate acetate (PMA) and 50 ^mol/L forskoiin (fors) for 8 hours In the presence or absence of 1 /xmol/L
LDL. Cells were extracted, and RNA was isolated as described in "Methods." TPA mRNA was measured by slot blot analysis; the values
presented in the text were derived from comparing the counts per minute in the bands representing the LDL-treated samples with that
of the respective control. Slots hybridized with a cDNA probe for actin are shown to demonstrate comparable loading of RNA.
Thus, abnormally high levels of Lp(a) and other lipoproteins may promote a prothrombotic state by manipulating the fibrinolytic capacity within the endothelial
cell environment.
Our results contrast with the previous report of
Etingin et al,20 who found that Lp(a) enhanced PAI-1
antigen secretion from human endothelial cells in culture by twofold while having no effect on TPA production. It is not clear why this discrepancy exists, although
the culture conditions under which these studies were
performed were quite different. Etingin et al performed
experiments in serum-free medium in contrast to the
1.25% serum used in the present studies. We have
determined previously that the level of constitutive and
stimulated TPA production and secretion in human
umbilical endothelial cells is dependent on the presence
of serum and that, in its absence, TPA secretion is
unresponsive to modulating compounds.21 Another potential source for the differences in results may be
related to the state of Iipoprotein oxidation. For example, Latron et al27 and Kugiyama et al28 reported that
oxidized but not nonoxidized LDL affects PAI-1 synthesis by endothelial cells. Our findings also are supported
to some extent by in vivo observations.29-30 In one study,
patients with coronary artery disease and levels of
Lp(a) 2.5 to 3.5 times higher than control subjects
showed a decreased ability to release TPA in response
to venous occlusion (50%), although basal levels of TPA
were increased 76%. PAI-1 levels increased an average
of only 25%. In the second study, a negative correlation
between Lp(a) levels and PAI-1 levels was observed.
The depression of TPA secretion from endothelial
cells can also be promoted by the cytokine interleukinl.31 Prolonged treatment of the cells with interleukin-1
results in a 50% decrease in the constitutive release of
TPA, whereas interleukin-1 stimulates PAI-1 expression. Addition of interleukin-1 to cells stimulated with
PMA also has a depressive effect (E.G. Levin, unpublished observations). In addition, we have demonstrated
recently that the disruption of microtubule organization
during treatment of cells with phorbol esters or hista-
mine depresses enhanced TPA secretion by approximately 50%.32 Thus, our observation that TPA secretion
can be inhibited by treatment of endothelial cells with
lipoproteins is not unusual.
The mechanism by which the three lipoproteins inhibit TPA secretion is not clear, eg, whether it occurs
through a protein or lipid moiety or whether it occurs
through a general perturbation of the endothelial cell
membrane or through a specific receptor or receptors.
The lack of specificity of the Iipoprotein effect shown in
Fig 1 suggests that it is the lipid components of the
lipoproteins that are responsible for the biologic activity, because the various lipoproteins do not contain
common apoproteins but do contain various common
lipid components, although at different relative proportions. A number of the lipid constituents are viable
candidates, including the lysolecithins, oxygenated sterols, and free fatty acids. The fact that all three lipoproteins have similar effects also suggests that the site of
interaction is similar in each case, a suggestion consistent with recent observations that LDL and HDL can
both inhibit Lp(a) binding to endothelial cells and that
gangliosides, which inhibit piasminogen binding to endothelial cells, also inhibit both Lp(a) and LDL binding.33 Although HDL and LDL generally exert counterbalancing effects, competition between HDL and LDL
for cellular binding sites on endothelial cells34 and other
cells35 has been noted. Tabas and Tall36 suggested that
the association of HDL with endothelial cells may be
dependent on lipid interactions that could be shared by
all Iipoprotein classes. Lipid transfer mechanisms28 may
also be involved and could be shared among the various
lipoproteins. The interaction of lipoproteins with certain shared cell surface binding sites such as lipids may
interfere with the proper production and/or secretion of
TPA.
Acknowledgments
This work was supported in part by National Institutes of
Health grants HL-40435, HL-43344, HL-35297, HL-38272,
and HL-14197 and MO1 RR0O833 to the General Clinical
442
Arteriosclerosis and Thrombosis
Vol 14, No 3 March 1994
Research Center of Scripps Clinic. This work was done during
the tenure of Established Investigatorship awards from the
American Heart Association and SmithKline Beecham to Dr
Miles and from the American Heart Association to Dr Levin.
References
Downloaded from http://atvb.ahajournals.org/ by guest on October 19, 2016
1. Koltringer P, Jurgens G. A dominant role of lipoprotein(a) in the
investigation and evaluation of parameters indicating the development of cervical atherosclerosis. Atherosclerosis. 1985;58:
187-198.
2. Armstrong VW, Cremer P, Eberle E, Manke A, Shulze H, Wieland
H, Kreuzer H, Seidel D. The association between serum Lp(a)
concentrations and angiographically assessed coronary atherosclerosis. Atherosclerosis. 1986;62:249-257.
3. Rhoads GG, Dahlen G, Berg K, Morton NE, Dannenberg AL.
Lp(a) lipoprotein as a risk factor for myocardial infarction. J Am
MedAssoc. 1986;256:2540-2544.
4. Hoefler G, Harnoncourt F, Paschke E, Mirti W, Pfeiffer KH,
Kostner GM. Lipoprotein Lp(a): a risk factor for myocardial
infarction. Arteriosclerosis. 1988;8:398-401.
5. Scanu AM. Lipoprotein(a): a genetically determined lipoprotein
containing a glycoprotein of the plasminogen family. Semin
Thromb Hemost. 1988;14:266-270.
6. Eaton DL, Fless GM, Kohr WJ, McLean JW, Xu Q-T, Miller CG,
Lawn RM, Scanu AM. Partial amino acid sequence of apolipoprotein(a) shows that it is homologous to plasminogen. Proc NatlAcad
SciUSA. 1987;84:3224-3228.
7. McLean JW, Tomlinson JE, Juang W-J, Eaton DL, Chen EY, Fless
GM, Scanu AM, Lawn RM. cDNA sequence of human apolipoprotein(a) is homologous to plasminogen. Nature. 1987;330:
132-137.
8. Miles LA, Plow EF. Lp(a): an interloper into the fibrinolytic
system? Thromb Haemost. 1990;63:331-335.
9. Miles LA, Gless GM, Levin EG, Scanu AM, Plow EF. A potential
basis for the thrombotic risks associated with lipoprotein(a).
Nature. 1989;339:301-303.
10. Harpel PC, Gordon BR, Parker TS. Plasmin catalyzes binding of
lipoprotein(a) to immobilized fibrinogen and fibrin. Proc NatlAcad
SciUSA. 1989;86:3847-3851.
11. Loscalzo J, Weinfeld M, Fless GM, Scanu AM. Lipoprotein(a),
fibrin binding, and plasminogen activation. Arteriosclerosis. 1990;
10:240-245.
12. Edelberg JM, Gonzales-Gronow M, Pizzo SV. Lipoprotein(a) inhibition of plasminogen activation by tissue-type plasminogen
activator. Thromb Res. 1990;57:155-162.
13. Karadi I, Kostner GM, Gries A, Nimpf J, Romics L, Malle E.
Lipoprotein(a) and plasminogen are immunochemically related.
Biochim Biophys Ada. 1988;960:91-97.
14. Edelberg JM, Gonzalez-Gronow M, Pizzo SV. Lipoprotein(a)
inhibits streptokinase-mediated activation of human plasminogen.
Biochemistry. 1989;28:2370-2374.
15. Kluft C, Jie AFH, Los P, deWit E, Havekes L. Functional analogy
between lipoprotein(a) and plasminogen in the binding to the
kringle 4 binding protein, tetranectin. Biochem Biophys Res
Commun. 1989;161:427-433.
16. Miles LA, Dahlberg CM, Plow EF. The cell-binding domains of
plasminogen and their function in plasma. J Biol Chem. 1988;263:
11928-11934.
17. Hajjar KA, Gavish D, Breslow JL, Nachman RL. Lipoprotein(a)
modulation of endothelial cell surface fibrinolysis and its potential
role in atherosclerosis. Nature. 1989;339:303-305.
18. Ezratty A, Simon DI, Loscalzo J. Lipoprotein(a) competitively
inhibits plasminogen activation on the platelet surface. Circulation.
1991;84(suppl II):II-566. Abstract.
19. Levin EG. Latent tissue plasminogen activator produced by human
endothelial cells in culture: evidence for an enzyme-inhibitor
complex. Proc Natl Acad Sci USA. 1983;80:6804-6808.
20. Etingin OR, Hajjar DP, Hajjar KA, Harpel PC, Nachman RL.
Lipoprotein(a) regulates plasminogen activator inhibitor-1
expression in endothelial cells. J Biol Chem. 1991;266:2459-2465.
21. Levin EG, Marzec U, Anderson J, Harker LA. Thrombin stimulates tissue plasminogen activator release from cultured human
endothelial cells. J Clin Invest. 1984;74:1988-1995.
22. Curtiss LK, Edgington TS. Immunochemical heterogeneity of
human plasma high density lipoproteins. / Biol Chem. 1985;260:
2982-2993.
23. Markwell MAK, Haas SM, Bieber LL, Tolbert NE. A modification
of the Lowry procedure to simplify protein determination in
membrane and lipoprotein samples. Anal Biochem. 1978;87:
206-210.
24. Chomczynski P, Sacchi N. Single-step method of RNA isolation by
acid guanidinium thiocyanate-phenol-chloroform extraction. Anal
Biochem. 1987;162:156-159.
25. Levin EG, Marotti KR, Santell L. Protein kinase C and the stimulation of tissue plasminogen activator release from human endothelial cells: dependence on the elevation of messenger RNA.
J Biol Chem. 1989;264:16030-16036.
26. Santell L, Levin EG. Cyclic AMP potentiates phorbol ester stimulation of tissue plasminogen activator release and inhibits
secretion of plasminogen activator inhibitor-1 from human endothelial cells. J Biol Chem. 1988;263:16802-16808.
27. Latron Y, Chautan M, Anfosso F, Alessi MC, Nalbone G, Lafont
H, Juhan-Vague I. Stimulating effect of oxidized low density lipoproteins on plasminogen activator inhibitor-1 synthesis by endothelial cells. Arterioscler Thromb. 1991;11:1821-1829.
28. Kugiyama K, Sakamoto T, Misumi I, Sugiyama S, Ohgushi M,
Ogawa H, Horiguchi M, Yasue H. Transferable lipids in oxidized
low-density lipoprotein stimulate plasminogen activator inhibitor-1
and inhibit tissue-type plasminogen activator release from endothelial cells. Circ Res. 1993;73:335-343.
29. Heinrich J, Sandkamp M, Kokott R, Schultz H, Assmann G. Relationship of lipoprotein(a) to variables of coagulation and fibrinolysis in a healthy population. Clin Chem. 1991;37:1950-1954.
30. Garcia Frade LJ, Alvarez JJ, Rayo I, Torrado MC, Lasuncion MA,
Garcia Avello A, Hernandez A, Marin E. Fibrinolytic parameters
and lipoprotein(a) levels in plasma of patients with coronary artery
disease. Thromb Res. 1991;63:407-418.
31. Bevilacqua MP, Schleef RR, Gimbrone MA, Loskutoff DJ. Regulation of the fibrinolytic system of cultured human vascular endothelium by interleukin 1.7 Clin Invest. 1986;78:587-591.
32. Santell L, Marotti K, Bartfeld NS, Baynham P, Levin EG. Disruption of microtubules inhibits the stimulation of tissue plasminogen activator expression and promotes plasminogen activator
inhibitor type 1 expression in human endothelial cells. Exp Cell
Res. 1992;201:358-365.
33. Plow EF, Fless GM, Scanu AM, Hickey DM, Levin EG, Curtiss
LK, Miles LA. Direct binding of Lp(a) particles to plasminogen
receptors on endothelial and monocytoid cells. Thromb Haemost.
1991;65:205a. Abstract.
34. Alexander JJ, Miguel R, Graham D. High density lipoprotein
inhibits low density lipoprotein binding and uptake by bovine
aortic endothelial cells. Angiology. 1990;41:1065-1069.
35. Miller NE, Weinstein DB, Carew TE, Koschinsky T, Steinberg D.
Interaction between high density and low density lipoproteins
during uptake and degradation by cultured human fibroblasts.
J Clin Invest. 1977;60:78-88.
36. Tabas I, Tall AR. Mechanism of the association of HDL, with
endothelial cells, smooth muscle cells, and fibroblasts. J Biol Chem.
1984;259:13897-13905.
Downloaded from http://atvb.ahajournals.org/ by guest on October 19, 2016
Lipoproteins inhibit the secretion of tissue plasminogen activator from human endothelial
cells.
E G Levin, L A Miles, G M Fless, A M Scanu, P Baynham, L K Curtiss and E F Plow
Arterioscler Thromb Vasc Biol. 1994;14:438-442
doi: 10.1161/01.ATV.14.3.438
Arteriosclerosis, Thrombosis, and Vascular Biology is published by the American Heart Association, 7272 Greenville
Avenue, Dallas, TX 75231
Copyright © 1994 American Heart Association, Inc. All rights reserved.
Print ISSN: 1079-5642. Online ISSN: 1524-4636
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://atvb.ahajournals.org/content/14/3/438
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in
Arteriosclerosis, Thrombosis, and Vascular Biology can be obtained via RightsLink, a service of the Copyright
Clearance Center, not the Editorial Office. Once the online version of the published article for which permission
is being requested is located, click Request Permissions in the middle column of the Web page under Services.
Further information about this process is available in the Permissions and Rights Question and Answerdocument.
Reprints: Information about reprints can be found online at:
http://www.lww.com/reprints
Subscriptions: Information about subscribing to Arteriosclerosis, Thrombosis, and Vascular Biology is online
at:
http://atvb.ahajournals.org//subscriptions/