Lipoprotein (A)

221 Pages·1990·4.293 MB·English

by  Angelo Scanu (Eds.)

Lipoprotein(a)
Edited by
Angelo M. Scanu
Department of Medicine, Biochemistry, and Molecular Biology
University of Chicago
Chicago, Illinois
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Library of Congress Cataloging-in-Publication Data
Scanu, Angelo M. Date.
Lipoprotein (a) / Angelo M. Scanu.
p. cm.
Includes bibliographical references.
ISBN 0-12-620990-1 (alk. paper)
1. Lipoprotein A-Pathophysiology. 2. Lipoprotein A-Metabolism.
3. Atherosclerosis-Pathophysiology. 4. Coronary heart disease-
-Pathophysiology. I. Title.
[DNLM: 1. Lipoproteins, LDL—genetics. 2. Lipoproteins, LDL-
-metabolism. 3. Plasminogen-physiology. QU 85 S283L]
QP552.L5S26 1990
612'.015754--dc20
DNLM/DLC
for Library of Congress 89-18248
CIP
Printed in the United States of America
90 91 92 93 9 8 7 6 5 4 3 2 1
Preface
Lipoprotein(a), usually referred to as Lp(a), made its entry into the
scientific field about 25 years ago through the original studies by
Norwegian geneticist Káre Berg, who first identified in human blood
a special lipoprotein genetically transmitted and associated with an
increased risk for atherosclerotic cardiovascular disease (ASCVD).
However, for many years the structural properties of Lp(a) escaped
clarification and, as a consequence, Lp(a) failed to receive the deserved
recognition by those working in the cardiovascular field. This scenario
was changed dramatically by the joint discovery by investigators at the
University of Chicago and Genentech in 1987 that the specific glyco-
protein determinant of Lp(a), apolipoprotein(a) or apo(a), has striking
structural similarities with plasminogen as well as a common genetic
determination. This discovery catalyzed a series of multidisciplinary
studies by workers in both the fields of atherosclerosis and thrombosis
resulting in a number of novel observations and new pathogenetic
views on the role of Lp(a) in ASCVD. The resulting explosion of
information called for an assessment of the state of the art in the field
and also for the identification of the most promising areas of future
research. To this end, an International Symposium was organized in
Chicago on December 2 and 3, 1988, under the sponsorship of the
University of Chicago, the National Institutes of Health (Grant R13
HL-41622), and several pharmaceutical companies.1 This book is an
account of the proceedings of that symposium. Chapters were written
by each of the speakers which provide an account of their presen-
tation. The topics discussed cover the several aspects of the research
on Lp(a) and go from a useful and authoritative historical coverage to
issues of structure, metabolism, comparative biology, epidemiology,
and treatment.
Several issues emerged. Lp(a) represents a class of plasma lipo-
proteins which differ in size and density but have apo(a) as the specific
marker. The apo(a) and plasminogen genes are both localized in the
long arm of chromosome 6 and may derive from the same ancestral
1 Sponsoring companies: Bristol-Myers Squibb, Ciba-Geigy, Genentech, Merck
Sharpe & Dohme, Merrel Dow, Pfizer, Sandoz, and Upjohn.
xi
Xll Preface
gene. Lp(a) has a metabolic behavior different from that of LDL from
which it differs by having apo B modified by a covalent attachment to
apo(a). Lp(a) is not confined to the human species. Only a small
percentage of people (10-15%) has high plasma levels of Lp(a). On an
epidemiological basis, high levels of Lp(a) in plasma are associated
with an increased risk for ASCVD by yet unknown mechanisms.
The pathogenicity of Lp(a) may be due to its cholesteryl ester
content contributing to the formation of the foam cells that are to be
precursors of the atherosclerotic plaque. At this time, however, there
are no data supportive of this mechanism. Lp(a) might be endowed of
special permeability properties and thus transverse the endothelial
and subendothelial layers and deposit in the arterial wall; in this
context apo(a) has been detected in arterial tissues but its pathogenic
significance has not been established. The fact that Lp(a) has a plas-
minogen-like component has stimulated research about its potential
pro-thrombotic action. As a result, in vitro studies have shown that
Lp(a) competes for the binding of plasminogen to fibrinogen or fibrin
and also interferes with other steps in the fibrinolytic and coagulation
system. Moreover, Lp(a) is a good competitor for the binding of plas-
minogen to plasminogen receptors which have been shown to occur in
several cell membranes. All of these findings are of obvious clinical
interest but have not yet seen application at this level.
In spite of all these advances, several questions remain unanswered:
What are the structure and biology of the apo(a) size-polymorphs and
what is their relation to Lp(a) size-density heterogeneity? What is the
regulation of apo(a) synthesis and its integration into a mature Lp(a)
particle? What is the catabolic fate of Lp(a)? What is the physiological
role of Lp(a)? How do we control Lp(a) levels in the plasma?
Lp(a) has become a challenging and attractive area for research in
the cardiovascular area and one which continues to call for multidisci-
plinary approaches. The multiauthorship of this book clearly docu-
ments this need. Each author has combined expertise and experience
in providing an up-to-date account of the various aspects of Lp(a)
research. As a whole, the book provides state-of-the-art coverage of
what has been accomplished and also identifies areas in which more
work remains to be done. I am grateful to my many colleagues for the
valuable job done and hope that readers find the efforts of the authors
beneficial to their future endeavors.
Angelo M. Scanu
Chapter 1
Lp(a) Lipoprotein: An Overview
Káre Berg
Department of Medical Genetics
Institute of Medical Genetics
University of Oslo
0315 Oslo 3, Norway
I. Introduction
II. Background
III. The Early Years
IV. Lp(a) Lipoprotein and Coronary Heart Disease
V. Concluding Remarks
References
I. Introduction
In this chapter I shall give an account of the work leading to the
detection of the Lp(a) lipoprotein, briefly comment on the association
between Lp(a) lipoprotein and coronary heart disease (CHD) as well as
some of the other findings and developments through the years, and
summarize results of recent studies in our own group. I will necessar-
ily be selective rather than exhaustive.
II. Background
Polymorphisms in human serum proteins were detected in the 1950s
(Smithies, 1955; Grubb and Laurell, 1956; Hirschfeld, 1959) by electro-
phoretic or immunological methods or their combination. By 1960, it
was well known to people studying serum proteins that ß-lipoprotein
was an excellent antigen toward which good antisera could easily be
prepared (Cramer, 1961). In 1961, the first system of inherited anti-
genic serum protein differences demonstrable by immune serum from
a patient who had received multiple blood transfusions was reported
(Allison and Blumberg, 1961). A heavily transfused patient had pro-
Lipoprotein(a) 1 Copyright ©1990 by Academic Press, Inc.
All rights of reproduction in any form reserved.
2 Káre Berg
duced antibodies to genetically determined protein antigens that he
himself lacked. Although the antigenic protein variation was initially
thought to reside in a-2 macroglobulin, it soon turned out that it
resided in ß-lipoprotein or low density lipoprotein (LDL). It was well
known to human immunogeneticists that immune sera raised in
animals could be used to study genetically determined structures on
human red blood cells if subjected to careful absorption procedures.
Thus, the time was ripe to search for genetic variation in human serum
lipoproteins using antisera raised in animals.
A. The Setting of the Early Lp(a) Lipoprotein Work
As a young doctor, I started to work at the Institute of Forensic
Medicine, University of Oslo, in the beginning of January 1962. Its
chairman was the late Professor Georg H. M. Waaler who had discov-
ered the two-locus control of color vision anomalies in humans some
35 years earlier and who throughout his life retained a strong interest
in genetics. With his co-workers he had created an active research
group in immunogenetics, focusing on blood group serology and
human serum protein polymorphisms. The group had a strong tradi-
tion for conducting experiments under rigorous conditions; there was
an absolute demand for experiments to be done blindly, the super-
visor keeping the code until the young researcher had handed him the
final results. Thus, in genetic analyses, the family connections be-
tween coded samples remained totally unknown to the researcher
until the whole series of families had been studied, and samples from
several families were always included in any single experiment.
The Institute was as poor in laboratory instruments and equipment
as it was rich in intellectual life. The reason it was at all possible to start
to work with LDL in this laboratory setting was that Hjertén (1959) had
shown that it was possible to purify serum /3-lipoprotein without
expensive instruments, by chromatography on calcium phosphate
columns as developed by Tiselius et al. (1956). Cramer and Brattsten
(1961) had reported that /3-lipoprotein prepared from such hydroxyap-
atite columns is homogeneous and contains the density classes 0.96-
1.006, 1.019-1.063, and, in preparations from hypercholesteremic se-
rum, also lipoproteins of the class 1.006-1.019. Cramer (1961) had
shown that the ß-lipoprotein fraction did not contain chylomicrons.
Hydroxyapatite was, to my knowledge, not commercially available at
that time, and it would hardly have mattered if it were. The prepara-
tion of hydroxyapatite, with numerous cooking procedures, was a
1. Lp(a) Lipoprotein: An Overview 3
time-consuming and nerve-wracking task with the equipment avail-
able. It goes without saying that the institute did not have a prepara-
tive ultracentrifuge. During the two and a half years that I worked at
the institute, I had access to a preparative ultracentrifuge for 24 hours.
This made it possible to float lipoproteins from four persons at three
different densities (Berg, 1964a), but I could do adequate density
studies only after joining Professor Alexander G. Beam's group at the
Rockefeller University in 1964.
B. The Discovery of the Lp(a) Lipoprotein
My attempts to uncover genetic lipoprotein variation in man by the
use of animal antiserum were started in early 1962. Rabbits were
immunized with the /3-lipoprotein fraction of human serum obtained
by hydroxy apatite chroma tography. As expected, the rabbit immune
sera initially reacted with all human sera examined. The rabbit sera
were then submitted to an absorption strategy aimed at uncovering
differences between individual human sera. In separate experiments,
each antiserum was absorbed at several ratios between immune serum
and individual human sera and tested against a panel of human sera.
When certain human sera were used for absorption, the antisera re-
tained precipitating capacity over a wide spectrum of absorption ratios
with 30-35% of individual human sera obviously containing a pre-
viously unknown antigen. At that time, the possibilities of conducting
quantitative immunological analyses were limited. The results of the
double immunodiffusion experiments in agar gel were interpreted as
most likely reflecting qualitative differences between human sera,
although the presence of small quantities of the new antigen in appar-
ently negative sera could not be ignored. The particle carrying the new
antigen shared antigenic properties with /3-lipoprotein but had an
additional antigenic structure (or structures) as evidenced by only
partial fusion of the precipitin bands in agar gel obtained when anti-
serum to ß-lipoprotein and the new, absorbed antiserum were placed
in adjacent wells to react with a positive human serum. The precipitin
bands produced by absorbed antiserum and positive sera could be
stained with Oil Red O, and the /3-lipoprotein fraction from positive
but not from negative sera reacted with absorbed antiserum in double
immunodiffusion experiments.
A family study was performed to test the hypothesis that the unique
antigenic structure(s) detectable with absorbed antiserum was geneti-
cally determined. With the exception of one positive child of two
4 Káre Berg
negative parents, the distribution of parents and offspring in this
blindly conducted study was in agreement with the expectations,
assuming autosomal dominant inheritance of the antigen (Berg, 1963;
Berg and Mohr, 1963). Having proved the genetic nature of the anti-
gen and that it resided in a lipoprotein particle, the term Lp(a) antigen
was introduced in agreement with existing traditions in human immu-
nogenetics.
It was observed in the early immunological studies that when ab-
sorbed and unabsorbed rabbit immune sera were placed in adjacent
wells to react with a positive human serum, a separate precipitin band
occurred against the well containing unabsorbed antiserum, and that
this band formed a reaction of complete immunological identity with
that produced with absorbed immune serum. It was, therefore, al-
ready expressed in the reports of the first studies that the Lp(a) antigen
was likely to reside in a separate class of lipoprotein particles (Berg,
1964a, 1965). Working with Finnish collaborators, we were later able to
demonstrate this lipoprotein particle by disc electrophoresis (Garoff et
a\. 1970). It was reasonable to use the term Lp(a) lipoprotein for the
f
particle carrying the unique Lp(a) antigen. [The decision to use the
Lp(a) term preceded the establishment of the present nomenclature
for apoproteins belonging to the major classes of serum lipoproteins.]
It was clearly shown in the early studies that the Lp(a) lipoprotein is
independent of the Ag polymorphism of LDL. Ag antigens and the
Lp(a) antigen were shown to reside in different lipoprotein particles,
and no genetic linkage could be detected in family studies (Berg,
1964b).
In conclusion, the studies conducted in 1962 and 1963 uncovered
genetic variation independent of the Ag polymorphism and any other
known genetic polymorphism. The antigenic structure(s) uncovered
resided in a lipoprotein that shared characteristics with LDL but was
likely to form a separate class of lipoprotein particles [Lp(a) lipo-
protein]. The prerequisite for studying this genetic variation was avail-
ability of adequate antiserum. Immunization protocols, absorption
strategies, and specificity control procedures were developed, and an
unbroken chain of specificity control has existed for over 25 years.
III. The Early Years
During the first few years after the original discovery had been re-
ported, several family studies were conducted in Europe. They con-
firmed the first genetic analyses, although occasional exceptions to the
1. Lp(a) Lipoprotein: An Overview 5
postulated mode of inheritance were also encountered. By October
1966, nearly 500 nuclear families had been studied in various laborato-
ries. The studies had "practically always confirmed the original hy-
pothesis of an autosomal dominant inheritance pattern" (Wendt,
1967).
During the early years, the Lp(a) lipoprotein variation was iden-
tified also in nonhuman primates, including chimpanzees, orang-
utans, baboons, and Rhesus monkeys (Berg, 1968,1969). The primate
studies uncovered that there were at least two antigenic structures in
human Lp(a) lipoprotein reacting with absorbed rabbit immune
serum—one that was shared with the Rhesus monkey and one that
was not present in the Rhesus monkey but was present in humans,
chimpanzees, and orangutans. Thus, more than one population of
Lp(a) antigen-containing particles was present in humans.
Not all antisera used during the first few years had an adequate
quality (Berg, 1979a). Cross-reactivity, particularly with LDL, was a
problem. The recent detection of a structural relationship between
Lp(a) lipoprotein and plasminogen indicates an additional reason for
cross-reactivity and specificity problems. A good many disease associ-
ations, most of which have never been confirmed and some of which
were believed to be secondary to disease, were claimed during the
early years. Antiserum problems, other problems with techniques,
or false positive results in small series may have caused some of these
associations and may underlie a more recent claim that Lp(a) lipo-
protein has characteristics of an acute phase reactant.
The detection by several workers of Lp(a) lipoprotein in small quan-
tities in the sera of people who typed as negative in the traditional
double immunodiffusion analysis led to some discussion in the late
1960s and early 1970s. Some workers felt that this argued against
single locus control of Lp(a) lipoprotein despite the fact that single
locus control of quantitative parameters was well known in genetics
(Berg, 1971).
Studies analyzing Lp(a) lipoprotein as a quantitative trait have in-
deed resulted in evidence that the level of Lp(a) lipoprotein is under
strict genetic control. Single locus determination and major gene(s)
were the conclusions in extensive and carefully conducted genetic
studies of Lp(a) lipoprotein as a quantitative parameter (Schultz et al,
1974; Sing et al, 1974; Morton et al, 1985). Thus, the evidence for
single locus control was very strong, even before the recent data at the
DNA level became available; the evidence is now irrefutable as will be
discussed subsequently.
In the late 1960s and early 1970s, several workers encountered
6 Káre Berg
"atypical" lipoproteins in electrophoretic analysis of serum lipo-
proteins. Several of these variants, including the "sinking pre-
ß-lipoprotein" of Rider et al. (1970) and the "pre-ßi-lipoprotein" of
Dahlén (1974), were identified as the Lp(a) lipoprotein and demon-
strated to segregate in families as autosomal dominant traits. Rittner
(1971) prepared concentrated fractions of lipoproteins of density
1.063-1.10 and found genetic variants using disc electrophoresis. A
strong, although not absolute, association with Lp(a) lipoprotein was
detected. It is unknown if the variation studied by Rittner is related to
the genetic isoforms of Lp(a) lipoprotein recently reported by Uter-
mann and co-workers (1987,1988a,b). The frequencies of "null alíeles"
in the two systems are similar (0.63 and 0.65, respectively).
IV. Lp(a) Lipoprotein and Coronary Heart Disease
Studies from the 1970s in Scandinavia (Berg et al., 1974; Dahlén et al.,
1976; Frick et al., 1978; Berg, 1979a, 1983) that established a definite
correlation between Lp(a) lipoprotein and premature coronary heart
disease (CHD) have been confirmed in many subsequent studies.
Lp(a) lipoprotein level is not strongly correlated with traditional risk
factors, such as cholesterol (Berg et al, 1974; Berg, 1979a, 1983; Rhoads
et al., 1986). It is evident that a high Lp(a) lipoprotein level is a signifi-
cant and independent genetic risk factor for CHD. This is well illus-
trated by the finding of Rhoads et al. (1986) of a 28% population:
attributable risk for men in the top quartile of Lp(a) lipoprotein con-
centrations of having myocardial infarction by age 60, and by the
observation of Durrington et al. (1988) that much of the genetic com-
ponent of cardiac ischemia that is not expressed through any of the
traditional risk factors operates through Lp(a) lipoprotein.
We have been very interested in the reason(s) for the association
between Lp(a) lipoprotein and CHD. On the basis of our own studies
we had to reject the possibility that Lp(a) lipoprotein interferes with
the LDL receptor (LDLR) pathway since we found no saturation char-
acteristics for Lp(a) lipoprotein in LDLR test systems, no competition
between Lp(a) lipoprotein and LDL for the LDLR, and no significant
difference between normals and homozygotes for hypercholesteremia
with respect to Lp(a) lipoprotein uptake by cells (Maartmann-Moe and
Berg, 1981). Based on the studies by Dahlén et al. (1978), we have
tended to believe that the Lp(a) lipoprotein particle itself is atherogenic
to a higher degree than LDL particles are because of its physical