Download INTRODUCTION

ROLE OF APOLIPOPROTEIN (a) AS DETERMINANT
RISK FACTOR FOR PREMATURE ACUTE
MYOCARDIAL INFARCTION
Djanggan Sargowo, Tinny Endang Hernowati
Brawijaya School of Medicine, Malang, Indonesia
Abstract
Introduction. Lipoprotein(a) as a coronary heart disease risk factor
consists of atherogenic molecule LDL and thrombogenic molecule apo(a).
There is still controversion regarding which of the (lp(a) serum level,
apo(a) isoform molecular weight, the affinity of apo(a) molecule towards
fibrin) determine the risk of AMI. This research was aimed to identify
which of the lp(a) components determine the risk of premature AMI.
Materials and Method. An observasional study was conducted on 52
males groups (aged 30 – 45 years) with premature AMI and 52 males
group without AMI. The diagnosis of premature AMI was determined
based WHO diagnosis criteria. Several measurement method was used to
determine the level of lp(a) components, i.e. ELISA for lp(a) serum level,
gel electrophoresis followed by Western Blot for apo( a) isoform molecular
weight, lysine binding assay method for LBS1 affinity, and plasmin modified fibrinogen assay method for LBS2 affinity.
Result. The result identify significant correlation between the four
components of lp(a) measurements and the risk of AMI with different
strengths and direction. LBS2 affinity level showed a highest strength of
correlation (r = 0.908, p = 0.000) as determinant for AMI, while apo(a)
isoform molecule weight (r = -0.265, p = 0.007) and LBS1 affinity (r =
0.0228, p = 0.020) have an equal strength of association with different
direction. The amino acid substitution in LBS1 domain correspond with
low affinity of LBS1 identified in kringle IV-6 and kringle IV-10 while
the amino acid substitution in LBS2 domain correspond with low affinity
of LBS2 identified in kringle IV-5 and kringle IV-7
Conclusion. The affinity of LBS2 on fibrin has more important role as
factor determining the risk of premature AMI compare to LBS1, lp(a)
levels, and apo(a) isoform. The LBS in kringle IV -10 and in kringle IV-6
apo (a) had a role as LBS1 domain, while LBS in kringle IV -5 and kringle
IV-7 had a role as LBS2 domain
[lp(a)] is one of risk factors that
INTRODUCTION
is hereditary for the premature
PJK/IMA and independent risk
Background
Coronary
heart
disease
factor such as: hyperlipidemia,
(PJK) is one of highest death
hypertension, diabetes mellitus,
causes either in developed or
and obesity
developing countries, including
Coronary heart disease /
Indonesia. Based on Household
IMA
Health Survey (SKRT) of 1972,
manifestation that is started by
PJK mortality in Indonesia at
endothelial damage, triggered by
position of XI, in 1986 the
some
mortality become at position of
oxidized
III from all mortalities. SKRT of
damage
1995 stated that Indonesia has
that trigger the occurrence of
high incident number of heart
aterogenesis since the formation
attack, that is 4.600 per 100.000
of
populations
formation. The plague formation
with
mortality
of
is
one
risk
clinical
factors
LDL.
cell
to
as
the
plague
at
PJK mortality increased to 26.4%
blood vessel will be worsen by
with premature PJK incidents
the thrombosis aggregation and
(age <45 years old) increased
fibrin
about 40% from all PJK.
(thrombogenesis)
caused
by
acute
of
components
24.5%. SKRT of 2000 stated that
High mortality at PJK is
surface
such
Endothelial
produced
foam
the
of
endothelial
coagulation
so
arterial
blood vessel clogging occurred
myocardial
that cause the necrosis of heart
infarction (IMA) that occurred
attack with clinical manifestation
because the clogging of coronary
the occurrence of IMA (Ridker
branching,
Hennenkens and Stamper, 1993)
so
produced
heart
muscle tissue necrosis. According
Fibrin
coagulation
is
to Adult Treatment Panel III
formed from fibrinogen through
Guidelines 2001, lipoprotein (a)
blood coagulation process. The
event
is
triggered
the
similar with plasminogen/plasmin
endothelial blood vessel layer
so apo(a) also has LBS sequence.
damage that occurred at one of
Like plasminogen, apo(a) able to
atherosclerosis
stages.
bind fibrin but different with
The fibrin coagulation can be
plasminogen, apo(a) unable to be
dissolved
again because of
inactivated become lack active
plasmin activities, protease that
protease, to with the presence of
able
apo(a),
to
by
process
break
fibrin
become
the
amount
of
soluble peptide fractions. Plasmin
plasminogen that bind the fibrin
come from plasminogen, plasmin
will decrease. The state means
pro enzyme form that exist in the
that
blood plasma. If fibrin be formed,
(competitive
inhibitor)
then plasminogen will bind the
plasminogen
/
fibrin
binding of apo(a) at fibrin will
through
amino
acid
apo(a)
is
antagonist
toward
plasmin.
sequence molecule that is called
decrease
by lysine binding site (LBS).
plasminogen to bind or with other
Plasminogen
activated
word, give greater chance for
activator
thrombus
become
be
plasmin
by
the
chance
The
of
formation
molecule, such as XIIa factor or
(thrombogenic
tissue
plasminogen
differ with ‘ordinary’ LDL that
(tPA),
so
activator
in
nature).
So
of
‘only’ atherogenic in nature, lp(a)
plasmin has been bound to fibrin
beside atherogenic in nature also
so
thrombogenic in nature. Apo(a) is
the
facilitate
occurrence
the
fibrinolisis
process (Harpel et al, 1995)
encoded
by
gene
in
the
Lipoprotein (a) is LDL
chromosome 6 at locus of 6q26-
variant that differ from ‘ordinary’
27, that is the same locus with the
LDL
plasminogen encoding gene. This
because
apoprotein
molecule
B100 (apoB100) that
apo(a) gene
is polymorphic in
exist at
LDL, bound to other
nature. It has been known there is
molecule that is apoprotein(a)
more than 34 alleles of apo(a)
(apo(a)). Apo(a) structure very
gene, each allele encodes apo(a)
isophorm with different molecule
lp(a)
weights
of
PJK/IMA beside the lp(a) content
is
and
(BM).
PJK/IMA
Mechanism
occurrence
that
for
the
occurrence
of
lp(a) isophom BM. The
triggered by lp(a) not clear fully.
factors possibly is the apo(a)
Some researchers stated that lp(a)
affinity toward fibrin, because the
content >30 mg/dl related
factors
with
will
determine
the
high risk of premature PJK/IMA
thrombogenic properties of lp(a)
(<45 years old) (Gazzaruso et al,
(Cano et al, 1997)
1999). Glader et al (2002) found
Apo(a)
affinity
toward
the inverse relation between lp(a)
fibrin is determined by amino
content with isophorm BM lp(a).
acid
High lp(a) content
amino
related with
sequence
of
acid
LBS.
sequence
is
low isophorm BM (BM <640
determined
kDa), in contrast, the low lp(a)
sequence at apo(a) gene that
content (<30 mg/dl) related with
encoding
lp(a) with high BM (>640kDa).
sequence at LBS. Lysine binding
Based
lp(a)
site (LBS) is the domain in the
isophorm with BM.
The low
apo(a) molecule that is the site
apo(a)
is
for
statement
isophorm
determining factor
above,
the
of PJK/IMA
occurrence.
lysine
by
LBS
the
nucleotide
amino
residue
acid
binding
(ligand) in the fibrin molecule or
cell membrane. Erns et al (1995)
Theory that PJK/IMA is
stated the presence of LBS I
triggered by lp(a) isophorm with
domain at the kringel 37 (K IV-
low BM not always true. It is
10) and LBS 2 that its place
proven that at Africa-America
unknown but it is expected at
race, the lp(a) content generally
kringel 32 to kringel 35 (K IV-5
high with BM of high apo(a), but
to KIV-8). LBS 1 at kringel IV-
the PJK/IMA frequency is low
10 is zwitterionic that consist of
(Klausen et al, 1992). It showed
aromatic ring hydrophobic region
that there is other factor that
of triptofan 60, phenylalanin62,
related
triptofan70.
with
prediction
value
Both
hydrophobic
ends are consist of
charged
old at Jakarta, found the content
amino acid: first end point with
limit value lp(a)
negative
with 87% sensitivity. Sargowo
charge
(anion)
of
aspartic54, aspartic 56, and the
(1996)
other end point
with positive
included 60 IMA patienst of 30-
charge (cation) of arginin 35,
50 years old at Malang obtained
arginin 69 (Harpel et al 1990).
that 80% has lp(a) content
Both
mg/dl
charged
end
points
are
at
the
was 30 mg/dl
with
research
90%
that
>30
sensitivity.
interact with carboxyl end point
Rambe (2004) at North Sumatra
and amino endpoint of ligand,
obtained significant difference of
while the aromatic ring
lp(a)
able to
content
between
stroke
nteract with hydrophobic region
sufferers and non sufferers. Until
at the ligand center. Scanu et al
today, the research about lp(a) at
(1993) reported that lp(a) rhesus
Indonesia only limited at the
ape bind the lysine sepharose
content determination of lp(a), no
with lower affinity
research
then human
about
molecule
lp(a). It is caused by the presence
component
of tiptofan substitution by arginin
example the isophorm molecule
at position of 72, LBS domain of
weight (BM), the LBS affinity or
Kringel IV-10 rhesus ape.
amino acid sequence of LBS
Wijaya (1993) investigated
37 IMA patients of 45-60 years
of
ptjer
kringel molecule
apo(a),
for
II. Conceptual Framework
Apo (a) molecule
Kringel IV-2
Apo(a) isophorm?
Kringel IV-10
Kringel IV 5 to 8
LBS 1 domain?
LBS 2 domain?
Lysine binding?
Lysine-prolin binding?
LBS Affinity
Plasminogen binding barrier with
fibrin
Plasmin formation barrier
Fibrinolisis barier
Thrombogensis
Figure 2.1. Conceptual framework
obtained
Conceptual framework
Lipoprotein
thrombogenity
(a)
or
lp(a)
the inverse relation
between the amount
of lp(a)
is
content with isophorm molecule
weight apo(a), but the relation
determined
by
lp(a)
content,
isophorm
molecule
weight
still in debate.
apo(a), and the affinity amount of
The molecule weight of
apo(a) that is called by lysine
apo(a)
binding site 1 and 2 (LBS1 and
determined by the kringel IV of
2). The increase of lp(a) content
type 2 copy whose the amount is
increases
varied at each individual in the
the
risk
of
atherosclerosis occurrence. It is
range
molecule
200
to
isophorm
900
kD.
is
The
thrombogenesis process is related
6,7,8 (K32, 33, 34, 35, Mc Lean,
with the molecule
weight of
1987). Based on the amino acid
apo(a), but the relation form still
sequence, kingel IV-5 to kringel
controversial.
argued
the
molecule
Some
researcher
IV-8, each has LBS potential that
smaller
isophorm
has hydrophobic cluster (Trp60,
weight
apo(a)
the
Tyr62, Trp70), anion
cluster
higher chance of thrombogenesis
(Asp54, Glu56) and cation cluster
occurrence,
other
(Arg35, Arg69). LBS1 and LBS2
researcher argued there is no
of apo(a) molecule compete with
relation between apo(a) isophorm
plasminogen in its binding with
with
fibrin through the inhibition
while
the
the
chance
of
thrombogenesis occurrence.
Lysine
binding
plasminogen change to plamin, so
site
1
(LBS1) is binding site between
apa(a)
molecule
with
of
inhibit fibrinolysis and then will
increase the thrombogensis.
lysine
At
LBS
domain,
the
residue from fibrin that has been
triptophan substitution at position
known located at kringel IV type
of 70 with arginin (trp70→Arg)
10 domain (K 37 Mc Lean, 1987).
will decrease it affinity toward
The site consist of hydrophobic
fibrin
cluster (Trp60, Phe62 Trp70) is
substitution of aspartate 56 amino
enclosed at either side by anion
acid
cluster (Asp54, Asp56) or cation
(Asp56→Asn)
cluster
apo(a) affinity
(Arg35
and
Arg69).
(Scanu,
cluster
1995).
The
with
asparagin
also
decrease
toward fibrin,
Lysine binding site 2 (LBS2)
while the substitution of amino
(Erns, 1995; Klezovitc, 1996)
acid at domain non LBS, such as
also be called by Lysine Proline
methionine
Sensitive Domain (LPSD) is the
threonine (Met66→Tre) do not
molecule binding site of apo(a) –
change the apo(a) affinity toward
proline with lysine residue of
fibrin (Chinivesse, 1998). The
fibrinongen
that still expected
binding affinity change of apo(a)
located at kringel IV of type 5,
toward fibrin then determines the
66
replaced
by
occurrence
of
thrombogenesis
process occurrence.
“Idlam Siti Aisyah” Malang, and
for control it is taken from
PKMRS program participants at
RSUD “Saiful Anwar” Malang, it
2.2. Hypothesis
1. There
is
between
relationship
lp(a) content of
is
the
reference
East
weight
determination
apo(a),
the
apo(a) LBS affinity level
with the premature IMA
occurrence.
2. LBS
for
Malang city and south area of
blood, isophorm molecule
of
hospital
Java.
The
criteria
of
in
sample
population as follow:
1. Sex
:
men
of
IMA
sufferers
affinity
more
2. Age : less than 45 years
determine the premature
old (Gazzaruso et al, 1999)
IMA occurrence
3. Ready to be sufferers: by
III. RESEARCH METHOD
signing
the
consent.
The
objective
It is analytic observational
design
with
with
cross
research
is
acute
myocardial infarction and
3.1. Research type/design
research
informed
sectional
correlational
and
for control it is taken non
IMA men with age < 45
ears old without IMA
explorative approach
The collected data as follows
3.2. Population, sample size and
1. Sufferers
characteristic:
sampling method
sex,
3.2.1. Sample population
marital status
The
sufferer
involved
in
research
is
inpatient
the
of
that
is
control
case
outpatient
and
RSUD
“Saiful
Anwar” Malang, inpatient of RS
age,
occupation,
2. Risk factors: lipid, smoker,
hypertension,
DM,
alcohol, and family history
3. Clinical
examination:
blood pressure, heart beat,
pulse,
breath
frequency,
body temperature
least 10 times of the researched
samples
at
the
research
is
4. EKG examination, 12 lead
determined the sample at least 50
5. Laboratory
persons (Aitchison et al, 2004)
examination:
complete data,
complete
urine,
renal
function,
hepatic
function,
sugar,
heart
blood
muscle
3.2.3. Sampling method
Sample
taking
is
done
based on inclusion and exclusion
enzyme, lipid, homosistein
content, clytomegalo virus
9CMV),
pylori,
helicobacter
lp(a)
content,
isophorm apo(a), affinity
3.2.3.1. Inclusion criteria
Inclusion criteria in the
research is men, 30-45 years old,
IMA sufferer.
LBS1 and LBS2, alkali
sequence,
nucleotide,
amino acid of LBS domain
at kringel IV of type 5,6,
7, 8, 10 of apo(a)
3.2.3.2. Exclusion criteria
Exclusion criteria in the
research as follow
1. Diabetes mellitus sufferer
2. Chronic
planned
disease
sufferer
3.2.2. Sample size
The
heart
sample
in
the
3. Hypertension sufferer
research is “purposive sampling”
4. Obesity
by taking saple based on certain
5. Smoker
purpose so fulfill the researcher’s
6. Hyperhomosysteinemia
hope and interest. The sample
size
is
determined
based
sufferer
on
7. Chronic
research variable, 1 independent
sufferer
variable (MA) and 4 independent
variable (lp(a) content, isophorm
BM of apo(a), LBS1 and LBS2
affinity). The sample members at
renal
disease
8. Cytomegalo virus infection
sufferer
9. Helicobacter
infection sufferer
pylori
myocardial infarction diagnosys
3.3. Research variables
(MA) is enforced based on WHO
3.3.1. Variable classification
criteria 1983 consist of: clinic
Dependent variables :
(special chest sharp pain), EKG
premature
IMA
disorder
(pathology
Independent variables:
elevation
segment
1. Lp(a) content
inversion
T
2. Apo(a) isophorm BM
enzyme increase of heart (LDH,
3. Alkali sequence of LBS1
CKMB, SGOT). Criteria 2 and 3
and LBS2 nucleotide
Q
wave,
ST,
wave)
and
and
the
positive mean the IMA diagnosis
can be ensured.
Premature IMA is acute
3.3.2. Operational definition
What that mean of risk
factor
determinant
occurrence
disease
of
for
coronary
(PJK)
the
heart
becase
of
lipoprotein risk factor (a) in the
research
is
isophorm
BM
:
myocardial
infarction
that
occurred at men < 45 years old,
while the women still in
fertile
age
and
not
the
using
hormonal contraception.
lp(a)
content,
Non IMA are person that
apo(a),
affinity
not suffer from chest sharp pain
LBS1 and LBS2 affinity and the
and EKG picture in the normal
nucleotide alkali sequence and
range, if men <45 years old, if
amino acid sequence
women <45 years old, and still in
of apo(a)
molecule LBS domain.
Acute
infarction
fertile
myocardial
(IMA)
is
one
age
and
not
using
hormonal contraception.
of
Hypertension is diagnosed
clinical manifestation of coronary
according to WHO criteria of
heart disease in the form of
1989, that is blood pressure >
disorder at the myocardium that
160/95
caused by the lack of coronary
hypertension
arterial blood
pressure >160 mmHg but the
flow because of
aatherosclerosis process. Acute
mm
Hg,
if
normal diastolic
the
systolic
systolic
pressure, mild
hypertension
if
the
diastolic
pressure 95-104 mmHg, medium
hypertension if diastolic pressure
105-115
mmHg
function failure.
The smokers are smoking
heavy
more than 1 stick per day about
The
one month (American Thoraxic
hypertension heart disease is hold
Society), the former smoker if
if the blood pressure more than
stop to smoke at least 3 months.
160/95 mmHg or normal blood
It is called heavy smoker if take
pressure but the sufferer take anti
cigarettes >20 sticks per day,
hypertension pill and marked by
while take 10-19 sticks per day is
the heart expansion, heart rhythm
called medium smoker, and light
hypertension
if
and
disorder, or experience the heart
>115.
smoker if 1-9 sticks per day..
Obesity criteria if RBW >120%
% RBW =
BB
x100%
TB  100
Obesity criteria if RBW > 120%
RBW : relative body weight
BB
: body weight (kg)
TB
: body height (cm)
Dyslipidemia consist of
Hypercholesterolemi
: if the cholesterol content > 200 mg/dl
Hypertrigliseridemi
: if triglyceride >150 mg/dl
Low HDL content
: if HDL content <35 mg/dl (men)
: < 45 mg/dl (women)
High LDL content
: if LDL content >130 mg%
Lp(a) improvement
: if Lp(a) content > 30 mg%
Diabetes mellitus diagnosis is
Kringel is series of 81-90
hold if the WHO criteria of 1985
amino acid that is part of apo(a)
consist
criteria:
molecule. Apo(a) molecule has
poliuri, polydipsi, polyfagi, and
kringel amount in range of 13-50
body weight decrease and fasting
kringels
sugar content >126 mg/dl with or
repeatability level.
of
clinical
without random sugar blood >200
mg/dl
depend
on
the
Lysine binding site (LBS)
is domain of certain amino acid
Chronic hepatic function
disorder
if found the clinical
evidence in the form of
queasy, feel
stomach
weak,
want to vomit,
filled
with
air,
that is the binding siter of aopa
molecule
residue.
with
LBS1
lysine/fibrin
is
located
at
kringel IV-10 while LBS2 may be
located at kringel IV-5, -6, -7, -8.
decreasing appetite, little eat or
Genetically,
nucleotide
without eat, yellow eyes and
alkali sequence of
LBS domain
accompanied with laboratory data
encoder located at exon 2 (consist
in the form of: SGOT and SGPT
of 200-300 nucleotide alkali),
> 40 mg/dl, BUN >20 mg/dl,
only the arginin 35 amino acid
creatinin
encoder (one of anion from LBS
>1.8
mg/dl
and
clearance creatinein <50%
The
molecule
apo(a)
weight
domain) that is located at exon 1.
isophorm
is
apo(a)
protein molecule weight
3.4. Research materials
in kD
Material for lipid content
by
measurement as follow: serum,
electrophoresis of polyacrilamid
cholesterol reagent, lipoprotein
gel.
reagent (a) (Strategic Diagnostic
that
is
determined
LBS1/LBS2 affinity is the
Kit), triglyceride reagen, HDL,
strength of apo(a) with lysine or
LDL,
fibrin that its level is determined
pack (Abbot)
by
double
antibody
anati-globulin ELISA.
sandwich
and
homosistein
Material
for
isophorm determination
reagen
apo(a)
with
western blot is serum, phosphate
Material
for
DNA
and
buffer saline (PEMBAHASAN),
PCR isolation is human DNA Kit
Na 2 HPO 4 ,
NaCl,
(Roche’s
Diagnostic),
Agarose
PAGE,
(Sigma),
ethidium
bromide
staining buffer, (BioRad), TBS
(Sigma),
ddH 2 O,
pH
NaH 2 PO 4 ,
EDTA,
5,5,
SDS
buffer
-
blotting,
lysosim,
polymerase, dNTP, mark DNA
bromphenol
(Sigma),
blue, clycerol, SDA, tris base,
(sigma),
boric acid, Na 2 EDTA, glysine,
(invitrogen) that is used:
mercaptoethanol,
NaCl,
NP-40,
methanol,
tag
bromphenol
isopropanol,
blue
primary
1. Kringel IV of type 5 (5’-
deocsicolic acid, apo(a) antibody,
TGAAATTTCAGTGGCC
anti human (Sigma), IgG anti
TGACCAG-3’) and R (5’-
rabbit
TAGACTTCCTACCTTCT
alkaline
phosphatase
(Sigma), mark protein(BioRad),
western blue (promega), skim
milk,
bovine
serum
TCAGAAG-3’)
2. Kringel IV of type 6 F (5’-
albumin
TGTAATTTGCAGTGGC
Tween
20
(Sigma),
CTGAC-3’) and R (5’-
Bromo-4-Chloro
3
indoxyl
AGACTTCTTACCTTGTT
(BSA),
phosphate (BCIP) and nitro blue
3. Kringel IV of type 7 (5’-
tetratzolium (NBT) (Promega)
Materials to measure LBS1
and LBS2 affinity
apo(a)
as
as follow
standard,
lysine,
fibrinogen, plasmin, PNS pH 7.2,
washing
solution,
(Sigma),
BSA
immunoglobuline
antirabbit
(Sigma),
CAGA-3’)
TGGAATTTGCAGTGGC
CTGAC-3’)
and
R(5’-
GGCTTCTTACCTTGTTC
AGAA-3’)
20
4. Kringel IV type 8 F (5’-
(promega),
CCTTGAATATTCTCCCA
G
TC-3’)
Tween
goat
alkaliphospatase
substrate
pnp
(p-
nitrophenyl phosphate), ELISA
cup and sterile aquades.
and
R(5’-
CCAGTATATAGTGTCT
AACC-3’)
5. Kringel IV type 10 (5’TGGAATTTCCAGTGGC
CTGACA-3’) and R (5’-
bath,
TCTTACCTTGTTCAGAA
transilluminator UVP chromato-
GGAGG-3’)
vue model NTM-20 with Polaroid
Sequencing
reagent
by
chloroform
(BioRad),
analytic
scale,
using
camerea Mamiya RB67 Program
absolute
SD 45 mm, micro ELISA Reader
alcohol, phenol – TNE saturated
type
(Sigma), agarose-L and agarose-S
sequncer DNA instrument set of
(Sigma),
ABI Prism 310 Genetic Analyzer
primary
sense
and
of
Nj-2-300
primary anti sense that has been
(Perkin
used at PCR (Proligo), big dye
software of PyMOL, HyperChem
terminator
v.7 and RasTop
v.1.1.
cycle
Elmer),
(Biorad),
computer
sequencing RR-100 (BioRad), big
dye terminator v.1.1 v.3.1 5X
sequencing buffer (BioRad)
3.6. Location and research time
Research
was
done
at
Saiful Anwar Hospital, Malang,
biomedical laboratory of Medical
3.5. Research instrument
The
used
instrument
is
Faculty of Brawijaya University
tube 1.5 ml and 2 ml, falcon tube
Malang,
TDC
15, 50 ml (lwaki), incubator, high
Medical
Faculty of
speed
University.
micro
centrifuge
vacuum
refrigerated
MRX-150
pump,
(Tomy),
Laboratory
Research
of
Airlangga
is
done
since 2002 to 2006.
micropipette
(Gilson) 2, 20, 50, 100, 5000 and
3.7. Data taking and collection
1000 ul, blue and yellow tips,
procedure
tube
glass,
electrophoresis
protein
research
subject
set
either premature IMA sufferers
(Biorad),
or control after make agreement
Visivle
to involve in the research with
PCR
informed consent, blood sample
PJ2000 instrument set of Perkin
taking of 10 ml (2 ml for DNA
Elmer, freezer-20 o C, shaker water
isolation,
and
instrument
Each
DNA
spectrophotometer
type
1601
UV
(Shimadzu),
8
ml
is
made
for
serum). All sufferers and control
get
same
treatment
in:
3.8.
Laboratory
examination
examination (physic, ECG, thorax
method
photo),
3.8.1. Blood examination
lipid
content
control
(cholesterol, triglyceride, HDL,
Blood
LDL, and homosistein)
used to know the blood chemical
Serum then be separated
examination
content
method
is
such as lipid fraction,
by centrifuge 2800 rpm, during
CK CKMB, troponin I, CMV and
15 minutes for clinical chemical
H.pylory
examination
cholesterol,
fraction examination, sufferer or
triglyceride, HDL, LDL, ureum,
control need fasting in 10 hours.
cretatinin, blood sugar, SGOT,
Cholesterol content is measured
SGPT, homosistein), cytomegalo
by enzymatic method CHOD-
virus examination, helicobacter
PAP, triglyceride is measured by
pylory
GPO-PAP,
(total
examination,
content,
apo(a)
lp(a)
isophorm
enzymatic.
content
toward fibrin.
homosistein
the
research
reagent
is measured
by IMZ
reagent
LDL
encoding gene analysis / LPSD is
measured
by
done as follows: DNA isolation
(mg/dl). CK and CKMB (NAC
with DNA isolation kit (Roche),
activated Boehringer), troponin 1
DNA amplification by using PCR
is measured by Status CS STAT
(amplification
(Dade
specific
content
pack
(Abbot),
with
LBS
with
precipitation method, homositein
molecule wight, and LBS affinity
In
HDL
Lipid
turbid
Behring).
CMV
examination
6, KIV-7, KIV-8, and KIV-10).
passive latex agglutination test,
Determining
/
helicobacter pylory is measured
sequencing
by helicobacteri pylori antibody
KIV-5, KIV-6, KIV-7, KIV-8,
IgG enzyme immunoassay test kit
and KIV-10, then be continued by
(BioCheck, Inc)
nucleotide
alkaline
amino determination.
sequence
measure
meter
primary for exon 2 KIV-5, KIV-
the
is
is
by
absorbent at 450 nm (Marcovina
3.8.2. Lp(a) measurement
by
Lp(a) content is measured
et al, 1995). This test is depend
using
on
sandwich
double-antibody
enzyme
plasminogen
content,
linked
cholesterol content, triglyceride
immunosorbent assay (Strategic
content. At this measurement, it
Diagnostic
has
Kit).
This
method
intraassay
variation
principle is examination material
coefficient value (CV) 3.5-6.3%
that contains antigen are reacted
(Kronenberg et al, 1999)
with first specific antibody that
is bound to solid phase, then
3.8.3.
added
determination by western blot
by
antibody
second
enzyme
isophorm
and
This method is used to
substrate label from the enzyme.
know the apo(a) molecule protein
The examination procedure is
band specifically at human serum
primary
and determine the isophorm type
rabbit
with
specific
Apo(a)
antibody
(monoclonal
anti-apo(a)
human
and apo(a) molecule weight. The
antibody) as antibody coating is
method principle is detection of
attached at the solid phase base
specific
and
electrophoresis
as
secondary
antibody
protein
results
from
that
is
(polyclonal goat anti-apoB-100
based on the binding between
antibody) with label horseradish
antigen
peroxidase-conjugated (HRP) and
examination procedure is apo(a)
O-phenylenediamine
protein
dihydrochloride
and
antibody.
separated
The
through
(OPD)
as
electrophoresis SDS PAGE then
chromogen. For substrate
by
be transferred to nitrocellulose
using H 2 O 2 in phsopate solution.
membrane (NS), incubated with
Lp(a) content is determined by
monoclonal
comparing linear standard curve
(monoclonal mouse anti-apo(a)
that is determined
human
in the same
apo(a)
antibody).
by
antibody
Then
it
is
anti-globulin
G
procedure with standard material
added
that is available and is read with
polyclonal (polyclonal rabit anti-
IgG mouse antibody) with label
procedure:
alkaline
molecule binding at
phospatase
(AP)
and
it is done by apo(a)
specific
western blue as substrate. The
rabbit
monoclonal
antibody
amount
toward
apo(a)
exist
of
each
isophorm
that
at
molecule weight is determined by
microplate, after the sample is
marker
entered, added by specific goat
molecule
weight
standard.
antibody
toward
LBS1
(immunopurified Kringel IV-10
3.8.4.
LBS1
affinity
goat antibody) and then added by
anti-globulin with label alkaline
determination
The determination of lp(a)
phosphatase, and the substrate of
affinity toward fibrin (Harpe et
p-nitrophenyl
al, 1986) is done by immunoassay
addition.
(double antibody sandwich anti
change
globulin ELISA / Indirect ELISA
spectrophotometer at wave length
(American Diagnosis Kit). The
of 405 nm (variation coefficient
examination principle: the will be
of
determined antigen
is reacted
immunoassay method, it needs
with coating antibody at the solid
fewer and more accurate sample
phase
and not influenced by the content
then added by specific
intra
antibody toward antigen, then the
of
addition of anti globulin with
bilirubin.
enzyme
label.
phosphate
The
color
is
assay
plasminogen,
(pnp)
intensity
read
=
5-8%).
by
At
cholesterol,
Examination
ELISA results is then converted in the Kd unit (constant
dissociation) with Leghmuir:
Kd = So x
(kx[lp (a)])
(1  kx[lp (a)])
Where
Kd
: dissociation constant (nm)
So
: apo(a) content at sample
K
: constant value = 3
[lp(a)] : lp(a) content of sample
The kd calculation then is determined its cut off. The normal limit
of LBS1 affinity is less then 27.7nM (Jane et al, 1996)
3.8.5.
LBS2
determination
affinity
(Plasmin
Modified Fibrinogen)
The
LBS2
affinity
and added by plasmin so there is
proteolitic
process
part
fibrinogen
from
expected
able
to
occurrence
that
is
increase
measurement method is done by
lp(a)binding
double antibody sandwich anti-
serum
globulin ELISA/Indirect ELISA
lp(a), it is added by primary
modification
Diagnostic
antibody toward apo(a) of rabbit.
method
The goat anti rabbit IgG with
Kit).
(Nunc
Immunoassay
modification
is done first
to fibrin. After
addition
that
contains
by
label of alkaliphospatase enzyme.
Harpel (1995) that is plasmin
The color change occurs when
modified Fibrinogen (PMF). The
added by p-nitrophenyl-pohspate
examination
(pnp) as substratet.
procedure
is
fibrinogen that attached to plate
ELISA results are convereted in the Kd uni with leghmuir formla. Kd = So
x Kd = So x
(kx[lp (a)])
(1  kx[lp (a)])
Where
Kd
: dissociation constant (nm)
So
: apo(a) content at sample
K
: constant value = 3
The normal value of LBS2 affinity less than 44.5 (Kang et al, 1997)
3.8.6. DNA Isolation and DNA
field becomes slower so at the
testing of isolation results.
end of process it will be formed
DNA isolation by using
DNA
Isolation
Singapore).
Kit
DNA
(Roche,
content
band that depict various size of
separated DNA molecule. DNA
at
results is viewed with ultra violet
certain solution can be tested by
light that will shoed the band, if
electrophoresis
the agarose gel field is empty it
at agarose gel
and measure its quantity by using
showed
spectrophotometer.
unsuccessful.
DNA
with
that
PCR
reaction
good quality will impact the
electrophoresis agarose as shiny
3.8.8.
DNA Sequencing and
thick band without tail (smear)
nucleotide
alkaline
sequence
3.8.7. DNA analysis
DNA analysis
Polymerase
/
amino acid.
by
using
PCR
product
according to
obtained
previous results, then conducted
DNA sample then be used for
the phenol-chloroform, then be
PCR with pre designed primary.
labeled
PCR is a method to amplify the
sequencing purpose
target
sequence
PRISM 310 (Perkin-Elmer). The
exponentially in vitro. At this
sequence results that will be
reaction, it needs target DNA,
obtained will be analyzed
primary pair, thermostable DNA
nucleotide
polymerase, reaction buffer and
profile, amino acid homology,
cycler thermal device (Perkin-
and the molecule model structure
Elmer).
with
method.
gene
The
The produced DNA
exposed
to
agarose
is
gel
electrophoresis 2%. The greater
size of DNA molecule then the
movement
at
electrophoresis
target
with
big
dye
the
for
with ABI
alkaline
HyperChem
from
that
Reaction
(PCR)
Chain
analysis
for
sequence
program
of
2005, ApE 2005, and PyMol v.
2005
The
use
of
nucleotide
alkaline sequence and amino acid
analysis at some apo(a) molecule
intra assay 3-10% and inter assay
kringel in the research o ensure
<15%
the
LBS1
and
LBS2
domain
location at each kringel and to
3.8.9.2. PCR
ensure that amount of LBS1 and
The control usage such as
LBS2 affinity by the existence of
positive control, negative control,
the nucleotide alkali change that
internal control
encodes the amino acid that has
control is used to ensure
role in the LBS domain.
testing quality
or inhibitor
the
of examination
with PCR. The importance of
3.8.9. Quality control
control usage in PCR
3.8.9.1. ELISA
that PCR that is worked has been
Each ELISA examination
done or run properly if the
always did the quality control at
amplified
the beginning of a research to
not
obtain the reliable results. At the
over.
ELISA
examination
accompanied
with
always
positive
to prove
is the required target
the contaminant or carry
Positive control is done by
pure
DNA
fragment
that
is
control and negative control. The
recognized by the used primary.
positive control results should
It is better to use positive control
give positive absorbent value and
that has been known
negative
is
contain less then 50 target copies
control
but more than 10 copies (the low
examination should be repeated
copy number of positive control)
by
the
(Dragon et al, 1993). Negative
deviation is, is it reagent kit
control able to use DNA fragment
deviation, control material that
that is unrecognized by primer
damaged
that is used
control.
If
then
the
deviation,
evaluation
or
instrument.
where
deviation
The
there
good
at
the
quality
water.
as true
or using deionized
Internal
control
or
control at ELISA examination if
inhibitor is done is don to ensure
Coefficient variation (CV) for
that if PCR give negative results
is
really
negative
negative. If
not
false
clinic specimen is
alkaline.
The
assessment
electrogram quality also can be
not amplified and it is expected
viewed
there is PCR inhibitor in the
sequencer
specimen,
displaying the DNA
entered in control
of
from
the
ability
instrument
of
in
pureness
sequence with low copy number
from template PCR based on
(such as 20 copies) in the clinic
signal expression.
specimen, then the PCR is run
again
3.9. Processing way and data
analysis
Frequency
3.8.9.3. DNA sequencing
Validation
and
DNA
spearman correlation test is used
sequencing validation, if viewed
to know the relation between
from
IMA (through clinic diagnosis)
its
of
analysis
electrophenogram.
Electrophenogram
is
graphical
with lp(a) content, apo(a) BM
picture of DNA sequencing, with
isophorm,
the
be
affinity. DNA sequence data is
good
analyzed by making adjustment
from
with gene bank of NCBI then it is
good
processed
quality
quality
further.
if
viewed
will
The
LBS1
the
and
amino
LBS2
nucleotide alkaline waves that
determined
acid
emerged but not overlap, and
homolog that appropriate with
there is no uncertain nucleotide
nucleotide alkaline.
3.10. Research operational framework
Subject
Non IMa
Chest sharp pain (-)
Normal EKG
DM(-)
Smoker (-)
Obes (-)
HT(-)
CMV(-)
H.pylory(-)
Hct, Chol, TG (N)
LFT (N)
RFT(N)
IMA
1.
2.
3.
Chest sharp pain
EKG
Lab.-CK; CKMB, nTI
Informed consent
Control
Non IMA
IMA
Fasting of 10
hours
[Lp(a)];
Apo(a) isophorm
PCR KIV
5,6,7,8 and 10
Data analysis
APO(A) RISK FACTOR
DETERMINANT
(PREDICTOR)
Figure 4.1. Research operational framework.
DNA Sequencing
Amino acid
sequence
persons
IV. RESEARCH RESULTS
with
averaged
age
(38.3±4.53 years old). The lipid
4.1.
Research
sample
content
measurement
results
such as cholesterol, triglyceride,
characteristic
The sample collection
at
LDL and homosistein in normal
the research is done since March
limit at all sample
2003 to March 2005. The sample
group or non IMA. The research
is taken from some hospitals in
is the amount of lp(a) content at
Malang. During two years, it has
IMA group (45.56±19.12 mg/dl),
been
and at statistical analysis, it is
obtained
clinical
with
diagnostic
myocardial
men
patients
less
acuter
infarction
than
45
(IMA),
years
ole
(38.33±5.33 years old) of 52
person. Non IMA sample
obtained
differences
group
and
either IMA
the
significant
between lp(a) IMA
non
IMA
group
(p<0.05)
is 52
Table 4.1. Sample characterization at IMA group (n = 52) and non IMA
(n=52)
Lipid profile
Age (years old)
Cholesterol (mg/dl)
Triglyceride (mg/dl)
HDL (mg/dl)
LDL (mg/dl)
Homosistein (mg/dl)
Lipoprotein(a) content
(mg/dl)
Apo(a) isophorm BM (kDa)
LBS1 affinity (nM)
LBS2 affinity (nM)
The
apo(a)
molecule weight
IMA group
(Mean ±SD)
38.33±4.53
147.87±27.67
106.81±19.74
64.21±7.37
90.72±19.20
6.64±1.30
17.63±19.12

<0.05
0.907
0.115
0.478
0.136
0.193
0.300
0.000
521.68±171.64 644.68±126.95
26.45±9.18
15.09±6.97
48.73±4.58
35.03±4.15
0.012
0.035
0.000
IMA group
(Mean ±SD)
38.33±5.33
159±26.95
115±30.10
62.36±11.44
132.22±49.06
6.93±1.35
45.56±19.12
isophorm
at IMA group
(521.68171.64 kDa) lower than
non
IMA
group
(644126.95
kDa). LBS1 affinity
higher at
4.2. Lipoprotein content (a) at
IMA group (26.459.18nM) of
IMA group
non IMA group (15.096.94) nM.
group
and non IMA
Analysis results of LBS2 affinity
In the research, from 52
showed that LBS2 affinity of
IMA sufferer, it is obtained 35
IMA group higher (48.734.58
(67%)
nM)
content >30 mg/dl and 17 (33%)
than
non
IMA
group
person
that
has
lp(a)
other persons that has lp(a)<30
(35.034.15 nM) table 5.1)
mg/dl.
At
all
non
IMA
(52
person) has lp(a) content <30
mg/dl (Table 4.2)
Table 4.2. Lipoprotein(a) content distribution at IMA and non IMA
Lipo(a) content
Non IMA
IMA
Total
>30 mg/dl
0
35
35
<30 mg/dl
52
17
69
Total
52
52
104
Based on the difference test result
there is increase of lp(a) content
toward
is
that will be followed by the
difference
increase of IMA occurrence. At
between IMA and non IMA (
this research, it is obtained the
=0.000), it means that at IMA
correlation between
group it is obtained lp(a) content
of lp(a) content with the LBS1
greater then non IMA group. The
affinity (r = 0.431,  =0.000), it
statistical analysis results through
can be meant that the increase of
spearman correlation test showed
lp(a) content
the positive correlation between
LBS1 affinity increase. There is
the amount of lipoprotein (a) with
strong correlation between lp(a)
IMA clinical
symptom IMA (r
content with LBS2 affinity (r =
=0.712,  = 0.000), this mean that
0.784, p = 0.000), so the increase
obtained
lp(a)
content,
significant
it
the amount
is followed by
of lp(a) is followed by the LBS2
0.292,  =0.003) showed the
affinity
increase of lp(a) will be followed
increase.
Negative
correlation between the amount
by
of lp(a) content with the amount
isophorm
the
decrease
of
apo(A)
BM
(table
4.3).
of apo(a) isophorm BM (r = -
Table 4.3. Analysis results of Spearman correlation between lp (a) content
with BM Isophorm apo(a), LBS1 affinity, and LBS2 at IMA
and Non IMA group
Correlation
IMA
Apo(a)
LBS1
LBS2
coefficient
Non IMA
isophorm
Affinity
Affinity
value
BM
(nM)
(nM)
(<0.01)
(kDa)
Lp(a)
R
0.712
-0.292
0.431
0.784
content
P
0.000
0.003
0.000
0.000
(mg/dl)
4.3.
Sample
distribution
according
to
isophorm
one
apo(a)
isophorm band or two isophorm
molecule
band either at IMA group or non
IMA group (Figure 4.1 and 4.2)
Examination results that is
by
using
electrophoresis
polyacrylamide
picture
apo(a)
content
conducted
obtaining
method
gel
and
imunobloting (western blot) is
with varied molecule weight 2001000 kDa
Figure 4.1. Electrophoresis results that is continued with western blot at
some IMA sufferer: No1, 3, 4 to 16
M: marker high protein (97.4-700kDA) (Sigma)
Figure 4.2. Electrophoresis results that is continued with western blot at
some non IMA sample: No1, 3, 4 to 16
M: marker high protein (97.4-700kDA) (Sigma)
The molecule weight of each
curve formulation
of marker
apo(a) isophorm is obtained from
molecule weight, that is y =
the electrophoresis results that
3.2575x+3.5675 with R 2 = 0.9942
then be determined with standard
(Figure 4.3)
Figure 4.3. Standard curve regression equation of protein marker molecule
weight. Rf is ratio results between sample band distance with
separation distance. Log BM is calculation results of BM
logarithm of standard protein.
Table 4.4. Distribution of apo(a) isophorm BM at IMA an non IMA
Apo(a) isophorm Non IMA
IMA
Total
BM
<640 kDa(low)
28
41
69
>640 kDa(low)
24
11
35
Total
52
52
104
Based on apo(a) isophorm BM
At this research results, from 53
level, it is consit of Low BM with
IMA sufferers, it is obtained 41
BM <640 kDa and high BM if
persons (78.8%) with BM<640
BM > 640 kDa (Utermann, 1997).
kDa while at non IMA group, it is
obtained 28 persons (53.8%) with
apo(a) isophorm BM with IMA
apo(a) isophorm BM <640 kDA
clinic symptoms
(table 4.4.). At difference test, it
0.265, p =0.007) or between
is
significant
apo(a) isophorm BM with lp(a)
apo(A)
content ( r = -0.292, p = 0.003).
obtained
differences
the
between
IMA (r = -
isophorm BM between IMA and
It is also obtained
non IMA (p = 0.012), the data
correlation
between
can
correlation
BM
be meant that
group,
it
is
at IMA
obtained
apo(a)
negative
apo(a)
with
LBS1
affinity (-0.197, p = 0.045) and
isophorm BM that lower then non
negative
IMA group. At the research , to
apo(a)
test
affinity (r = -0.320, p =0.001).
the
correlation
between
correlation
isophorm
apo(a) isophorm BM with lp(a)
The
content,
correlation
LBS1
affinity,
LBS2
presence
between
with
of
negative
between
apo(a)
affinity or correlation between
isophorm
apo(a) isophorm BM with the
parameters, mean that the apo(a)
IMA
isophorm
occurrence
Spearman
by
correlation
using
BM
LBS2
BM
with
above
decrease
analysis.
accompanied by the lp(a) content
The analysis results showed the
increase, and followed by LBS 1
negative
or LBS2 increase (table 4.5)
correlation
between
Table 4.5. Analysis results of Spearman correlation between apo(a)
isophorm BM size with lp(a) content, LBS1 affinity, and
LBS2 affinity at IMA and non IMA groups.
Correlation IMA
Apo(a)
LBS1
LBS2
coefficient
isophorm
Affinity
Affinity
value
BM
(nM)
(nM)
(<0.01)
(kDa)
Non IMA
Apo(a)
R
-0.265
-0.292
-0.197
-0.320
isophorm
P
0.007
0.003
0.045
0.001
BM (kDa)
-1
Apolipoprotein(a) is used
lysine
as LBS1 affinity measurement
binding site 2 (LBS2) at IMA
standard with some concentration
and non IMA group
20, 40, 60, 80, 100, 120, and 140
4.4.
Lysine
(LBS)
binding
affinity
site
and
The LBS 1 and LBS2
nM,
while
for
affinity
affinity at the research is by
measurement standard of LBS2
using double antibody sandwich
by using apolipoprotein(a) with
anti
some concentration 5, 10, 20, 40,
globulin
Before
ELISA
conducting
LBS2 measurement
method.
LBS1
and
at group
80, and 12 nM. At LBS1 affinity
determination
standard,
it
is
IMA an non IMA, it is preceded
produced exponential curve with
by
R2=0.9537 value, while
making
standard.
This
LBS2
research using apolipoprotein(a)
affinity determination standard, it
as standard to measure LBS1 and
is produced exponential curve
LBS2 affinity.
with R2 = 0.9287 (figure 4.4)
Figure 4.4. (A) Apo(a) standard curve for LBS1 affinity measurement a nd
B. apo(a) standard curve for LBS2 affinity measurement.
At table 4.6 showed the LBS1
nM (Jane et al, 1995). Based on
affinity level with dissociation
the amount of LBS1 at this
(Kd) constant limit value of 27.7
research, it is obtained the LBS
1affinity increase at IMA group
affinity increase for 7 person
of 17 persons (32.5%) while
(13.5%)
at
non IMA group only get LBS1
Table 4.6. Distribution frequency of LBS1 affinity at IMA and non IMA.
LBS1 affinity
Non IMA
IMA
Total
<27.7 nM (low)
45
35
80
>27.7 nm (high)
7
17
24
total
52
52
104
At LBS2 affinity, the limit value
LBS2 at 47 persons (90%) at
to measure LBS2 affinity level at
IMA group while at non IMA
the research is 44.5 nM Kang et
group no LBS2 affinity increase
al, 1997). Distribution of LBS2
(table 4.7)
affinity size showed increase of
Table 4.7. Distribution frequency of LBS2 affinity at IMA and non IMA.
LBS1 affinity
Non IMA
IMA
Total
<44.5 nM (low)
52
5
57
>44.5 nm (high)
0
47
47
total
52
52
104
The correlation analysis results
analysis also showed the presence
showed
of
the
correlation
either
LBS1
affinity
and
lp(a)
between IMA with LBS2 affinity
content correlation (r =0.431, p =
(r = 0.908, p = 0.000), while
0.000), between LBS1 affinity
between IMA and LBS1 affinity,
with LBS2 affinity (r = 0.282, p =
it is obtained weak correlation (r
0.004), between LBS1 affinity
= 0.228, p = 0.020). From the
with apo(a) isophorm BM (r = -
0.197, p = 0.045). There is strong
affinity and apo(a) isophorm BM
correlation
size correlation (r = -0.320, p=
between
LBS2
affinity with lp(a) content (r =
0.001) (table 4.8)
0.784, p = 0.000) and LBS2
Table 4.8 Spearman correlation analysis between LBS1, LBS2 and Lp(a),
Affinity LBS1, affinity LBS2.
Correlation IMA
Lp(a)
Apo(a)
coefficient
Non
content
isophorm Affinity
Affinity
value
IMA
(mg/dl)
BM
(nM)
(nM)
(<0.01)
LBS1
LBS2
(kDA)
LBS1
affinity R
0.228
0.431
-0.197
1.000
0.282
(nM)
P
0.020
0.000
0.045
-
0.004
LBS2
affinity R
0.908
0.784
-0.320
0.282
1.000
(nM)
P
0.000
0.000
0.001
0.004
-
Correlation analysis results above
4.5.
Comparison
analysis
of
can be stated the relation between
nucleotide
IMA and the increase of LBS2,
sequence
the relation of lp(a) content level
type 5, 6, 7, 8 and 10 exon
and the LBS2 affinity increase.
2
Based
bank gene
on
correlation
results
above, it means that the high
of kringel IV
with
Based
alkaline
apo(a)
on
lp(a)
molecule
content
lp(a) content is followed by the
variation, LBS1 and LBS2 at
high LBS2 affinity will increase
IMA group then in the research,
premature IMA event (table 4.8)
to analyze nucleotide alkaline
sequence, some apo(a) molecule
kringel is grouped into 8 groups
such as in table 4.9
Table 4.9. Group division according to clinic diagnosis, lp(a) content,
LBS1 and LBS2 content.
Group
1
2
3
4
5
6
7
8
Criteria
IMA, lp (a) ≥ 30 mg/dl, LBS1 ≥ 27.7, LBS2 ≥ 44.5
IMA, lp (a) ≥ 30 mg/dl, LBS1 < 27.7, LBS2 ≥ 44.5
IMA, lp (a) ≥ 30 mg/dl, LBS1 ≥ 27.7, LBS2 < 44.5
IMA, lp (a) < 30 mg/dl, LBS1 ≥ 27.7, LBS2 ≥ 44.5
IMA, lp (a) < 30 mg/dl, LBS1 < 27.7, LBS2 ≥ 44.5
IMA, lp (a) < 30 mg/dl, LBS1 ≥ 27.7, LBS2 < 44.5
IMA, lp (a) < 30 mg/dl, LBS1 < 27.7, LBS2 < 44.5
Non IMA, lp (a) < 30 mg/dl, LBS1 < 27.7, LBS2 < 44.5
At eight group
obtained
the
above, it is
most
sample
2 affinity high of 17 persons.
at
Group 5 with lp(a) content low
group 2, that is IMA sufferers
but have LBS2 affinity high of 12
with high lp(a) hat have low
person. At group 8 (non IMA) it
LBS1 affinity and LBS2 affinity
is obtained lp(a) low with LBS1
high consist of 18 persons (table
or LBS2 affinity low of 45
4.9 and table 4.10). Group 1
persons, while group 3, 4, and 6
consist of IMA sufferer with lp(a)
its distributions is zero (figure
high, LBS1 affinity high and LBS
4.5)
Table 4.10. Distribution sample according to lp(a) content, a ffinity LBS1
and LBS2.
Group
Group 1
Group 2
Group 3
Group 4
Group 5
Group 6
Group 7
Group 8
Total
Sum of Sample
17
18
0
0
12
0
5
45
104
Percentage (%)
16.4
17.3
0
0
11.5
0
4.8
43.3
100
S
U
M
O
F
A
M
P
L
E
Group
Figure 4.5. Frequency analysis results at group according to clinic
diagnosis (IMA and non IMA), lp(a) and LBS affinity
Then
for
nucleotide
alkaline
kDA, LBS1 affinity = 34.63 nM
sequence of Kringel IV type 5,
and LBS2 = 49.41 nM), group 5
6,7,8 and 10 exon 2 at the
is represented by sample
research is one at
4 groups
(IMA sufferer of content lp(a)
according to the sample size that
26.7 mg/dl, apo(a) isophorm BM
is represented by group1, group
apo(a)
2, and group 5 and group8.
affinity = 26.21 nM and LBS2 =
Groupo1 is represented by sample
54.35 nM) while for group 8
5a (IMA sufferer, lp(a) content =
(nonIMA) is represented by 3
53.4 mg/dl, BM apo(A) = 437.7
(non IMA, content lp(a) = 21.6
kDa, LBS1 affinity = 39.55 nM
mg/dl, isophorm BM = 1051.7
and LBS2 = 48.78 nM). Group 2
kDA, LBS1 affinity =17.69 nM
is represented by sample with 5B
and LBS2 = 34.05 nM)
(IMA sufferer, lp(a) content =
58.6 mg/dl, Apo(a) BM = 544.7
=
484.8
kDa,
73
LBS1
4.6. Detection result of Kingel
AAT
TTG
CAG
TGG
CCT
IV type 5, 6, 7, 8 and 10
GACT CAG-3’) and R (5’-TGT
exon
TCC TAC CTT CTT CAG AAG-
2
with
PCR
3’), kringel IV type 6 : F(5’-TGT
technique.
In this research, it is done
ATT TTG CAG TGG CCT GAC-
DNA amplification by using PCR
3’) and R (5’-TGG AAT TTG
machine Pekin Elmer at four
CAG TGG CCT GAC-3’) and R
groups that is group 1, 2, 5, and 8
(GGC TCC TTA CCT TGT TCA
(Sample 5A, sample 5B, and
GAA-3’), kringel IV of type 8:
sample 73 for IMA group while
F(5’-CCT TGA ATA TTC TCC
sample N3 for group non IMA)
CAT C-3’) and R (5’-CCA GTA
each one sample. At each sample,
TAT AGT GTC TAA CC-3’),
it is done DNA amplification to
kringel IV of type 10: F (5’-TGG
detect Kringel IV of type 5,
AAT TTC CAG TGG CCT GAC
kringel IV type 6, kringel IV
A-3’) and R (5’-TCT TAC CTT
trype 7, kringel IV trype 8, and
GTT CAG AAG GAG G-3’)
kringel IV type 10 of apo(a)
PCR results showed that at
molecule. At this stage, it should
sample
isolated the DNA first at the
kringel IV of type 5 showed
blood sample of
positive results with 200 alkaline
IMA and non
5A,
5B,
N3
pairs
done by gel agarose. While for
control did not show the DNA
negative
using
band presence. At KR IV type 6,
demonized water. Then DNA that
it is obtained nucleotide of 181
is obtained
alkaline pairs
dNTP,
by
added by primary,
buffer
while
and
IMA then DNA qualitative is
control
length
73
negative
length. Based on
compound,
PCR KR IV type 7, it is obtained
polymerase Tag enzyme, mineral
nucleotide of 200 alkaline pairs
oil
length. At Kringel IV of type 8
for
DNA
sample
multiplication with PCR machine.
showed
The primary that is used is
nucleotide
KringelIV of type 5: F (5’- TGA
positive
length
results
with
about
267
alkaline pairs. PCR results of
Kringel IV of type 10.
Control negatif
Figure 4.6. Amplification of DNA sample PCR of group IMA and non
IMA. PCR results showed nucleotide with 200 bp target at
Kringel IV of type 5(A), 181 bp at kringel IV of type 6 (B),
200 bp at kringel IV of type 7, (C) 267 bp of kringel IV of
type 8 (D) and 200 bp at kringel IV of type 10
4.7.
Nucleotide
alkaline
4.7.1. Results of nucleotide acid
sequence results of Kringel
sequence of Kringel KIV-
type 5, 6, 7, 8 (LBS2) and
5 exon 2 (158 bp) (LBS2)
10 (LBS1) at group 1, 2, 5,
Results of nucleotide acid
sequence of Kringel KIV-5 exon
and 9
Amino acid sequence can
be done
The
through some stages.
first
stage
alkaline sequence according to
DNA
NCBI
PCR
replacement of timin nucleotide
technique. The PCR results can
alkalin (T) 144 cytosine (c )
be purified
four groups. At sample N3 that
multiplication
nucleotide
analysis
is
2 is compared with nucleotide
with
and multiplied for
is
obtained
with
alkaline
sequence
represent non IMA group (group
(nucleotide
sequence
8), there is some adenine (A) 13
DNA
nucleotide alkaline replacement
mapping)
by
using
sequencing
technique
of DNA

timin
(T),
timin
(T)
54
be
guanine (G), guanine 55 (G) 
acid
timin T) and adenine (A)77 
sequence prediction, homology
timin (T). At sample 5a, 5B, and
and
apo(a)
73 there is no nucleotide alkaline
biomolecule by using Genetyx
replacement. At non IMA sample,
Mac Version 8.0
it is obtained nucleotide alkaline
(ABI
PRISM
continued
310)
by
visualization
then
amino
of
Sequence analysis results
replacement of adenine (A) 13 
at each sample then be compared
timin (T) and guanine (G) 55 
with data of apo(a) nucleotide
timin (T)
alkaline sequence that is obtained
from gene bank NCBI 2006.
Comparison results of nucleotide
alkaline sequence is as follow:
Results of kringel
IV-5
nucleotide alkaline sequence at
the research, that is appropriate
with
target is the nucleotide
alkaline sequence no 3 (C ) until
NCBI will disturb the binding
No 62 (A). If research results of
functions with lysine.
nucleotide alkaline sequence at
the
research
adjusted
with
4.7.2.
Nucleotide
alkaline
sequence data from gene bank of
sequence result of kingel
NCBI then it is appeared that the
KIV 6 exon 2 (158 bp)
nucleotide alkaline sequence at
(LBS 2)
sample
5A, 5B, and 73 has
If the nucleotide alkaline
nucleotide alkaline sequence that
sequence at kringel IV of type 6
is same with gene bank NCBI
exon-2 is compared with gene
where there is no nucleotide
bank (NCBI), it is obtained the
alkaline replacement. Not only
nucleotide alkaline replacement
nucleotide alkaline replacement
cytosine (C) 32  timin (T) at
at gent target means
the three
four groups. At sample 5B, 73,
domain
and non IMA, it is obtained the
at
N3
nucleotide alkaline replacement
sample (non IMA) has some
adenine (A) 7  cytosine (C). At
nucleotide alkaline replacement
group N3 that represent non IMA
especially target gene so not
group,
appropriate
nucleotide
sample
have
LBS2
encoding gene. While
with
gene
bank
it
is
obtained
the
alkaline replacement
NCBI, but the nucleotide alkaline
guanine (G) 55  cytosine (C),
replacement should be translated
adenine (A) 74  guanine (G),
first in its amino acid sequence.
and guanine (G)  cytosine. At
If
amino
results
acid
of
translation
included the amino acid
that play role in the lysine biding
site (LBS) then the nucleotide
alkaline
replacement
is
not
appropriate with the nucleotide
alkaline element from gene bank
sample 5A, that represent IMA
group with lp(a) > 30 mg/dl,
occur
nucleotide
replacement
alkaline
guanine
(G)95adenine(A). At group 5,
there
is
insertion
nucleotide
of
adenine
alkaline
(A)
at
nucleotide alkaline sequence of
70
while
nucleotide
alkaline
4.7.3.
Nucleotide
alkaline
replacement also exist at adenine
sequence
(A)73 guanine (G). At sample
kringel
73 that represent IMA with lp(a)
(200 bp) (LBS2)
content <30 mg/dl, and sample
The
results
KIV-7
nucleotide
of
exon
2
alkaline
N3 that represent non IMA group
sequence of Kringel IV of type 7
at each nucleotide alkaline 121, it
exon
is
nucleotide
obtained
the
nucleotide
alkaline insertion of guanine (G)
The
nucleotide
alkaline
2
according
if
compared
alkaline
to
with
sequence
NCBI
provide
nucleotide alkaline replacement
at
of adenine (A) 45guanine (G),
kringel V-6 that appropriate with
adenine (A) 53guanine (G),
gene bank NCBI is nucleotide
timin (T)80  cytosine (C) at all
alkaline
sequence
cytosine
(C)
sequence
target
for
LBS2
3
of
sample. At sample 5A and 5B
NO
61
beside there is nucleotide alkaline
Adenine (A). At the research, it is
replacement such as above also
obtained the nucleotide alkaline
ther
replacement at four sample that is
replacement
not appropriate with nucleotide
 timin (T). At sample 73 also
alkaline sequence of gene bank
occurs the nucleotide alkaline
NCBI, it is depend on the amino
replacement of adenine (A) 13
acid translation, is the amino acid
guanine (G) while at sample
included in amino acid that has
N3 that represent normal group
role in the lysine binding site or
also ther is nucleotide alkaline
not. If the amino acid translation
replacement
up
no
to
has no role in LBS then there is
no LBS function disturbance.
is
nucleotide
alkaline
of cytosine (C) 81
of
guanine(G)
55timin (T). At sample 5A, 73,
and N3, there is replacement,
timin(T) 104 cytosine (C ) and
timin (T) 104  adenine (A)
replacement at sample 5B
The
sequence
nucleotide
target
for
alkaline
nucleotide
at
alkaline sequence of 96 at sample
kringel V-7 that appropriate with
5B, 73, and N3. Also there is
gene bank NCBI is the nucleotide
deletion of nucleotide alkaline of
alkaline
cysteine(C) at nucleotide alkaline
sequence
LBS2
of adenine (A) at
of
no
3
cytosine (C) to no 61 adenine
(A).
The
nucleotide
replacement
at
the
sequence of 119 at group 5.
alkaline
research
The
sequence
nucleotide
target
for
alkaline
LBS2
at
sample that is not appropriate
kringel V-8 that appropriate with
with nucleotide
gene bank NCBI is the nucleotide
alkaline gene
bank encoding NCBI
for LBS
sequence no 2 of Guanine (G)
kringel-7 not always cause the
with
LBS 2 function disturbance, the
nucleotide alkaline replacement
replacement influence the amino
of kringel IV-8 that is obtained
acid
at some sample depend on its
that
has
role
at
LBS2
domain.
4.7.4.
is
no.82
guanine
(G).
the
amino acid translation
Nucleotide
alkaline
4.7.5.
Nucleotide
alkaline
sequence results of KIV-
sequence
8 exon 2 (267 bp) (LBS2)
kringel KIV-10 exon 2
At sample 5A (group 1), it
(200 bp) (LBS1)
obtained
the
nucleotide
results
of
At sample 5A (group 1) it
alkaline replacement cytosine (C
is
) 10  adenine (A), for other
alkaline sequence that is same
group at cytosine (C) 10 that not
with nucleotide alkaline sequence
obtain
of
nucleotide
alkaline
obtained
NCBI.
If
the
nucleotide
compared
with
5B
nucleotide alkaline sequence of
(group 2), there is nucleotide
NCBI, it is obtained nucleotide
alkaline deletion of guanine (G)
alkaline replacement at kringel
at nucleotide alkaline sequence of
KIV-10 exon 2. At sample N3,
83. At group 8, there is insertion
the represent non IMA group, it
replacement.
At
sample
is obtained the adenine (A) 8 
sequence at each sample and each
timine (T) and the insertion of
type of kringel IV. Amino acid
Guanine
(G), cytosine (C) at
sequence data is obtained from
alkaline sequence of nucleotide
nucleotide alkaline sequence that
130 and 131 respectively. At
is translated by using Program
group
the
Software of ApE. The translation
timine (T) 49
results then suited with homology
cytosine (C), adenine 87 (A)
of amino acid sequence that is
guanine (G). At sample 73,
reported
there is replacement of guanine
Appropriate
(G) 50 cytosine (C), Adenine62
amino acid sequence Gene Bank
(A) cytosine (C), adenine 87
NCBI 2006, the reading of amino
2,
it
is
replacement of
(A)
timin
there
is
(A)
at
nucleotide alkaline sequence
of
insertion
(T)
obtained
of
and
Adenine
99 also insertion of guanine (G)
Gene
with
Bank.
the
apo(a)
acid is begin from cysteine (C)
fist cluster from the amino acid
sequence of each kringel. At the
research, amino acid sequence, it
is given priority to the amino acid
at sequence of 122.
Nucleotide
at
alkaline
sequence
target for LBS2 at kringel IV-10
that is appropriate with gene bank
NCBI is the nucleotide alkaline
sequence of no 1 guanine (G) up
that has role in the lysine binding
site NCBI: apo (a) protein kringel
IV: Arg54 and Asp56/Glue 56 as
anion
cluster
pairs,
Trp
60-
Phe62/Tyr 62-Trp70 is 3 aromatic
cluster is the zwitterions area.
to no 67 cytosine (C)
According to LBS domain at
4.8. Amino acid homolog if
kringel IV type 5, 6, 7, 8 and 10
2006,
lysine
binding
domain at the research results
located at amino acid sequence
exon 2
Homolog
analysis
(translation) of amino acid aimed
at
NCBI
determining
the
protein
46
until
81.
Amino
acid
homology results at the research
can be seen as follow
4.8.1. Amino acid sequence of kringel –IV 5
At Kringel IV of type 5 sample
lysine-proline
5A, 5B, and 73, there is no amino
(LBS2), that is Glu56val and
acid
Trp70Val.
replacement
(appropriate
binding
Amino
acid
site
of
with NCBI) except at sample N3
glutamine (Glu) is amino acid as
(group non IMA), then it can be
one
translated at sample 5A, 5B, and
triptophan (trp) is a zwitterions
73 has amino acid sequence that
cluster where each has role at
appropriate with NCBI and has
LBS2 domain, where the amino
role at LBS2 domain. Based on
acid replacement will disturb the
the finding at non IMA sample,
LBS function and cause the LBS2
where there is replacement of
affinity decrease.
some amino acid that serve as
of
anion
cluster
and
4.8.2. Amino acid sequence of Kringel –IV 6
Analysis results of protein
replacement (according to amino
sequence of Kringel IV of type
acid arrangement
showed that the sample 5B (group
there
2), and sample 73 (group 5) it is
disturbance and then will cause
obtained
acid
the LBS2 affinity increase. At
replacement Asp 54Ala. Amino
sample N 3 (group 8), beside
acid aspartat 54 is one of anion
there is amino acid replacement
cluster has role at LBS2 domain
of Asp 54 as mentioned above, it
then the amino acid replacement
is
at the sample 5B and non IMA
replacement
sample will disturb the LBS2
zwitterions cluster that has role at
function so will decrease the
LBS2 function so the replacement
LBS2 affinity. At sample 5A
of both amino acid will decrease
there
the LBS 2 affinity
the
is
no
amino
amino
acid
is
also
no
of NCBI), so
LBS
obtained
that
2
function
the
is
Trp70
the
4.8.3. Amino acid sequence of kringel IV 7
At kringel IV of type 7 sample
disturb the LBS3
5A (group 1), 5B (group 2), 73
there is LBS2 affinity increase.
(group 5) and N3 (group 8) there
While non IMA sample, it is
is amino acid replacement that is
obtained amino acid replacement
Asn 67 Ser but the amino acid
of Trp70Leu, while triptopan is
replacement is not amino that act
one of Zwitterion cluster so will
as amino acid cluster that has role
disturb the LBS function and then
in LBS2, so the amino acid
will decrease the LBS 2 affinity
replacement of asparagines not
function and
4.8.4. Amino acid sequence of kringel IV 8
At kringel IV of type 8,
amino cluster at domain LBS 2 so
either sample 5A (group 1) and
there
sample 5B (group 2), there is no
disturbance. At non IMA sample,
amino acid replacement, so there
there is amino acid replacement
is no LBS 2 function disturbance.
Arg60Thr that is one of cation
At sample 73 (group 5), it is
cluster from LBS2 domain, so the
obtained
replacement will disturb LBS2
the
amino
acid
is
LBS2
replacement Glu56 Gly, while
function
amino acid of glutamine is one of
affinity decrease.
and
there
function
is
LBS2
4.8.5. Amino acid sequence of kringel IV 10
Based on translation analysis of
replacement of Trp  Ser and
protein, it is obtained that sample
sample N3 obtain the replacement
5A (group 1) has amino acid of
of
Arg 35, Asp 54, Asp 56, Trp 60,
triptophan
Phe62, Arg69, and Trp70 that is
zwitterions cluster that has role in
not
LBS1
LBS1 function so at the three
domain of NCBI, so it is obtained
sample, there will be LBS 1
the Trp70Arg replacement, at
affinity
appropriate
with
sample 73 it is obtained
the
Trp70Ser,
70
while
is
one
the
of
Table 4.11. Amino acid replacement summary of LBS1 domain and LBS2
domain at kringel IV-5, 6, 7, 8, and 10 group 1, 2, 5, and 8.
Lp(a)
Amino acid
content
replacement
LBS1
LBS2
Clinic
Gazzaruso et al (1999), reported
V. DISCUSSION
that lp(a) is independent predictor
This
research
is
factors of PJK at age less than 45
observational research aimed at
years old (premature PJK). Lp(a)
knowing
particle
the
relation
of
beside
consist
of
lipoprotein (a) content, isophorm
atherogenic LDL
molecule weight apo(a), LBS1
apo(a)
and LBS2 affinity toward fibrin
thrombogenic in nature so it is
and the picture
of some apo(a)
the risk factors of PJK occurrence
molecule kringel with premature
that is important especially at
acute
myocardial
premature PJK. To date there is
(IMA)
(<45
infarction
old).
molecule
that
The
still argument differences about
should be conducted
lp(a) content or the size of apo(a)
because there is still controversy
molcule affinity toward fibrin /
between some risk factors.
fibrinogen
research
years
also contain
The coronary heart disease
(PJK)
with
infarction
acute
(IMA)
some
conventional
risk
can
be
the
predictor of PJK occurrence, so it
myocardial
need further research to find
manifestation
molecular risk factor that is more
occurred because of the influence
from
that
factors
and
accurate.
of
non
The
observational
research
is
research that is
conventional. The conventional
done at premature IMA patient
risk factos
of PJK is Diabetes
(<45 years old) related with lp(a)
Mellitus, dislipedime, smoking,
to know the relation between
hypertension and obesity, while
some
the non conventional risk factos
particles, also be analyzed the
such
hyperhomosistinemia,
relation between apo(a) isophorm
pillory,
molecule weight, LBS1 affinity
cytomegalovirus, and hiperlipo-
and LBS2 and nucleotide alkaline
protein(a). Bostom et al (1996),
sequence LBS1 and LBS2 so it
as
helicobacter
risk
factors
in
lp(a)
can be known which factor that is
sufferers (<45 years old) has
strongest
lp(a) content that is higher than
with
the
IMA
occurrence.
control group and it limit value
is 30 mg/dl. In 1996, Alber et al
5.1. Relation of lp(a) content,
reported there is relation between
apo(a) isophorm with IMA
lp(a) content
5.1.1. Relation of lp(a) content
PJK
with IMA
research, Sankand et al (1990)
Lp(a)
is
complex
increase with the
occurrence.
found that
At
other
risk relative toward
lipoprotein particle with unique
myocardium
properties
LDL
individual with lp(a) content >30
structure and apo(a) molecule
mg/dl, it is 2-5 times higher
where each of it atherognic in
compared with
nature and thrombogenic
so
has lp(a) content <30 mg/dl.
accelerate the PJK occurrence
Some prospective research stated
with IMA clinic manifestation.
that there is no relation between
Although more than 30 years has
the height of lp(a) content with
been done research that related
the
with lp(a) content of serum with
(Jauhian et al, 1991; Cantin et al,
diseases
1998). Stein et al (1997) proved
because
has
that
related
infarction
individual that
atherosclerosis
occurrence
atherosclerosis, but the exact role
that
of lp(a) in the
atherosclerosis
although lp(a) content three times
[Mitropoulus
higher compared with Caucasian
unknown
yet
(1994);
Marcovina
and
Koschinsky (1997), Karniawati
(2001)]
that
there
Africa-America
race,
race but the PJK prevalence is
lower.
is
In the research, it is done
statement that contrary with the
examination of lp(a) content by
relation
double antibody sandwich ELISA
with
stated
at
at
of lp(a) content level
the
increase
of
IMA
and
then to test the relation of
occurrence. Reblin et al (1992)
lp(a) content with IMA premature
stated
clinical symptoms by spearman
that
premature
IMA
correlation. The presence of 17
between
premature IMA sufferer from 52
isophorm BM
premature
IMA
sufferers
lp(a)
with
apo(a)
at
research that showed lp(a) <30
5.1.2.
mg/dl may be caused by the risk
apo(a) isophorm with IMA
factors
beside
increase,
lp(a)
The
relation
between
content
Apo(a) molecule that is
for example, the LBS
protein in the lp(a) particle, has
affinity role or infection factors
polymorphism
that is non excluded (Klebsiella
with
pneumoniea,
variation (200-900 kDa). There is
streptoccosus
mutans,
porphyromonas
gingivalis) (Vojdani, 2002)
The
apo(a)
molecule
isophorm
determined
weight
difference
genetically
that
sample
related with copy of kringel IV-2
showed that there is significant
that varied between 3 to 40
differences between lp(a) content
copies, so the apo(a) isophorm
of
molecule weight
IMA
research
some
form (isophorm)
sufferers
with
lp(a)
differences is
content of non IMA person (p =
determined by apo(a) mRNA size
0.000)
difference. Relative mobility of
and
the
presence
of
positive relation between lp(a)
specific apo(a) isophorm
in the
content and IMA (r = 0.712, p =
electophoresis
gel
related
0.000).
directly
apo(a)
mRNA
The
appropriate
research
with
previous
length at liver. Harpel et al
research (Sankand et al, 1990;
(1989) by using polyacrylamide
Albers et al, 1996) that stated
gel electrophoresis (PAGE) then
that the high lp(a) content will
be
increase the IMA occurrence. At
immunoblotting obtain 11 apo(a)
the research, it is also get the
isophorm band between 415 kD
relation between the lp(a) content
until
with
LBS2
individual only get one to two
affinity. There is inverse relation
protein band. Some researcher
LBS1
with
is
affinity or
stated
continued
838kD,
the
but
inverse
with
at
each
relation
between isophorm BM with lp(a)
00265, = 0.007). There is inverse
content,
apo(a)
relation between apo(a) isophorm
isophorm BM, the higher lp(a)
BM with lp(a) content (r = 0.292,
content
1994;
p = 0.003), or apo(a) isophorm
Keltzovitch and Scannu, 1995).
BM with LBS affinity LBS1 (-
Islam et al (1994) stated that
0.197, p = 0.045) and LBS2
apo(a) isophorm of low molecule
affinity LBS2 (r = -0.320, p =
weight related with premature
0.001). The relation between the
PJK risk but not related with
amount of apo(a) isophorm BM
lp(a)
with lp(a)
the
(Marcovina,
content.
(1994)
smaller
stated
statement
that
Klausen
the
et
al
content
appropriate
different
with Marcovina (1994) that state
Africa-America
the smaller apo(a) isophorm BM
race has lp(a) content higher with
the greater lp(a)
apo(a) isophorm BM great but
PJK/IM
occurrence
frequency
low.
5.2. Lysine binding site affinity
toward fibrin
The research is by using
electrophoresis
IMA
pathogenesis
and
mechanism because of lp(a) risk
immunoblotting, it is obtained on
factors is expected through apo(a)
or two band at each individual
competition with plasminogen in
with BM varied in range 200-
binding fibrin, so lp(a) inhibit
1000 kDA either in non IMA
plasminogen
group or IMA group.
plasmen
In the
activities
then
will
become
inhibit
research also get the significant
fibrinolysis. The inhibition of
differences
apo(a)
fibrinolysis is a foundation of
isophorm BM of IMA group an
thrombogenesis. Apo(a) molecule
non IMA group but from the
binding with fibrin is determined
Spearman
analysis
with LBS affinity that is depend
results, it showed that apo(a)
on the lysine binding site domain
isophorm
that
between
correlation
BM
related directly
with the IMA occurrence (r = -
exist
at
molecule kringel.
some
apo(a)
Lysine binding site (LBS)
plasmin
modified
fibrinogen
is one of domain in the apo(a)
prove the presence of two binding
molecule kringel consist of some
sites with fibrinogen (LBS) that
amino acid sequence that form
is different with LBS 1 at apo(a)
zwitterions are: three triptofan 60
molecule
aromatic
cluster,
and LBS2 at kringel-32 to 36 (K
fenilalanin62/tirosin and triptofan
IV-5 to 9). The LBS finding open
70 that is hydrophobic area, one
chance to discover
of its end points surrounded by
role of LBS as the determinant of
anion cluster of aspartat 54 and
IMA occurrence.
kringel-37
(KIV-10)
the possible
aspartat56/glutamate, while the
other end points is surrounded
5.2.1. Relation between lysine
by cation cluster of arginin 35
binding
and arginin 71. There is change
toward fibrin with lp (a)
from amino acid sequence in LBS
content
domain will cause LBS function
Molecular
distucbance and will decrease the
toward
LBS
atherosclerosis
affinity.
Guevara
et
al
site
affinity
approach
patogenesys
as
basic
(1993) stated that beside kringel
mechanism of IMA related with
37 (K IV-10), kringel 32 to
lp(a) particle more direct to lp(a)
kringel 35 (KIV-5 to KIV -8) has
competition with plasminogen in
LBS potential while kringel 36
its binding with fibrin. The prior
(KIV-9)
LBS
research has proven the presence
potential because kringel 36 bind
of relation between lp(a) content
with
molecule
binding with the IMA occurrence
fromLDL. Ernst et al (1994) by
increase, the higher lp(a) content
using apo(a) recombinant (normal
will increase the apo(a) molecule
or
binding affinity toward fibrin that
do
not
apoB-100
mutant)
from
have
cell
line
hepatocarsinoma of human with
lysine
sepharose
affinity
chromatography examination and
will increase thrombogensis
The research results will
showed
that the LBS 1 affinity
increase showed that the LBS1
occurrence then LBS1. The prior
increase more at IMA group than
researcher Goevara et al (1993)
non IMA group and based on
and Erns et al (1994) only stated
correlation analysis results, it
that the presence of lysine praline
showed
binding
sites
content with LBS1 affinity or
(lysine
binding
LBS2 affinity. So it is expected
stated the affinity amount or the
the
relation
relation between lp(a)
lp(a)
content
that
is
outside
site)
between
LBS1
but
not
LBS1 and
accompanied with LBS1 affinity
LBS2 affinity. The statement of
increase ot LBS2 affinity will
LBS 2 affinity
increase
than
the
premature
IMA
occurrence.
1
determinant
In the research, it is also
obtained
LBS
the
LBS2
affinity
has more role
as
risk
factors
of premature IMA
occurrence is new finding from
the research.
increase more at IMA group then
non
IMA
group,
and
from
5.2.2.
Relation between lysine
correlation test, it is obtained
binding
site
strong relation between the lp(a)
toward
fibrin
content with LBS2 affinity (r =
apo(a) isophorm BNM
0.784) and the presence of strong
relation
between
The
apo(a)
affinity
with
molecule
IMA
isophorm BM varied depend on
occurrence with LBS2 affinity (r
the copy of kringel IV-2 that is
= 0.908), it mean the increase of
not same among individuals that
lp(a) content and LBS2 affinity
is regulated genetically. Some
increase
researcher
will
increase
the
stated
the
inverse
occurrence of premature IMA.
relation between the amount of
Based on consideration above and
apo(a) isophorm BM with lp(a)
the presence of weak relation
relation,
between
isophorm BM the higher lp(a)
LBS
1
and
LBS2
the
and
smaller
the
higher
apo(a)
affinity, it means that LBS2 has
content,
PJK
more role at the premature IMA
occurrence. The relationof LBS
affinity toward fibrin with apo(a)
weight
isophorm molecule weight still
interaction
of
controversial. Klezovitch (1996)
molecule
between
did
not obtain relation between
kringel in the apo(a) molecule.
LBS affinity with the amount of
The interaction able to cause the
apo(a)
molecule
covering of LBS series so inhibit
weight, but Leerink et al (1994),
the binding between LBS with
Angles-Cano (1997) reported that
fibrin that may be influenced by
apo(a) LBS affinity toward fibrin
the apo(a) isophorm molecule
related
weight.
isophorm
inversely
with
apo(a)
isophorm BM.
may
be
caused
inter
Further
various
research
is
of contribution inter or intra
weak relation (r = 0.282) between
molecular that cover
affinity LBS1 or LBS2 toward
lp(a) particle.
with
intra
needed to evaluate the presence
The research showed the
fibrin
or
by
apo(a)
LBS in
isophorm
Based on the research and
molecule weight wither at IMA
correlation statistical test among
group or non IMA group. The
each lp(a) content
finding is not appropriate with
apo(a) isophorm BM
research results of Rahman et al
0.265), LBS1 affinity (r = 0.228)
(2001)
ap(a)
LBS 2 affinity (r = 0.908) at IMA
recombinant of kringel IV type 2
sample and non IMA means that
that concluded that kringel IV
LBS2
affinity
type 2 of apo(a) molecule is not
toward
the
related with fibrin, so the size of
occurrence and can be considered
apo(a) isophorm molecule weight
that LBS2 affinity as the risk
depend on the copy of kringel IV
factor
of type
occurrence.
by
using
2 not correlated with
LBS affinity toward fibrin.
The
presence
of
weak
relation between LBS affinity
with apo(a) isophorm molecule
of
has
(r = 0.712),
(r
very
premature
premature
=-
role
IMA
IMA
5.3.
Nucleotide
alkaline
function, and also the triptophan
and
70 that is the non polar aromatic
amino acid at KIV-5, KIV-
cluster if replaced by valin of
6, KIV-7, KIV-8, and KIV-
aliphatic cluster of non polar will
10
disturb
sequence
analysis
In the research, nucleotide
alkaline sequence analysis
or
the
stability
of
binding
will
hydrophobic
influence
the
LBS
function
LBS1 and LBS2 domain amino
because either Glu56 or Trp70 is
acid sequence at some apo(a)
amino acid cluster that has role in
kringel compared with nucleotide
the LBS function. It means that
alkaline
the
arrangement
/
NCB
replacement
of
one
of
amino acid (2006). The presence
zwitterions cluster at non IMA
of
of
group will decrease the LBS
nucleotide alkaline or change at
affinity, it is appropriate with the
LBS amino acid domain from
low LBS affinity
at non IMA
NCBI means the presence of
group
1998).
apo(a) molecule binding function
research results according to the
disturbance apo(A) with fibrin
Rahman research results
that caused the occurrence of
that stated Trp70Phe decrease
LBS1/LBS2 affinity decrease at
the LBS affinity. IMA group of
the
nucleotide
high or low lp(a) has high LBS2
alkaline replacement/amino acid
affinity, and has LBS domain
of each LBS domain (Scanu et al,
amino
1994)
same with at kringel IV-5. At non
change
of
presence
replacement
of
(Wilcox,
acid
The
(2001)
arrangement
that
At kringel IV-5 non IMA
IMA has LBS2 affinity smaller,
sample, there is replacement of
ant do not has glutamic56 cluster
Glu 56(GAG) Val (GTG) and
and triptophan 70 at LBS domain
Trp70
(TGG)
Val
(GTG).
that has role at LBS function. The
if
finding showed that amino acid
replaced by valin with non polar
arrangement of LBS domain at
Glutamic
as
anion
cluster
properties will disturb the LBS
kringel IV-5 related with LBS2
substitution
role.
alanine but in the research not
According
to
nucleotide
alkaline sequence results
kringel
IV-6,
it
is
of
of
Asp
56
with
call its role toward LBS1 or
LBS2.
obtained
nucleotide alkaline replacement
6.
TAC42TAT at all sample but
SUGGESTION
CONCLUSION
AND
not change the amino acid of
tyrosine62 so the replacement do
6.1. Conclusion
not disturb the LBS function. At
Based
sample of 5B and 73 IMA of high
discussion
or low lp(a)
lp(a)
with high LBS2
on
the
results
and
that investigate the
content
paramaters
and
affinity and non IMA with low
some apo(a) components such as
LBS
apo(a) isophorm BM, LBS1 and
affinity
occur
the
replacement of Asp54 (GAT) 
LBS2
Ala (GCT). Aspartic that is polar
arrangement
anion cluster if replaced with
amino acid at the premature IMA
alanine cluster that is non polar
sufferers then it can be concluded
aliphatic in nature will disturb the
as follows:
LBS function. Because at the
affinity
of
and
LBS
the
domain
1. There is relation between
three sample (5b, 73, and non IM
lp(a)
sample) there is LBS1 affinity
isophorm
decrease
affinity with the premature
while
LBS2
affinity
content,
apo(a)
BM,
LBS
inconsistent then the replacement
acute
will influence the LBS1 than
infarction occurrence. The
LBS2. The research results will
lp(a) content increase will
strengthen
increase
the
argument
of
myocardial
the
premature
LoGrasso and Bonnmark (1997)
IMA. The LBS1 or LBS 2
that proved the decrease of apo(a)
affinity
increase
binding of recombinant toward
increase
the
plasmin modified fibrinogen after
IMA
will
premature
occurrence.
The
smaller
BM
apo(a) isophorm
will
increase
while lysine binding site at
the
kringel IV-5 and kringel
occurrence of premature
IV-7 has role as LBS2
IMA.
domain.
2. The LBS2 affinity level
toward fibrin has
more
role as the risk factors the
premature
acute
myocardial
infarction
6.2. Suggestion
1. Because apo(a) molecule
isophorm is determined by
genetic
factors, it needs
(IMA) compared with the
polymorphism
lp(a)
type (isophorm) of apo(a)
content,
apo(a)
isophorm BM or the level
of LBS1 affinity.
binding
at
site
molecule in Indonesia.
2. It needs special formula at
3. The amino acid sequence
arrangement
phenotype
lysine
domain
the
diagnostic
reagent
provision of lp(a) content
diagnostic
(ELISA)
(arg35, asp 54, asp/glu56,
especially the usage
trp 60, phe/tyr62, arg69,
apo(a) antibody as coating
trp70) determine the LBS
antibody
affinity toward fibrin. At
appropriate
premature
isophorm
IMA,
it
is
obtained the LBS domain
amino acid arrangement,
of
that
is
with
apo(a)
BM
of
allele
type
Indonesian
3. It
needs
while at non IMA, it is
determination with method
obtained replacement one
of
or some amino acid of
electrophoresis
LBS
continued
domain.
Lysine
pulsed
by
binding site at Kringel IV-
blotting
10 and kringel
confirmation
apoprotein
IV-6 of
molecule
(a)
has role as LBS1 domain,
field
for
gel
then
southern
suitability
between
alleles with the copy of
kringel
IV-2
or
apo(a)
isophorm BM
4. It need further research
about
the
amino
acid
sequence of LBS domain
at apo(a) molecule kringel
stereometry
high
resolution crystal structure
to ensure accurately the
chemical
binding
interaction either intra or
inter molecule in various
apo (a) molecule kringel,
so knowing
character
exactly the
and
affinity
power of each LBS domain
in the apo(a) molecule.
5. It
need
lp(a)
content
examination accompanied
by
LBS
affinity:
at
premature IMA sufferers
(<45 years old) that do not
have
PJK risk factors
(hyperthension,
diabetes,
obesity,
smoking,
dislipidemi)
individuals
family
and
that
have
history
with
premature IMA sufferers.