terolemic patients [14]. It is elucidated that the antico- agulant activity of lipoprotein-associated TFPI is
markedly lower than that of fTFPI [15]. Therefore, fTFPI antigen was analyzed in plasma in hyperlipi-
demic patients and the relation between fTFPI and lipoproteins was further investigated.
Moreover, it is also elucidated that TFPI activity and total TFPI antigen normalize during cholesterol-lower-
ing therapy because of a specific drop in LDL-associ- ated TFPI in plasma [16]. Almus et al. have found that
reducing plasma TFPI by 50 causes a similar increase in the factor VIIa-TF catalytic activity toward factor
IX in an umbilical vein model of coagulation [17]. The question then arises whether the changes observed in
tTFPI concentrations after cholesterol-lowering therapy should be considered beneficial or not. Therefore, in the
present study, some parameters of the hemostatic sys- tem were monitored in order to determine whether
decreases in tTFPI induced by cholesterol-lowering therapy reduce the anticoagulant potency of plasma in
hyperlipidemic patients.
2. Subjects and methods
2
.
1
. Subjects
2
.
1
.
1
. Study Group I Eighty-one subjects, 38 healthy controls and 32 pa-
tients with type II primary hyperlipoproteinemia were recruited from a health center Asanogawa Hospital,
Kanazawa, Japan, to which they had been referred for a periodic health check-up. Twenty patients 19 men, 13
women were classified as type IIa and 12 as type IIb hyperlipoproteinemia, according to the World Health
Organization’s WHO classification for hyperlipi- demia. They had never been treated. Patients with
secondary hyperlipoproteinemia were excluded from the study. Familial hypercholesterolemia FH was clin-
ically diagnosed according to the following criteria [18];
1. Primary hypercholesterolemia of more than 260 mg dl with an increase in Achilles tendon thickness.
2. Primary hypercholesterolemia of more than 260 mg dl and the presence of one or more direct relatives
with hypercholesterolemia of more than 260 mgdl and an increase in Achilles tendon thickness.
Achilles tendon thickness was determined by xerora- diography. A thickness of more than 9 mm was desig-
nated as abnormal tendon thickening. In Japan, serum total cholesterol levels are lower than those in Western
countries because the Japanese diet has far less fat than the Western diet. Therefore, patients with abnormal
tendon thickening and hypercholesterolemia of more than 260 mgdl are reported to have decreased LDL
receptor activity [18].
The control subjects 22 men, 16 women, matched for age and gender, were apparently healthy according
to their medical history and the results of periodic health check-up. Relevant characteristics of the control
group and the hyperlipidemic patients are shown in Table 1. No subjects had clinical evidence of diabetes,
hypertension, hypothyroidism, or cardiovascular, neu- rologic or renal diseases. None of them took drugs
known to affect lipid metabolism or the coagulation system.
Overnight fasting, venous blood samples were ob- tained from patients after 24 h of abstaining from
alcohol. The blood for the measurement of hemostatic parameters was collected into siliconized glass tubes
containing 3.8 wv trisodium citrate. Plasma was obtained from blood centrifuged at 2500 × g for 10 min
at 4°C and stored frozen at − 80°C until use the time
Table 1 Characteristics of patients with primary hyperlipidemia and normolipemic control subjects
a
Variable Controls n = 38
Hyperlipidemia IIa n = 20
IIb n = 12 44 40–55
51 45–57 46 41–58
Age years 22.5 21.5–23.8
Body mass index kgm
2
22.6 21.5–23.8 24.5 23.4–26.0
6.35 6.11–6.97 4.56 4.14–5.15
6.29 6.16–7.38 Total cholesterol mmoll
2.75 2.36–3.20 LDL-cholesterol mmoll
4.55 4.27–4.99 3.94 3.56–4.55
1.35 1.11–1.58 HDL-cholesterol mmoll
1.37 1.06–1.71 1.32 0.98–1.40
0.91 0.68–1.29 Triglycerides mmoll
1.14 0.87–1.40 2.86 2.31–3.97
Apoprotein AI mgdl 102 95–107
107 87–116 122 92–128
70 58–88 Apoprotein B mgdl
108 94–122 127 107–169
Fasting blood sugar mgdl 94 89–98
92 90–98 107 99–113
Lipoprotein a mgdl 15.1 5.5–26.2
16.4 7.7–20.7 22.9 11.7–40.3
a
Values are shown as the median 25–75th percentiles. PB0.05;
PB0.01; PB0.001, compared to normolipemic control subjects.
of assay. Serum was prepared by clotting of whole blood in glass tube at room temperature for 1 h and
then centrifuging it at 1200 × g for 10 min for the measurement of cholesterol and triglycerides.
2
.
1
.
2
. Study Group II In addition, 25 consecutive outpatients were stud-
ied, aged 35 – 71 years, including 14 men and 11 women with hyperlipidemia 12 had type IIa and 13
had type IIb hyperlipoproteinemia, not included in Study Group I. These patients were recruited in the
Department of Internal Medicine, University Hospital of Kanazawa, Kanazawa, Japan. Five patients had
hypertension; five had diabetes mellitus. The patients were followed for more than 6 months during treat-
ment with 3-hydroxy-3-methylglutaryl coenzyme A HMG-CoA reductase inhibitors. Eleven patients
were treated with 5 mgday simvastatin Banyu, Tokyo, Japan, and 14 patients were treated with 10
mgday atorvastatin Yamanouchi, Tokyo, Japan. Fasting blood samples were obtained before and after
6 months of treatment. Informed consent was ob- tained from all patients.
2
.
2
. Methods Serum lipids were measured by standard enzymatic
colorimetric methods using commercially available kits for total cholesterol Kyowa Medex, Tokyo,
Japan and triglycerides Toyobo, Osaka, Japan. HDL-cholesterol
was measured
using the
same method after the VLDL and LDL were removed by
precipitation with dextran sulphate and phospho- tungstic acid in the presence of magnesium ions De-
terminer HDL;
Kyowa Medex.
Plasma LDL
cholesterol was calculated using the Friedewald equa- tion. If the triglyceride measurement was \ 4.52
mmoll, LDL cholesterol was measured by enzymatic colorimetric method using commercially available kits
Daiichi Chemical, Tokyo, Japan. Apolipoprotein apo AI and apo B were measured using a nephelo-
metric method Eiken, Tokyo, Japan. Plasma concen- trations of lipoprotein a Lp a were assayed using
an
enzyme-linked immunosorbent
assay ELISA
Biopool AB, Umea˚, Sweden. The inter-and intra-as- say coefficients of variation CV of serum lipids and
apolipoproteins were as follows: total cholesterol, 0.9 and 0.9; tryglyseride, 0.8 and 0.6;
HDL-cholesterol, 1.1 and
1.8; LDL-choles- terol, 1.1 and 0.9; apo AI, 0.6 and 0.3;
apo B, 0.6 and 0.5; Lp a, 1.2 and 1.4, respectively.
The following hemostatic parameters were mea- sured: tTFPI, fTFPI, tissue factor TF antigen, fac-
tor VII activity FVIIc and prothrombin fragment 1 + 2 F1 + 2. Plasma TF antigen was measured us-
ing the IMUBIND Tissue Factor ELISA American Diagnostica, Greenwich, CT [19]. The test specimen
was diluted 1:5 in 0.05 M Tris, pH7.5, with 2 bovine serum albumin and 0.05 Tween 20, to elimi-
nate the matrix effect of undiluted plasma. The mon- oclonal antibody used in this kit was the murine
immunoglobulin G1 raised against tissue factor from the human brain, which detects the TF – apoprotein
complex, TF and TF – VIIa complex. Plasma tTFPI concentration was measured using an ELISA kit
American Diagnostica [20]. The murine monoclonal antibody used in this assay binds near Kunitz domain
1, detecting native, complexed and truncated TFPI. Plasma fTFPI concentration was measured by the
ELISA kit Free TFPI ELISA kit ‘KAKETUKEN’; Chemo-Sera-Therapeutics
Research Institute,
Ku- mamoto, Japan, which was based on the one-step
sandwich ELISA method. The kit uses two different monoclonal antibodies against human TFPI obtained
by the cell fusion method [8]. FVIIc was determined with a one-stage clotting assay using Owren’s veronal
buffer Diagnostica Stago, Asnieres, France, human FVII
deficient plasma
Baxter Diagnostics
AG, Deerfield, IL and thromboplastin reagent Baxter Di-
agnostics AG, Miami, FL. The FVIIc levels were expressed as percent activity compared with a com-
mercially available standardized calibration plasma Baxter Diagnostics AG. Plasma F1 + 2 was mea-
sured using a commercially available ELISA kit En- zygnost F1 + 2 micro, Behringwerke AG, Marburg,
Germany [21]. The inter-and intra-assay CV of he- matological parameters were as follows: tTFPI, 2.5
and 3; fTFPI, 3.7 and 3.2; TF antigen,
2.7 and 3.9; FVIIc, 3.9 and 5; F1 + 2, 5.6 and 5.3, respectively.
2
.
3
. Statistics Data are presented as the median and interquartile
ranges. Because normal distributions were not found for some parameters, statistical analysis was per-
formed by nonparametric tests the Mann – Whitney two-sample rank test and the Kruskal – Wallis one-
way analysis of variance. The changes in the plasma concentrations of tTFPI, fTFPI, FVIIc, F1 + 2 and
serum lipids were analyzed using the Wilcoxon’s signed-rank test for matched pairs. Correlations be-
tween laboratory parameters were calculated using Spearman rank correlation coefficients. P values B
0.05 were regarded as statistically significant. All cal- culations
were performed
using software
for a
personal-computer Stat View 4.0; Abacus Concepts, Berkeley, CA.
Table 2 Hemostatic parameters in patients with hyperlipidemic and normolipemic control subjects
a
Hyperlipidemia Controls n = 38
Variable IIa n = 20
IIb n = 12 165 130–206
Total TFPI ngml 151 140–197
113 96–141 31 27–34
Free-form TFPI ngml 41 38–45
21 17–27 93 77–130
90 50–116 103 70–147
TF pgml 92 79–100
FVIIc 112 97–118
121 109–128 1.00 0.67–1.21
0.74 0.56–0.92 0.94 0.79–1.71
F1+2 nmoll
a
TF, tissue factor; TFPI, tissue factor pathway inhibitor; FVIIc, factor VII activity; F1+2, prothrombin fragment 1+2. Values are shown as the median 25–75th percentiles.
PB0.05; PB0.01;
PB0.001, compared to normolipemic control subjects. Table 3
Serum lipids, apoprotein and hemostatic parameters in patients with hyperlipidemia before and after treatment with 3-hydroxy-3-methylglutaryl coenzyme A HMG-CoA reductase inhibitors
a
After treatment n = 25 Variable
Statistical significance Before treatment n = 25
5.03 4.66–5.49 Total cholesterol mmoll
PB0.0001 7.07 6.52–7.29
2.75 2.41–3.49 LDL-cholesterol mmoll
PB0.0001 4.75 3.96–5.14
1.45 1.28–1.77 1.32 1.17–1.68
PB0.05 HDL-cholesterol mmoll
1.20 0.91–2.23 Triglycerides mmoll
PB0.01 1.73 1.31–2.59
142 130–173 138 123–163
NS Apoprotein AI mgdl
157 140–173 Apoprotein B mgdl
94 89–122 PB0.001
21.0 8.5–36.0 Lipoprotein a mgdl
30.0 3.8–38.3 NS
162 149–217 200 168–235
PB0.005 Total TFPI ngml
Free-form TFPI ngml 31 27–37
31 28–39 NS
118 105–124 128 112–150
PB0.005 FVIIc
0.89 0.72–1.21 PB0.05
F1+2 nmoll 1.13 0.94–1.27
a
TFPI, tissue factor pathway inhibitor; FVIIc, factor VII activity; F1+2, prothrombin fragment 1+2. Values are shown as the median 25–75th percentiles.
3. Results
