Method for purifying thrombomodulin

The present invention permits human urinary thrombomodulin to be purified by a simple and practical procedure, while it enables even a starting material with a lowered content of thrombomodulin to be purified by greater purification rate than the one with a high content of thrombomodulin.

The present invention relates to a method for purifying through 
concentration thrombomodulin contained in human urine. Thrombomodulin is 
an anticoagulant and is expected to find application as a prophylactic and 
therapeutic agent against diseases being associated with abnormalities in 
blood coagulation capability. 
BACKGROUND OF THE INVENTION 
Thrombomodulin (hereinafter referred to briefly as "TM"), which is a 
high-affinity thrombin-receptor being present on the angioendothelial 
cells, was discovered by N. L. Esmon et al. (J. Biol. Chem., 257: 859-864, 
1982) as a protein being capable of promoting the activation of protein C. 
TM binds to thrombin at a molar ratio of 1:1 to thereby activate protein C 
in the presence of Ca.sup.2+, while, on the other hand, the TM-bound 
thrombin loses almost entirely its own coagulation activities, such as 
fibrinogen-agglutinating activity and platelet activating activity. TM, 
with its endogenous heparin activity, is also known to promote the 
thrombin suppression by anti-thrombin III, and is expected to find 
clinical application as a novel type of drugs. 
Human TM was initially purified from the human placenta by H. H. Salem et 
al. (J. Biol. Chem., 259: 12246-12251, 1984), but the human placenta was 
not suited as a starting material for the large-scale production of TM 
(EMBO J. 6: 1891-1897, 1987). Though the genetic structure of human TM was 
identified by Suzuki et al. (EMBO J. 6: 1891-1897, 1987), furthermore, it 
is difficult to synthesize TM having the same sugar chains as those of the 
naturally occurring one, since TM contains lots of sugar chains. 
Subsequently, human urinary TM was isolated and purified by Yamamoto et al. 
(J. Biochem. 113: 433-440, 1993) and D. E. Jackson et al. (Eur. J. 
Biochem. 221: 1079-1087, 1994), demonstrating that human urine is suitable 
as a starting material for isolating the naturally occurring type of TM. 
Referring to the method for purifying TM from urine, Yamamoto et al. 
produced purified TM by use of QAE Sephadex, anti-TM monoclonal antibody 
column, affinity column in which thrombin was used as a ligand, and 
Sephadex G-200, but because the method requires preparation of the 
monoclonal antibody, its procedure is therefore complicated and 
troublesome. D. E. Jackson et al. purified TM by the combined use of DEAE 
Sepharose, affinity column with thrombin employed as a ligand and 
reverse-phase HPLC, wherein the method encountered the problems in that 
the purification yield attainable by reverse-phase HPLC is very poor and 
that HPLC is not suited for large-scale purification. Furthermore, Aoki et 
al. (Japanese Patent Application Laid-Open No. Sho-63-30423) and Kunihiro 
et al. (Japanese Patent Application Laid-Open No. Sho-63-218399) reported 
that TM was purified from fresh urine by ion exchange chromatography, 
thrombin-bound affinity column and gel permeation. From the inventors' 
experience, nevertheless, it has been learnt that the content of TM in 
urine is not always constant, while in the case of lowered TM contents, 
especially, such method often does not work satisfactorily. Consequently, 
there is strongly demanded the development of a practically expedient and 
efficient purification means that can be applied even when urinary TM 
contents are low. 
SUMMARY OF THE INVENTION 
In view of the above, the inventors investigated into a purification means 
which would be able to permit TM with an enhanced degree of purity to be 
produced, irrespective of the magnitude of TM contents in urine. 
In order to solve the above-mentioned problem, the present inventors 
conducted repeatedly extensive investigation and as a result, found that 
adsorption chromatography with hydroxyapatite used as an adsorbent is 
highly effective for the purification of thrombomodulin, resulting in 
completion of the present invention. 
The present invention relates to a method for purifying human urinary 
thrombomodulin, which comprises purifying human urinary thrombomodulin by 
employing adsorption chromatography with hydroxyapatite used as an 
adsorbent. 
DESCRIPTION OF THE PREFERRED EMBODIMENTS 
Referring to a material containing human urinary thrombomodulin which is a 
starting material in the method of the present invention, there may be 
used, for example, raw human urine as such or after being concentrated by 
fractionation with ammonium sulfate, desirably preliminarily purified 
human urine obtained by means of anion-exchange chromatography, 
thrombin-bound affinity chromatography, etc., if possible. though no 
particular limitation is posed. 
With specific reference to hydroxyapatite, there may be utilized any 
commercially available products (for example, those manufactured by 
Seikagaku Kogyo K. K. of Japan and Wako Pure Chemicals Co. of Japan). 
In cases where a starting material is available in small quantities, it is 
possible to employ HPLC column for fractionation or analytical uses, such 
as TSK gel HA-1000 (manufactured by Tosoh Inc. of Japan) and KBC columns 
(manufactured by Kohken Co., Ltd. of Japan). 
A column is packed with hydroxyapatite and then equilibrated with a 1 to 30 
mM, preferably 5 to 10 mM phosphate buffer adjusted at pH 5 to 8, 
preferably pH 6 to 7, whereby it is effective to add salts, such as 
calcium chloride, sodium chloride, potassium chloride and magnesium 
chloride, to the equilibrating solution used, with calcium chloride of 
less than 1 mM in concentration being particularly preferably utilized. 
Then, a starting material is applied to the thus buffer-replaced column, 
followed by washing thoroughly with the same buffer. After the effluent 
shows fully decreased absorption at a wavelength of 280 nm, elution is 
conducted with phosphoric acid at a linear concentration gradient, whereby 
a concentration gradient in the range of 5 to 300 mM is suited for the 
purification of TM. In the step of elution, TM is eluted in the first 
place (the phosphoric acid concentration is in the region of 50 to 100 
mM), with impurities being eluted subsequently. In cases where an open 
column is used, meanwhile, such open column can desirably be shaped in the 
long and narrow form to achieve improved separation. When impurities are 
still detected even after completion of this step, the final, ultimate 
purification can be carried out by means of gel permeation, etc. The 
procedure of adsorption chromatography with use of hydroxyapatite 
according to the present invention offers the characteristic feature that 
even when a starting material with lowered specific activity is applied to 
such chromatographic procedure, there can always be produced the purified 
product with the same degree of purity as in the case of the ones with 
high specific activity. 
The method of the present invention can be used in combination with other 
chromatographic procedures, such as ion exchange chromatography, 
thrombin-bound affinity chromatography and gel filtration chromatography 
so as to purify human urinary thrombomodulin. 
In each purification step, the specific activity of TM can be assayed by 
the following procedure: 30 .mu.l of Buffer A (20 mM Tris-HCl buffer (pH 
7.5) containing 0.1M sodium chloride and 3.5 mM calcium chloride), 10 
.mu.l of a human Protein C solution a solution being prepared with Buffer 
B (a solution of 0.1% BSA in Buffer A) to a concentration of 100 
.mu.g/ml!, 10 .mu.l each of a sample and the standard sample are mixed, 
and 50 .mu.l of a human thrombin solution (a solution being prepared with 
Buffer B to a concentration of 4 U/ml) is added to the mixture solution, 
followed by incubation at 37.degree. C. for 30 min. Then, 150 .mu.l of a 
human antithrombin III solution a solution being prepared with Buffer C 
(a solution being prepared with 50 mM tris-HCl solution (pH 7.5) 
containing 0.1M sodium chloride and 1 mM calcium chloride) to a 
concentration of 50 .mu.g/ml! and 50 .mu.l of a heparin solution (a 
solution being prepared with Buffer C to a concentration of 2 U/ml) are 
added to the reaction solution, followed by incubation at 37.degree. C. 
for 15 min. 600 ml of a substrate solution {a solution of 25 mg of Test 
Zyme S-2366 (produced by Daiichi Kagaku Inc. of Japan) in 23.19 ml of 
Buffer C} is added to the solution, followed by incubation at 37.degree. 
C. for 20 min, and then, the reaction is stopped by adding 100 .mu.l of a 
50% acetic acid solution. The reaction solution is subjected to 
measurement of absorption at a wavelength of 405 nm to calculate a TM 
content in the sample on the basis of the absorption measurements obtained 
with the standard samples (solutions of TM originated from rabbit lungs 
diluted with Buffer B to concentrations of 2, 5, 10, 20 and 40 mU/ml, 
respectively, are used as the standard sample). 
As may be evident from the above, the present invention, by use of the 
easy-to-operate procedure of adsorption chromatography, permits not only 
human urinary thrombomodulin to be concentrated and purified by raised 
purification rate but also even a starting material with lowered specific 
activity of thrombomodulin to be purified by greater purification rate 
than the ones with high specific activity.

EXAMPLE 1 
Outlined below are the purification results obtained with two different 
lots each of 2000 liters of human urine. A 2,000-liters volume of fresh 
human urine was adjusted to pH 5.5 and admixed with a 0.5% chitosan 
solution (w/v) serving as an adsorbent, followed by stirring. The solution 
mixture was maintained at pH 5.5 for 30 min, filtered, washed with water 
and suspended in a 5% ammonium sulfate solution (pH 10.5), followed by 
stirring at room temperature for 1 hour. After the suspension was 
filtered, followed by washing to elute the adsorbed component, 
fractionation with 60% ammonium sulfate was effected, and the precipitate 
was recovered by filtration. The separated precipitate was dissolved in 20 
mM Tris-HCl buffer (pH 8.0), and the solution was dialyzed against the 
same buffer, followed by application onto a column of DEAE-Toyopearl 650M 
(the resin amount of 1.2 liters; measuring 115 by 11 cm) (manufactured by 
Tosoh Inc. of Japan) equilibrated in advance with the same buffer. The 
column was washed with the same buffer, and elution was conducted with the 
same buffer containing 0.3M sodium chloride. The elution fractions were 
pooled and diluted 3-fold with 20 mM Tris-HCl buffer (pH 8.0), and the 
solution was admixed with calcium chloride to a final concentration of 5 
mM. The mixture was applied to a DIP-thrombin bound Sepharose column (the 
resin amount of 40 ml; measuring 2.5 by 8 cm) equilibrated in advance with 
20 mM Tris-HCl buffer (pH 8.0) containing 0.1M sodium chloride and 5 mM 
calcium chloride, and after the column was washed with the same buffer, 
elution was conducted with 20 mM Tris-HCl buffer (pH 8.0) containing 2M 
sodium chloride and 10 mM EDTA. The elution fractions were dialyzed 
against 5 mM sodium-phosphate buffer (pH 6.8) containing 0.4 mM calcium 
chloride and 0.9% sodium chloride and applied to a column of 
hydroxyapatite (the adsorbent amount of 20 ml; measuring 1.0 by 25 cm) 
(manufactured by Seikagaku Kogyo K. K. of Japan) equilibrated in advance 
with the same buffer. The column was washed with the same buffer, and 
elution was conducted with a linear concentration gradient of sodium 
phosphate from 5 to 300 mM, whereby the TM activity was firstly eluted as 
a sharp peak, as illustrated in FIG. 1 (Lot No. 1) and FIG. 2 (Lot No. 2), 
with impurities being eluted subsequently. The active fraction was 
concentrated, and the concentrate was applied onto a column of Sephacryl 
S-300 (the resin amount of 80 ml; measuring 1.5 cm by 90 cm) (manufactured 
by Pharmacia Co. of Sweden) equilibrated in advance with 25 mM 
sodium-phosphate buffer (pH 7.0) containing 0.9% sodium chloride, followed 
by elution with the same buffer. Tabulated in Table 1 are the results 
after purification was carried out twice, which indicates that even 
purification of a starting material exhibiting low specific activity 
before hydroxyapatite-chromatography was able to provide the purified 
product with a satisfactorily high degree of specific activity. FIG. 3 
illustrates the results of SDS polyacrylamide gel electrophoresis of the 
final-purified product under reducing and non-reducing conditions, and the 
figure reveals two neighboring bands (in the neighborhood of 70,000 in 
molecular weight under reducing conditions) characteristic to TM observed 
in both of the two lots of the starting material. 
TABLE 1 
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Total Yield Specific Purification 
activity, U 
% activity, U/mg 
rate 
______________________________________ 
Before hydroxyapatite- 
5,556 100 446.3 1.0 
chromatography 
(Lot No. 1) 
After hydroxyapatite- 
4,483 81 1,306.9 2.9 
chromatography 
(Lot No. 1) 
Before hydroxyapatite- 
3,071 100 28.7 1.0 
chromatography 
(Lot No. 1) 
After hydroxyapatite- 
2,565 84 674.6 23.5 
chromatography 
(Lot No. 1) 
______________________________________ 
EXAMPLE 2 
In order to confirm that the purified products each consist of TM, 
furthermore, investigation was conducted to identify the amino acid 
composition and N-terminal amino acid sequence. The amino acid composition 
was analyzed by use of an amino-acid analysis device (manufactured by 
Beckmann Co. of USA) with a hydrolysate with 6N hydrochloric acid, while 
the N-terminal amino acid sequence was analyzed with Protein Sequencer 
473A (manufactured by Applied Bio-System Co. of USA). The results of amino 
acid analysis are tabulated in Table 2, with the identified N-terminal 
amino acid sequence being shown below: 
N-Terminal amino acid sequence: 
Ala-Pro-Ala-Glu-Pro-=Gln-Pro-Gly-Gly-SEr-Gln- 
TABLE 2 
______________________________________ 
Purified TM 
Ref. 
______________________________________ 
Asx 47.0 47 
Thr 18.2 19 
Ser 23.2 24 
Glx 55.1 50 
Pro 49.6 43 
Gly 50.8 46 
Ala 52.7 52 
Cys N.D. 46 
Val 24.6 25 
Met 4.9 4 
Ile 11.3 13 
Leu 34.0 32 
Tyr 9.5 10 
Phe 16.5 16 
Lys 3.0 3 
His 11.0 12 
Arg 17.2 20 
Trp N.D. 6 
______________________________________ 
Notes: N.D. stands for "not detected". 
Values in Ref. designate those as calculated from the amino acid sequence 
of the portion of from Ala 1 to Asp 468 of TM. 
Asx stands for (Asp + Asn), while Glx for (Glu + Gln).