JP2645774B2 - TAT stabilization method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an method for stabilizing T AT, the A Rugatoroba on to T AT containing plasma is more
About the stabilization how of TAT due to be added.

[0002]

2. Description of the Related Art Thrombin, a major enzyme in the blood coagulation system, is a physiological inhibitor, antithrombin I.
Its activity is inhibited by II (AT-III). At this time, thrombin and AT-III are in a complex (hereinafter referred to as “T
AT "), and this T
AT is an indicator for diagnosing various hypercoagulable states such as thrombosis and DIC, and its usefulness in measurement is being recognized.

However, if blood collection for measuring TAT is not performed smoothly, or if a tissue factor is aspirated during blood collection due to tissue damage or the like, coagulation is caused by its coagulation promoting action, and TAT increases rapidly. When measuring,
The exact amount of TAT in the sample cannot be known. Also,
After blood collection, if plasma is separated and stored, T
AT is easily decomposed and it is difficult to perform accurate quantification.

Therefore, anticoagulants such as sodium salt of EDTA and sodium citrate have been added to plasma. However, it is difficult to stabilize TAT by this method. It is difficult to get.

[0005]

The present invention 0005] the hand upon TAT measurement <br/>, an object of tomb Rukoto stabilization of TAT between after blood collection until measurement.

[0006]

The present invention provides a TAT comprising adding argatroban to plasma containing TAT.
It is a method of stabilizing.

Argatroban used in the present invention is (2)
R, 4R) - methyl-1- ^[N 2 - ((RS) -3- methyl-1,2,3,4-tetrahydro-8-quinoline sulfonyl) -L- arginyl] -2-piperidinecarboxylic acid solution It is a compound represented by Formula 1 below.

[0008]

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As argatroban, Novastan (registered trademark, Tokyo Tanabe Seiyaku Co., Ltd.), Slonnon (registered trademark, Daiichi Pharmaceutical Co., Ltd.) and the like can be used.

In the present invention, argatroban is added in advance to a blood collection tube containing an anticoagulant such as sodium citrate, and blood is collected in the blood collection tube. Due to the method of adding argatroban, TA in plasma was measured before TAT measurement.
It is possible to know the exact amount of TAT in the body by preventing T from being decomposed or newly formed.

The amount of argatroban added is preferably at least 0.005 mM as a final concentration,
More preferably, it is 0.01 mM or more. If the added amount of argatroban is less than 0.005 mM, the stabilization of TAT is not sufficient. The upper limit of the amount to be added is determined from common sense and economic viewpoints, and is about 1 mM as the final concentration.

When argatroban is added to blood or plasma, argatroban is added to the desired final concentration of 10
1 to 10 times of blood or plasma volume
/ 50 can be added.

[0013] Further, the present invention relates to the stabilization of the plasma containing TAT of the present invention.
In this case , a known protease inhibitor such as benzamidine, aprotinin, phenylmethylsulfonyl fluoride, EDTA, or N-ethylmaleimide may be added. In addition, sodium citrate, ED
TA, heparin, gabexate mesylate (FOY) and the like may be added. These can be used alone or in combination of two or more with argatroban. Above all, the combined use of FOY not only inhibits thrombin finally produced by coagulation activation but also inhibits F-Xa
Excellent because it can inhibit.

The present invention provides a method for stabilizing TAT-containing plasma.
Thus , for example, a 3.8% by weight sodium citrate solution 0.1%.
Blood 4.5ml added to blood collection tubes by adding 5ml and 1mM argatroban solution 0.05 ml, was mixed well, you to prepare by centrifugation (2000 G × 10 minutes). Further, 4.5 ml of blood was collected in a blood collection tube to which 0.5 ml of 3.8% by weight of sodium citrate had been added, mixed, and then centrifuged.
It is also possible to add 0.05 ml of a mM argatroban solution.

[0015]

The present invention will now be described more specifically with reference to examples. In the examples, the identification method and the quantification method were performed as follows. SDS polyacrylamide gel electrophoresis (SDS-PA
GE) Using a gradient slab gel of 2 to 40% by weight (Daiichi Pure Chemicals Co., Ltd.) according to the method of Laemmli et al. Silver staining was performed according to the method of Oakley et al., And a carburetion kit for electrophoresis (Pharmacia) was used as a molecular weight marker.

TAT was measured using a sandwich EIA method (TAT test kit “Teijin”, manufactured by Teijin Limited).

In the examples, the following materials were used. Argatroban Novastane (Tokyo Tanabe Seiyaku) injection 1 mM solution was diluted with physiological saline and used. Thrombin 1,000 NIH units of human thrombin (manufactured by Sigma) are diluted with Tris-HCl buffer to 250 NIH units / ml.
It adjusted and used. 500 units of AT-III dry concentrated human AT-III (manufactured by Hoechst) were adjusted to 1 unit / ml with Tris-HCl buffer (pH 7.4, μ = 0.15) and used. 1,000 units of heparin sodium for heparin injection (manufactured by Novo) was used after adjusting with physiological saline.

EXAMPLE 1 (Effect of Argatroban on Inhibition of TAT Production) To 900 μl of fresh plasma, 50 μl of argatroban (final concentration 0.01 mM) was previously added (final concentration 0.01 mM).
M), coagulation activator [Erajin acid 200 [mu] l 1 to × ^10- 6 M) cephalin 0.025MCaCl ₂
Was mixed at 37 ° C. for 60 minutes, and TAT was quantified. The results are shown in FIG.

Comparative Example 1 TAT was quantified in the same manner as in Example 1 except that physiological saline was used instead of argatroban. The results are shown in FIG.

As shown in FIG. 1, in the case of Example 1 to which argatroban was added, T
The AT amount was 3.0 to 7.0 ng / ml, with no significant change. Comparative Example 1 without addition of argatroban
In case (1), the amount of TAT was increased after the start of incubation, indicating that argatroban suppressed the increase in TAT.

Example 2 (Effect of Argatroban on TAT Decomposition in Purification System) 900 μl of AT-III (1.0 unit / ml) and 900 μl of thrombin (250 NIH unit / m 2)
1) was mixed and incubated at 37 ° C. for 1 hour, then 200 μl of argatroban (1.0 mM) was added, further incubation was continued at 37 ° C. for 72 hours, sampling was performed with time, and 0.2 M diisopropylfluoro Stop the reaction with phosphate (DFP)
TAT was quantified. The results are shown in FIG. In addition, at the same time as the quantification of TAT, analysis was performed by SDS-PAGE. Fig. 3 shows the results.
It is shown in (B).

Comparative Example 2 TAT was quantified in the same manner as in Example 2 except that physiological saline was used instead of argatroban, and SDS-PAGE was performed.
Analysis by E was performed. The results are shown in FIG. 2 and FIG. 3 (A). When thrombin and AT-III were independently incubated, no change was observed in their molecular weights until 72 hours.

As shown in FIG. 2, in Comparative Example 2 containing no argatroban, the amount of TAT gradually decreased from 67 hours after the incubation (67 to 22.8 ng / ml).
In contrast, in Example 2 containing argatroban, no change was observed until after 24 hours, and TAT
Is suppressed, and the effect is maintained four times longer than that of Comparative Example 2.

As shown in the results of SDS-PAGE,
In Comparative Example 2 (FIG. 3 (A)), a plurality of complexes having a molecular weight of 78,000 to 93,000 were observed with the incubation time, and the complexes formed at the latter stage of the incubation were compared to the complexes formed at the beginning of the incubation. The body has a low molecular weight. In contrast, Example 2
In FIG. 3 (B), only a single complex having a molecular weight of 90,000 (estimated as TAT) is observed until late in the incubation.

COMPARATIVE EXAMPLE 3 (Conventional Anticoagulant) From five healthy subjects, three syringes were used with three anticoagulants (3.13% by weight of sodium citrate, EDTA-2Na, and NaF). Blood was collected and the plasma obtained therefrom was incubated at 37 ° C. for 96 hours, and the T
The AT amount was measured over time. FIG. 4 shows the results.

As shown in FIG. 4, the plasma using any of the coagulants showed a value approximately 45% lower than the TAT value immediately after collection in 24-48 hours of incubation, and thereafter turned to rise. It can be seen that the variation is large and is not preferable for use in quantifying TAT in the body.

Example 3 (Argatroban Inhibition of TAT Production in Purification System) 245 μl of AT-III (1.0 unit / ml) and 10 μl of argatroban (final concentration 0.1 mM) were mixed at 37 ° C. for 5 minutes. After incubation, 245
μl of thrombin (250 NIH units / ml) was added and incubated at 37 ° C. for 60 minutes. During this time, samples were taken over time, the reaction was stopped by adding 0.2 M DFP, and analyzed by SDS-PAGE. The results are shown in FIG.

Comparative Example 4 The formation of a complex was examined in the same manner as in Example 3 except that physiological saline was used instead of argatroban. Fig. 5 shows the results.
It is shown in (B).

As shown in FIG. 5, in Example 3 in which argatroban was present, no complex (TAT) was formed after 60 minutes of incubation. On the other hand, in Comparative Example 4 in the absence of argatroban, the complex (TAT)
Was observed, and T of Argatroban in the purification system was observed.
It is understood that the AT production is suppressed.

Example 4 (Addition amount of argatroban) To a mixture of 360 μl of fresh human plasma and 80 μl of ellagic acid, 20 μl of various concentrations of argatroban were added, and the mixture was incubated at 37 ° C. for 60 minutes.
The reaction was stopped by adding 0 μl of 0.2 MDFP, and TAT in plasma was quantified. FIG. 6 shows the results.

As is apparent from FIG. 6, when the added amount of argatroban exceeds 0.004 mM in the final concentration, TA
Although the increase in the amount of T is slight, it can be seen that the increase in TAT is significant when 0.004 mM is added.

[0032]

According to the present invention, it is possible to stabilize TAT in plasma between the time after blood collection and the time of measurement, and thus to accurately measure the amount of TAT in the human body. Various hypercoagulable states such as thrombosis and DIC can be diagnosed.

[Brief description of the drawings]

FIG. 1 is a graph showing the inhibitory effect of argatroban on TAT production in plasma.

FIG. 2 is a graph showing the inhibitory effect of argatroban on TAT degradation.

FIG. 3 shows S showing the inhibitory effect of argatroban on TAT degradation.
It is a result of DS-PAGE. A is the result of Comparative Example 2, and B is the result of Example 2.

FIG. 4 is a graph showing changes in the amount of TAT in plasma to which a conventional anticoagulant has been added.

FIG. 5 shows S showing the inhibitory effect of argatroban on TAT production.
It is a result of DS-PAGE. A is the result of Example 3 and B is the result of Comparative Example 4.

FIG. 6 is a graph showing the amount of argatroban added.

 ──────────────────────────────────────────────────続 き Continuation of the front page (56) References THROMBOSIS RESEARCH CH 59, p. 967-977 (1990)

Claims (2)

(57) [Claims] 1. A method for stabilizing TAT, comprising adding argatroban to plasma containing TAT. 2. The method for stabilizing TAT according to claim 1, wherein the added amount of argatroban is at least 0.005 mM in final concentration.