JPS60186760A - Blood coagulation accelerator - Google Patents

Abstract

PURPOSE:To improve separability of a serum by incorporating specific oxalate and hydrolytic enzyme into a titled agent to activate the XII factor thereby reducing the time required for blood coagulation and stabilizing the effect of accelerating the blood coagulation. CONSTITUTION:The XII factor activator for blood coagulation consisting of oxalate of bivalent or higher valency metallic ions and the hydrolytic enzyme of the conjugation between the residual Arg residue or Lys residue and optional amino acid residue in an amino acid arrangement are incorporated into a titled accelerator. One among Cu<2+>, Co<2+>, Fe<2+>, Ni<2+>, Mg<2+>, Sn<2+> and Ce<2+> is used as the metallic ions of the above-mentioned oxalate. Protease,etc. are preferably used as the above-mentioned hydrolytic enzyme. The proteaze alone can activate blood coagulation but the activating power for blood coagulation is remarkably improved when the protease is added to the blood in conjunction with the above-described oxalate. The thorough reduction in the coagulating time after drawing of the blood and shrinkage of the blood-clot component, the good separation of the serum and the blood-clot and the increase in the yield of the serum are effected. The accelerator is thus widely used in clinical inspection fields and for hemostasia of a bleeding wound, etc. as well.

Description

DETAILED DESCRIPTION OF THE INVENTION (Technical Field) The present invention relates to a blood coagulation promoter, and more particularly to a blood coagulation promoter having the ability to activate factor XN.

(Prior art) In recent years, with the remarkable progress in testing technology, blood tests such as serum biochemistry tests, serum immunology tests, and blood cell tests have become widespread and have greatly contributed to disease prevention and early diagnosis. Among them, serological tests are the main body of blood tests.
The serum required for this test is usually obtained by collecting blood into a blood test container, coagulating it, and then centrifuging it to separate blood clots with different specific gravities, i.e., gel-like lumps that are a mixture of fibrin and blood cells. The serum portion is collected by pipetting or by decantation.

However, blood generally takes a considerable amount of time to coagulate, making it difficult to conduct tests quickly. Even with glass blood test containers, which are said to have the shortest blood coagulation time, it takes 40 to 60 minutes for blood to coagulate after blood is injected.As for blood test containers made of synthetic resin, It takes 4 hours or more for blood to coagulate.

For this reason, glass, kaolin, bentonite, silica, ellagic acid, etc., which have the ability to activate blood coagulation factor Although it has been put to practical use as a component of a reagent for measuring activated partial thromboplastin time, there is a problem that its activation ability is unstable depending on its purity, composition, etc.

Furthermore, when these blood coagulation promoters are used, blood clots are generally separated from serum after blood coagulation, and when serum is collected, the separability of serum is not good, and contamination of blood clot components with serum is unavoidable. .

(Objective of the Invention) The present invention was made to solve the above-mentioned problems, and it activates factor The object of the present invention is to provide a blood coagulation promoter that is extremely stable and has excellent serum separation properties.

(Structure of the Invention) The blood coagulation promoter of the present invention is a metal salt of oxalic acid, and the blood coagulation factor It is characterized by containing an enzyme that hydrolyzes the bond between Arg or Lys residue and any amino acid residue.

In the blood coagulation promoter of the present invention, the divalent or higher metal ion in the oxalate, which is a component thereof, is at least one selected from alkaline earth metals, transition metals, and rare earth metals, preferably Cu2 +, Co”,
Fe2′″, Ni″, M g 2 +, Sn2′ and C
At least one type selected from the group consisting of e3+.

The present inventor already has carbonyl groups adjacent to each other in the molecule,
Furthermore, it was revealed that a group of cyclic organic compounds, which are three-dimensionally located on substantially the same plane, have a specific effect on blood coagulation factors, which are proteins (Japanese Patent Application No. 1983). -114659), the metal ion forming oxalate is divalent or higher, and the oxalate ion forms a chelate-like salt structure via the metal ion, and carbon-
When the free rotation of the carbon bond is inhibited, oxalate has a steric structure in which it has adjacent carbonyl groups in the molecule and these are sterically located on substantially the same plane. Therefore, although the mechanism of action is not clear, it appears to have a specific effect on blood coagulation factors in the same way as above.

In addition, when the metal ion is monovalent, oxalate is carbon-
Carbon-carbon bonds can rotate freely, and adjacent carbonyl groups are sterically virtually identical due to steric repulsion between carbonyl groups and electrostatic repulsion between two metal ions. Since it is not on a flat surface, it probably does not have a specific effect on blood coagulation. Incidentally, monovalent metal salts of oxalic acid, such as potassium oxalate and sodium sulfate #I, dissociate in the blood and are important blood coagulation factors, and have a higher tendency to ionize than K' or Na'. Since it forms a poorly soluble chelate compound with small Ca'', C
Blood that has lost a Z+ has its coagulation mechanism inhibited, and therefore, the above-mentioned monovalent metal salts have been widely used as blood anticoagulants.

Next, the hydrolase, which is another component of the blood coagulation promoter according to the present invention, has an amino acid sequence of Arg or Lys.
It is an enzyme that hydrolyzes a bond between a residue and an arbitrary amino acid residue, and protease is particularly preferably used as such a hydrolase. Specific examples of this protease include serine proteases such as trypsin, thrombin, snake venom thrombin-like enzymes, and cathepsin B.
1. Thiol protease such as ficin, kininase I
Examples include metalloproteases such as
Serine proteases are also readily available and therefore suitable for use. These proteases can activate blood coagulation even when used alone, but according to the present invention, when used in combination with the above-mentioned activator of blood coagulation factor It improves.

In using the blood coagulation promoter of the present invention, for example, blood is collected into a blood test container and the promoter is present in the blood when coagulating the blood. In this case, the blood coagulation accelerator may be present in the blood as it is in powder form, but preferably the blood coagulation accelerator is dissolved or dispersed in an appropriate solvent and then added to the blood. do. The blood test container is not particularly limited, and conventional glass or resin containers may be used as appropriate.

In addition, if blood comes into contact with these blood coagulation promoters momentarily or partially in a blood test container and there is a risk that protein components in the blood may be denatured, use a carrier with a large specific surface area. A blood coagulation promoter may be supported and added to the blood in the blood test container. As the carrier, various carriers can be used without particular limitation as long as they do not have a harmful effect on blood tests and have a large specific surface area. For example, nonwoven fabrics, woven fabrics, resins, etc. Beads and the like can be suitably used.

In order to support a blood coagulation promoter on such a carrier, for example, the carrier may be coated with a solution or dispersion thereof, or immersed therein, and then dried and attached to the carrier. Further, particulate matter carrying a blood coagulation promoter can also be obtained by mixing with an appropriate auxiliary agent such as gum arabic to form an aqueous dispersion, and then rapidly freeze-drying the resulting aqueous dispersion.

The amount of blood coagulation promoter present in blood is 1ml of blood.
When the amount is less than this, the effect of promoting blood coagulation is poor. However,
If it is present in too large a quantity, it may cause various problems in blood tests, so it is preferable to limit the amount to 10-'g or less.

(Effects of the Invention) According to the blood coagulation promoter of the present invention, when it is present in blood, factor X is rapidly activated, and the time required for coagulation after blood is collected into a container is significantly shortened. At the same time, as the blood clot components are sufficiently contracted, the separability of serum and blood clot is improved, and the separated and collected serum is not contaminated with blood clot components, and furthermore, the yield of serum is significantly increased. do. Therefore, the blood coagulation promoter of the present invention can be widely used in the field of clinical testing, and can also be used to stop bleeding.

The present invention will be explained below with reference to Examples, but the present invention is not limited to these Examples in any way.

(Example) Example 1 Copper oxalate, magnesium oxalate, cobalt oxalate,
A blood coagulation promoter according to the present invention was prepared using iron oxalate, nickel oxalate, tin oxalate, and cerous oxalate, and trypsin, thrombin, and a snake venom thrombin-like enzyme as proteases, respectively. still,
The content of each component in each blood coagulation promoter is 0.5% by weight of oxalate, 0% of protease, trypsin, thrombin, and snake venom thrombin-like enzyme.
．． 05% by weight, 500 units/ml and 0.005% by weight
Closed.

Using commercially available polyethylene braces, 30 μp of the blood coagulation promoter according to the present invention was added to 3 ml of large fresh blood, and the time required for the blood to lose its fluidity was measured as the coagulation time. The mixture was centrifuged at 3,000 rpm for 5 minutes, and the state of separation was observed.

The results are shown in Table 1.

Comparative Example 1 For comparison, Table 2 shows the clotting time and separation state when each of the oxalate and protease used in Example 1 was used alone.

Comparative Example 2 Table 3 shows the clotting time and separation state when blood was treated in the same manner as in Example 1, except that no blood coagulation promoter was used.

Patent applicant: Sekisui Chemical Co., Ltd. Representative Mototoshi Fujinuma Procedural amendment (voluntary) June 3, 1985 1. Indication of the case 1988 Patent application No. 42660 2. Name of the invention Blood coagulation promoter 3 , Relationship with the case of the person making the amendment Patent applicant Postal code: 530 Address: 2-4-4 Nishitemma, Kita-ku, Osaka Patent Department Tokyo based T[! L Tokyo (03) 434-95524, Column for detailed description of the invention in the specification subject to amendment ζe, 4=5, Contents of amendment (1) "Table 1" on page 10 of the specification as attached. to correct.

6. Attached list of class 111 (1) Document stating the amended Table 1 on one board

Claims (1)

[Scope of Claims] (A metal salt of 11 oxalic acid, wherein the metal ion is 2
It is characterized by containing a blood coagulation factor A blood coagulation promoter. (2) Metal ions include Cu2+, Co", Fe2+,
The blood coagulation promoter according to claim 1, which is at least one selected from the group consisting of Ni", M gZ +, Sn", and Ce'+.