JPH0515437B2 - - Google Patents
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- Publication number
- JPH0515437B2 JPH0515437B2 JP4266084A JP4266084A JPH0515437B2 JP H0515437 B2 JPH0515437 B2 JP H0515437B2 JP 4266084 A JP4266084 A JP 4266084A JP 4266084 A JP4266084 A JP 4266084A JP H0515437 B2 JPH0515437 B2 JP H0515437B2
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- Prior art keywords
- blood
- blood coagulation
- oxalate
- present
- serum
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
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- Life Sciences & Earth Sciences (AREA)
- Hematology (AREA)
- Engineering & Computer Science (AREA)
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- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Description
（技術分野）
本発明は血液凝固促進剤に関し、詳しくはＸ
因子活性化能を有する血液凝固促進剤に関する。
（従来技術）
近年、検査技術の目覚しい進歩と相俟つて、血
清生化学検査、血清免疫学検査、血球検査等の血
液検査が広く普及し、病気予防や早期診断に大き
く貢献するい至つている。なかでも血清検査は血
液検査の主体をなしており、この検査において必
要な血清は、通常、血液を血液検査用容器に採取
し、これを凝固させた後、遠心分離によつて比重
の異なる血餅、即ち、フイブリンと血球が混合し
たゲル様塊状物を分離させ、血清部分をピペツト
で吸い上げたり、或いはデカンテーシヨンして採
取している。
しかしながら、一般に血液は凝固するまでにか
なりの時間を要し、従来、迅速に検査を実施する
ことが困難である。最も血液凝固時間が短いとさ
れているガラス製血液検査用容器でさえ、血液を
注入した後、凝固に至るまでに40分乃至60分を必
要とし、合成樹脂製血液検査用容器に至つては、
血液凝固までに４時間以上の放置を必要とする。
このため、血液凝固第Ｘ因子活性化能を有
し、血液凝固を促進するガラス、カオリン、ベン
トナイト、シリカ、エラジン酸等が血清検査にお
ける血液凝固促進剤としてのほか、血液の凝固機
能検査の一つである活性化部分トロンボプラスチ
ン時間の測定試薬の一成分として実用に供されて
いるが、その純度や組成等によつてその活性化能
が安定しない問題がある。
更に、これらの血液凝固促進剤による場合、一
般に血液凝固後に血清から血餅を分離し、血清を
採取する際に血清の分離性がよくなく、血清に血
餅成分が混入するのを避けられない。
（発明の目的）
本発明は上記した問題を解決するためになされ
たものであつて、Ｘ因子を活性化させ、血液凝
固に要する時間を大幅に短縮させると共に、その
血液凝固の促進効果が極めて安定しており、更
に、血清の分離性にもすぐれた血液凝固促進剤を
提供することを目的とするものである。
（発明の構成）
本発明の血液凝固促進剤は、シユウ酸の金属塩
であつて、当該金属イオンが２価以上であるシユ
ウ酸塩からなる血液凝固第Ｘ因子活性化剤と、
アミノ酸配列においてArg又はLys残基と任意の
アミノ酸残基との間の結合の加水分解酵素とを含
有することを特徴とするものである。
本発明の血液凝固促進剤において、その一成分
である上記シユウ酸塩における２価以上の金属イ
オンは、アルカリ土類金属、遷移金属及び希土類
金属から選ばれる少なくとも１種であるが、好ま
しくはCu2+，Co2+，Fe2+，Ni2+，Mg2+，Sn2+及
びCe3+よりなる群から選ばれる少なくとも１種
である。
本発明は既に、分子内に相隣るカルボニル基を
有し、且つ、これらが立体的に実質的に同一の平
面上にある一群の環式有機化合物がタンパク質で
ある血液の凝固因子に対して特異的な作用をもつ
ことを明らかにしたが（特願昭58−114659号）、
シユウ酸塩を形成する金属イオンが２価以上であ
つて、シユウ酸イオンが当該金属イオンを介して
キレート様の塩構造を形成し、炭素−炭素結合が
自由回転を阻害されるとき、シユウ酸塩は分子内
に相隣るカルボニル基を有し、且つ、これらが立
体的に実質的に同一の平面上に位置するような立
体構造をとるために、作用機序は明らかではない
が、上記と同様にして血液凝固因子に対して特異
的な効果を有するとみられる。
尚、金属イオンが１価であるとき、シユウ酸塩
は炭素−炭素間結合が自由に回転することができ
ると共に、カルボニル基相互の立体的な反発や二
つの金属イオンの静電的な反発のために、相隣る
カルボニル基が立体的に実質的に同一の平面上に
ないために、血液凝固に対して特異的な作用を有
しないのであろう。因にシユウ酸の１価の金属
塩、例えば、シユウ酸カリウムやシユウ酸ナトリ
ウムは血液中において解離して、重要な血液凝固
因子であり、且つ、K+やNa+よりもイオン化傾
向の小さいCa2+と難溶性のキレート化合物を形
成するので、Ca2+を失なつた血液は凝固機構を
阻害され、従つて、上記１価金属塩は従来より血
液の抗凝固剤として広く使用されている。
次に、本発明による血液凝固促進剤の他の成分
である加水分解酵素は、アミノ酸配列において
Arg又はLys残基と任意のアミノ酸残基との間の
結合の加水分解酵素であり、このような加水分解
酵素としては、特にプロテアーゼが好ましく用い
られる。このプロテアーゼの具体例として、例え
ば、トリプシン、トロンビン、ヘビ毒トロンビン
様酵素等のセリンプロテアーゼ、カテプシンB1、
フイシン等のチオールプロテアーゼ、キニナーゼ
のような金属プロテアーゼ等を挙げることがで
きるが、セリンプロテアーゼが入手容易であるの
で、使用するのに好適である。これらプロテアー
ゼは、単独でも血液凝固を活性化し得るが、本発
明に従つて、前記したシユウ酸金属塩からなる血
液凝固第Ｘ因子の活性化剤と併用することによ
つて、血液凝固の活性化能が飛躍的に向上するの
である。
本発明の血液凝固促進剤の使用においては、例
えば、血液を血液検査用容器に採取し、これを凝
固させる際に促進剤を血液中に存在させる。この
場合、上記血液凝固促進剤は、これをそのままの
粉末状で血液中に存在させてもよいが、好ましく
は、血液凝固促進剤を適宜の溶剤に溶解若しくは
分散させて、血液中に添加する。上記血液検査用
容器は特に制限されず、従来より普通に使用され
ているガラス製又は樹脂製の容器が適宜に用いら
れる。
尚、血液が血液検査用容器内において瞬間的
に、又は部分的に高濃度のこれら血液凝固促進剤
と接触し、血液中のタンパク質成分が変質するお
それがあるときは、比表面積の大きい担体に血液
凝固促進剤を担持させ、これを血液検査用容器中
の血液に添加してもよい。上記担体としては、血
液検査に有害な影響を与えず、大きい比表面積を
有するものであれば、特に制限されることなく、
種々のものを用いることができるが、例えば、不
織布、織布、樹脂ビーズ等を好適に用いることが
できる。このような担体に血液凝固促進剤を担持
させるには、例えば、その溶液や分散液を塗布
し、又はこれに浸漬した後、乾燥して、担体に付
着させればよい。また、アラビアゴム等の適宜の
助剤と混合して水分散液とし、これを急速凍結乾
燥する等の方法により、血液凝固促進剤を担持し
た粒子状物を得ることもできる。
血液凝固促進剤の血液中における存在量は、血
液１mlについて少なくとも１×10-10ｇであり、
これよりも少ないときは、血液凝固の促進効果が
乏しい。しかし、余りに多量に存在させるとき
は、却つて血液検査に種々の支障を来すおそれが
あるので、10-1ｇ以下とするのが好ましい。
（発明の効果）
本発明の血液凝固促進剤によれば、これを血液
中に存在させるとき、Ｘ因子が迅速に活性化さ
れ、容器に血液を採取後の凝固に要する時間が著
しく短縮されると共に、血餅成分の収縮が十分に
行なわれる結果、血清と血餅との分離性にすぐ
れ、分離採取した血清に血餅成分が混入すること
がなく、更に、血清の収量も著しく増大する。従
つて、本発明の血液凝固促進剤は、臨床検査分野
において広く用いることができるほか、出血創の
止血等にも使用することができる。
以下に実施例を挙げて本発明を説明するが、本
発明はこれら実施例により何ら限定されるもので
はない。
（実施例）
実施例 １
血液凝固第Ｘ因子活性化剤であるシユウ酸塩
としてシユウ酸銅、シユウ酸マグネシウム、シユ
ウ酸コバルト、シユウ酸鉄、シユウ酸ニツケル、
シユウ酸スズ及びシユウ酸第１セリウムを、ま
た、プロテアーゼとしてトリプシン、トロンビン
及び蛇毒トロンビン様酵素をそれぞれ用いて、本
発明による血液凝固促進剤を調製した。尚、各血
液凝固促進剤における各成分の含有量は、シユウ
酸塩が0.5重量％、プロテアーゼは、トリプシン、
トロンビン及び蛇毒トロンビン様酵素がそれぞれ
について0.05重量％、500単位／ml及び0.005重量
％とした。
市販のポリエチレンプレーンスピツツを用い
て、本発明による血液凝固促進剤30μlを人新鮮血
(Technical field) The present invention relates to a blood coagulation promoter, and more specifically to X
The present invention relates to a blood coagulation promoter having factor activating ability. (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 widely used, and have greatly contributed to disease prevention and early diagnosis. . Among these, serum tests are the main body of blood tests, and the serum required for this test is usually obtained by collecting blood into a blood test container, coagulating it, and then centrifuging it to collect blood with different specific gravities. The mochi, that is, a gel-like mass containing a mixture of fibrin and blood cells, is separated, and the serum portion is collected by pipetting or decanting. 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, and synthetic resin blood test containers do not. ,
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 is in 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. . (Purpose 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 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 amino acid sequence. In the blood coagulation promoter of the present invention, the divalent or higher metal ion in the oxalate, which is one of its components, is at least one selected from alkaline earth metals, transition metals, and rare earth metals, preferably Cu 2+ , Co 2+ , Fe 2+ , Ni 2+ , Mg 2+ , Sn 2+ and Ce 3+ . The present invention has already been applied to blood coagulation factors in which proteins are a group of cyclic organic compounds having adjacent carbonyl groups in the molecule and in which these groups are sterically substantially on the same plane. It was revealed that it had a specific action (Patent Application No. 114659/1982),
When the metal ion forming oxalate is divalent or more, and the oxalate ion forms a chelate-like salt structure via the metal ion, and the free rotation of the carbon-carbon bond is inhibited, oxalate The mechanism of action is not clear because salts have adjacent carbonyl groups in the molecule and have a steric structure in which they are located on substantially the same plane, but the above-mentioned It appears to have a specific effect on blood coagulation factors in the same way. In addition, when the metal ion is monovalent, the carbon-carbon bond in oxalate can freely rotate, and the steric repulsion between carbonyl groups and the electrostatic repulsion between two metal ions can occur. Therefore, adjacent carbonyl groups are not sterically located on substantially the same plane, and therefore do not have a specific effect on blood coagulation. Incidentally, monovalent metal salts of oxalic acid, such as potassium oxalate and sodium oxalate, dissociate in the blood and form Ca, which is an important blood coagulation factor and has a smaller tendency to ionize than K + and Na + . Since it forms a poorly soluble chelate compound with Ca 2+ , the coagulation mechanism of blood depleted of Ca 2+ is inhibited, and therefore, the above 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
It is an enzyme that hydrolyzes the bond between an Arg or Lys residue and any 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, and snake venom thrombin-like enzymes, cathepsin B 1 ,
Examples include thiol proteases such as fuicin, metalloproteases such as kininase, and serine proteases are preferred because they are easily available. Although these proteases can activate blood coagulation alone, according to the present invention, they can activate blood coagulation by using them in combination with the above-mentioned blood coagulation factor X activator consisting of a metal oxalate salt. This results in a dramatic improvement in performance. 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 promoter may be present in the blood as it is in powder form, but preferably, the blood coagulation promoter is dissolved or dispersed in an appropriate solvent and then added to the blood. . 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. The carrier is not particularly limited as long as it does not have a harmful effect on blood tests and has a large specific surface area.
Although various materials can be used, for example, nonwoven fabrics, woven fabrics, resin beads, etc. 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 the blood coagulation promoter present in the blood is at least 1 x 10 -10 g per ml 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 -1 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 a result of sufficient contraction of blood clot components, the separability of serum and blood clot is excellent, the blood clot components are not mixed in the separated and collected serum, and furthermore, the yield of serum is significantly increased. Therefore, the blood coagulation promoter of the present invention can be widely used in the field of clinical testing, and can also be used for hemostasis of bleeding wounds. 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 Oxalates which are blood coagulation factor X activators include copper oxalate, magnesium oxalate, cobalt oxalate, iron oxalate, nickel oxalate,
A blood coagulation promoter according to the present invention was prepared using tin oxalate and cerous oxalate, and trypsin, thrombin, and a snake venom thrombin-like enzyme as proteases, respectively. The content of each component in each blood coagulation promoter is 0.5% by weight of oxalate, trypsin, and protease.
Thrombin and snake venom thrombin-like enzyme were 0.05% by weight, 500 units/ml and 0.005% by weight, respectively. Using a commercially available polyethylene plain spit, 30 μl of the blood coagulation promoter of the present invention was added to fresh human blood.
【表】【table】
【表】【table】
【表】【table】
【表】
３mlに加え、血液が流動性を失なうまでに要し
た時間を凝固時間として測定し、また、凝固後、
3000回転／分で５分間遠心分離して、分離状態を
観察した。
結果を第１表に示す。
比較例 １
比較のために、実施例１で用いたシユウ酸塩及
びプロテアーゼをそれぞれ単独で用いた場合の凝
固時間及び分離状態を第２表に示す。
比較例 ２
血液凝固促進剤を用いないほかは、実施例１と
同様に血液処理したときの凝固時間及び分離状態
を第３表に示す。[Table] In addition to 3 ml, the time required for blood to lose its fluidity was measured as the coagulation time, and after coagulation,
The mixture was centrifuged at 3000 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.
Claims (1)
が２価以上であるシユウ酸塩からなる血液凝固第
Ｘ因子活性化剤と、アミノ酸配列においてArg
又はLys残基と任意のアミノ酸残基との間の結合
の加水分解酵素とを含有することを特徴とする血
液凝固促進剤。 ２ 金属イオンがCu2+，Co2+，Fe2+，Ni2+，
Mg2+，Sn2+及びCe3+よりなる群から選ばれる少
なくとも１種であることを特徴とする特許請求の
範囲第１項記載の血液凝固促進剤。[Scope of Claims] 1. A blood coagulation factor
Or a blood coagulation promoter characterized by containing an enzyme that hydrolyzes the bond between a Lys residue and any amino acid residue. 2 The metal ions are Cu 2+ , Co 2+ , Fe 2+ , Ni 2+ ,
The blood coagulation promoter according to claim 1, which is at least one member selected from the group consisting of Mg 2+ , Sn 2+ and Ce 3+ .
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP59042660A JPS60186760A (en) | 1984-03-05 | 1984-03-05 | Blood coagulation accelerator |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP59042660A JPS60186760A (en) | 1984-03-05 | 1984-03-05 | Blood coagulation accelerator |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS60186760A JPS60186760A (en) | 1985-09-24 |
| JPH0515437B2 true JPH0515437B2 (en) | 1993-03-01 |
Family
ID=12642166
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP59042660A Granted JPS60186760A (en) | 1984-03-05 | 1984-03-05 | Blood coagulation accelerator |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS60186760A (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA1313997C (en) * | 1986-04-11 | 1993-03-02 | Hideo Anraku | Accelerator of the activity of hydrolase |
| JP2000187032A (en) * | 1998-12-21 | 2000-07-04 | Nagase & Co Ltd | Method and device for separating blood serum |
| FR2908892B1 (en) * | 2006-11-21 | 2009-02-20 | Hyphen Biomed Soc Par Actions | PROCESS FOR THE CHROMOGENIC ASSAY OF FACTOR VIII: C ACTIVITY AND NEED FOR CHROMOGENIC ASSAY OF FACTOR VIII: C ACTIVITY IN PLASMAS OR THERAPEUTIC FRACTIONS |
-
1984
- 1984-03-05 JP JP59042660A patent/JPS60186760A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS60186760A (en) | 1985-09-24 |
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