JPH099995A - Measurement of clot forming tendency of blood - Google Patents

Abstract

PROBLEM TO BE SOLVED: To measure a clot forming tendency(CFT) of blood useful for diagnosis, etc., of thrombosis by measuring the total fibrinolytic activities in a sample derived from the blood. SOLUTION: The activities or fibrinolytic activities of a protein participating in fibrinolysis (a tissue plasminogen activating factor, a plasminogen activating factor inhibitor 1, etc.) in a sample derived from blood (preferably the blood in a pseudocoagulated state) are measured. The fibrinolytic activities in the sample derived from the blood and the CFT are regarded as having a positive correlation and the CFT can simply be measured from the measurement of the fibrinolytic activities.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention can be applied to the diagnosis of thrombosis and the like, and the tendency of blood thrombus formation [hereinafter referred to as CFT (Clot
Forming Tendency)]].
The present invention also relates to a method for measuring the activity of a protein involved in fibrinolysis or fibrinolytic activity. In the present invention, thrombosis refers to a pathological phenomenon in which blood flow coagulates in the heart or blood vessels of a living body, and includes diseases such as myocardial infarction, angina, and cerebral thrombosis.

[0002]

As an index of fibrinolytic activity, tissue plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1), α2 in blood are used.
Plasmin inhibitor (α2PI), plasminogen, fibrinogen-fibrin degradation product (FD
P), D-dimer and other molecules involved in fibrinolysis, tPA
-PAI, PIC and their complexes. Therefore, thrombosis or DIC (Disseminated intravascular) is measured by measuring the enzyme activity of these individual molecules or measuring the concentration of each individual molecule immunologically.
coagulation: generalized intravascular coagulation) is being considered. In fact, in the group of patients with myocardial infarction, t
It has been reported that PA release decreases and PAI-1 shows a high value (Hamaten AU. Et al., N. Engl. J. Med. 31.
3,1557,1985). In addition, PAI-1 (Tomohiro Sakamoto et al., Clinical Pathology, 42,1,1994), in patients with acute myocardial infarction,
PA-PAI (Nobuhiro Ito, Equipment / Reagents, 16,1110,1993),
It has been reported that PIC (Yasuo Kama et al., Clinical Pathology, 42, 22, 1994) shows a significantly higher value than that of a healthy person. Also,
It has been reported that plasminogen has a significantly low value. However, none of these findings can be directly applied to the diagnosis of thrombosis and the like. Moreover,
The measurement of these molecules was carried out only in samples taken when the blood flow in the living body was in a normal state.

[0003]

The present invention can be applied to the diagnosis of thrombosis and the like, and the tendency of blood clot formation (CFT).
The task is to measure. Another object of the present invention is to measure the activity of a protein involved in fibrinolysis or fibrinolytic activity using a sample having a high correlation with thrombosis, and to provide data closely related to thrombosis.

[0004]

[Means for Solving the Problems] It is considered that normal blood retains its normal action because the equilibrium between coagulation activity and fibrinolytic activity is maintained. The term "coagulation activity" as used herein refers to the activity of coagulating blood at the site in order to repair the tissue when it is damaged by trauma or the like and to prevent the blood from flowing out of the blood vessel. Is. The term "fibrinolytic activity" refers to the activity of dissolving and eliminating blood coagulation products (thrombus) generated by the coagulation activity after the tissue is repaired.

[0005] If the equilibrium state between the coagulation activity and the fibrinolytic activity is broken for some reason, an abnormal state will appear. For example, if the fibrinolytic activity exceeds the coagulation activity, the blood will not coagulate easily, that is, the blood will easily bleed (bleeding tendency). Further, when the coagulation activity exceeds the fibrinolytic activity, the blood easily coagulates, that is, a thrombus is easily formed. In the present specification, the strength of the state where the thrombus is easily formed is referred to as “thrombogenic tendency (CFT)”. That is, CFT can be referred to as an index of thrombosis susceptibility, and is useful for diagnosing thrombosis, monitoring the morbidity of patients with thrombosis, determining the therapeutic effect on the prognosis of thrombosis, and preventing thrombosis.

As a result of extensive studies on an effective method for measuring CFT, the present inventors have found that the change in CFT mainly depends on the change in fibrinolytic activity. Furthermore, CFT
As a result of examining from various angles an effective method for measuring the fibrinolytic activity reflected in, when the fibrinolytic activity is measured, individual molecules or complexes involved in fibrinolysis, such as t-PA, are detected. The total fibrinolytic activity in a blood-derived sample is measured rather than measuring the activity individually and judging the fibrinolytic activity from the individual values, or integrating the individual values to judge the fibrinolytic activity. It was found that this is effective, and completed the present invention.

That is, the present invention comprises a method for measuring CFT, which comprises measuring fibrinolytic activity in a blood-derived sample.

Usually, it is considered that the fibrinolytic activity in a sample derived from blood and CFT have a positive correlation. That is, it is generally considered that the CFT is high when the fibrinolytic activity in the blood-derived sample is high and the CFT is low when the fibrinolytic activity in the blood-derived sample is low. In particular, when fibrinolytic activity in a blood-derived sample is increased with time when the same person is sampled with time, CFT is increased (that is, there is a high possibility of thrombosis). Has become). Further, when fibrinolytic activity in a blood-derived sample is decreased with time when the same person is sampled with time, CFT is decreased (that is, the possibility of thrombosis is low. Is considered to be).

The present inventors also considered that whether or not a thrombus is actually formed in a living body depends on the ease with which a thrombus is formed when the fluidity of blood is impaired. Then, the activity or fibrinolytic activity of the protein involved in fibrinolysis in a blood-derived sample in a pseudocoagulated state (state in which blood flow is stopped or flow is deteriorated by an operation such as binding the arm) is The present invention has been completed by finding that it is more closely related to the formation of thrombus in a living body than the value of a sample derived from blood in a state (a state where normal blood flow is secured).

That is, the present invention is characterized by measuring the activity of a protein involved in fibrinolysis or the activity of a protein involved in fibrinolysis in a sample derived from blood in a pseudo-coagulated state. A method for measuring fibrinolytic activity.
The proteins involved in fibrinolysis here are tPA and PA.
I-1, α2PI, plasminogen, FDP, molecules involved in fibrinolysis such as D-dimer, and tPA-PAI, P
Any compound may be used as long as it is involved in fibrinolysis, such as a complex thereof such as IC. The blood in the pseudo-coagulated state may be any blood as long as the blood flow is stopped or the flow is deteriorated. For example, blood collected by tying an arm to artificially stop or worsen blood flow or blood retained around the thrombus site is used.

Furthermore, the inventors of the present invention determine whether or not a thrombus is actually formed in a living body by determining the difference in blood state between a state in which blood fluidity is impaired and a state in which blood flow is normal. Thought to be related to. Then, the activity or the fibrinolytic activity of the protein involved in fibrinolysis in the "sample derived from blood in a pseudo-coagulated state" measured in the same individual, and, as it were, a reference value for each person, "derived from blood in a static state" The inventors have found that the difference in the activity of the proteins involved in fibrinolysis or the fibrinolytic activity in the “sample” is closely related to the formation of thrombus in the living body, and completed the present invention.

That is, the present invention relates to the activity or fibrinolytic activity of a protein involved in fibrinolysis in a sample derived from blood in a pseudo-coagulated state and to fibrinolysis in a sample derived from blood in a static state. It relates to a method for measuring the tendency of blood clot formation (CFT), which comprises comparing the activity of a protein or the fibrinolytic activity. The proteins involved in fibrinolysis here are
tPA, PAI-1, α2PI, plasminogen, F
Molecules involved in fibrinolysis such as DP and D-dimer, and tPA
-A complex such as PAI or PIC may be used as long as it is involved in fibrinolysis. The blood in the pseudo-coagulated state may be any blood as long as the blood flow is stopped or the flow is deteriorated. For example, blood collected by tying an arm to artificially stop or worsen blood flow or blood retained around the thrombus site is used.

Usually, "the activity of a protein involved in fibrinolysis or fibrinolytic activity in a sample derived from blood in a pseudocoagulant state and the activity of a protein involved in fibrinolysis in a sample derived from blood in a static state" Difference in activity or fibrinolytic activity "and CFT
Are considered to have a negative correlation with. That is, it is usually considered that the CFT is high when the difference is small and the CFT is low when the difference is large. In particular, if the difference becomes smaller over time when the same person is sampled over time, it is considered that the CFT is elevated (that is, the possibility of thrombosis is increased). Be done. In addition, if the difference increases with time when the same person is sampled over time, it is considered that the CFT is decreasing (that is, the possibility of thrombosis is low). To be

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, a blood-derived sample is any sample derived from blood such as plasma, serum, whole blood, or those obtained by subjecting them to some treatment (such as centrifugation or separation by filtration). Although anything can be used, it is particularly preferable to use plasma.

Further, in the present invention, as a method for measuring fibrinolytic activity, any known method such as the method described in JP-A-3-123498 can be used, as well as any method. As the substrate, any substrate can be used as long as it is a substrate that is decomposed by plasmin, such as fibrin clot and plasmin synthetic substrate. In addition, the substrate may be a lump, a colloid, or a fixed one. For example, a method of observing a change in substrate weight by immersing a lump substrate in a blood-derived sample for a certain period of time, or passing a blood-derived sample through a substrate housed or immobilized in a container and observing a change in substrate weight. The method etc. are used.
Alternatively, a bulk or immobilized substrate labeled with an enzyme or a radioisotope is contacted with a sample derived from blood, and the concentration of the decomposed substance of the substrate that is decomposed and flows out into the sample is determined by measuring the enzyme activity or the radiation in the sample. A method of detecting by quantity or the like is used. Further, it is also possible to bring a plasmin synthetic substrate that develops color upon decomposition into contact with a blood-derived sample and observe the absorbance. Since the fibrinolytic reaction is affected by the presence of fibrin, in the present invention, it is desirable that fibrin is present in the test solution in some form as in the case of the living body.

The following method is mentioned as an example of a suitable method for measuring fibrinolytic activity. Add the sample solution (neat solution or diluted with buffer) and standard solution (plasmin solution etc.) to the fibrin immobilization phase labeled with an enzyme such as peroxidase (immobilized fibrin on plate, beads etc.) And incubate at about 37 ° C. After a certain period of time, the reaction solution is sampled, the enzyme substrate solution is added thereto, and the mixture is further incubated. The fibrinolytic activity is measured by investigating the concentration of the reaction substance generated by changing the substrate due to the enzymatic reaction by a method such as measuring the absorbance of the solution.

[0017]

INDUSTRIAL APPLICABILITY According to the present invention, thrombus formation tendency (FC) which is useful for diagnosis of thrombosis, monitoring of a patient's body condition of thrombosis, determination of therapeutic effect on prognosis of thrombosis, prevention of thrombosis, etc.
It has become possible to easily and simply measure T). Further, the present invention has made it possible to provide data closely related to thrombosis regarding the activity of proteins involved in fibrinolysis or fibrinolytic activity.

Claims (5)

[Claims] 1. A thrombus formation tendency (CFT) of blood, characterized by measuring fibrinolytic activity in a blood-derived sample.
Measuring method. 2. The activity of a protein involved in fibrinolysis or the fibrinolytic activity in a sample derived from a blood in a pseudo-coagulated state is measured. Method. 3. The activity or fibrinolytic activity of a protein involved in fibrinolysis in a pseudocoagulated blood-derived sample and the activity or fibrinolysis of a protein involved in fibrinolysis in a static-state blood-derived sample. A method for measuring thrombus formation tendency (CFT) of blood, which comprises comparing with activity. 4. The method according to claim 1, wherein the blood-derived sample is plasma. 5. The method according to claim 1, wherein the activity of a protein involved in fibrinolysis or fibrinolytic activity is measured in the presence of fibrin in a test solution.