JP2012008044A - Measurement method of platelet activation capacity and anti-platelet drug - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a measurement method of platelet activation capacity that is the basis of platelet aggregation with excellent sensitivity to evaluate the effect of an anti-platelet drug.SOLUTION: A measurement method of platelet activation capacity and an evaluation method of an anti-platelet drug using the result of the measurement method as an index are provided. The measurement method comprises the steps of: retaining whole blood collected from a human in a non-aggregation state; contacting a platelet stimulator to the whole blood; fixing activated platelets resulting from the previous process; performing immune reaction using a fluorescent-labeled anti-CD61 antibody and a fluorescent-labeled CD62P antibody; and measuring the CD62P value using flow cytometry.

Description

  TECHNICAL FIELD The present invention relates to a method for measuring platelet activation ability and a method for evaluating antiplatelet drugs, and more specifically, a method for measuring platelet activation ability by measuring blood P-selectin (CD62P), and an antiplatelet using the measurement method. The present invention relates to a method for evaluating drugs.

  In recent years, with lifestyles becoming westernized, vascular disorder diseases such as ischemic heart disease and cerebrovascular disorder have become the leading cause of death in Japan. Increasing the incidence of these vascular disorder diseases is deeply related to an increase in lifestyle-related diseases such as diabetes, hyperlipidemia, hypertension and gout, which has become a social problem. In diabetes and hyperlipidemia, vascular endothelial cells are injured under high LDL cholesterol and hyperglycemic conditions, and activate circulating platelets to cause aggregation and promote thrombus formation. As described above, the thrombus includes a platelet thrombus formed by activating platelets and a fibrin thrombus formed by stimulating a coagulation factor. Anti-platelet therapy with aspirin is effective for the former, and anticoagulant therapy with warfarin or heparin is used for the latter. When treating thrombosis such as antiplatelet therapy and anticoagulant therapy, monitoring the drug effect is indispensable to increase the therapeutic effect, but antiplatelet therapy is performed without special monitoring. This is the current situation. Although Sato et al. Have a high necessity for monitoring the antiplatelet drug therapeutic effect by the platelet aggregation test, the standardization of the platelet aggregation test has not been performed, and the importance of the realization was reported (Non-patent Document 1). ).

  The platelet agglutination ability test includes a conventionally used platelet aggregation test (Born method) and a recently developed laser light scattering method. The principle of these measurement methods is to detect platelet aggregates generated by activating platelets with stimulating substances. In this method, since the activation state of platelets is determined by the aggregation state of platelets, a dedicated instrument and a large amount of sample are required for measurement, and there are many problems in operational complexity, reproducibility, and sensitivity. The current situation is that it is not performed at the hospital.

  In recent years, a measurement method at a molecular level has been proposed as a method for measuring platelet aggregation. Patent Document 1 clarifies that a substance that appears specifically on the platelet surface by activation is measured by an immunological technique. Here, a method of detecting activated platelets by measuring PAC-1, which is an antibody that binds to activated glycoprotein IIb / IIIa, is shown. Patent Document 2 discloses a method for determining platelet aggregation by measuring CD40L appearing on the platelet surface by immunoblotting and measuring by Western blotting. Patent Document 3 discloses a method for evaluating an antiplatelet drug by monitoring membrane-bound and / or soluble P-selectin. However, high platelet content plasma (PRP) or low platelet content plasma (PPP). Therefore, there is a problem that a large amount of blood for testing is required. Patent Document 4 discloses a method for measuring the degree of platelet activation by measuring the expression level of P-selectin using a whole blood sample. In addition to whole blood to which acid Na is added, a conventional activation method for measuring the degree of activation of platelets is used, keeping the collected whole blood in a non-activated state, and how much it is activated with an activation stimulating substance It does not clarify how to measure.

  Flow cytometry has been used as an instrument for measuring platelet aggregates in a state close to a living body by capturing and discriminating a weak platelet aggregation particle image. Introducing into flow cytometry and specifying based on forward scattered light information and fluorescence information, platelet aggregation and measurement method (Patent Document 5) that can accurately classify and count myeloblasts, using flow cytometry A method and apparatus for staining and analyzing CD4 and CD8 of lymphocyte surface antigens of human peripheral blood (Patent Document 6) has been disclosed, but activation of platelets by detecting CD62P on the activated platelet surface is disclosed. It is not a measure of performance.

JP 2004-117152 A (Table 1) JP-T-2009-516201 (Example 5, FIG. 2) JP-T-2001-526774 (Table 4) JP 10-512951 A (Example 7, Example 8) JP 2007-263894 A (Example 1, FIGS. 13 and 14) JP 2002-310886 A (FIGS. 5 and 6)

Japanese Society for Laboratory Hematology 9: 167-177,2008

  The main object of the present invention is to provide a method for measuring the platelet activation ability, which is the basis of platelet aggregation, with high sensitivity in order to evaluate the effects of antiplatelet drugs.

  The present inventors have developed a method for detecting the CD62P value expressed on the activated platelet surface and measuring the platelet activation ability with high sensitivity. Furthermore, an evaluation method for antiplatelet drugs using this measurement method was found, and the present invention was completed.

  That is, the present invention provides the following (1) to (6).

(1) A method for measuring platelet activation ability comprising the following steps (a) to (e):
(A) a step of keeping whole blood collected from a human in a state where it is not aggregated;
(B) contacting the whole blood with a platelet stimulating substance;
(C) fixing activated platelets produced by the above step;
(D) performing an immune reaction using a fluorescently labeled anti-CD61 antibody and a fluorescently labeled CD62P antibody; and (e) measuring a CD62P value using flow cytometry.

  (2) The measuring method according to the above (1), wherein the step of keeping the agglomerated state is a step of adding an EDTA salt.

  (3) The platelet stimulating substance is ADP, collagen, epinephrine, ristocetin, convulxin, serotonin, vasopressin, carbazochrome, blood coagulation factor (FVIII, FIX), thrombin, epsilon-aminocaproic acid, tranexamic acid, protamine sulfate, ethanesylate, phytonadione The measuring method in any one of said (1) or (2) which is a stimulating substance chosen from sodium estrone sulfate and sodium equilin sulfate.

  (4) A method for evaluating an antiplatelet drug, using the result of the measurement method according to any one of (1) to (3) as an index.

  (5) The antiplatelet drug is aspirin, cilostazol, dipyridamole, ticlopidine hydrochloride, clofibrate, ethyl icosapentate, sulfinpyrazone, clopidogrel, beraprost, thrombomodulin, prostaglandin E, antiserotonin drug, coumarin anticoagulant The method according to (4) above, which is a drug selected from drugs and active protein C.

  (6) A kit comprising a reagent and a container for use in the measurement method according to any one of (1) to (3) above.

  The method for measuring the ability to activate platelet drugs of the present invention is a highly specific and highly sensitive method, and therefore, the measurement is performed with a very small amount of blood. .

It is the figure which showed the positive rate of CD62P when the stimulating substance of each density | concentration was added.

  In contrast to the conventional method, the present invention takes advantage of the fact that platelets must be activated before aggregation, which is a characteristic of platelets. Measure how much it has the ability to be activated by stimulating substances. Under these measurement conditions, the antiplatelet drug is evaluated by measuring how much the drug called antiplatelet drug can suppress the activation of platelets. Furthermore, the route of platelet activation is predicted based on the type of stimulating substance, and selection of antiplatelet drugs is proposed.

  The mechanism of platelet activation that is the basis of platelet aggregation can be explained mainly by two routes. In the first route, cyclooxygenase acts on arachidonic acid released from phospholipid by the action of phospholipase by stimulation of platelets by peptide chains such as collagen, and prostaglandin G2 and thromboxane A2 are biosynthesized. Thromboxane A2 increases platelet Ca concentration and activates platelets. In the second route, stimulation of platelets by ADP suppresses adenylate cyclase and decreases the conversion of adenosine triphosphate (ATP) to 3 ', 5' cyclic AMP (c-AMP). Furthermore, c-AMP is hydrolyzed and decreased by phosphodiesterase (PDE), thereby suppressing the passage of Ca ions from the cell membrane, increasing the Ca concentration in platelets, and activating platelets.

  When activated, platelets release various substances from alpha granules and darkly stained granules in the platelets. CD62P is one of them. CD62P was discovered by Johnston GI et al. In 1984 as a marker of platelet activity (Johnston GI, et al, J Biol. Chem. 264: 1816-1823, 1989). CD62P is a type 1 glycoprotein with a molecular weight of 140 kDa, which is present in the α-granule of platelets and the granule membrane of Weibel-Plade body of vascular endothelial cells. Activation of platelets reveals the surface of the platelet membrane, promotes adhesion of platelets, and plays an important role in platelet aggregation. In the present invention, the platelet activation ability is measured from the CD62P value expressed by activation. Specifically, it can be expressed as an increase ratio of how much the CD62P value increases depending on the presence or absence of a stimulating substance.

  In order to measure the CD62P value in the activated state, the present invention first maintains the “non-aggregated state” of the platelets as the first step. The “non-aggregated state” of the present invention represents a state immediately after blood collection, that is, a state in which the blood does not proceed to coagulation. It is to perform a treatment that does not increase the Ca concentration in platelets, or to decalcify. Examples of the chelating agent include citrates such as sodium citrate, ethylenediaminetetraacetate (EDTA) such as ethylenediaminetetraacetic acid dipotassium (EDTA-2K), ethylenediaminetetraacetic acid disodium (EDTA-2Na), and diaminopropanol 4 Examples thereof include acetate and ethylenediamine dipropionate. The chelating agent used for the evaluation of platelet drugs is based on the condition that the scattered light is stable and that it is difficult to produce platelet waste, but it is preferable to use an EDTA salt rather than Na citrate or heparin. . When using an effective chelating agent for inhibiting platelet aggregation, in the case of EDTA-2K, about 0.05 to 1% of the blood sample, and in the case of EDTA-2Na, about 0.1 to 2% Although it is used, it is most desirable to use EDTA-2K in an amount of 0.1 to 0.5% as a means for maintaining a state in which it does not aggregate.

  The step of bringing the platelet stimulating substance into contact with the whole blood in the “non-aggregated state” of the present invention is a step for producing activated platelets in an unaggregated state. In the present invention, activated platelets refer to “platelets in which CD62P that promotes platelet adhesion appears on the platelet membrane surface from the α granules in the platelets”. By measuring CD62P expressed on the platelet membrane surface, it is possible to measure to what extent platelets have the ability to be activated by platelet stimulating substances. In this case, blood that is not brought into contact with the stimulating substance is also handled as a comparison target. Platelet stimulants include ADP, collagen, epinephrine, ristocetin, convulxin, serotonin, vasopressin, carbazochrome, blood coagulation factors (FVIII, FIX), thrombin, epsilon-aminocaproic acid, tranexamic acid, protamine sulfate, ethanesylate, phytonadione, estrone sulfate Examples thereof include sodium and sodium equilin sulfate. In the process, one kind is selected from the above stimulating substances. In using a platelet stimulating substance effective for stimulating platelets, when adding ADP, about 0.01 to 10 μM / L with respect to the test solution, and when adding collagen, 0.01% with respect to the test solution. It is desirable to add about 10 μg / ml. Moreover, although reaction with these platelet stimulating substances can be performed for 5 to 30 minutes, a more effective reaction time is 10 minutes.

  The step of fixing activated platelets of the present invention is a step performed for measuring CD62P generated in the previous step in a stable state. As the fixing solution, formaldehyde, paraformaldehyde, glyoxal, and the like can be used alone or in combination of two or more. However, it is preferable to use a mixture of formaldehyde and glyoxal. These fixatives can be used, for example, by dissolving them in a buffer solution such as PBS at a concentration of about 100 mM to 2000 mM / L. However, the CD62P fixative is preferably a mixture of 666 mM / L formaldehyde and 167 mM / L glyoxal. The time is 1 to 10 minutes, preferably 1 to 5 minutes.

  The present invention includes a step of performing an immune reaction using a fluorescently labeled anti-CD61 antibody and a fluorescently labeled CD62P antibody. In this step, the CD62P value expressed by platelet activation and the CD61 (platelet glycoprotein GPIIIa) value present in all platelets are measured by a fluorescent immunoassay. The CD62P value is preferably measured by PE (Physotritrin). A CD62P antibody labeled with fluorescence is used, and the CD61 value is preferably measured using a CD61 antibody fluorescently labeled with FITC (fluorescent isothiocyanate). The combination of FITC and PE double labels that simultaneously carry out a fluorescence reaction is excited at 488 nm, FITC emits green fluorescence around 530 nm, and PE emits red fluorescence around 570 nm, so be clearly distinguished Can do. As a negative control for CD62P value, mouse-immunoglobulin G (IgG) bound to FITC and PE, respectively, is used.

  The present invention uses flow cytometry as a means of measuring CD62P values. That is, the final step of the platelet activation ability measurement of the present invention is performed using computer analysis of the measured values of CD62P positive rate, CD61 positive rate and IgG positive rate in which the above immune reaction was performed using flow cytometry. It is a process of measuring a value.

  What is flow cytometry? Scattered light (forward scattered light: FSC, side scattered light: SSC) and fluorescence generated by passing cells and other biological particles through a narrow flow and irradiating with laser light Is measured as a voltage pulse to measure the physical and chemical properties of the particles. In 1947, the principle was devised by the Wallace Coulter. In 1953, a fluid cell number measuring instrument was put into practical use. In 1969, a flow cytometer equipped with an argon laser was developed.

  In the measurement of activated platelets, it is desirable to subject 10000 to 90000 platelets to flow cytometry, and the obtained data is analyzed by suitable computer software. For example, the measurement parameters of all platelets are SSC that reflects the internal structure and FSC that reflects the size of the sample on two axes, and the created dot plot is gated (limitation and setting of enclosure), Activated platelets and aggregates and cell debris can be separated and analyzed. Gating is performed from the negative control data, and then a histogram is generated. The CD62P value can be determined from the percentage of platelets in which both CD61 and CD62P are positive by measuring the CD62P positive ratio from the CD61 positive ratio. That is, the CD62P positive rate using flow cytometry reflects the CD62P value expressed on the platelet surface.

  Furthermore, the present invention provides a method for evaluating the antiplatelet drug using the result of the CD62P value as an index. Antiplatelet drugs can be evaluated as follows. That is, blood is collected before and after taking antiplatelet drugs and the following test is performed. The CD62P positive rate (A) is measured by using no stimulating substance added as a control. At the same time, a stimulating substance such as ADP or collagen is added to the blood, and the CD62P positive rate (B) is measured. Next, the platelet activation ability was calculated from the increase ratio of CD62P positive rate when stimulating substances such as ADP or collagen were added from B ÷ A, and the increase ratio before and after taking was compared, showing a significantly low value Find out if it is. If the value is significantly low, it is evaluated that the antiplatelet drug was effective. In addition, the method for evaluating an antiplatelet drug includes predicting the route of platelet activation from the platelet activation ability of a specific platelet stimulating substance and selecting an antiplatelet drug.

  Examples of the antiplatelet drug of the present invention include aspirin, cilostazol, dipyridamole, ticlopidine hydrochloride, clofibrate, ethyl icosapentate, sulfinpyrazone, clopidogrel, beraprost, thrombomodulin, prostaglandin E, antiserotonin drug, coumarin anticancer drug Coagulants and active protein C are included.

  The blood used as a sample for evaluating the antiplatelet drug of the present invention is blood collected from a human at a time when the effect is recognized after taking the antiplatelet drug of interest, and the effectiveness of the drug after taking the drug. Blood collected during the period is determined.

  In addition, the method for evaluating an antiplatelet drug of the present invention includes selection of an antiplatelet drug suitable for the human who provided the sample.

  The present invention also provides a kit in which reagents for measuring platelet activation ability are combined in advance. In addition to the antibody, the kit may contain a container in addition to an immobilized carrier, a labeling substance, and a reagent such as a substrate compound used for detection of the label.

  Hereinafter, examples will be given to describe the present invention in more detail, but the present invention is not limited thereto.

[Blood sample]
Blood samples were obtained from 30 healthy adults (ages 21-23, 23 women, 7 men). In addition, blood samples were obtained from 7 healthy adults (aged 21-23 years, 4 women, 3 men) who took aspirin (manufactured by Bayer) at a daily dose of 100 mg for 3 days. None of the participants in the study had a history of any other medications. All study participants were given informed consent and the study was approved by the Yamaguchi University School of Medicine Ethics Committee. In accordance with international guidelines regarding collection, including hemostasis, blood was collected in 2 tubes of 2 ml each via veins. The blood in the first tube was discarded due to contamination of tissues and the like, and the second was used as test blood.

[Measurement of CD62P value]
Platelet stimulating substance ADP 0.1-100 μM / L or collagen was added to 45 μl of peripheral blood whole blood containing 1.5 mg / ml EDTA-2K obtained in Example 1 or 0.1 mol / L Na citrate. Add 5 μl each of 0.1-100 μg / ml solution, react for 10 minutes with ADP 0.01-10 μM / L or collagen 0.01-10 μg / ml final concentration, fixative solution [in PBS, 666 mM (2%) formaldehyde, 167 mM (1%) glyoxal]] 5 μl is mixed and fixed for 5 minutes, then washed with 4 ml of phosphate buffer (PBS), and centrifuged at 3000 rpm for 5 minutes. The supernatant PBS is removed by aspiration so as to leave 100 μl of the supernatant PBS, and the fixed platelets are suspended in 100 μl of the supernatant PBS and divided into 50 μl for measuring the CD62P value and 50 μl for the negative control. For measuring the CD62P value, 5 μl of fluorescent isothiocyanate (FITC) labeled anti-CD61 antibody (Caltag Laboratories) and 20 μl of CD62P-conjugated phycoerythrin (PE) (EBioscience Laboratories) were added and allowed to react for 20 minutes. For negative control, 50 μl of platelet solution was reacted with mouse-immunoglobulin G-binding FITC and PE. The reaction was stopped by adding 300 μl of 333 mM (1%) formaldehyde in PBS, 10000 platelets were measured by flow cytometry (Cytomics FC500, manufactured by Bechman Coulter), and parameters were measured using visual statistical software (Stat Flex Ver. 5.). The CD62P value was determined by computer analysis using 0 (manufactured by Artec).

  Fig. 1 shows stimulation with 3 concentrations of ADP or collagen using whole blood supplemented with Na citrate, which is generally used as an anticoagulant for peripheral blood, and EDTA-2K of the present invention. The CD62P positive rate reflecting the CD62P value of each sample was shown. Since the addition of each stimulating substance formed platelet aggregates and platelet waste, Na citrate blood addition significantly decreased the CD62P positive rate compared to EDTA-2K blood addition that did not cause these changes.

[Evaluation of antiplatelet drugs]
Blood samples were collected from 7 healthy adults 10 days before aspirin, 1 day before and 1 day after stopping aspirin for 3 days (1 day after stopping) and 10 days after stopping, and each was immediately subjected to the test. Blood was collected in an EDTA-2K blood collection tube, an immune reaction was performed in the same manner as in Example 2, and the CD62P positive rate was determined by flow cytometry.

  The results are shown in Table 1. Aspirin was taken for 3 days, and one day after stopping the administration, the CD62P positive rate reflecting the CD62P value decreased in all blood. However, ADP stimulation showed the same increase rate of CD62P positive rate as before taking aspirin, whereas collagen stimulated blood had a lower increase rate of CD62P positive rate than before taking, and antiplatelet drugs It became clear that platelet activation by (aspirin) was suppressed. Furthermore, the increase rate of CD62P positive rate before taking and the CD62P positive rate in collagen stimulation returned to the original on the 10th day after stopping taking, which is considered that the effect of taking aspirin disappeared. From this, it was confirmed that the effect of the antiplatelet drug aspirin, which is effective for platelet aggregation by collagen, can be proved by the measurement method of the present invention. In addition, when taking antiplatelet drugs such as ticlopidine, the rate of increase in the CD62P positive rate in ADP stimulation is predicted to be low. In such healthy adults, the positive rate of each CD62P and the rate of increase in the CD62P positive rate do not change 10 days before and 1 day before taking aspirin, so CD62P in the blood of the patient is targeted for the positive rate of healthy adults. By measuring the positive rate and the rate of increase in CD62P positive rate in various stimulants, it was estimated that the thrombus formation (enhancement / suppression) state of the patient can also be estimated.

  The present invention is a method for measuring platelet activation ability by measuring the CD62P value in whole blood, and since it has excellent sensitivity and reproducibility with a small amount of sample, it can be mounted on an automatic blood cell analyzer. Is the law. For this reason, there is a possibility that it may become an effective method as a clinical test method, such as monitoring pharmacokinetics in patients with antiplatelet drugs and evaluating the effectiveness of antiplatelet drugs.

Claims (6)

A method for measuring platelet activation ability comprising the following steps (a) to (e):
(A) a step of keeping whole blood collected from a human in a state where it is not aggregated;
(B) contacting the whole blood with a platelet stimulating substance;
(C) fixing activated platelets produced by the above step;
(D) a step of performing an immune reaction using a fluorescently labeled anti-CD61 antibody and a fluorescently labeled CD62P antibody; and (e) a step of measuring a CD62P value using flow cytometry. The measuring method according to claim 1, wherein the step of keeping the state of not aggregating is a step of adding an EDTA salt. The platelet stimulating substance is ADP, collagen, epinephrine, ristocetin, convulxin, serotonin, vasopressin, carbazochrome, blood coagulation factor (FVIII, FIX), thrombin, epsilon-aminocaproic acid, tranexamic acid, protamine sulfate, ethanesylate, phytonadione, estrone sulfate The measuring method according to claim 1, wherein the measuring substance is a stimulating substance selected from sodium and sodium equilin sulfate. The evaluation method of an antiplatelet drug which uses as a parameter | index the result by the measuring method of any one of Claims 1-3. The antiplatelet drug is aspirin, cilostazol, dipyridamole, ticlopidine hydrochloride, clofibrate, ethyl icosapentate, sulfinpyrazone, clopidogrel, beraprost, thrombomodulin, prostaglandin E, antiserotonin drug, coumarin anticoagulant or activity The method according to claim 4, which is a drug selected from protein C. A kit comprising a reagent and a container for use in the measurement method according to any one of claims 1 to 3.

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