JP4369143B2 - Detection method of von Willebrand factor cleaving enzyme function - Google Patents

Description

【００３８】
血小板中のＡＤＡＭＴＳ１３ｍＲＮＡの絶対量を計算するために、本発明者等は既知濃度のプラスミドｃＤＮＡサンプルの分析に基づく検量線（ｌｉｎｅａｒｉｔｙ ｓｔｕｄｙ）を用いた（図２）。この検量線により、１ｎｇ全ＲＮＡ中にＡＤＡＭＴＳ１３ ｍＲＮＡが５．５±２．５×１０^−８ｎｇ存在していることを示した。
サンプル１：６．９±２．１、
サンプル２：９．１±０．２、
サンプル３：５．２±１．０、
サンプル４：３．５±０．３、
サンプル５：６．０±０．９、
サンプル６：２．０±０．４。
これらの値は、３回の独立した測定の平均と標準偏差である。
【００３９】
【発明の効果】
本発明は、血小板中でのＡＤＡＭＴＳ１３ｍＲＮＡの発現を初めて見出した。さらに、ＡＤＡＭＴＳ１３ｍＲＮＡ発現の定量評価をするための有用な方法としてＲｅａｌ−ｔｉｍｅＰＣＲを確立した。本発明の結果は、血小板がＡＤＡＭＴＳ１３ｍＲＮＡを明らかに恒常的に持っていることを明確にし、量比が変化すれば何等かの血栓性疾患が疑える。また、間接的にはＡＤＡＭＴＳ１３蛋白質が血小板に存在していることを示した。このことに基づき、血小板中のｖＷＦ−ＣＰ活性を測定することの生理学的意義を見出すことの基盤を確立した。
【図面の簡単な説明】
【図１】蛍光を用いたＲｅａｌ−ｔｉｍｅＰＣＲを用いた血小板中のＡＤＡＭＴＳ１３ｍＲＮＡ発現の定量。
【図２】ＡＤＡＭＴＳ１３ｃＤＮＡ（×１０^−８ｎｇ／３００ｎｇＲＮＡ）を含むプラスミドの検量線を基にした絶対量。
【配列表】

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for detecting von Willebrand factor cleaving enzyme function. In particular, mRNA of von Willebrand factor cleaving enzyme (hereinafter sometimes abbreviated as vWF-CP) is characterized by using blood as a specimen, specifically platelet fraction obtained from blood, more specifically platelet. This is a method for detecting a vWF-CP function as a marker, and relates to a diagnostic method and an inspection means using this method.
[0002]
[Prior art]
Von Willebrand factor (hereinafter sometimes abbreviated as vWF) is a protein produced by vascular endothelial cells and megakaryocytes and present not only in blood but also in the matrix under endothelial cells. It plays an important role in hemostasis as an adhesion factor that intervenes and binds between vascular endothelial tissues. In addition, vWF forms a complex with blood coagulation factor VIII and functions as a carrier protein.
[0003]
Between 15% and 25% of vWF in the circulatory bloodstream is stored in platelet alpha granules. Stimulation of endothelial cells or platelets by thrombin or ADP releases highly polymerized vWF (hereinafter sometimes abbreviated as UL-vWF) into the blood. High polymerization vWF exhibits higher activity than low polymerization vWF, which is smaller in size. This indicates that low polymerized vWF has a weak ability to induce platelet aggregation, whereas highly polymerized vWF has a strong ability to induce platelet aggregation. That is, when the proportion of low polymerized vWF in the blood is high (relative to the highly polymerized vWF), damage to the microvessel causes a weak platelet aggregation action and tends to prolong bleeding time. On the contrary, in a state where the ratio of highly polymerized vWF in the blood is high, a thrombus is generated in the microvessels throughout the body, resulting in various diseases accompanied by symptoms of thrombosis.
[0004]
The ratio of the polymerized size of vWF in the blood depends on the function and / or expression level of von Willebrand factor cleaving enzyme (vWF-CP). The cleaving enzyme cleaves a peptide bond between tyrosine 842 and methionine 843 residues of vWF, thereby degrading the polymer into a size range of 500 to 20000 kDa (Non-patent Documents 1 and 2).
[0005]
As a disease caused by quantitative and qualitative abnormality of vWF, von Willebrand disease can be mentioned. Clinical findings of von Willebrand disease include prolonged bleeding time, decreased factor VIII activity, decreased platelet adhesion, and the like. There are two types of von Willebrand disease: type 1 which is a quantitative abnormality of vWF and type 2 with qualitative abnormality. In type 1, blood vWF is quantitatively reduced, but the structure of the polymer is normal, and the blood of type 1 patients is characterized by a lack of high vWF polymer.
[0006]
Recent studies have reported that the proportion of highly polymerized vWF is high in the blood of patients with thrombotic thrombocytopenic purpura (hereinafter TTP). This is a state in which highly polymerized vWF has increased due to a decrease in vWF-cleaving enzyme activity, and forms platelet aggregation in the circulating blood stream and subsequent microthrombus formation. A predisposition to a high proportion of highly polymerized vWF is due to loss of vWF-CP activity caused by acquired autoantibodies to vWF-CP (Non-Patent Documents 3 and 4) or in patients with an innate form of TTP. This is due to an innate defect of vWF-CP (Non-patent Document 5). TTP is a disease characterized by five signs of thrombocytopenia, microangiopathic hemolytic anemia, psychiatric symptoms, renal dysfunction, and fever.
[0007]
In the study of von Willebrand factor cleaving enzyme, the partial purification of vWF-CP and the properties of the protein were specified (Non-patent Documents 1 and 2), and human vWF-CP was immediately purified. This allowed the determination of the N-terminal amino acid sequence and the determination of the cDNA sequence encoding vWF-CP. vWF-CP has been classified into the metalloprotease ADAMTS family and was named ADAMTS13. The reason for the nomenclature is that it is disintegrin-like with a thrombospondin type 1 (TSP1) motif and is composed of a metalloprotease domain (Non-patent Documents 6 and 7).
[0008]
Zheng et al., Levy et al., And Soeima et al. Reported the mRNA expression distribution of ADAMTS13 using Northern blot analysis (Non-Patent Documents 6, 8, and 9). The 4.7 kb ADAMTS13 transcript was specifically detected in the liver. In addition, a transcript close to 2.3 kb was slightly detected in the placenta (Non-patent Document 8). These data suggest that vWF-CP in the blood may be derived from the liver. The partial cDNA of ADAMTS13 is isolated from the brain and prostate. Strong RT-PCR signals in the ovary and random expression in other organs may be the basis for other potential functions in ADAMTS13 (Non-Patent Document 8). Weak mRNA expression of ADAMTS13 was demonstrated in kidney, pancreas, spleen, thymus, prostate, testis, small intestine, and peripheral blood leukocytes. The level of ADAMTS13 mRNA expression in the human organ panel by semi-quantitative PCR analysis has been reported. Expression in bone marrow is seen at a low level, but expression in peripheral blood leukocytes was denied (Non-patent Document 10). None of the reports are known for expression in platelets. ADAMTS13 degrades vWF but is not known for other substrates. The physiological relationship of ADAMTS13 expressed in the above-mentioned organs is not yet clear.
[0009]
The documents cited here are listed below.
[Prior literature]
[Non-Patent Document 1]
Blood. 87: 4223-4234, 1996
[Non-Patent Document 2]
Blood. 87: 4235-4244, 1996
[Non-Patent Document 3]
N Engl J Med. 339: 1585-1594, 1998
[Non-Patent Document 4]
N Engl J Med. 339: 1578-1584, 1998
[Non-Patent Document 5]
Blood. 89: 3097-3103, 1997
[Non-Patent Document 6]
J Biol Chem. 276: 41059-41063,2001
[Non-Patent Document 7]
Blood. 98: 1662-1666, 2001
[Non-Patent Document 8]
Nature. 413: 488-494, 2001
[Non-patent document 9]
J Biochem (Tokyo). 130: 475-480, 2001
[Non-Patent Document 10]
Blood. 100: 3626-3632, 2002
[Non-Patent Document 11]
Br. J. et al. Haematol. 102: 829-840, 1998
[0010]
[Problems of the Invention]
The present inventors examined the relationship between the expression of vWF-CP (ADAMTS13) and the tissue as described above, and made it a task to find a more reliable physiological function and significance of vWF-CP (ADAMTS13). In addition, a detection method capable of measuring the function and expression level of vWF-CP in blood was studied, and an object was to establish a diagnosis method and a diagnostic means for diseases predisposed to the function and expression level of vWF-CP. .
[0011]
[Means for solving problems]
The inventors focused on the presence and function of vWF-CP in platelets from the viewpoint of platelet thrombus formation. As a result, the presence of mRNA encoding vWF-CP in platelets in blood was confirmed and the present invention was completed.
[0012]
That is, this invention consists of the following.
1. A method for detecting vWF-CP function using von Willebrand factor cleaving enzyme (hereinafter referred to as vWF-CP) mRNA as a marker, characterized by using blood as a specimen.
2. A method for detecting a vWF-CP function using vWF-CP mRNA as a marker, wherein a platelet fraction obtained from peripheral blood is used as a specimen.
3. 1. The method according to 1 above, wherein the mRNA of vWF-CP in platelets is a marker.
4). 4. The method according to any one of 1 to 3 above by a PCR method.
5. The following diagnostic method using the method of said 1-4.
1) Diagnosis of easy thrombosis.
2) Predictive diagnosis of predisposition to thrombosis.
3) Predictive diagnosis of predisposition to thrombotic microangiopathy.
4) Genetic abnormality diagnosis.
6). A means for examining whether the mRNA transcript of vWF-CP is produced as a functional protein, characterized by using mRNA of vWF-CP in platelets.
[0013]
BEST MODE FOR CARRYING OUT THE INVENTION
A feature of the present invention is that blood is used as a specimen for measurement of vWF-CP, and in particular, mRNA of vWF-CP in platelets is used as a marker. Examples of a method for obtaining a platelet fraction and platelets from blood include a centrifugal separation method, a gel filtration method, a method using a density difference, a filter method, and the like. In addition, platelet rich plasma prepared from blood can be used as a specimen. The fraction of platelets in the blood is fractionated by centrifugation, the platelet pellet is taken and subjected to the test. From platelet pellets, platelet RNA fractionation is described in H.C. Anbo et al. [Br. J. et al. Haematol. 102 (1998), 829-840]. The sorted RNA is then amplified by reverse transcription PCR. It is sufficient that the amplification method is performed according to a commercially available reagent kit. Primers for reverse transcription are shown in Sequence Listings 1 and 2. This primer corresponds to exons 1 to 7 of vWF-CP and is used for amplification of cDNA. These amplified cDNAs are further amplified by PCR using commercially available reagents. The nucleotides shown in Sequence Table 3 were used for the forward primer and Sequence Table 4 was used for the reverse primer. In the present invention, good results were obtained by amplification by Real-time PCR. As a marker primer, a fluorescent oligonucleotide (sequence table 5) in which a reporter dye and a quencher dye were bound was used.
[0014]
In the present invention, primers corresponding to exons 1 to 7 of vWF-CP were used for RT-PCR, and the base sequence of the amplified DNA fragment was determined. In addition, fluorescent oligonucleotides located in exons 15-16 were used in Real-time PCR. These results proved that there was no possibility of contamination of the genomic DNA in the test sample and that ADAMTS13 mRNA at least consistent with exon 15-16 was expressed in platelets.
[0015]
Real-time PCR is a method for monitoring and analyzing in real time the process of generating the amount of amplified product by PCR using an apparatus in which a thermal cycler and a spectrofluorometer are integrated. In the present invention, PCR amplification was performed using a fluorescent oligonucleotide having a reporter dye (FAM) 5 ′ upstream of a specific probe and a quencher dye (TAMRA) 3 ′ downstream. In addition, if a fluorescent substance such as TET having a wavelength different from that of FAM is used as a probe for detecting mutant vWF-CP mRNA, it can be applied to detection of point mutations in genes, and gene abnormality diagnosis of vWF-CP mRNA can be performed. .
[0016]
By the means as described above, the present invention has found for the first time the expression of ADAMTS13 mRNA in blood, particularly in platelets. Furthermore, Real-time PCR was established as a useful method for quantitative evaluation of ADAMTS13 mRNA expression. The data of the present invention showed that ADAMTS13 mRNA is expressed in platelets in six healthy subjects with little constitutive difference.
[0017]
The results of the present invention clarified that platelets retain ADAMTS13 mRNA, and indirectly indicated that ADAMTS13 protein is present in platelets. Since vWF-CP in platelets is considered to be a regulator for determining the polymer structure of vWF, it is easy to measure the vWF-CP activity in platelets, particularly the amount of mRNA of vWF-CP. I think it will be sex diagnosis.
[0018]
Platelet thrombus formation in local tissues is due to the nature of platelet-derived vWF, and the establishment of a method for evaluating the activity of vWF-CP in platelets of the present invention, in particular the amount of mRNA of vWF-CP of the present invention. Measuring is useful for predicting the predisposition to thrombosis. Furthermore, a predictive diagnosis of a disease predisposition to thrombotic microangiopathy can be performed. Examples of the predisposition here include vWF-CP activity abnormality, vascular endothelial cell disorder, and blood coagulation abnormality.
[0019]
In the diagnostic method and test means according to the present invention, the amount of vWF-CP mRNA in the blood can be quantitatively measured, so that the amount of vWF-CP mRNA in the blood of the patient and the amount of vWF-CP mRNA in the blood of the healthy sample are determined. By comparison, it becomes possible to diagnose and test the amount of vWF polymer in the blood. Therefore, as described above, diagnosis of thrombosis easily due to quantitative abnormality of vWF, prediction diagnosis of thrombosis disease predisposition, and prediction diagnosis of thrombosis microvascular disorder disease predisposition have become possible.
[0020]
When platelet-derived vWF-CP is incompletely expressed, there is a possibility of promoting thrombus formation in the local tissue by competitive inhibition against vWF-CP of blood which is normally expressed. Incompletely expressed vWF-CP derived from platelets may be used as a diagnostic target marker in diseases. Incompletely expressed vWF-CP can be determined by, for example, expressing a protein from mRNA or cDNA encoding platelet-derived vWF-CP using a known gene recombination technique and determining its function. Moreover, gene abnormality can be confirmed by the function. As one of the means, it is a simple method to test in combination with wheat germ cell-free protein synthesis means such as Endo. Of course, it is useful to express the protein in a system such as Escherichia coli, yeast, animal cells, and insect cells to determine its function.
[0021]
Further investigation is meaningful to determine whether full-length ADAMTS13 mRNA is expressed in platelets. In addition, morphological studies on the localization of ADAMTS13 in platelet structures provide clues for obtaining detailed knowledge of ADAMTS13 in platelets. The present invention has also been found to be a good tool for examining whether the transcript is produced as a functional protein in platelets.
[0022]
The presence of vWF-CP autoantibodies that bind to platelets of TTP patients underlies the possibility of autoimmune thrombocytopenia that complicates the etiology of TTP patients. The presence of the antibody is an important focus in therapy. Anti-vWF-CP antibodies that bind to platelets may be an important tool in the diagnosis of TTP.
[0023]
According to the present invention, the fact that vWF-CP mRNA can be easily obtained from peripheral blood makes it possible to easily examine the significance of genetic abnormalities in patients with vWF-CP deficiency. For example, when a mutation causing splicing abnormality is found in the genome, it is possible to determine what kind of abnormality is caused at the RNA level by examining platelet mRNA.
[0024]
In conclusion, the confirmation of the presence of vWF-CP in platelets according to the present invention is undoubtedly a meaningful fact for analyzing the etiology of platelet thrombus formation.
[0025]
【Example】
Hereinafter, the present invention will be described in detail with reference to production examples and experimental examples, but the present invention is not limited thereto.
[0026]
Example 1:
1) Blood was used as the material measurement specimen for analysis. As blood, blood collected from six subjects with no past medical history was used. In all experiments, six subjects were given informed consent.
[0027]
2) Analysis of the mRNA of platelets Anbo et al. [Br. J. et al. Haematol. 102 (1998), 829-840]. Platelet rich plasma (PRP) was prepared by collecting venous blood collected at a 3.8% sodium citrate concentration of 1/10 of the final volume, followed by centrifugation at 700 rpm for 15 minutes at room temperature. Platelet pellets (platelet fraction) were obtained by further centrifuging PRP at 2000 rpm for 7 minutes at room temperature. Platelet pellets were treated with TRIZOL (Invitrogen).
[0028]
Total RNA was extracted from platelet pellets dissolved in chloroform and precipitated with isopropanol. The dried platelet RNA was dissolved in RNase-free water. To avoid RNA degradation, platelet RNA was amplified by reverse transcription PCR as soon as possible. 3 μg of platelet RNA is heated at 70 ° C. for 10 minutes, and reverse transcription reaction is performed at 42 ° C. for 65 minutes using the TrueScript II Reverse Transcriptase kit (Sawady Technology, Tokyo) with a final volume of 20 μl according to the manufacturer's instructions. It was.
[0029]
For reverse transcription PCR, special oligonucleotide primers for reverse transcription were used. The primer corresponding to exon 1-7 of vWF-CP, the primer for amplification of cDNA is a sense primer that is RT1 (5′-ATGCACCAGCGTCACCCCCG-3 ′) (SEQ ID NO: 1), and RT2 (5′-AATGGTACTACTCCAGGTCCGA-3 ') An antisense primer which is (SEQ ID NO: 2).
[0030]
The cDNA solution was subjected to PCR using TaKaRa LA Taq (Takara Shuzo. CO., LTD, Shiga, JAPAN) by GC buffer. PCR was performed in a total volume of 50 μl containing 0.5 U polymerase in GC buffer, 4 μM dNTP, 0.5 μM forward primer and reverse primer, respectively. The sample was incubated at 96 ° C. for 5 minutes and amplified in 35 cycles under the following cycle conditions (cycle conditions: 96 ° C. for 1 minute, 52 ° C. for 1 minute, 72 ° C. for 1 minute).
Samples were fractionated on a 2% agarose gel stained with ethidium bromide. Amplified DNA was purified and re-recovered from agarose gel using GENECLEAN II kit (Bio 101, La Jolla, Calif.) According to the manufacturer's procedure. Nucleotide sequences were determined by the ABI PRISM Dye Terminator Cycle Sequencing Ready Reactions (Perkin-Eb method by Perkin-Eb, directly using 310 DNA antosequencer (Applied Biosystems Inc, Foster City, CA).
[0031]
3) Real-time PCR
After separating RNA from each sample and preparing cDNA, the target cDNA and the reference β-actin cDNA are separately separated from the 5 'upstream reporter dye (FAM) and the 3' downstream quencher dye (TAMRA). Amplification by PCR was performed using the oligonucleotide. During PCR, the 5 ′ to 3 ′ nuclease activity of Taq DNA polymerase liberated the reporter, the fluorescence of the reporter dye increased, and the fluorescence was detected by the ABI Model 7700 Sequence Detection System fluorescence detector. After the fluorescence threshold was crossed, the PCR-amplification resulted in a linear curve consistent with the PCR product. The threshold fluorescence intensity was set and the point of the cycle where this occurred was determined as the starting point of the threshold cycle. Internal positive or negative controls (samples without polymerase) were run in parallel during different quantitative measurements to confirm comparable measurement conditions.
[0032]
The oligonucleotides used are shown below.
forward ADAMTS 13 primer: 5′-CCCAACCTGACCAGTGTCTACA-3 ′ (exon 15) (SEQ ID NO: 3),
reverse ADAMTS 13 primer: 5′-CTTCCCAGCCACGACATAGC-3 ′ (exon 16) (SEQ ID NO: 4),
Probe: 5′-FAM-ACAGGCCTCTCTTCACACACTTGGGCG-TAMRA-3 ′ (exon 15-16) (SEQ ID NO: 5).
[0033]
The PCR reaction mixture (50 μl) consists of 0.5 μM each primer, 0.25 μM probe, TaqMan Universal Master Mix (2 ×). The following cycle conditions were used (cycle conditions: first 96 ° C for 10 minutes, followed by 95 cycles of 15 ° C for 15 seconds, 60 ° C for 1 minute, 72 ° C for 1 minute for 40 cycles), and the ABI from Applied Biosystems. Measurements were made using a Prism 7700 sequence detection system. Data obtained from Real-time PCR was expressed as a relative amount based on the rate of fluorescence change. A standard curve was obtained based on the molecular count of the recombinant plasmid containing ADAMTS13 cDNA.
[0034]
4) Results (1) RT-PCR analysis of ADAMTS13 Platelet RNA isolated from the platelet fraction of citrated platelet-rich plasma was amplified by RT-PCR. The amplified DNA was purified and recollected from an agarose gel. A cDNA fragment of the expected size (660 bp) was obtained and analyzed by direct sequencing. The sequenced nucleotide sequence was identified as ADAMTS13 cDNA by searching using the BLAST program.
[0035]
(2) Real-time PCR
The ADAMTS13 mRNA expression in the platelet fraction was quantitatively analyzed by Real-time PCR using fluorescence. It was shown that ADAMTS13 mRNA exists in six healthy subjects with almost no difference in the constant amount (FIG. 1). PCR amplification was performed individually on mRNA from six subjects using special primers and fluorescent probes for ADAMTS13. The relative normalized fluorescence change (relative normalized fluorescence change ΔRn) against time (number of PCR cycles) was plotted for each sample. After setting the threshold fluorescence intensity, the threshold cycle was determined. Relative gene expression was determined by gene targeting and internal standard threshold cycle. The threshold cycle of ADAMTS13 (3 times) is as follows.
Sample 1: 23.38, 29.02, 28.18,
Sample 2: 28.46, 27.83, 28.19,
Sample 3: 28.79, 29.05, 29.39,
Sample 4: 29.78, 29.60, 29.47,
Sample 5: 28.92, 29.01, 28.56,
Sample 6: 29.98, 30.85, 30.59.
[0036]
The ratio between the threshold cycle of the internal standard β-actin and that of ADAMTS13 is shown below.
Sample 1: 0.563 ± 0.007,
Sample 2: 0.570 ± 0.005
Sample 3: 0.567 ± 0.005
Sample 4: 0.572 ± 0.003,
Sample 5: 0.583 ± 0.004,
Sample 6: 0.563 ± 0.007.
[0037]
These values are the mean and standard deviation of three independent measurements (Table 1).
[Table 1]
Ratio of threshold cycle of internal standard β-actin to that of ADAMTS13

[0038]
In order to calculate the absolute amount of ADAMTS13 mRNA in platelets, we used a linearity study based on the analysis of plasmid cDNA samples at known concentrations (FIG. 2). This calibration curve indicated that ADAMTS13 mRNA was present in 1 ng total RNA in 5.5 ± 2.5 × 10 ⁻⁸ ng.
Sample 1: 6.9 ± 2.1,
Sample 2: 9.1 ± 0.2,
Sample 3: 5.2 ± 1.0
Sample 4: 3.5 ± 0.3,
Sample 5: 6.0 ± 0.9,
Sample 6: 2.0 ± 0.4.
These values are the average and standard deviation of three independent measurements.
[0039]
【The invention's effect】
The present invention has found for the first time the expression of ADAMTS13 mRNA in platelets. Furthermore, Real-time PCR was established as a useful method for quantitative evaluation of ADAMTS13 mRNA expression. The results of the present invention clarify that platelets clearly have ADAMTS13 mRNA, and suspect any thrombotic disease if the quantitative ratio changes. Indirectly, it was shown that ADAMTS13 protein is present in platelets. Based on this, the foundation for finding the physiological significance of measuring vWF-CP activity in platelets was established.
[Brief description of the drawings]
FIG. 1. Quantification of ADAMTS13 mRNA expression in platelets using real-time PCR with fluorescence.
FIG. 2 shows absolute amounts based on a standard curve of a plasmid containing ADAMTS13 cDNA (× 10 ⁻⁸ ng / 300 ng RNA).
[Sequence Listing]

Claims (3)

A method for detecting a vWF-CP function using vWF-CP mRNA as a marker, wherein a platelet fraction obtained from peripheral blood is used as a specimen. The method according to claim 1 , wherein vWF-CP mRNA in platelets is used as a marker. The method of Claim 1 or 2 by PCR method.

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