An ancylostoma kunitz polypeptide and its anticoagulant applications

Abstract

The present application relates to a hookworm Kunitz polypeptide and its anticoagulant application, the amino acid sequence of the polypeptide is shown as SEQ ID NO. 1. The present application also relates to the following application of the polypeptide or the nucleotide fragment encoding the polypeptide: (1) preparing an anti-FXIa preparation; (2) preparing an anticoagulant drug.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medical biotechnology, and in particular, relates to a hookworm Kunitz polypeptide and its anticoagulant application. BACKGROUND

[0002] Thromboembolic disease refers to the production of excessive blood clots in the blood circulation system, which exceeds the need of physiological healthy hemostatic reaction, leading to the production of thrombus to block the blood flow to the downstream tissue, and causing pathological symptoms of the body. According to the type of blood vessels forming thrombus, thromboembolic disease can be divided into arterial thromboembolic disease (ATD), venous thromboembolism (VTE) and thrombotic microangiopathy (TMA) [2]. The treatment of thromboembolic disease has two means of surgical treatment and drug treatment. Since drug treatment has the advantages of convenient approach and low requirement for blood detection of drug activity, drug treatment of thromboembolic disease is the most basic and extensive treatment means [3].

[0003] Anticoagulants are drugs that prevent and reduce the formation of thrombus by inhibiting coagulation factors. Since its advent, anticoagulants have been widely used in the prevention and treatment of VTE due to its convenience and obvious effect. With the continuous deepening of the research on anticoagulants, the types of anticoagulants are also increasing. Traditional anticoagulants are multi-target anticoagulants, which have an impact on multiple factors of the coagulation pathway. The representative drug is heparin and warfarin. Heparin is the earliest traditional anticoagulant applied in clinical practice, which opens a new chapter in the history of drug anticoagulant therapy and greatly changes the treatment concept of thrombotic diseases. Warfarin is the first oral traditional anticoagulant. The development of new oral anticoagulants (NOACs) mainly includes thrombin inhibitors and factor Xa inhibitors. Compared with traditional anticoagulants, NOACs have the advantages of fast onset, relatively fast metabolism, less drug interaction, and no need for close clinical laboratory monitoring. Currently available NOACs include direct thrombin (FIIa) inhibitors dabigatran, and direct factor Xa inhibitors apixaban, edoxaban, and rivaroxaban. Dabigatran is the first NOAC applied in clinical practice, which exerts its effect by competing with the active site of thrombin. Therefore, dabigatran can prevent thrombin from converting fibrinogen into its active form fibrin, thereby inhibiting the development of thrombus. Due to the complexity of the coagulation homeostatic system and the diversity of physiological and pathological conditions, these anticoagulants still have varying degrees of bleeding risk. Therefore, the discovery of new anticoagulant lead molecules is still of great significance.

[0004] Hookworm is a common blood-sucking human parasite that mainly parasitizes the small intestine and duodenum of the host. It feeds on blood by attaching its buccal capsule to the intestinal mucosa. The hookworms that parasitize the human body mainly include Ancylostoma americanum and Ancylostoma duodenale. During the long evolution process, hookworms have evolved an effective anticoagulant mechanism to adapt to feeding and survival. The secretions of hookworms contain anticoagulant substances that make the intestinal mucosa of the host bitten by hookworms less likely to coagulate, which is beneficial to the hookworms to suck the blood of the host. This characteristic suggests that the anticoagulant polypeptides secreted by hookworms are promising anticoagulant polypeptide drugs.

[0005] So far, 20 hookworm anticoagulant polypeptides have been found, which are derived from Ancylostoma caninum, Ancylostoma duodenale, and Ancylostoma ceylanicum. Among them, 14 anticoagulant polypeptides derived from Ancylostoma caninum include AcAP, AcAPc2, NAPc2, AcAPc3, AcAPc4, AcAP5

[11] AcAP5, NAP5, AcAP6, NAP6, AcaNAP7, AcaNAP8, AduNAP1, AduNAP7

[12] ; there are 3 kinds of anti-coagulation polypeptides from duodenum: AduNAP1

[13] , AduNAP4, AduNAP7

[14] ; there are 4 kinds of anti-coagulation polypeptides from Ceylon hookworm: AceAP, AceKI, AceKI-1, Ace-HPI

[15] Structurally, the hookworm anti-coagulation polypeptides reported so far all contain 10 cysteine residues, can form 5 pairs of intramolecular disulfide bonds, and have a typical Ascaris type structural motif. These highly similar hookworm anti-coagulation polypeptides exert anti-coagulation effects by inhibiting key factors such as FXa, FXIa, TF / VIIa complex in the coagulation process. Among them, there are 10 FXa inhibitors: AcAP, AcAP5, NAP5, AcAP6, NAP6, Ac-AP-12, AduNAP4, AceAP1, AceKI and AceKI-1; there are 3 FXIa inhibitors: AduNAP4, AcaNAP10 and AcaNAP11, which are all dual-target inhibitors; there are 7 TF / VIIa inhibitors: AcAPc2, NAPc2, AcAPc3, AcAPc4, AcaNAP10, AcaNAP11 and AceAP1. AcaNAP7, AcaNAP8, AduNAP1 and AduNAP7 were found to significantly prolong PT and APTT in in vitro coagulation time determination, and have anti-coagulation function, but the mechanism is unclear and needs further study. Among the 21 hookworm anti-coagulation polypeptides, the best anti-coagulation effect is AcAPc2 and NAP5. The hookworm anti-coagulation polypeptides found so far are all serine protease inhibitors of the Ascaris family, have high sequence homology and the same disulfide bond pairing, similar beta-chain, and contain a C-terminal extension

[16] Research shows that this kind of anti-coagulation agent has a high risk of bleeding.

[0006] Kunitz type polypeptides are a kind of widely distributed serine protease inhibitors. Bioinformatics analysis based on hookworm genome and transcriptome shows that there are a large number of Kunitz type polypeptide sequence resources in hookworm, but the anti-coagulation function research has not been reported, which indicates that the research on the function and mechanism of Kunitz type polypeptides in hookworm has great potential and research prospect. Based on this, the present application is proposed.

[0007] Reference

[0008] 1. Palazzolo JS, Westein E, Hagemeyer CE, Wang TY: Targeting Nanotechnologies for the Treatment of Thrombosis and Cardiovascular Disease. Semin Thromb Hemost 2020, 46(5):606-621.

[0009] 2. Mackman N: Triggers, targets and treatments for thrombosis. Nature 2008, 451(7181):914-918.

[0010] 3. Liu FQ, Chen X: Advances in Anticoagulant Therapy for Pediatric Pulmonary Thromboembolism. Medical Review 2021, 27(06):1157-1164.

[0011] 4. Barkun AN, Almadi M, Kuipers EJ, Laine L, Sung J, Tse F, Leontiadis GI, Abraham NS, Calvet X, Chan FKL et al: Management of Nonvariceal Upper Gastrointestinal Bleeding: Guideline Recommendations From the International Consensus Group. Ann Intern Med 2019, 171(11):805-822.

[0012] 5. Chan YH, Lee HF, See LC, Tu HT, Chao TF, Yeh YH, Wu LS, Kuo CT, Chang SH, Lip GYH: Effectiveness and Safety of Four Direct Oral Anticoagulants in Asian Patients With Nonvalvular Atrial Fibrillation. Chest 2019, 156(3):529-543.

[0013] 6. Inoue H, Uchiyama S, Atarashi H, Okumura K, Koretsune Y, Yasaka M, Yamashita T, Taniguchi A, Fukaya T, Investigators JDS: Effectiveness and safety of long-term dabigatran among patients with non-valvular atrial fibrillation in clinical practice: J-dabigatran surveillance. J Cardiol 2019, 73(6):507-514.

[0014] 7. Jansson M, Sjalander S, Sjogren V, Renlund H, Norrving B, Sjalander A: Direct comparisons of effectiveness and safety of treatment with Apixaban, Dabigatran and Rivaroxaban in atrial fibrillation. Thromb Res 2020, 185:135-141.

[0015] 8. Kjerpeseth LJ, Selmer R, Ariansen I, Karlstad O, Ellekjaer H, Skovlund E: Comparative effectiveness of warfarin, dabigatran, rivaroxaban and apixaban in non-valvular atrial fibrillation: A nationwide pharmacoepidemiological study. PLoS One 2019, 14(8):e0221500.

[0016] 9. Shalash AO, Hussein WM, Skwarczynski M, Toth I: Hookworm infection: Toward development of safe and effective peptide vaccines. J Allergy Clin Immunol 2021, 148(6): 1394-1419e1396.

[0017] 10. Abuzeid AMI, Zhou X, Huang Y, Li G: Twenty-five-year research progress in hookworm excretory / secretory products. Parasit Vectors 2020, 13(1): 136.

[0018] 11. Zhu Y, Lin Y, Liu X, Hu W, Wang Y: Identification of AcAP5 as a novel factor Xa inhibitor with both direct and allosteric inhibition. Biochem Biophys Res Commun 2017, 483(1): 495-501.

[0019] 12. Jiang D, Zhan B, Mayor RS, Gillespie P, Keegan B, Bottazzi ME, Hotez P: Ac-AP-12, a novel factor Xa anticoagulant peptide from the esophageal glands of adult Ancylostoma caninum. Mol Biochem Parasitol 2011, 177(1): 42-48.

[0020] 13. Chen YZ, Deng L, Shao Z, He QF, Situ YL, Zhou Y, Peng LF: Prokaryotic expression of Ancylostoma duodenale anticoagulant peptide AduNAP1 and its anticoagulant activity. Chinese Journal of Parasitology & Parasitic Diseases 2016, 34(07): 611-614+619.

[0021] 14. Peng LF, Deng L, Yang C, Hu J, Gan WQ, Wu YM, Fu HW: Prokaryotic expression, purification and anticoagulant activity identification of Ancylostoma duodenale anti-coagulation protein AduNAP7. Chinese Journal of Zoonoses 2007(10): 1021-1025.

[0022] 15. Huang Y, Abuzeid AMI, Liu Y, He L, Zhao Q, Yan X, Hang J, Ran R, Sun Y, Li X et al: Identification and localization of hookworm platelet inhibitor in Ancylostoma ceylanicum. Infect Genet Evol 2020, 77: 104102.

[0023] 16. Zhu W, Gao H, Luo X, Ye X, Ding L, Hao J, Shu Z, Li S, Li J, Chen Z: Cloning and identification of a new multifunctional Ascaris-type peptide from the hemolymph of Buthus martensii Karsch. Toxicon 2020, 184: 167-174. SUMMARY

[0024] The present application firstly relates to a group of Kunitz type anticoagulant polypeptides Namp48, characterized in that the amino acid sequence of the Namp48 polypeptide is shown as SEQ ID NO. 1.

[0025] The present application also relates to a nucleotide fragment encoding the anticoagulant polypeptide Namp48, preferably the sequence of the nucleotide fragment is shown as SEQ ID NO. 2.

[0026] The present application also relates to a medicine or a pharmaceutical composition comprising the anticoagulant polypeptide Namp48 or the nucleotide fragment encoding the anticoagulant polypeptide Namp48, which contains a therapeutically effective amount of the polypeptide or the nucleotide fragment encoding the polypeptide, and necessary pharmaceutical adjuvants.

[0027] The present application also relates to the following applications of the anticoagulant polypeptide Namp48 or the nucleotide fragment encoding the anticoagulant polypeptide Namp48:

[0028] (1) preparing an anti-FXIa preparation;

[0029] (2) Preparation of an anticoagulant drug.

[0030] The present application has the following beneficial effects:

[0031] (1) A new high-activity anticoagulant polypeptide Namp48 is discovered from A. americana, which has inhibitory activity on three coagulation activation pathways of APTT, PT and TT;

[0032] (2) Mechanism research results show that the anticoagulant polypeptide Namp48 has very specific inhibition on the activity of FXIa;

[0033] (3) Safety experiments show that the anticoagulant polypeptide Namp48 has good safety.

[0034] The above results show that the anticoagulant polypeptide Namp48 has the development potential of a more specific anticoagulant drug. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 、 Seven A parasitic hookworm Kunitz polypeptide sequence, wherein,

[0036] Namp2 is derived from NCBI: XP_013308967.1;

[0037] Namp3 is derived from NCBI: XP_013308916.1;

[0038] Namp15 is derived from XP_013305030.1;

[0039] Namp21 is derived from NCBI: XP_013302552.1;

[0040] Namp31 is derived from NCBI: XP_013302471.1;

[0041] Namp34 is derived from NCBI: XP_013298401.1;

[0042] Namp48 is derived from NCBI: XP_013295982.1.

[0043] Figure 2 、 Four RP-HPLC elution chromatogram of 2 parasitic hookworm Kunitz polypeptides, wavelength 230 nm. 2A: Namp2, 2B: Namp31, 2C: Namp34, 2D: Namp48.

[0044] Figure 3 、 FourFigure 3A shows the difference between wild-type polypeptides Namp2 and Namp31 in prolonging the APTT time fold at a final concentration of 20 μg / mL; Figure 3B shows the difference between wild-type polypeptides Namp34 and Namp48 in prolonging the APTT time fold at a final concentration of 5 μg / mL.

[0045] Figure 4 Figure 4A, 4B, 4C show the time fold of polypeptide Namp34 in prolonging APTT, PT and TT; Figure 4D, 4E and 4F show the time fold of polypeptide Namp48 in prolonging APTT, PT and TT.

[0046] Figure 5 Figure 5 shows the erythrocyte toxicity of anticoagulant polypeptide Namp48.

[0047] Figure 6 Figure 6 shows the thrombelastogram of anticoagulant polypeptide Namp48, where curves a, b and c represent the thrombelastogram of polypeptide Namp48 at a concentration of 0 μg / mL, 5 μg / mL (519.11 nM) and 10 μg / mL (1.04 μM), respectively.

[0048] Figure 7 Figure 7 shows the thrombelastogram parameters of anticoagulant polypeptide Namp48, where the four parameters of the thrombelastogram of three different concentrations of Namp48 are compared, and the X-axis represents the four parameters R, K, Angle deg and MA of the thrombelastogram, and the Y-axis represents the ratio of the parameters of the polypeptide group to the parameters of the physiological saline group.

[0049] Figure 8 Figure 8A, 8B, 8C, 8D, 8E and 8F show the evaluation of the 6 coagulation factor inhibition activity of anticoagulant polypeptide Namp48, where the change curves of the residual enzyme activity of the 6 coagulation factors after incubation of polypeptide Namp48 at the set concentration gradient with Kallikrein, FXIIa, FXIa, FXa, FIIa and Kallikrein are shown. In the figure, the X-axis represents the concentration gradient of polypeptide Namp48 (0, 78.125 nM, 156.25 nM, 312.5 nM, 625 nM, 1250 nM, 2500 nM), and the concentration gradient of coagulation factor FXIa polypeptide is set as 0, 10 nM, 20 nM, 40 nM, 80 nM, 160 nM, 320 nM; and the Y-axis represents the residual enzyme activity of the different coagulation factors.

[0050] Figure 9His pull down gel electrophoresis of the anticoagulant polypeptide Namp48 and FXIa and FXa, 9A is the interaction of Namp48 and FXIa, Namp48 and FXIa were incubated with Ni Beads in turn, the mixture was denatured by heating and then centrifuged, and the supernatant was loaded into a 15% SDS-PAGE gel electrophoresis; 9B is the interaction of Namp48 and FXa, Namp48 and FXa were incubated with Ni Beads in turn, and the supernatant was loaded into a 15% SDS-PAGE gel electrophoresis. DETAILED DESCRIPTION

[0051] Namp48, sequence as SEQ ID NO. 1:

[0052] DQNLFCLMPPDPGFCRAILRRWAWNPVEERCERFEYGGCGGNRNNFKTQKECLYECWNKFV

[0053] Optimized coding nucleic acid, sequence as SEQ ID NO. 2:

[0054] GatcagaacctgttttgcctgatgccgccggatccgggcttttgccgcgcgattctgcgccgctgggcgtggaacccggtggaagaacgctgcgaacgctttgaatatggcggctgcggcggcaaccgcaacaactttaaaacccagaaagaatgcctgtatgaatgctggaacaaatttgtgTGA

[0055] Namp2, sequence as SEQ ID NO. 3:

[0056] QGYDRSGDICNMKEDEGPCKSLQTRWRWDFNEGNCVKFNYGGCGGNKNNFETEEKCLERCTF

[0057] Optimized coding nucleic acid, sequence as SEQ ID NO. 4:

[0058] cagggctatg atcgcagcgg cgatatttgc aacatgaaag aagatgaagg cccgtgcaaa agcctgcaga ccgctggcgc tgggatttaa cgaaggcaac tgcgtgaaat ttaactatgg cggctgcggc ggcaacaaaa acaactttga accgaagaaa atgcctggaac gctgcacctt TGA

[0059] Namp31, sequence as SEQ ID NO. 5:

[0060] WPKRVREECPETEWYDFCFNGCEPTCYNPEPKCSKLCGWGGCTCRGGYVREDEGECILDSECR

[0061] Optimized coding nucleic acid, sequence as SEQ ID NO. 6:

[0062] tggccgaaac gcgtgcgcga agaatgcccc gaaaccgaat ggtatgattt ttgctttaac ggctgcgaac cgacctgcta taacccggaa cgaaatgcag caaactgtgc ggctggggcg gctgcacctg ccgcggcggc tatgtgcgcg aagatgaagg cgaatgcatt ctggatagcg aatgccgcTGA

[0063] Namp34, sequence as SEQ ID NO. 7:

[0064] NIDSRCLQPVNGPVCRALVKRWSYDPTTNKCKTFMYGGCGGTENNFLSEEECIKTCVRRSH

[0065] Optimized coding nucleic acid, sequence as SEQ ID NO. 8:

[0066] aacattgatagccgctgcctgcagccggtgaacggcccggtgtgccgcgcgctggtgaaacgctggagctatgatccgaccaccaacaaatgcaaaacctttatgtatggcggctgcggcggcaccgaaaacaactttctgagcgaagaagaatgcattaaaacctgcgtgcgccgcagccatTGA

[0067] Example 1, bioinformatics analysis to obtain Kunitz family sequences of parasitic hookworm

[0068] Search Kunitz sequences of A. americanum from NCBI website, remove duplicate amino acid sequences, obtain Kunitz sequence information resource library of A. americanum. Analyze the signal peptide of the protein to obtain the amino acid sequence of the mature peptide, and analyze and compare the amino acid sequence. Perform local blast comparison of A. americanum Kunitz sequences with polypeptides reported in the literature that have both anticoagulant function and Kunitz structural motifs, and obtain candidate A. americanum Kunitz sequences according to homology and number and name.

[0069] Seven subsequent Kunitz polypeptide sequences were found, named Namp2, Namp3, Namp15, Namp21, Namp31, Namp34 and Namp48. These Kunitz polypeptides all contain 6 conserved Cys, whose sulfhydryl dehydrogenation forms three pairs of disulfide bonds (-S-S-), with pairing modes of Cys I-Cys VI, Cys II-Cys IV and Cys III-Cys V, which are the classic structural characteristics of Kunitz type proteins. Figure 1 ).

[0070] Example 2, expression and purification of hookworm Kunitz family polypeptides and their analogs

[0071] Using pET-28a as a vector, construct a plasmid of recombinant human parasitic hookworm Kunitz family polypeptides and their analogs, and transform the plasmid into BL21 competent cells and store in a -20°C refrigerator.

[0072] 100 μL of Namp series protein expression bacterial solution was inoculated into 0.003% Kana Luria-Bertani liquid medium, and placed in a gas bath constant temperature oscillator, cultured at 37°C, 150 rpm for 11-12 hours.

[0073] Use a measuring cylinder to take 50 mL of overnight activated expression bacterial solution, inoculate into 1000 mL of Luria-Bertani liquid medium containing 0.003% Kana, and culture at 37°C, 210 rpm for about 2 hours. When the OD value of the bacterial solution is about 0.4, add IPTG for induction, and continue to culture at 37°C, 210 rpm for 4 hours.

[0074] After the induction, the bacteria were collected at 4°C, 6000 rpm for 6-8 min. 25 mL ice PBS was added to the collected bacteria to resuspend the bacteria. The bacteria were broken by ultrasonic wave for 22 min at 80 W, 3 s on and 8 s off. The bacteria were centrifuged at 4°C, 12000 rpm for 20 min after the ultrasonic wave. The supernatant was discarded and the precipitate was collected, which was the inclusion body.

[0075] 10 mL ice PBST was added to the collected inclusion body and mixed well. The mixture was ice-bathed for 10-30 min and centrifuged at 4°C, 5000 g for 15 min. The supernatant was discarded. The step was repeated once to obtain the inclusion body protein with high purity.

[0076] Renaturation of the inclusion body protein: 0.15 g GSH was added to 5 mL denaturing solution and stirred well. The mixture was denatured at 25°C for 2 h. Renaturation of the inclusion body protein: the denatured inclusion body was centrifuged at 20°C, 12000 rpm for 20 min. The precipitate was discarded and the supernatant was collected. 0.06 GSSG was added to 500 mL ice renaturation solution. The supernatant after centrifugation was added dropwise to the 500 mL renaturation solution while stirring. After the dropwise addition, the renaturation solution was renatured at 16°C for 16 h.

[0077] The renatured protein solution was centrifuged at 4°C, 12000 rpm for 20 min and the precipitate was discarded.

[0078] The supernatant of the renaturation solution was centrifuged at 4°C, 4000 rpm for 40 min using a 3 KD ultrafiltration tube until the volume of the protein concentrate was <5 mL. The protein concentrate was divided into 1 mL / tube. 10 μL 10% TFA was added to each 1 mL protein concentrate. The mixture was centrifuged at 4°C, 12000 rpm for 20 min. The supernatant after centrifugation was transferred to a new EP tube. The tube was labeled and centrifuged again for 5 min. The protein concentrate was purified by RP-HPLC.

[0079] The flow rate of the HPLC instrument (Agilent liquid chromatograph 1260) was 4 mL / min and the sample volume was <5 mL. The HPLC instrument used a C18 reverse column (10x250 mm, 5 μm, Elite-HPLC). The mobile phase was solution B-0.1% TFA and solution D-90% acetonitrile+0.1% TFA. The elution gradient was a linear elution gradient for 60 min. The initial B solution was 95% and the initial D solution was 5%. The final B solution was 5% and the final D solution was 95%. The detection wavelength was 230 nm. After the purified protein solution was collected, it was labeled clearly and stored in a -80°C refrigerator.

[0080] The HPLC purified protein solution was pre-frozen in a -80°C freezer for more than 8 hours, and then the pre-frozen protein solution was freeze-dried into powder using a vacuum freeze-dryer. Sterilized ice ddH2O was added to the protein powder. The sterilized ice ddH2O was added according to the principle of 50,000 peak area plus 1 mL sterilized ice ddH2O, wherein the peak area of the protein was automatically calculated by the HPLC sample collection program.

[0081] The re-dissolved protein solution was sub-packaged into 1.8 mL cryogenic tubes, each containing 500 μL. The sub-packaged protein solution was freeze-dried into protein powder again using a freeze-dryer, and the protein name, sub-packaging time and other information were labeled. For the remaining protein solution, SDS-PAGE gel electrophoresis and BCA quantitative experiments were performed.

[0082] Seven Kunitz polypeptides of the parasitic hookworm, four of which, Namp2, Namp31, Namp34 and Namp48, were successfully prepared. When the protein concentrate was purified by RP-HPLC, the protein peak was collected at 10-30 min, and the HPLC spectrum of the collected protein fraction is shown in Figure 2 , wherein Namp31 was collected from the two fractions (Peak 1, Peak 2) of the spectrum shown in Figure 2 . Namp3, Namp15 and Namp21 were not obtained.

[0083] Example 3, detection of the anticoagulant function of the polypeptide

[0084] Activated partial thromboplastin time (APTT) is a coagulation function test index reflecting the comprehensive activity of coagulation factors in the endogenous coagulation pathway, especially the first stage. It is widely used to determine the defects of coagulation factors in the endogenous pathway, such as factors XI, VIII and IX, and can also be used for initial screening diagnosis of bleeding diseases and laboratory monitoring of heparin anticoagulant therapy.

[0085] Prothrombin time (PT) refers to the time required for the conversion of prothrombin to thrombin, leading to plasma clotting, in the presence of excess tissue thromboplastin and calcium ions in platelet-free plasma. PT is an index reflecting the activity of coagulation factors in plasma. Prothrombin time determination is a screening test to check whether the body's extrinsic coagulation system is functioning properly, and is also an important monitoring index for clinical anticoagulant therapy.

[0086] Thrombin time (TT) refers to the time required for blood to clot after the addition of standardized thrombin to plasma. In the common coagulation pathway, the generated thrombin converts fibrinogen to fibrin, which can be reflected by thrombin time (TT).

[0087] 1. APTT function detection of the polypeptide

[0088] Open the MD Pacific TSA 9000C fully automatic coagulation instrument, calculate the required cleaning solution, APTT reagent, Cacl2 solution according to the number of samples, among which APTT reagent 50 μL / time, CaCl2 solution 50 μL / time, and put these reagents into the specified interval. Take 25 μL and 50 μL of polypeptide with the prepared concentration and 50 μL of healthy human plasma, mix well, and incubate on ice for 15 min. Input sample information on the fully automatic blood coagulation instrument, and set to perform APTT detection. After 15 min ice bath, place the samples one by one according to the set position. Import the data detected by the instrument into Graph Pad Prism, and draw the concentration-dependent curve graph of the polypeptide.

[0089] The results are shown in Figure 3 PBS is the negative control, and the APTT time multiple of PBS is set to 1.

[0090] (1) When the final concentration of polypeptides Namp2 and Namp31 is 20 μg / mL, the APTT time multiple of Namp2 is 1.73±0.03, the APTT time multiple of Namp31 Peak1 is 1.01±0.009, and the APTT time multiple of Namp31 Peak2 is 1.85±0.03 Figure 3 A).

[0091] (2) Because the APTT time of Namp34 and Namp48 does not stop at 20 μg / mL, the concentration of polypeptide is reduced to 5 μg / mL, and the APTT detection of Namp34 and Namp48 is performed. When the final concentration of polypeptides Namp34 and Namp48 is 5 μg / mL, the APTT time multiple of Namp34 is 12.12±0.57, and the APTT time multiple of Namp48 is 35.12±1.21 Figure 3 B).

[0092] After APTT activity screening, two polypeptides Namp34 and Namp48 with good APTT activity were selected from the four Kunitz polypeptides for APTT, PT and TT concentration-dependent test.

[0093] 2. Polypeptide PT function detection method:

[0094] Open the MD Pacific TSA 9000C automatic coagulation instrument, calculate the required cleaning solution, PT reagent according to the number of samples, among which the PT reagent is 100 μL / time, and put these reagents into the specified interval. Prepare polypeptide of the right concentration, take 25 μL and 50 μL of healthy human plasma for mixing, and then incubate on ice for 15 min. Input sample information on the automatic blood coagulation instrument, and set to perform PT detection. After 15 min of ice bath, place the samples one by one according to the set position. Import the data detected by the instrument into Graph Pad Prism, and draw the concentration-dependent curve graph of the polypeptide.

[0095] 3. TT function detection method of polypeptide:

[0096] Open the MD Pacific TSA 9000C automatic coagulation instrument, calculate the required cleaning solution, PT reagent according to the number of samples, among which the TT reagent is 100 μL / time, and put these reagents into the specified interval. Prepare polypeptide of the right concentration, take 25 μL and 100 μL of healthy human plasma for mixing, and then incubate on ice for 15 min. Input sample information on the automatic blood coagulation instrument, and set to perform TT detection. After 15 min of ice bath, place the samples one by one according to the set position. Import the data detected by the instrument into Graph Pad Prism, and draw the concentration-dependent curve graph of the polypeptide.

[0097] The polypeptide Namp34 is set to 0, 31.25 nM, 62.5 nM, 125 nM, 250 nM, 500 nM, and 1000 nM, and the results show that Namp34 significantly prolongs the APTT, PT and TT time in a concentration-dependent manner, and at the highest concentration of 1000 nM, the APTT, PT and TT time is prolonged by 20.2, 8 and 2.7 times, respectively.

[0098] The polypeptide Namp48 is set to 0, 15.625 nM, 31.25 nM, 62.5 nM, 125 nM, 250 nM, and 500 nM, and the results show that Namp48 significantly prolongs the APTT, PT and TT time in a concentration-dependent manner, and at the highest concentration of 500 nM, the APTT, PT and TT time is prolonged by 35.1, 9.1 and 3.3 times, respectively. Figure 4 ).

[0099] Namp34 and Namp48 are two polypeptides that have inhibitory effects on the APTT, PT and TT coagulation pathways, but Namp48 has stronger anticoagulation effect Therefore, our subsequent research focuses on the polypeptide Namp48.

[0100] Example 4, Effect of polypeptide Namp48 on human whole blood

[0101] To study whether the polypeptide Namp48 also has an anticoagulant effect on human whole blood, we use thrombelastograph instrument to evaluate its whole blood anticoagulant effect.

[0102] 1. First, perform the red blood cell hemolysis experiment to evaluate the toxicity of the polypeptide Namp48, and the experimental steps are as follows:

[0103] Extract fresh whole blood from healthy people with sodium citrate anticoagulant tubes, then suck out part of it into a 1.5 mL EP tube, then add 1 mL of sterile 0.9% normal saline to the EP tube, centrifuge at 1000g for 10 min, then discard the supernatant. Repeat the above steps, and wash with sterile normal saline for 2-3 times until the supernatant is colorless and transparent.

[0104] Prepare a 4% (V / V) solution of the washed red blood cells with normal saline, mix well and reserve. Add 100 μL of 4% red blood cell suspension and 100 μL of different concentrations of polypeptide to a 1.5 mL EP tube, and perform water bath at 37°C for 1 hour.

[0105] Centrifuge the EP tube after water bath at 3000g for 10 min, and suck 100 μL of supernatant into a 96-well plate well. After the sample addition is completed, place the 96-well plate in the enzyme marker, set the wavelength to 540 nm for detection, and obtain the sample absorbance value data. The negative control is 100 μL of normal saline, and the positive control is 100 μL of 2% Triton X-100.

[0106] Hemolysis rate (%) = 100 x (A sample - A negative) / (A positive - A negative).

[0107] Normal saline is set as the negative control group and the hemolysis rate is 0%, and 0.5% Triton X-100 is set as the positive control group and the hemolysis rate is 100% ( Figure 5 ).

[0108] The polypeptide Namp48 is set at 50 μg / mL (5.19 μM) and 100 μg / mL (10.38 μM),

[0109] The experimental results show that, The hemolysis rate of polypeptide Namp48 is 0% at two different concentrations , which preliminarily indicates that the polypeptide Namp48 has low hemolysis toxicity.

[0110] 2. Thrombelastogram detection

[0111] To study the effect of the anticoagulant polypeptide Namp48 on the blood coagulation mechanism of human whole blood. The thrombelastogram instrument experimental parameters include coagulation time (R), blood clot formation rate (K), arc tangent and horizontal line angle Angel deg, and blood clot strength (MA). The experimental method is as follows:

[0112] Turn on the thrombelastograph (CFMS LEPU-8800), first perform a horizontal test, then select the test channel for detection, input the sample number at the corresponding position, and set the test type as "CK-Citrated kaolin".

[0113] Load the normal cup on the two channels to be detected according to the requirements, then add 1 mL of sodium citrate anticoagulated whole blood to the kaolin tube and mix well, dilute the polypeptide with normal saline to the detection concentration, then take 50 μL of the polypeptide solution of the detection concentration and add it to the kaolin tube and mix gently, then place it on ice for 4 minutes of ice bath activation.

[0114] First add 20 μL of standard CaCl2 solution to the test cup, then add 340 μL of activated whole blood.

[0115] Quickly lift the test cup, draw the test rod to the test position, click start in the computer program, and start the test.

[0116] Determine whether to end according to the trend of the thrombelastogram, click stop in the program after ending, open the test rod, and remove the test cup.

[0117] Export the thrombelastogram and data, and turn off the instrument according to the program.

[0118] The final concentration of the polypeptide is calculated as follows: (340 / 360)*(50 / 1050)

[0119] The anticoagulant polypeptide Namp48 is set to three concentrations: 0, 5 μg / mL (519.11 nM), and 10 μg / mL (1.04 μM), and each concentration is repeated twice to detect the effect of different concentrations of Namp48 on the clotting time of human whole blood.

[0120] The thrombelastogram results show that:

[0121] Compared with the 0 μg / mL Namp48 group (normal saline group), the 5 μg / mL (519.11 nM) Namp48 group and the 10 μg / mL Namp48 (1.04 μM) group showed a significant inhibitory effect on whole blood clotting.

[0122] The clotting time (R) and the blood clot formation rate (K) increased in a concentration-dependent manner, while the alpha angle (Angel deg) and the blood clot strength (MA) decreased in a concentration-dependent manner. Figure 6 ).

[0123] In the 5 μg / mL (519.11 nM) Namp48 group, the R value increased by 3.8 times, the K value increased by 3.4 times, the Angel value decreased by 0.4 times, and the MA value decreased by 0.8 times;

[0124] 10 μg / mL (1.04 μM) Namp48 group: R value increased to 6.3 times, K value increased to 5.9 times, Angel value decreased to 0.2 times, MA value decreased to 0.8 times Figure 7 ).

[0125] The above results show that the R value and K value of the polypeptide Namp48 group increased significantly in a concentration-dependent manner, while the alpha angle and MA value decreased significantly in a concentration-dependent manner. The increase in R value indicates an increase in the time to start clotting, reflecting the weakening of the function of clotting factors; the increase in K value and the decrease in alpha angle indicate a decrease in the rate of blood clot formation, reflecting a decrease in the formation of fibrin; the decrease in MA value indicates a decrease in the strength of the formed blood clot, reflecting a decrease in the number of platelets involved in blood clotting. The changes in these four parameters are consistent in reflecting that The sample added with Namp48 is inhibited in coagulation function, and the inhibition degree is positively correlated with the concentration of Namp48 Figure 8 .

[0126] Example 5, Anticoagulant Mechanism of Anticoagulant Polypeptide Namp48

[0127] 1. Detection of the inhibitory activity of polypeptides on clotting factors

[0128] Detection of Kallikrein, FXIIa, FXIa, FXa, FIIa and Plasmin inhibitory activity

[0129] Take 25 μL of the working concentration of enzyme and 25 μL of different concentrations of polypeptide and add them to the wells of a 96-well plate in turn. The negative control is the clotting factor Buffer.

[0130] Add 80 μL of the working concentration of luminescent substrate solution to another row of wells.

[0131] After the addition is complete, incubate the 96-well plate at 37°C, 90 rpm for 15 min, and then place it on the microplate reader.

[0132] Add 50 μL of the working concentration of luminescent substrate solution to each [E]-[I] mixture well using the row gun, set the microplate reader detection wavelength to 405 nm, and set the detection time to 6 min, detecting once every 1 min, for a total of 7 times.

[0133] Import the measured data into Graph Pad Prism and plot the concentration-dependent curve to calculate the inhibition rate.

[0134] The results show that

[0135] The anticoagulant polypeptide Namp48 has no obvious inhibitory effect on FIIa, but has significant inhibitory effect on Kallikrein, FXIIa, FXIa, FXa and Plasmin, and the IC50 is 139.8±2.15nM, 1661±3.22nM, 9.95±0.99nM, 151.1±2.18nM and 17.21±1.24nM respectively. The activity of FXIa can be almost completely inhibited by polypeptide Namp48 at a concentration of 160 nM Namp48 has inhibitory effect on Plasmin, FXa, FXIa, FXIIa and Kallikrein, and the inhibitory effect on coagulation factor FXIa is the strongest. Figure 9 ​ .

[0136] 2. Detection of the binding of Kunitz polypeptide Namp48 to FXIa and FXa by His pull down

[0137] Pull down is a technology similar to immunoprecipitation, and we can use this technology to detect whether the polypeptide and the enzyme have specific binding. The detection principle is that the labeled protein can be captured by Ni Beeds, and after the specified incubation and washing steps, the interacting polypeptide and enzyme are eluted and identified by SDS-PAGE gel electrophoresis. The Kunitz type anticoagulant polypeptide Namp48 prepared by E. coli heterologous expression technology contains a His (histidine) fusion tag, which can specifically bind to nickel ions (Ni 2+ ). Therefore, the Namp48 with His tag is used as bait to capture possible partners that can bind to it, and after incubation and elution, it is identified by SDS-PAGE gel electrophoresis. The SDS-PAGE gel electrophoresis result shows that in the experimental group, after the Namp48 and the coagulation factor are incubated together, the Namp48 and the coagulation factor are pulled together by the Ni Beeds; while the coagulation factor is not pulled by the Ni Beeds when the coagulation factor is added alone ​ . This shows that Namp48 and coagulation factor FXIa, FXa have specific binding.

[0138] Finally, it should be noted that the above examples are only used to help those skilled in the art to understand the essence of the present application, and are not used to limit the protection scope of the present application.

Claims

1. A Kunitz-type anticoagulant polypeptide Namp48, characterized in that The amino acid sequence of the Namp48 polypeptide is shown in SEQ ID NO.

1.

2. A nucleotide fragment encoding the anticoagulant polypeptide Namp48 according to claim 1, characterized in that: The sequence of the nucleotide fragment is shown in SEQ ID NO.

2.

3. A drug or pharmaceutical composition comprising the anticoagulant polypeptide Namp48 according to claim 1 or the nucleotide fragment encoding the anticoagulant polypeptide Namp48 according to claim 2, wherein the drug or pharmaceutical composition contains a therapeutically effective amount of the polypeptide or the nucleotide fragment encoding the polypeptide, and necessary pharmaceutical excipients.

4. Use of the anticoagulant polypeptide Namp48 according to claim 1 or the nucleotide fragment encoding the anticoagulant polypeptide Namp48 according to claim 2 in the preparation of anticoagulant drugs.

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