An anticoagulant polypeptide and its application in preparing plasma kallikrein activity inhibitor

Abstract

The present invention belongs to the field of biomedical technology and specifically relates to an anticoagulant polypeptide and its use in the preparation of a plasma kallikrein activity inhibitor. The anticoagulant polypeptide of the present invention comprises the amino acid sequence shown in SEQ ID No. 2. The anticoagulant polypeptide of the present invention can significantly inhibit plasma recalcification time and activated partial thromboplastin time, exhibits excellent anti-inflammatory thrombotic and stroke drug activity, has low bleeding risk and cytotoxicity, and is highly safe.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to an anticoagulant polypeptide and its application in preparing a plasma kallikrein activity inhibitor. Background Art

[0002] Thrombosis is a common cause of myocardial infarction, ischemic stroke, and venous thromboembolism. In 2010, thromboembolic conditions accounted for a quarter of global deaths, and antithrombotic therapy has become a major challenge in the healthcare field. Currently, the clinical use of tissue plasminogen activator (t-PA), heparin, and hirudin can effectively target and inhibit the coagulation cascade and platelet aggregation, but they are also associated with the risk of perioperative bleeding. In addition, these therapies cannot completely address the risk of re-thrombosis, highlighting a third mechanism that has not been fully addressed: inflammation.

[0003] Plasma kallikrein (PKa) is a multifunctional serine protease that plays a crucial role in thrombotic inflammation and cardiovascular events. Traditionally, PKa triggers inflammation by cleaving high-molecular-weight kininogen (KNG) to release bradykinin (BK), promoting the release of inflammatory factors such as tumor necrosis factor (TNF) and interleukins (IL). Furthermore, PKa plays a crucial role in the coagulation cascade, converting coagulation factor FXII to its active form (FXIIa). This activation leads to autoactivation of the contact pathway (intrinsic coagulation cascade) and thrombosis. Inhibition of the intrinsic coagulation pathway has been reported to be unrelated to hemostasis (bleeding), and the majority of patients with PKa deficiency do not have hemostatic defects. Therefore, PKa inhibition provides a theoretical target for the development of anti-inflammatory thrombotic drugs; currently, there is no potent anti-inflammatory thrombotic peptide drug that inhibits PKa with a low bleeding risk. Summary of the Invention

[0004] The purpose of the present invention is to provide an anticoagulant polypeptide and its use in the preparation of a plasma kallikrein activity inhibitor, which can inhibit plasma kallikrein activity, has good anti-inflammatory thrombotic and stroke drug activity, low bleeding risk and low cytotoxicity, and high safety.

[0005] The present invention provides an anticoagulant polypeptide, which comprises the amino acid sequence shown in SEQ ID No. 2.

[0006] Preferably, the amino acid sequence of the anticoagulant polypeptide is shown as SEQ ID No. 2 and SEQ ID No. 1.

[0007] The present invention also provides the use of the anticoagulant polypeptide described in the above technical solution in the preparation of a plasma kallikrein activity inhibitor.

[0008] The present invention also provides the use of the anticoagulant polypeptide described in the above technical solution in the preparation of a product for treating psychiatric diseases related to plasma kallikrein and inflammatory pathways.

[0009] The present invention also provides the use of the anticoagulant polypeptide described in the above technical solution in the preparation of anti-inflammatory products.

[0010] The present invention also provides the functional application of the anticoagulant polypeptide described in the above technical solution in the preparation of antithrombotic products.

[0011] The present invention also provides the use of the anticoagulant polypeptide described in the above technical solution in the preparation of anti-stroke products.

[0012] Preferably, the stroke includes ischemic stroke and / or hemorrhagic stroke.

[0013] Preferably, the ischemic stroke includes one or more of thrombotic cerebral infarction, embolic cerebral infarction, lacunar cerebral infarction, multiple cerebral infarction and transient ischemic attack.

[0014] The present invention also provides an anti-stroke drug, the active ingredient of which includes the anticoagulant polypeptide described in the above technical solution. Calculated by the dosage of the anticoagulant polypeptide, the dosage of the drug in the human body is 0.027 to 0.444 mg / kg.

[0015] Beneficial effects

[0016] The present invention provides an anticoagulant polypeptide and its use in the preparation of a plasma kallikrein activity inhibitor. The anticoagulant polypeptide comprises one or more of the following: 1) the amino acid sequence shown in SEQ ID No. 1; 2) a partial fragment of the amino acid sequence shown in SEQ ID No. 1, having less than 14 amino acid residues and comprising the amino acid sequence shown in SEQ ID No. 2. The anticoagulant polypeptide of the present invention can significantly inhibit plasma recalcification time and activated partial thromboplastin time, exhibits excellent anti-inflammatory, thrombotic, and stroke drug activity, and has low bleeding risk, low cytotoxicity, and a high safety profile.

[0017] Furthermore, in vitro experiments demonstrated that the amino acid sequence SD6, as shown in SEQ ID No. 2, can prolong plasma recalcification time and activated partial thromboplastin time, demonstrating a specific inhibitory effect on the intrinsic coagulation pathway. Within a dose range of 0.25 to 4 mg / kg, SD6 can mitigate arterial and cortical venous thrombosis in FeCl3- and photochemically induced mice without affecting bleeding. Furthermore, SD6 also reduced brain inflammation and edema in a mouse model of transient middle cerebral artery occlusion ischemic stroke. This suggests that SD6 has promise as a safer therapeutic agent for thrombotic inflammation, is readily available, and is amenable to future industrial production.

[0018] In the present invention, Fisher-t test and one-way analysis of variance were used to determine the significant differences in the data, which were expressed as *p<0.05; **p<0.01; ***p<0.001; and ****p<0.0001. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0020] Figure 1 The results of enzyme kinetic analysis of the effects of CW15 and SD6 on PKa activity using the chromogenic substrate method;

[0021] Figure 2 The results of SPR analysis of the binding strength of SD6 and PKa;

[0022] Figure 3 The effect of SD6 on PRT in vitro;

[0023] Figure 4 The effect of SD6 on APTT in vitro;

[0024] Figure 5 The effect of SD6 on PT in vitro;

[0025] Figure 6 The experimental results of the effect of SD6 on cytotoxicity;

[0026] Figure 7 The experimental results show the effect of SD6 on HEK293T cell proliferation;

[0027] Figure 8 The experimental results of the effect of SD6 on the proliferation of mouse spleen cells;

[0028] Figure 9 This is the result of the effect of SD6 on the bleeding time of mouse tail;

[0029] Figure 10The antithrombotic function of SD6 was evaluated in the carrageenan-induced rat tail thrombosis model;

[0030] Figure 11 The antithrombotic function of SD6 was evaluated in the FeCl3-induced mouse common carotid artery model;

[0031] Figure 12 The effect of SD6 on photochemically induced cerebral cortical thrombosis in mice;

[0032] Figure 13 This is the result of SD6's effect on restoring neurological function in tMCAO mice;

[0033] Figure 14 The results show the effect of SD6 on the infarct size of tMCAO mice with cerebral ischemia-reperfusion;

[0034] Figure 15 ELISA analysis of the effect of SD6 on reducing the release of inflammatory factors in the brain of tMCAO mice;

[0035] Figure 16 The dry and wet weighing method was used to analyze the effect of SD6 on brain edema in tMCAO mice;

[0036] Figure 17 Evans blue staining was used to analyze the blood-brain barrier function in tMCAO mice after SD6 injection. DETAILED DESCRIPTION

[0037] The present invention provides an anticoagulant polypeptide, comprising one or more of the following:

[0038] 1) the amino acid sequence shown in SEQ ID No. 1;

[0039] 2) A partial fragment of the amino acid sequence shown in SEQ ID No. 1, having less than 14 amino acid residues and including the amino acid sequence shown in SEQ ID No. 2.

[0040] In the present invention, the amino acid sequence of the partial fragment is preferably as shown in SEQ ID No. 2. The amino acid sequence information of SEQ ID No. 1 and SEQ ID No. 2 of the present invention is as follows:

[0041] SEQ ID No.1: CTSVSLGASDPFDY; SEQ ID No.2: SLGASD.

[0042] The present invention obtained the amino acid sequence CW15 as shown in SEQ ID No. 1 from the influenza-related immunoglobulin heavy chain connecting region (derived from the immunoglobulin heavy chain connecting domain sequence library on NCBI, Sequence ID: MBB2084429.1), and further optimized it to obtain the amino acid sequence SD6 as shown in SEQ ID No. 2. The effects of SD6 and CW15 on the activity of PKa were analyzed by chromogenic substrate method and enzyme kinetics. The results showed that SD6 and CW15 inhibited the activity of PKa in a dose-dependent manner, and IC 50 The molecular weights of SD6 and CW15 were approximately 200 μM and 69 μM, respectively. SD6 has a smaller molecular weight than CW15 and exhibits superior PKa inhibitory activity.

[0043] The present invention also provides the use of the anticoagulant polypeptide described in the above technical solution in the preparation of a plasma kallikrein activity inhibitor.

[0044] The present invention also provides the use of the anticoagulant polypeptide described in the above technical solution in the preparation of a product for treating psychiatric diseases related to plasma kallikrein and inflammatory pathways.

[0045] The present invention also provides the use of the anticoagulant polypeptide described in the above technical solution in the preparation of anti-inflammatory products.

[0046] The present invention also provides the functional application of the anticoagulant polypeptide described in the above technical solution in the preparation of antithrombotic products.

[0047] The present invention also provides the use of the anticoagulant polypeptide described in the above technical solution in the preparation of an anti-stroke product. In the present invention, the stroke preferably includes ischemic stroke and / or hemorrhagic stroke, more preferably ischemic stroke; the ischemic stroke preferably includes one or more of thrombotic cerebral infarction, embolic cerebral infarction, lacunar cerebral infarction, multiple cerebral infarctions, and transient ischemic attack. The anticoagulant polypeptide provided by the present invention can effectively reduce cerebral ischemic infarction damage, reduce blood-brain barrier damage, reduce brain edema, and reduce the content of brain inflammatory factors, and has good anti-stroke activity.

[0048] The present invention also provides an anti-stroke product, the active ingredient of which includes the anticoagulant polypeptide described in the above technical solution. Calculated by the dosage of the anticoagulant polypeptide, the dosage of the drug in the human body is 0.027 to 0.444 mg / kg, more preferably 0.05 to 0.2 mg / kg, and even more preferably 0.1 mg / kg.

[0049] To further illustrate the present invention, an anticoagulant polypeptide provided by the present invention and its application in the preparation of a plasma kallikrein activity inhibitor are described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0050] The materials used in the examples are as follows:

[0051] CW15: CTSVSLGASDPFDY (SEQ ID No. 1), commissioned by GLBiochem (China) to synthesize (purity 98%); SD6: SLGASD (SEQ ID No. 2), commissioned by GLBiochem (China) to synthesize (purity 98%); PKa: purchased from Enzyme Research, USA, HPKa1303.

[0052] Example 1

[0053] In order to detect the effects of SD6 and CW15 on PKa substrate hydrolysis, CW15-50μM, CW15-100μM, CW15-150μM, SD6-34μM, SD6-69μM, SD6-138μM, SD6-276μM7 and control groups were set up and treated as follows:

[0054] CW15-50 μM group: 50 μM CW15 was incubated with 35 nM PKa at 37°C for 3 min;

[0055] CW15-100 μM group: 100 μM CW15 was incubated with 35 nM PKa at 37°C for 3 min;

[0056] CW15-150 μM group: 150 μM CW15 was incubated with 35 nM PKa at 37°C for 3 min;

[0057] SD6-34 μM group: 34 μM SD6 was incubated with 35 nM PKa at 37°C for 3 min;

[0058] SD6-69 μM group: 69 μM SD6 was incubated with 35 nM PKa at 37°C for 3 min;

[0059] SD6-138 μM group: 138 μM SD6 was incubated with 35 nM PKa at 37°C for 3 min;

[0060] SD6-276 μM group: 276 μM SD6 was incubated with 35 nM PKa at 37°C for 3 min;

[0061] Control group: PBS and 35 nM PKa were incubated at 37°C for 3 min.

[0062] After the incubation, 50 μL of 30 nM PKa chromogenic substrate (S-2302; Chromogenix; USA) was added to 50 μL of each mixture, and the OD value was immediately measured using a microplate reader. 405nmKinetic monitoring was performed at the wavelength. Figure 1 A is the activity analysis diagram of CW15 on PKa, and B is the activity analysis diagram of SD6 on PKa.

[0063] To determine the SD6 inhibition constant, two 50 μL aliquots of the above mixture containing SD6 and the control solution were taken. 50 μL of 60 nM PKa chromogenic substrate was added to one aliquot and 50 μL of 30 nM PKa chromogenic substrate was added to the other aliquot. The OD values ​​were immediately measured using a microplate reader. 405nm After obtaining the maximum reaction rate automatically generated by the microplate reader activity analysis, the inhibition constant analysis was performed using the Dixon double reciprocal plot method. The concentration of the inhibitors (CW15 and SD6) was plotted on the x-axis and the reciprocal of the reaction rate was plotted on the y-axis to generate a Dixon calculation image, such as Figure 1 Shown in C is the Dixon calculation diagram of SD6 and PKa used to obtain the inhibition constant Ki.

[0064] Depend on Figure 1 It can be seen that the IC of CW15 50 The half-maximal inhibitory concentration (50%) was about 200 μM ( Figure 1 A), while the IC of SD6 50 The value is about 69μM ( Figure 1 (B) with a molecular weight of only 549 Da; this indicates that SD6 has better PKa inhibitory activity and a smaller molecular weight than CW15. In addition, Dixon diagram analysis showed that SD6 non-competitively inhibited PKa activity with an inhibition constant Ki value of 36.34 μM ( Figure 1 Middle C).

[0065] Example 2

[0066] PKa was immobilized on a CM5 chip (29149603, Cytiva, USA) with a level of 6000 RU. The mobile phase consisted of 0-600 μM SD6 at a flow rate of 30 μL / min and a binding time of 3 min. An increase in the RU value indicated that PKa bound to SD6. Figure 2 shown.

[0067] Depend on Figure 2 It can be seen that compared with the PBS control, the RU of SD6 at concentrations of 37.5μM, 75μM, 150μM, 300μM and 600μM increased by 7.6, 18.61, 41.33, 85.74 and 224.82, respectively. The association rate constant (Ka), dissociation rate constant (Kd) and equilibrium dissociation constant (KD) of the interaction between SD6 and PKa were 8×10 3 M -1 s -1 , 5.11×10-2 s -1 and 6.39 μM, showing a strong binding ability.

[0068] Example 3

[0069] The extrinsic and intrinsic coagulation pathways in the coagulation cascade are involved in hemostasis and thrombosis, respectively. In coagulation assays, PRT and APTT show intrinsic intrinsic coagulation activity, while prothrombin time (PT) measures extrinsic coagulation activity.

[0070] Fresh plasma was randomly divided into four treatment groups: 26 μM, 52 μM, 104 μM and 208 μM, and processed as follows:

[0071] 26 μM group: 10 μL of 26 μM SD6 and 20 μL of fresh plasma were mixed and added to a 96-well plate;

[0072] 52 μM group: 10 μL of 52 μM SD6 and 20 μL of fresh plasma were mixed and added to a 96-well plate;

[0073] 104 μM group: 10 μL of 104 μM SD6 and 20 μL of fresh plasma were mixed and added to a 96-well plate;

[0074] 208 μM group: 10 μL of 208 μM SD6 and 20 μL of fresh plasma were mixed and added to a 96-well plate;

[0075] Then, 50 μL of HEPES (pH 7.4) buffer solution containing 150 mM NaCl was added to each group and incubated at 37°C for 10 min;

[0076] After the incubation, 60 μL of HEPES buffer solution containing 25 mM CaCl2 was added to each group, and the kinetics were immediately monitored in a microplate reader at an absorbance of 650 nm for 30 min. The results are shown in Figure 2. Figure 3 As shown in Table 1, Med represents the solvent control group, the upper figure is the kinetic absorbance record value, which reflects the degree of blood coagulation; the lower figure is the quantitative data of the upper figure, which is expressed at the time point corresponding to the maximum reaction rate.

[0077] Table 1 Effects of SD6 on PRT in vitro

[0078]

[0079]

[0080] The effects of 26 μM SD6, 52 μM SD6, 104 μM SD6 and 208 μM SD6 on APTT were determined according to the commercial kit (GMS10178.2, GenMed, USA) and the instructions provided therein. Figure 4 As shown in Table 2, Med represents the solvent control group, the upper figure is the kinetic absorbance record value, which reflects the degree of blood coagulation; the lower figure is the quantitative data of the upper figure, expressed at the time point corresponding to the maximum reaction rate.

[0081] Table 2 Effects of SD6 on APTT in vitro

[0082] Group average value Number of cases Standard Deviation Standard error of the mean Med 23.00 3 1.73 1.00 26 μM group 41.33 3 1.53 0.88 52 μM group 34.33 3 1.15 0.67 104 μM group 30.00 3 1.00 0.58 208 μM group 25.33 3 2.51 1.45 total 30.80 15 6.93 1.79 - sum of squares degrees of freedom mean square F Between groups 644.40 4 161.10 57.54 Within the group 28.00 10 2.80 / total 672.40 14 / / Significance 0.0001 / / /

[0083] The effects of 52 μM SD6, 104 μM SD6, and 208 μM SD6 on PT were determined according to the commercial kit (STY50101, Steelex, China) and the instructions provided. Figure 5 As shown in Table 3, Med represents the solvent control group, the upper figure is the kinetic absorbance record value, which reflects the degree of blood coagulation; the lower figure is the quantitative data of the upper figure, expressed at the time point corresponding to the maximum reaction rate.

[0084] Table 3 Effects of SD6 on PT in vitro

[0085]

[0086]

[0087] Depend on Figures 3 to 5 As shown in Tables 1 to 3, the PRT values ​​of the solvent control group (Med) ( Figure 3 ) was 234±4.04s, while the PRT value of the 208μM SD6-treated group was 406.3±6.44s; the APTT value of the solvent control group (Med) ( Figure 4 ) was 23±1s, while the PRT value of the 208μM SD6-treated group was 41.33±0.88s; the PT value of the solvent control group (Med) ( Figure 5 ) was 30.67±0.33s, while the PRT value of the 208μM SD6-treated group was 30±0.58s; it can be seen that SD6 prolonged the PRT (F 4，10 =133.87, p<0.0001) and APTT (F 4，10 =57.54, p<0.0001), but had no effect on PT (F 3，8 =0.60, p=0.63), indicating that SD6 inhibits the intrinsic coagulation pathway and has the potential not to affect bleeding.

[0088] Example 4

[0089] To evaluate the effect of SD6 on hemolysis, fresh blood samples were centrifuged at 3500 rpm for 10 min at room temperature. The supernatant was discarded and the obtained red blood cells were divided into 7 groups: positive control (Triton-X100) group, negative control (Control) group, SD6-5 group, SD6-10 group, SD6-20 group, SD6-40 group and SD6-80 group. The red blood cells in each group were 1×10 7 Cells were resuspended in 0.01 M PBS (pH 7.2) at a concentration of 10 cells / mL and treated as follows:

[0090] Positive control group (Triton-X100): incubate the resuspended red blood cells with the positive control Triton-X100 (0.1% v / v) at 37°C for 30 min;

[0091] Negative control group (Control): 200 μL of resuspended red blood cells were incubated with 20 μL of PBS at 37°C for 30 min;

[0092] SD6-5 group: resuspended red blood cells were incubated with 5 μg / mL SD6 at 37°C for 30 min;

[0093] SD6-10 group: resuspended red blood cells were incubated with 10 μg / mL SD6 at 37°C for 30 min;

[0094] SD6-20 group: resuspended red blood cells were incubated with 20 μg / mL SD6 at 37°C for 30 min;

[0095] SD6-40 group: resuspended red blood cells were incubated with 40 μg / mL SD6 at 37°C for 30 min;

[0096] SD6-80 group: The resuspended erythrocytes were incubated with 80 μg / mL SD6 at 37°C for 30 min.

[0097] After the incubation, each group of cells was centrifuged at 3500 rpm for 5 min, and then the light absorbance of the supernatant was measured at a wavelength of 540 nm using an enzyme-labeled instrument. The results are shown in Figure 2. Figure 6 and as shown in Table 4.

[0098] Table 4 Effect of SD6 on cytotoxicity

[0099]

[0100]

[0101] Effect of SD6 on HEK293T cell proliferation

[0102] HEK293T cells (1.5 × 10 4 After culturing at 37°C in a 5% CO2 incubator for 12 hours, the HEK293T cells were divided into seven groups: a positive control (10% v / v DMSO), a negative control (Control), SD6-5, SD6-10, SD6-20, SD6-40, and SD6-80. The cells were then treated as follows:

[0103] Positive control group (10% DMSO): 10% DMSO was added to the cultured HEK293T cells and cultured in a 37°C 5% CO2 cell culture incubator for 10 h;

[0104] Negative control group (Control): Add 20 μL PBS to the cultured HEK293T cells and culture in a 37°C 5% CO2 cell culture incubator for 10 h;

[0105] SD6-5 group: 5 μg / mL SD6 was added to the cultured HEK293T cells and cultured in a 37°C 5% CO2 cell culture incubator for 10 h;

[0106] SD6-10 group: 10 μg / mL SD6 was added to the cultured HEK293T cells and cultured in a 37°C 5% CO2 cell culture incubator for 10 h;

[0107] SD6-20 group: 20 μg / mL SD6 was added to the cultured HEK293T cells and cultured in a 37°C 5% CO2 cell culture incubator for 10 h;

[0108] SD6-40 group: 40 μg / mL SD6 was added to the cultured HEK293T cells and cultured in a 37°C 5% CO2 cell culture incubator for 10 h;

[0109] SD6-80 group: 80 μg / mL SD6 was added to the cultured HEK293T cells and cultured in a 37°C 5% CO2 cell culture incubator for 10 h.

[0110] Afterwards, 10 μL of CCK-8 reagent was added to each group of cells and cultured for another 3 h. Cell viability was counted and expressed as a percentage relative to the control group. Figure 7 and as shown in Table 5.

[0111] Table 5 Effect of SD6 on HEK293T cell proliferation

[0112]

[0113]

[0114] Effects of SD6 on the proliferation of mouse spleen cells

[0115] Primary mouse splenocytes (1.5×10 5 Cells / well) were seeded into 96-well plates. After culturing for 12 hours in a 37°C 5% CO2 cell culture incubator, the cultured primary mouse splenocytes were divided into seven groups: positive control (10% DMSO), negative control (Control), SD6-5, SD6-10, SD6-20, SD6-40, and SD6-80. They were then treated as follows:

[0116] Positive control group (10% DMSO): 10% DMSO was added to the cultured primary mouse spleen cells and cultured in a 37°C 5% CO2 cell culture incubator for 10 h;

[0117] Negative control group (Control): Add 20 μL PBS to the cultured primary mouse spleen cells and culture in a 37°C 5% CO2 cell culture incubator for 10 h;

[0118] SD6-5 group: 5 μg / mL SD6 was added to the cultured primary mouse spleen cells and cultured in a 37°C 5% CO2 cell culture incubator for 10 h;

[0119] SD6-10 group: 10 μg / mL SD6 was added to the cultured primary mouse spleen cells and cultured in a 37°C 5% CO2 cell culture incubator for 10 h;

[0120] SD6-20 group: 20 μg / mL SD6 was added to the cultured primary mouse spleen cells and cultured in a 37°C 5% CO2 cell culture incubator for 10 h;

[0121] SD6-40 group: 40 μg / mL SD6 was added to the cultured primary mouse spleen cells and cultured in a 37°C 5% CO2 cell culture incubator for 10 h;

[0122] SD6-80 group: 80 μg / mL SD6 was added to the cultured primary mouse spleen cells and cultured in a 37°C 5% CO2 cell culture incubator for 10 h.

[0123] After 10 h of treatment, 10 μL of CCK-8 reagent was added to each group of cells and cultured for another 3 h. Cell viability was counted and expressed as a percentage relative to the control group. The results are shown in Figure 8 and as shown in Table 6.

[0124] Table 6 Effects of SD6 on mouse spleen cell proliferation

[0125]

[0126]

[0127] Depend on Figures 6-8 As shown in Tables 4 to 6, compared with the PBS-treated group, 5-80 μg / mL SD6 had a significant effect on erythrocyte hemolysis (F 5，12 =1.243, p=0.349, Figure 6 ) and HEK293T cells (F 5，11 =1.918, p=0.171, Figure 7 ) and mouse spleen cells (F 5，12 =1.282, p=0.334, Figure 8 ) had no effect on proliferation.

[0128] Example 5

[0129] The bleeding risk of SD6 was assessed using a tail-docking bleeding mouse model: 8-week-old male C57BL / 6J mice were divided into five groups (positive control (Heparin-1000 U / kg), negative control (Control), SD6-0.25, SD6-1, and SD6-4), with six mice in each group. The mice were treated as follows:

[0130] Positive control (Heparin-1000U / kg) group: intravenous injection of 1000U / kg heparin;

[0131] Negative control (Control) group: intravenous injection of 200 μL PBS;

[0132] SD6-0.25 group: intravenous injection of 0.25 mg / kg SD6;

[0133] SD6-1 group: intravenous injection of 1 mg / kg SD6;

[0134] SD6-4 group: intravenous injection of 4 mg / kg SD6;

[0135] Five minutes after the injection, the tail of each mouse was cut off 2 mm from the tip of the tail and placed in 37°C preheated sterile saline. The time from bleeding to cessation of bleeding was observed and recorded. The results are shown in Table 1. Figure 9 and as shown in Table 7.

[0136] Table 7 Effect of SD6 on the bleeding time of mouse tail

[0137] Group average value Number of cases Standard Deviation Standard error of skewness Standard error of the mean Control 113.000 6 19.473 0.845 7.950 SD6-4 group 120.667 6 11.961 0.845 4.883 SD6-1 group 99.667 6 19.190 0.845 7.834 SD6-0.25 group 119.333 6 19.211 0.845 7.843 total 113.167 24 18.600 0.472 3.797 - sum of squares degrees of freedom mean square F Significance Between groups 1659.333 3 553.111 1.756 0.188 Within the group 6298.000 20 314.900 / / total 7957.333 23 / / /

[0138] Depend on Figure 9As shown in Table 7, the tail bleeding time of heparin-treated mice was significantly prolonged (201.5±18.81s) compared with the control mice (113±7.95s), but there was no significant difference in the tail bleeding time of mice treated with 0.25-4 mg / kg SD6 (F 3，20 =1.756, p=0.188), indicating that SD6 has a higher hemostatic safety compared with heparin, an anticoagulant positive drug targeting antithrombin III (AT-III).

[0139] Example 6

[0140] To evaluate the effects of SD6 on thrombosis, three different mouse models were used: a carrageenan-induced tail thrombosis model, a ferric chloride-induced carotid artery thrombosis model, and a Rose Bengal photochemically induced cerebral cortex thrombosis model. These models trigger thrombosis by inducing endothelial cell damage and inflammatory responses.

[0141] 1. Effect of SD6 on carrageenan-induced tail thrombosis in mice

[0142] Six-week-old male BALB / c mice were divided into five groups: positive control (Heparin-1000 U / kg), negative control (Control), SD6-0.25, SD6-1, and SD6-4, with six mice in each group. The mice were treated as follows:

[0143] Positive control (Heparin-1000 U / kg) group: 200 μL of 1% type I carrageenan (CC3171, Coolaber, China) dissolved in 0.9% saline was intraperitoneally injected, followed by an immediate intravenous injection of 1000 U / kg heparin sodium, supplemented every 12 h for a total of three injections;

[0144] Negative control group: 200 μL of 1% type I carrageenan (CC3171, Coolaber, China) dissolved in 0.9% saline was injected intraperitoneally, followed by immediate intravenous injection of PBS, which was replenished every 12 h for a total of three injections;

[0145] SD6-0.25 group: 200 μL of 1% type I carrageenan (CC3171, Coolaber, China) dissolved in 0.9% saline was injected intraperitoneally, followed by an immediate intravenous injection of 0.25 mg / kg SD6, supplemented every 12 h for a total of three injections;

[0146] SD6-1 group: 200 μL of 1% type I carrageenan (CC3171, Coolaber, China) dissolved in 0.9% saline was injected intraperitoneally, followed by an immediate intravenous injection of 1 mg / kg SD6, supplemented every 12 h for a total of three injections;

[0147] SD6-4 group: 200 μL of 1% type I carrageenan (CC3171, Coolaber, China) dissolved in 0.9% saline was injected intraperitoneally, followed by an immediate intravenous injection of 4 mg / kg SD6, supplemented every 12 h for a total of three injections;

[0148] Twelve hours after the last injection of each treatment group, the length of the tail thrombus of each mouse was observed and recorded. Figure 10 As shown in Table 8.

[0149] Table 8 Effects of SD6 on carrageenan-induced tail thrombosis in mice

[0150] Group average value Number of cases Standard Deviation Standard error of the mean Control 4.933 6 1.080 0.441 SD6-4 group 1.983 6 0.685 0.280 SD6-1 group 2.683 6 1.044 0.426 SD6-0.25 group 2.717 6 0.879 0.359 Heparin-1000U / kg 1.607 6 0.986 0.403 total 2.785 30 1.466 0.268 - sum of squares degrees of freedom mean square F Between groups 29.561 3 9.854 11.26 Within the group 17.498 20 0.875 / total 47.060 23 / / Significance 0.00015 / / /

[0151] Figure 10 The results in Table 8 showed that compared with the control group, intravenous injection of 0.25-4 mg / kg SD6 significantly reduced the length of the tail thrombus in mice in a dose-dependent manner (F 3，20 =11.26, p=0.00015). The reduction in the 4 mg / kg SD6 group was comparable to that in the 1000 U / kg heparin group (p=0.5).

[0152] 2. Effect of SD6 on Ferric Chloride-Induced Carotid Artery Thrombosis

[0153] Eight-week-old male C57BL / 6J mice were divided into five groups (6 mice each): a positive control (Heparin-1000 U / kg), a negative control (Control), an SD6-0.25 group, an SD6-1 group, and an SD6-4 group. After anesthesia, a midline incision was performed on the neck to expose and isolate the left common carotid artery (CCA). A 4 mm diameter light shield was placed beneath the artery to reduce background blood flow interference. Subsequently, the following procedures were performed:

[0154] Positive control (Heparin-1000U / kg) group: intravenous injection of 1000U / kg heparin sodium;

[0155] Negative control (Control) group: intravenous injection of PBS;

[0156] SD6-0.25 group: intravenous injection of 0.25 mg / kg SD6;

[0157] SD6-1 group: intravenous injection of 1 mg / kg SD6;

[0158] SD6-4 group: intravenous injection of 4 mg / kg SD6;

[0159] Two minutes after intravenous injection, a piece of filter paper with a diameter of 4 mm was taken from each mouse and soaked in 10% ferric chloride solution and placed on the artery to induce arterial wall lesions for 2 minutes; then the filter paper was removed, and the blood flow was measured and imaged using a real-time laser speckle perfusion (LSP) imaging system (RWD, China) and SIM BFI software (SIM BFI HRPro, Simopto, China). When the color of the blood vessels in the image changed from red to blue, thrombus occlusion was determined. The continuous recording time was fixed at 30 minutes. Mice with an operation time of more than 10 minutes or mice with severe surgical bleeding were excluded from further data analysis. The results are shown in Figure 2. Figure 11 As shown in Table 9, Figure 11 A is the LSP imaging result, and B is the quantitative image of the corresponding blood flow in Figure A.

[0160] Table 9 Blood flow of mouse common carotid artery model in 10 minutes

[0161] Group average value Number of cases Standard Deviation Standard error of the mean Control 32.515 6 16.922 6.909 SD6-0.25 group 29.999 6 8.984 3.668 SD6-1 group 13.132 6 9.545 3.897 SD6-4 group 9.991 6 6.340 2.588 Heparin-1000U / kg 6.553 6 5.015 2.047 total 18.438 30 14.447 2.638

[0162] Table 10 Blood flow of mouse common carotid artery model at 13 min

[0163]

[0164]

[0165] Depend on Figure 11 As shown in Tables 9 and 10, the reduction in blood flow in the carotid artery of mice injected intravenously with 1 mg / kg SD6 and 4 mg / kg SD6 at 10 and 13 minutes after ferric chloride induction was less than that in the control group ( Figure 11 A and B), indicating less thrombosis. The reduction rate of arterial thrombosis in mice treated with 4 mg / kg SD6 was also comparable to that in the heparin group ( Figure 11 (A, B).

[0166] 4. Effects of SD6 on photothrombosis in mouse cerebral cortex

[0167] Eight-week-old male C57BL / 6J mice were divided into five groups: positive control (Heparin-1000 U / kg), negative control (Control), SD6-0.25, SD6-1, and SD6-4, with six mice in each group. The mice were treated as follows:

[0168] Positive control (Heparin-1000U / kg) group: intravenous injection of 1000U / kg heparin, once every 12 hours, for a total of six injections;

[0169] Negative control group: intravenous injection of PBS 200 μL, once every 12 hours, for a total of six injections;

[0170] SD6-0.25 group: intravenous injection of 0.25 mg / kg SD6, once every 12 hours, for a total of six injections;

[0171] SD6-1 group: intravenous injection of 1 mg / kg SD6, once every 12 hours, for a total of six injections;

[0172] SD6-4 group: intravenous injection of 4 mg / kg SD6, once every 12 hours, for a total of six injections;

[0173] Twelve hours after the last injection (day 4), each mouse was intravenously injected with 50 mg / kg of Rose Bengal (330000, Sigma-Aldrich, USA); 1 hour later, the mice were anesthetized and the epidermis was incised along the midline from the eyes down to the neck to expose the skull. A 15.5 mW 562 nm yellow-green laser (R-LG561-100-A5, RWD, China) was used to irradiate the designated area for 6 minutes. On the second day of the CPS process, the skull was exposed again for LSP imaging to select mice with similar blood flow in the target irradiated brain area. The results are shown in Figure 2. Figure 12 As shown in Table 11, A is the LPS imaging result, and B is the LPS imaging blood flow quantitative graph.

[0174] Table 11 Effects of SD6 on photochemically induced cerebral cortical thrombosis in mice

[0175] Group average value Number of cases Standard Deviation Standard error of the mean Control 90.267 6 10.681 4.360 SD6-0.25 group 121.000 6 6.870 2.805 SD6-1 group 143.167 6 9.538 3.894 SD6-4 group 122.333 6 22.456 9.168 Heparin-1000U / kg 169.000 6 10.412 4.250 total 129.153 30 29.232 5.337

[0176] Depend on Figure 12 As shown in Table 11, severe thrombosis and brain tissue damage occurred on day 1 of photochemically induced thrombosis, likely due to brain inflammation and subsequent activation of the coagulation cascade, which hindered the repair of intracranial damage. However, SD6 and heparin sodium alleviated these effects and accelerated blood flow recovery on day 4. Although blood flow recovery on day 4 was improved in the SD6-treated group (121±2.8 for 0.25 mg / kg, 143.17±3.89 for 1 mg / kg, and 122.33±9.17 for 4 mg / kg) compared to the control group (90.27±4.36), recovery was even better in the heparin-treated group (1000 U / kg), at 169±4.25. Despite these comparisons, precise quantitative analysis to assess the effects on brain tissue recovery was not possible. Overall, SD6 demonstrated a positive effect in reducing thrombosis and the progression of thrombosis.

[0177] Example 7

[0178] Effects of SD6 on tMCAO-induced cerebral ischemia injury

[0179] (1) Eight-week-old male C57BL / 6J mice were divided into five groups: positive control (Ecallantide-4 mg / kg), negative control (Control), SD6-0.25, SD6-1, and SD6-4, with 6 to 8 mice in each group. Each mouse was anesthetized and fixed on a 37°C constant temperature pad. The right CCA, external carotid artery (ECA), and internal carotid artery (ICA) were dissected. The upper end of the right ECA was tied, and a suture (diameter 0.23±0.02 mm, CNONTECH, China) was passed down through the ECA and guided into the ICA to occlude the MCA. 50 minutes after MCAO, the mice were treated as follows:

[0180] Positive control (Ecallantide-4 mg / kg) group: intravenous injection of 4 mg / kg Ecallantide (MCE, USA);

[0181] Negative control (Control) group: intravenous injection of 200 μL PBS;

[0182] SD6-0.25 group: intravenous injection of 0.25 mg / kg SD6;

[0183] SD6-1 group: intravenous injection of 1 mg / kg SD6;

[0184] SD6-4 group: intravenous injection of 4 mg / kg SD6;

[0185] Ten minutes after intravenous injection, the suture was slowly withdrawn to allow blood reperfusion. If mice died within 24 hours after tMCAO or the operation lasted longer than 15 minutes, they were excluded from the endpoint analysis.

[0186] To evaluate neurological function and infarct size 24 hours after tMCAO, the mice were first subjected to Bederson score and grip strength test. Figure 13 As shown in Tables 12 and 13, A is the grip test result and B is the Bederson score result.

[0187] The Bederson neurological function scoring system is as follows: 0 points, no behavioral disorder; 1 point, inability to extend the right forelimb; 2 points, rotation to the right; 3 points, tilt to the right; 4 points, no spontaneous activity with impaired consciousness; 5 points, death.

[0188] Grip strength test: A grip dynamometer (DS2-50N, Sansbio, China) was used to test the grip strength according to the manufacturer's instructions.

[0189] Table 12 Grip test results

[0190] Group average value Number of cases Standard Deviation Standard error of the mean Control 98.667 6 19.253 7.860 SD6-4 group 160.625 8 28.086 9.930 SD6-1 group 154.625 8 30.425 10.757 SD6-0.25 group 140.286 7 34.067 12.876 Ecallantide-4mg / kg 168.375 8 29.554 10.449 total 147.108 37 36.082 5.932

[0191] Table 13 Bederson score

[0192]

[0193]

[0194] Figure 13 The results in Tables 12 and 13 show that compared with the control group (98.67±7.85gf), the grip strength of the mice in the 0.25mg / kg SD6-treated group (140.29±12.88gf), the 1mg / kg SD6-treated group (154.63±10.76gf), the 4mg / kg SD6-treated group (160.63±9.93gf), and the 4mg / kg Ecallantide-treated group (168.38±10.45gf) was significantly increased ( Figure 13 There was no significant difference between the low-dose (0.25 mg / kg) SD6 group and the high-dose (4 mg / kg) Ecallantide group (p=0.071), indicating that the efficacy was better than the positive control ( Figure 13 In addition, the Bederson neurological function scores of the mice in the 1mg / kg SD6 group and the 4mg / kg SD6 group (1.22±0.55 and 1.44±0.58, respectively) and the Bederson neurological function score of the mice in the 4mg / kg ecallantide group (1.33±0.58) were lower than those in the control group (3.33±0.5), indicating that the neurological function of the mice had recovered ( Figure 13 Middle B).

[0195] (2) After the Bederson score and grip strength test, the mice were killed and the brain sections were stained with 2,3,5-triphenyltetrazolium chloride (TTC). Specifically, after the mice were euthanized, the intact brain was immediately removed and placed in a -20°C environment for 3 minutes. Subsequently, 2 mm thick coronal sections were cut using a mouse brain slice mold (Harvard Apparatus, Holliston, MA, USA). These sections were stained with 2% TTC (T-8877, Sigma-Aldrich, USA) and imaged after incubation at 37°C for 20 minutes. The cerebral infarction volume was calculated using ImageJ software (National Institutes of Health, USA) according to the following formula. The results are shown in Figure 2. Figure 14 As shown in Figure 14.

[0196] Cerebral infarction volume (V) = Sd / T 2, where S represents infarct size, d represents thickness, and T represents linear magnification.

[0197] Table 14 Infarct size of tMCAO mice with cerebral ischemia-reperfusion

[0198] Group average value Number of cases Standard Deviation Standard error of the mean Control 32.968 6 12.679 5.176 SD6-4 group 11.438 8 6.167 2.180 SD6-1 group 17.051 8 6.040 2.135 SD6-0.25 group 23.179 7 10.424 3.940 Ecallantide-4mg / kg 15.604 8 8.536 3.018 Sham 4.591 7 1.776 0.671 total 16.931 44 11.457 1.727

[0199] Depend on Figure 14 As shown in Table 14, TTC staining of mouse brain sections revealed that the infarct area (white area) in the 4 mg / kg SD6-treated group (11.43% ± 2.18%) was not significantly different from the sham-operated group (4.59% ± 0.67%) (p = 0.112), and was more effective than the 4 mg / kg ecallantide-treated group (15.6% ± 3.02%), indicating that SD6 has a substantial protective effect on brain tissue.

[0200] (3) Another part of the brain tissue samples was taken to determine the brain water content by weighing the dry and wet weights or after homogenization, the IL-1β, IL-6 and TNF-α levels were analyzed by enzyme-linked immunosorbent assay (ELISA), and the blood-brain barrier function of tMCAO mice after SD6 injection was analyzed by Evans blue staining. Figures 15 to 17 As shown in Tables 15 to 17.

[0201] ELISA: ELISA detection of IL-6 (DG30062M), IL-1β (DG30045M), and TNF-α (DG30048M) were performed using commercial kits (Dogesce, China) according to the instructions.

[0202] Evans blue staining: To evaluate the protective effect of SD6 on the blood-brain barrier, mice were injected with 200 μL of 0.5% Evans blue staining solution (R20616, China Yuanye Biotechnology Co., Ltd.) immediately after tMCAO surgery and sacrificed 24 hours later. Brain slices were homogenized after imaging, and the supernatant was quantitatively analyzed at 650 nm using a microplate reader. The imaging results are shown in Figure 2. Figure 17 As shown in A, the results of quantitative analysis by enzyme marker at 650nm are as follows Figure 17 As shown in B.

[0203] Table 15 1L1b release in the brain of tMCAO mice

[0204] Group average value Number of cases Standard Deviation Standard error of the mean Control 125.167 6 14.508 5.923 SD6-4 group 84.900 8 8.291 2.931 SD6-1 group 86.950 8 9.144 3.233 SD6-0.25 group 88.257 7 16.382 6.192 Ecallantide-4mg / kg 100.775 8 15.919 5.628 Sham 60.250 6 15.317 6.253 total 90.960 43 22.146 3.377

[0205] Table 16 IL6 release in the brain of tMCAO mice

[0206] Group average value Number of cases Standard Deviation Standard error of the mean Control 182.000 6 31.061 12.681 SD6-4 group 91.500 8 28.097 9.934 SD6-1 group 80.500 8 17.881 6.322 SD6-0.25 group 115.143 7 16.382 6.192 Ecallantide-4mg / kg 100.775 8 15.919 5.628 Sham 60.250 6 15.317 6.253 total 90.960 43 22.146 3.377

[0207] Table 17 TNFα release in the brain of tMCAO mice

[0208] Group average value Number of cases Standard Deviation Standard error of the mean Control 149.750 6 21.538 8.793 SD6-4 group 54.750 8 14.587 5.157 SD6-1 group 71.250 8 18.889 6.678 SD6-0.25 group 84.429 7 32.121 12.141 Ecallantide-4mg / kg 117.125 8 30.736 10.867 Sham 24.200 6 12.982 5.300 total 83.249 43 44.787 6.830

[0209] Table 18 Evans blue staining of tMCAO mouse brain sections

[0210]

[0211] Table 19 Brain water content of tMCAO mice

[0212] Group average value Number of cases Standard Deviation Standard error of the mean Control 87.210 7 1.532 0.579 SD6-4 group 81.703 8 1.451 0.513 SD6-1 group 83.119 7 0.967 0.365 SD6-0.25 group 84.630 7 1.091 0.412 Ecallantide-4mg / kg 82.630 8 1.665 0.589 Sham 79.872 6 1.118 0.456 total 83.223 43 2.587 0.395

[0213] Depend on Figure 15 As shown in Tables 15 to 17, 24 hours after tMCAO surgery, the inflammatory factors IL-1β, IL-6, and TNF-α in the mouse brain increased, but after intravenous injection of SD6, the inflammatory factors IL-1β, IL-6, and TNF-α were significantly reduced; it can be seen that SD6 can also reduce tMCAO brain inflammation and brain edema and protect the blood-brain barrier.

[0214] Reperfusion injury exacerbates inflammation and may lead to further thrombosis. The mutual deterioration of inflammation and thrombosis in the brains of tMCAO mice exacerbates brain edema and BBB disruption. As a PKa activity inhibitor, SD6 has considerable therapeutic potential. Intravenous injection of 0.25, 1, and 4 mg / kg can reduce brain water content by 1.5, 2.3, and 4 times, respectively ( Figure 16 , Table 19); the amount of Evans blue staining decreased by 1.9, 4.6 and 15.4 times, respectively ( Figure 17 (A-B, Table 18). Brain water content (p = 0.18) and Evans blue staining (p = 0.52) in the 1 mg / kg SD6-treated group were comparable to those in the 4 mg / kg ecallantide-treated group. These findings suggest that SD6 protects mice from tMCAO by inhibiting inflammation and thrombosis, highlighting its potential as a therapeutic agent for ischemic stroke.

[0215] It can be seen from the above examples that CW15 and SD6 can effectively inhibit PKa activity, thereby achieving the effect of anti-thrombotic inflammation. In vitro, SD6 can prolong the plasma recalcification time and activated partial thromboplastin time, and has a specific inhibitory effect on the intrinsic coagulation pathway. Within the dosage range of 0.25 to 4 mg / kg, SD6 can reduce arterial thrombosis and cortical venous thrombosis in FeCl3-induced and photochemically induced mice, and does not affect bleeding. In addition, SD6 also reduces brain inflammatory damage and cerebral edema in the transient middle cerebral artery occlusion ischemic stroke mouse model. It shows that the anticoagulant polypeptide provided by the present invention has good anti-inflammatory thrombotic and stroke drug activity, and has low bleeding risk and low cytotoxicity, and is relatively safe.

[0216] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. An anticoagulant polypeptide, characterized in that The amino acid sequence of the anticoagulant polypeptide is shown as SEQ ID No. 2 or SEQ ID No.

1.

2. The functional application of the anticoagulant polypeptide according to claim 1 in the preparation of antithrombotic drugs.

3. Use of the anticoagulant polypeptide according to claim 1 in the preparation of anti-ischemic stroke drugs.

4. The use according to claim 3, characterized in that The ischemic stroke is one or more of thrombotic cerebral infarction, embolic cerebral infarction, lacunar cerebral infarction, multiple cerebral infarction and transient ischemic attack.

5. A drug for preventing ischemic stroke, characterized in that: The active ingredient of the drug includes the anticoagulant polypeptide according to claim 1. Calculated by the dosage of the anticoagulant polypeptide, the dosage of the drug in the human body is 0.027 to 0.444 mg / kg.

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