Plasmin-targeted complement C3 derived anticoagulant polypeptide and application thereof

Abstract

The invention provides plasmin-targeted complement C3 derived anticoagulant polypeptide and application thereof, and belongs to the technical field of biological medicine. The present invention provides a complement C3-derived anticoagulant polypeptide (C3-HK10) and a modified peptide (C3-moHK10), which reduce nerve injury by enhancing the activity of plasmin in degrading fibrin thrombus and promoting the production of matBDNF (matured brain-derived neurotrophic factor), thereby protecting a host from dual threats of inflammatory thrombosis and neurological dysfunction, and also provides a method for preparing the anticoagulant polypeptide (C3-HK10) and a modified peptide (C3-moHK10). And good safety is shown. In conclusion, it is found for the first time that the complement C3 polypeptides C3-HK10 and C3-moHK10 have the characteristic of promoting plasmin activity, have the functions of inhibiting inflammation, thrombus and ischemic cerebral apoplexy injury and relieving nerve injury, and have the potential of becoming anti-inflammatory, thrombus and stroke treatment and neurological function drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a complement C3-derived anticoagulant polypeptide that targets plasmin and its applications. Background Technology

[0002] Thrombosis is a major health threat worldwide. It is not only a common core cause of serious cardiovascular and cerebrovascular diseases such as myocardial infarction, ischemic stroke, and venous thromboembolism, but also accounts for a significant proportion of deaths globally. Meanwhile, serious bacterial infections are also a leading cause of death worldwide. It is noteworthy that the fatal risk of such infections is often not directly driven by the pathogen itself, but rather stems from a dysregulation of the host's own immune response. Their threat to patients' lives and health is comparable to that of thrombosis-related diseases.

[0003] Currently used antithrombotic treatments in clinical practice, such as tissue plasminogen activator (tPA), heparin, and hirudin, can effectively target and inhibit the coagulation cascade and platelet aggregation to a certain extent, thus exerting an antithrombotic effect. However, these therapies have significant limitations. On the one hand, they are generally closely related to the risk of perioperative bleeding, severely limiting their application in some high-risk patients. On the other hand, these therapies cannot fundamentally address the risk of thrombus reformation, highlighting the unresolved key mechanism of inflammation in antithrombotic therapy. In severe bacterial infections leading to immune dysregulation, the key pathological manifestation is precisely the synergistic effect between excessive inflammation and the prothrombotic cascade. This harmful interaction often ultimately leads to disseminated intravascular coagulation (DIC), significantly increasing the risk of multiple organ failure and even death. Although the clinical impact of DIC is extremely significant, the complex network of peptide mediators regulating the progression of this fatal disease remains insufficiently understood, and this gap in understanding severely hinders the development and application of effective treatment strategies. Summary of the Invention

[0004] This invention provides a complement C3-derived anticoagulant polypeptide that targets plasmin and its application. The complement C3-derived anticoagulant polypeptide can bind to plasmin and enhance plasmin activity, and counteract lethal immune thrombosis.

[0005] This invention provides a complement C3-derived anticoagulant polypeptide that targets plasmin, the complement C3-derived anticoagulant polypeptide comprising C3-HK10 and a polypeptide formed by partial amino acid mutations based on said C3-HK10; The amino acid sequence of C3-HK10 is as described in SEQ ID No. 1.

[0006] In one specific embodiment of the present invention, the polypeptide formed by partial amino acid mutation includes C3-moHK10, the amino acid sequence of which is shown in SEQ ID No. 2.

[0007] This invention also provides the application of the above-mentioned complement C3-derived anticoagulant polypeptide in the preparation of plasmin promoters.

[0008] This invention also provides the application of the above-mentioned complement C3-derived anticoagulant polypeptide in the preparation of anti-inflammatory drugs.

[0009] This invention also provides the application of the above-mentioned complement C3-derived anticoagulant polypeptide in the preparation of antithrombotic drugs.

[0010] This invention also provides the application of the above-mentioned complement C3-derived anticoagulant polypeptide in the preparation of anti-stroke drugs.

[0011] In one specific embodiment of the present invention, the stroke includes ischemic stroke or hemorrhagic stroke.

[0012] This invention also provides the application of the above-mentioned complement C3-derived anticoagulant polypeptide in the preparation of drugs for mental illnesses that target kinin-releasing enzymes and inflammatory pathways.

[0013] The present invention also provides the application of the above-mentioned complement C3-derived anticoagulant polypeptide in the preparation of vaccines with anticoagulant, anti-inflammatory, antithrombotic and stroke functions.

[0014] The present invention also provides a medicament comprising the above-mentioned complement C3-derived anticoagulant polypeptide or a pharmaceutically acceptable excipient; The drug has at least one of the following functions: targeting plasmin, enhancing plasmin activity, anticoagulation, anti-inflammation, antithrombosis, and anti-stroke.

[0015] Beneficial Effects: This invention, based on bat C3 homologous peptides, developed complement C3-derived anticoagulant peptides (C3-HK10) and modified peptides (C3-moHK10). In a mouse model of staphylococcal sepsis, both C3-HK10 and C3-moHK10 effectively reduced inflammation, thrombosis, and infection-related neuropsychiatric sequelae (such as depressive-like behavior). This invention reveals that C3-HK10 and C3-moHK10 exert a dual protective function through interaction with plasmin: enhancing the activity of plasmin in degrading fibrin thrombi and promoting the production of mature brain-derived neurotrophic factor (matBDNF) to reduce neurological damage, thereby protecting the host from the dual threats of inflammatory thrombosis and neurological dysfunction. Furthermore, experiments have confirmed that compared with traditional plasminogen activator tissue plasminogen activator (tPA), C3-HK10 and C3-moHK10 have a lower risk of bleeding, exhibiting superior safety characteristics, while maintaining comparable protective effects against ischemic brain injury. In summary, this invention is the first to discover that complement C3 polypeptides C3-HK10 and C3-moHK10 have the characteristic of promoting plasmin activity, and have the functions of inhibiting inflammation, thrombosis, ischemic stroke injury and reducing nerve damage, and have the potential to become anti-inflammatory, thrombotic, stroke treatment and neurofunctional drugs. Attached Figure Description

[0016] Figure 1 The SPR analysis results for determining the binding strength between C3-HK10 and Palmsin are shown in the figure. Figure 2 Figure 1 shows the enzyme activity analysis results for detecting the effect of C3-HK10 on plasmin-dependent fibrinolysis; Figure A: Effect of C3-HK10 on plasmin activity; Figure B: Fibrin clot dissolution rate. Figure 3 The results show the effects of C3-HK10 and C3-moHK10 on the activity of Pramsin. Figure 4 The figure shows the effect of C3-HK10 on the in vitro formation of MatBDNF; Figure 5 The thrombolytic effects of C3-HK10 and C3-moHK10 are shown in the diagram. Figure 6 Figure showing the effect of C3-HK10 and C3-moHK10 on tail hemorrhage time in mice; Figure 7 Immunoblot images to detect the effects of C3-HK10 and C3-moHK10 on the production of MatBDNF in the brain; Figure 8Figure 1 shows the results of C3-HK10 and C3-moHK10 reducing the cerebral ischemia-reperfusion injury in the tMCAO model. In the figure, A: muscle strength test; B: neurological behavioral score; C: Evans blue staining; D: cerebral hemorrhage area percentage; E: cerebral edema content; F: quantitative Evans blue staining; G: brain slice TTC staining; H: cerebral infarction volume. Figure 9 The figure shows the results of ELISA analysis of the effects of C3-HK10 and C3-moHK10 on reducing the release of inflammatory factors in the brain of tMCAO mice. In the figure, A and E: IL-6; B and F: IL-1β; C and G: CXCL8; D and H: LBP. Figure 10 The following figures show the effects of C3-HK10 and C3-moHK10 on systemic damage caused by MRSA. In the figure, A: weight change; B: survival rate; C: muscle strength test; D: social interaction test; E: forced swimming test; F: tail suspension test; G: liver, lung and other tissue samples stained with hematoxylin and eosin (H&E). Detailed Implementation

[0017] This invention provides a complement C3-derived anticoagulant polypeptide that targets plasmin, the complement C3-derived anticoagulant polypeptide comprising C3-HK10 and a polypeptide formed by partial amino acid mutations based on said C3-HK10; The amino acid sequence of C3-HK10 is as described in SEQ ID No. 1.

[0018] The present invention does not specifically limit the amino acid mutation sites and the mutated sequence in C3-HK10, as long as the mutation can still target Plasmin and enhance the activity of Plasmin in degrading fibrin thrombi. In one embodiment of the present invention, the polypeptide formed after mutation includes C3-moHK10, and the amino acid sequence of C3-moHK10 is shown in SEQ ID No. 2.

[0019] C3-HK10: His-Gln-Gln-Thr-Val-Thr-Ile-Pro-Pro-Lys (HQQTVTIPPK); C3-moHK10: His-Leu-Gln-Thr-Val-Thr-Ile-Pro-Pro-Lys (HLQTVTIPPK).

[0020] In one embodiment of the present invention, the interaction between C3-HK10 and Plamsin is analyzed using surface plasmon resonance (SPR). The results show that the equilibrium dissociation constant of C3-HK10 and Plamsin ( KDThe M value was 47.9 μM, indicating a strong targeting binding ability.

[0021] This invention also provides the application of the above-mentioned complement C3-derived anticoagulant polypeptide in the preparation of plasmin promoters.

[0022] In this embodiment of the invention, the effects of C3-HK10 and C3-moHK10 on plasmin-dependent fibrinolysis were analyzed using enzyme activity analysis. The results showed that C3-HK10 and C3-moHK10 could promote plasmin-dependent fibrinolysis. The effects of C3-HK10 and C3-moHK10 on the activity of Plamsin were analyzed using a chromogenic substrate method. The results showed that C3-HK10 and C3-moHK10 could significantly enhance the activity of Plamsin.

[0023] This invention also provides the application of the above-mentioned complement C3-derived anticoagulant polypeptide in the preparation of anti-inflammatory drugs.

[0024] In this embodiment of the invention, C3-HK10 and C3-moHK10 can also alleviate intracranial inflammation and cerebral edema in a transient middle cerebral artery occlusion (tMCAO) model and protect the blood-brain barrier. As a pramsin activity enhancer, C3-HK10 and C3-moHK10 have considerable therapeutic potential, significantly reducing brain water content and Evans blue staining after intravenous injection. This indicates that C3-HK10 and C3-moHK10 protect mice from the effects of tMCAO by inhibiting inflammation and thrombosis, highlighting their potential as therapeutic agents for ischemic stroke. ELISA analysis of the effects of C3-HK10 and C3-moHK10 on inflammatory factor levels showed that they could reduce the levels of CXCL8, IL-6, IL-1β, and LBP, and alleviate MRSA DNA-induced systemic and neuroinflammatory responses.

[0025] This invention also provides the application of the above-mentioned complement C3-derived anticoagulant polypeptide in the preparation of antithrombotic drugs.

[0026] The embodiments of the present invention demonstrate that both C3-HK10 and C3-moHK10 can promote thrombolysis, and that C3-HK10 and C3-moHK10 have the potential to reduce the risk of bleeding.

[0027] This invention also provides the application of the above-mentioned complement C3-derived anticoagulant polypeptide in the preparation of anti-stroke drugs.

[0028] The stroke described in this invention includes ischemic stroke or hemorrhagic stroke, wherein the ischemic stroke includes thrombotic cerebral infarction, embolic cerebral infarction, lacunar infarction, multiple cerebral infarctions, or transient ischemic attack. In this embodiment of the invention, a thrombotic-reperfusion stroke model (tMCAO) was used to evaluate the therapeutic potential of C3-HK10 and C3-moHK10 for stroke by occlusion of the middle cerebral artery in mice for 60 min using the suture occlusion method. The results showed that 1–4 mg / kg C3-HK10 and C3-moHK10 dose-dependently reduced the area of ​​cerebral ischemic infarction, brain water content, blood-brain barrier damage, and the content of inflammatory factors in the brain in tMCAO mice. Therefore, C3-HK10 and C3-moHK10 can alleviate the effects of stroke injury and reduce neurological damage.

[0029] This invention also provides the application of the above-mentioned complement C3-derived anticoagulant polypeptide in the preparation of drugs for mental illnesses that target kinin-releasing enzymes and inflammatory pathways.

[0030] In this embodiment of the invention, the effect of C3-HK10 on the in vitro generation of MatBDNF was detected by immunoblotting. The results showed that C3-HK10 could promote the in vitro generation of MatBDNF. The effects of C3-HK10 and C3-moHK10 on the generation of MatBDNF in the brain were detected by immunoblotting. The results showed that C3-HK10 and C3-moHK10 promoted the generation of MatBDNF in the brain and reduced neurological function damage.

[0031] The present invention also found through an exploratory experiment on the depressive phenotype that the C3-HK10 and C3-moHK10 can alleviate MRSA-induced systemic damage.

[0032] The present invention also provides the application of the above-mentioned complement C3-derived anticoagulant polypeptide in the preparation of vaccines with anticoagulant, anti-inflammatory, antithrombotic and stroke functions.

[0033] The present invention also provides a medicament comprising the above-mentioned complement C3-derived anticoagulant polypeptide or a pharmaceutically acceptable excipient; The drug has at least one of the following functions: targeting plasmin, enhancing plasmin activity, anticoagulation, anti-inflammation, antithrombosis, and anti-stroke.

[0034] The present invention does not impose any particular limitation on the dosage form of the drug; conventional drug dosage forms in the art can be used.

[0035] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, of a complement C3-derived anticoagulant polypeptide targeting plasmin and its applications, should not be construed as limiting the scope of protection of the present invention.

[0036] Example 1 1. Strength analysis of the binding between C3-HK10 and Plamsin The binding affinity of C3-HK10 to Plamsin was evaluated using SPR experiments: 10 μg of Plamsin (HPlasmin, Enzyme Research, USA) was immobilized on a chip at a level of 1000 RU, the mobile phase was 0–500 μM Plamsin, the flow rate was 20 μL / min, and the binding time was 50 s. An increase in the RU value indicates binding of C3-HK10 to Plamsin.

[0037] The results are as follows Figure 1 As shown, the equilibrium dissociation constant of the interaction between C3-HK10 and Pramsin ( KD The molecular weight (M) was 47.9 μM, indicating strong binding ability.

[0038] 2. Effects of C3-HK10 on plasmin-dependent fibrinolysis To investigate the plasmin-dependent fibrinolysis of C3-HK10, fibrin clots were formed in 96-well microplates by incubating 8 mM fibrinogen (F3879-250MG, Sigma-Aldrich, USA) and 5 nM thrombin (HT1002a, Enzyme Research, USA) in 100 µL buffer A (50 mM Tris-HCl, pH 7.4, 100 mM sodium chloride, 5 mM calcium chloride) at 25 °C. After the clot turbidity stabilized, 50 µL buffer A containing 50 nM plasmin and 0–120 µg / mL C3-HK10 was layered on the fibrin clot matrix. The kinetics of clot dissolution were monitored at 405 nm using a multimode microplate reader for 5 hours.

[0039] The results are as follows Figure 2 As shown, C3-HK10 promotes plasmin-dependent fibrinolysis.

[0040] 3. Effects of C3-HK10 and C3-moHK10 on Palmsin activity To investigate the effects of C3-HK10 and C3-moHK10 on the substrates hydrolyzed by Plamsin, 0–400 μg / mL of complement C3-derived peptide HK10 was first incubated with 50 nM plasmin (HPlasmin, Enzyme Research, USA) at 37°C for 5 min. Then, 50 μL of the chromogenic substrate HD-isoleucyl-L-prolyl-L-arginine-p-nitroaniline dihydrochloride (Chromogenix S-2288™, Diapharma, USA) of 200 μM Plamsin was added, and kinetics were immediately monitored at OD405 nm using a microplate reader.

[0041] To determine the activity intensity, the maximum enzyme reaction rate (Vt) is used. max Quantitative analysis revealed that the rate was derived by fitting the initial rate data to the Michaelis equation: V = V max [S] / (Km+[S]), where V is the observed reaction rate and [S] is the substrate concentration.

[0042] The results are as follows Figure 3 As shown, both C3-HK10 and C3-moHK10 can promote Palmsin activity.

[0043] 4. C3-HK10 promotes the in vitro generation of MatBDNF. To assess the effect of C3-HK10 on plasmin-mediated proBDNF cleavage, 0–120 μg / mL of C3-HK10 was incubated with mouse brain homogenate (10 mg / mL) in 0.01 M PBS for 3 h. Subsequently, 5× loading buffer (10 μL, CW0027A, CWBIO, China) was added to the reaction mixture at a 1:4 ratio (buffer to sample), followed by heating at 95 °C for 5 min. Then, 10 μL of each sample was subjected to 12% SDS-PAGE electrophoresis. Finally, MatBDNF (approximately 10 kDa) was detected by Western blotting using an anti-BDNF antibody (ab108319; Abcam, UK). β-actin was used as a loading control.

[0044] The results are as follows Figure 4 As shown, C3-HK10 promotes the conversion of ProBDNF to MatBDNF and promotes the in vitro generation of MatBDNF.

[0045] 5. C3-HK10 and C3-moHK10 promote thrombolysis To evaluate the thrombolytic effect of C3-HK10 and its variant C3-moHK10, venous blood samples were incubated at 37 °C for 24 hours to promote thrombus formation. After removing the supernatant, each tube was reweighed to determine the initial thrombus mass. Serum containing 0–100 μg / mL C3-HK10 or C3-moHK10 was added to the thrombus and incubated at 37 °C for 12 hours. After incubation, the supernatant was removed and the tubes were reweighed to quantify the degree of thrombolysis. The percentage of thrombolysis was calculated as (weight of dissolved thrombus / weight of initial thrombus) × 100%. Results are as follows: Figure 5 As shown, C3-HK10 and C3-moHK10 have the effect of promoting thrombolysis.

[0046] 6. C3-HK10 and C3-moHK10 did not affect the tail hemorrhage time in mice. A tail-hemorrhage mouse model was used to assess the hemorrhage risk of C3-HK10 and C3-moHK10. Eight-week-old male C57BL / 6J mice were intravenously injected with 1 mg / kg tPA (TP09775, GeneCodex, China), C3-HK10 (0~4 mg / kg), or C3-moHK10 (0~4 mg / kg) for 5 minutes. Then, the tail was cut off 2 mm from the tip and placed in preheated sterile saline at 37 ℃. The time from the onset of bleeding to the cessation of bleeding was observed and recorded.

[0047] The results are as follows Figure 6 As shown, compared with the control group mice (100.67±17.67 s), the tail bleeding time of tPA-treated mice was significantly prolonged (220.83±20.83 s), but there was no significant difference in tail bleeding time between mice treated with 0~4 mg / kg C3-HK10 and C3-moHK10, indicating that C3-HK10 and C3-moHK10 have higher hemostatic safety than tPA.

[0048] 7. C3-HK10 and C3-moHK10 promote the production of MatBDNF in the brain. To assess the effects of C3-HK10 and C3-moHK10 on MatBDNF production in the brain, brain homogenates were collected 1 hour after the hemorrhage time in a tail-cutting hemorrhage mouse model. The homogenates were prepared in PBS containing a mixture of protease inhibitors (EDTA-free, 100× dissolved in DMSO, HY-K0010, MedChemExpress, USA) for immunoblotting analysis to quantify MatBDNF levels. The brain homogenates were separated by 12% SDS-PAGE and then transferred to a polyvinylidene fluoride (PVDF) membrane by electrophoresis. The membrane was blocked for 1 hour at room temperature with TBST (50 mM Tris, 150 mM sodium chloride, 0.1% Tween-20) containing 10% bovine serum albumin (BSA), followed by incubation at 4°C for 16 hours with an anti-BDNF antibody. After washing with TBST, the membrane was incubated for 1 hour at room temperature with horseradish peroxidase (HRP)-labeled anti-rabbit secondary antibody (7074s, CST, USA). Subsequently, development was performed using an enhanced chemiluminescence kit (PA112, Tiangen, China), and imaging was performed using an ImageQuant LAS 4000 mini system (GE Healthcare, USA). Results are as follows: Figure 7 As shown, C3-HK10 and C3-moHK10 promote the generation of MatBDNF in the brain.

[0049] 8. C3-HK10 and C3-moHK10 reduce cerebral ischemia-reperfusion injury in the tMCAO model. To investigate the effects of C3-HK10 and C3-moHK10 on tMCAO cerebral ischemia-reperfusion injury, 8-week-old male C57BL / 6J mice were anesthetized with isoflurane (RWD, China) by inhalation and fixed on a 37 ℃ constant temperature pad for right middle cerebral artery (MCA) occlusion surgery (tMCAO). The simplified steps are as follows: dissection of the right CCA, external carotid artery (ECA), and internal carotid artery (ICA). The upper end of the right ECA was bound, and a suture (0.23±0.02 mm in diameter, CNONTECH, China) was passed downward through the ECA to guide it into the blood-brain barrier for protection. Immediately after tMCAO, mice were injected with 200 μL of 0.5% Evans blue staining solution (R2) into the ICA to occlude the MCA. Fifty minutes after tMCAO, mice were intravenously injected with 1 mg / kg tissue plasminogen activator (tPA), C3-HK10 (0–4 mg / kg), or C3-moHK10 (0–4 mg / kg). The suture was slowly withdrawn after another 10 minutes to allow for blood reperfusion. Mice with severe intraoperative hemorrhage or whose surgery lasted more than 15 minutes were excluded from endpoint analysis.

[0050] To assess neurological function and infarct size 24 hours after tMCAO surgery, mice were first assessed using the Bederson score and grip strength test. The mice were then euthanized, and brain sections were stained with 2,3,5-triphenyltetrazolium chloride (TTC) to evaluate infarct area. Additionally, another portion of brain tissue was weighed (both wet and dry) to determine brain water content.

[0051] The Bederson Neurological Assessment Criteria are as follows: 0 points, no behavioral impairment; 1 point, inability to extend the right forelimb; 2 points, rotation to the right; 3 points, tilting to the right; 4 points, no voluntary movement, accompanied by impaired consciousness; 5 points, death.

[0052] Grip test: The grip test was performed using a grip tester (DS2-50N, Sansbio, China) according to the manufacturer's instructions.

[0053] TTC staining: Immediately after euthanasia, the intact brain of mice was removed and placed at -20 °C for 3 min. Subsequently, 2 mm thick coronal sections were cut using a mouse brain section mold (Harvard Apparatus, Holliston, MA, USA). These sections were stained with 2% TTC (T-8877, Sigma-Aldrich, USA) and incubated at 37 °C for 20 min before imaging. Infarct volume was quantified using ImageJ software (NIH, USA) with the formula V = Sd / T. 2 Where S represents the infarct area, d represents the thickness, and T represents the linear magnification factor.

[0054] Evans blue staining: To assess the effects of C3-HK10 and C3-moHK10 on blood-brain barrier damage, mice were injected with 200 μL of 0.5% Evans blue staining solution (R20616, China Yuanye Biotechnology Co., Ltd.) immediately after tMCAO surgery. Neurological assessments were completed 24 h later, and mice were then sacrificed. Brain sections were imaged to quantify Evans blue extravasation (blood-brain barrier damage) and hemorrhage areas (hemorrhagic transformation). The ipsilateral hemisphere tissue was then weighed (wet weight), and the dry weight was obtained after drying for calculation of brain water content.

[0055] The results are as follows Figure 8 As shown, compared with the control group (89±34 gf), mice treated with 1, 2, and 4 mg / kg C3-HK10 and 1, 2, and 4 mg / kg C3-moHK10, and mice treated with 1 mg / kg tPA (140.56±51.56 gf) showed significantly increased grip strength. Figure 8 (A). Furthermore, there was no significant difference between the low-dose (1 mg / kg) C3-HK10 and C3-moHK10 groups and the (1 mg / kg) tPA group. p<0.001 indicates that the therapeutic effect is better than that of the positive control. Figure 8 (A). Furthermore, the Bederson neurological function scores of mice in the 1 (2.42±2.58), 2 (2.17±2.83), 4 (1.83±3.17) mg / kg C3-HK10 group and the 1 (2.25±2.75), 2 (1.75±3.25), 4 (1.25±2.75) mg / kg C3-moHK10 group, as well as the Bederson neurological function score of mice in the 1 mg / kg tPA group (2.42±2.58), were all lower than those in the control group (3.08±2.08), indicating that the mice's neurological function had recovered. Figure 8 (B)

[0056] The infarct area (white area) of TTC-stained brain sections from sacrificed mice showed ( Figure 8 The efficacy rates of C3-HK10 in the 1 mg / kg C3-HK10 treatment group (23.06%±9.94%), the 1 mg / kg C3-moHK10 treatment group (18.94%±12.66%), and the 1 mg / kg tPA positive drug treatment group (17.92%±9.92%) were all lower than those in the control group (39%±7.8%). Among them, the 4 mg / kg C3-moHK10 treatment group (11.65%±9.35%) was more effective than the 1 mg / kg tPA positive drug treatment group, indicating that C3-HK10 and C3-moHK10 have a substantial protective effect on brain tissue.

[0057] C3-HK10 and C3-moHK10 also alleviated intracerebral inflammation and cerebral edema in tMCAO mice and protected the blood-brain barrier. The mutual aggravation of inflammation and thrombosis in the MCAO mouse brain exacerbated cerebral edema and BBB disruption. As a pramsin activity enhancer, C3-HK10 and C3-moHK10 have considerable therapeutic potential; intravenous injection of 1, 2, and 4 mg / kg significantly reduced brain water content. p <0.001)( Figure 8 In the middle E), the Evans blue staining amount was significantly reduced ( p <0.001)( Figure 8 (CD and F). This indicates that C3-HK10 and C3-moHK10 protect mice from the effects of tMCAO by inhibiting inflammation and thrombosis, highlighting their potential as therapeutic agents for ischemic stroke.

[0058] 9. ELISA analysis of the effects of C3-HK10 and C3-moHK10 on inflammatory factor levels To investigate the effects of C3-HK10 and C3-moHK10 on inflammatory cytokine levels, genomic DNA was extracted from MRSA strain ATCC 33591 using a commercial kit (DP302-02, Tiangen, China). Eight-week-old male C57BL / 6J mice were intravenously injected with MRSA DNA at a dose of 50 ng per mouse, either alone or in combination with C3-HK10 or C3-438 moHK10, at doses of 2 mg / kg and 4 mg / kg, respectively. The control group did not receive DNA injection. Serum and brain homogenates (prepared with PBS, 10 mg / mL) were collected two hours after injection. The levels of CXCL8 (DG30028M, Dogesce, China), IL-6 (DG30062M, Dogesce, China), IL-1β (DG30045M, Dogesce, China), and LBP (DG95654Q, Dogesce, China) in these samples were quantitatively analyzed by enzyme-linked immunosorbent assay (ELISA). Results are as follows: Figure 9 As shown, C3-HK10 and C3-moHK10 can alleviate MRSADNA-induced systemic and neuroinflammatory responses.

[0059] 10. Effects of C3-HK10 and C3-moHK10 on systemic injury induced by MRSA To investigate the effects of therapeutic intervention on systemic damage induced by MRSA, mice were intraperitoneally injected with 1×10 6 CFU MRSA infection. Starting 2 hours post-infection, mice were administered intravenously daily with tPA (1 mg / kg), C3-HK10 (4 mg / kg), C3-moHK10 (4 mg / kg), or a PBS control. Survival and body weight were monitored for 14 days. Surviving mice were then subjected to behavioral tests for neurological and depressive-like phenotypes, including grip strength tests, social interaction tests, forced swimming tests, and tail suspension tests, with a 3-hour rest period between each assessment. On day 15, tissues (liver and lung) were harvested after cardiac PBS perfusion, fixed in 4% paraformaldehyde, and stained with H&E. Bederson neurological scores and grip strength tests were performed as described above.

[0060] The Social Interaction Test (SIT) consisted of two 2.5-minute phases. Mice were placed in an open area (45×45×45cm) with a visible and ventilated circular isolation cage (10 cm in diameter). In the first phase (without a target) and the second phase (with a target) containing an unfamiliar CD1 mouse, researchers measured the time mice spent in the interaction area surrounding the isolation cage for 2.5 minutes. Between the two phases, the mice were returned to their main cage for a 30-second rest. The time spent in the social area was analyzed using the SMART video tracking system v3.0.

[0061] Tail suspension test: The mouse's tail was fixed to a shelf 30 cm above the table and suspended. Video recording was conducted during a 6-minute test period (1 minute adaptation period, 5 minutes recording). The resting time was automatically determined using the SMART video tracking system v3.0.

[0062] Forced swimming test: Mice were placed in a round, transparent beaker with a diameter of 11.2 cm and a water depth of 11 cm for 6 minutes at room temperature (25°C). The resting time was automatically measured using the SMART video tracking system v3.0.

[0063] The results are as follows Figure 10 As shown, compared with mice injected with 1 mg / kg tPA, mice injected with 4 mg / kg C3-HK10 and C3-moHK10 experienced a slower daily weight loss. Figure 10 (Among A), the survival rate increases ( Figure 10 (B) Figure 10 The CF test in the middle refers to the GST, SIT, TST and FST behavioral tests performed sequentially on mice that survived 14 days after MRSA infection. Figure 10 Image G represents a representative H&E staining image of lung (lower) and liver (upper) tissues collected on day 15 after pericardial PBS perfusion. The image shows that MRSA infection leads to enlargement of alveolar spaces in the lungs and inflammatory infiltration and cellular vacuolation in the liver. Perfusion results show widespread microthrombi in the hepatic sinusoids of infected control mice, which decreased after peptide treatment.

[0064] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A complement C3-derived anticoagulant polypeptide targeting plasmin, characterized in that, The complement C3-derived anticoagulant polypeptide includes C3-HK10 and a polypeptide formed by partial amino acid mutations on the basis of C3-HK10; The amino acid sequence of C3-HK10 is as described in SEQ ID No.

1.

2. The complement C3-derived anticoagulant polypeptide according to claim 1, characterized in that, The polypeptide formed by partial amino acid mutation includes C3-moHK10, the amino acid sequence of which is shown in SEQ ID No.

2.

3. The use of the complement C3-derived anticoagulant polypeptide of claim 1 or 2 in the preparation of plasmin promoters.

4. The use of the complement C3-derived anticoagulant polypeptide of claim 1 or 2 in the preparation of anti-inflammatory drugs.

5. The use of the complement C3-derived anticoagulant polypeptide of claim 1 or 2 in the preparation of antithrombotic drugs.

6. The use of the complement C3-derived anticoagulant polypeptide of claim 1 or 2 in the preparation of anti-stroke drugs.

7. The application according to claim 6, characterized in that, The stroke includes ischemic stroke or hemorrhagic stroke.

8. The use of the complement C3-derived anticoagulant polypeptide of claim 1 or 2 in the preparation of a drug for mental illnesses targeting kallikrein and inflammatory pathways.

9. The use of the complement C3-derived anticoagulant polypeptide of claim 1 or 2 in the preparation of a vaccine with anticoagulant, anti-inflammatory, antithrombotic and stroke functions.

10. A drug, characterized in that, Includes the complement C3-derived anticoagulant peptide as described in claim 1 or 2, or a pharmaceutically acceptable excipient; The drug has at least one of the following functions: targeting plasmin, enhancing plasmin activity, anticoagulation, anti-inflammation, antithrombosis, and anti-stroke.

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