Use of a polypeptide in preventing or treating thrombotic diseases

Abstract

The present invention discloses the application of a polypeptide in the prevention or treatment of thrombotic diseases, belonging to the field of biomedicine. The amino acid sequence of the polypeptide is: SEQ ID NO.1 or SEQ ID NO.2. The polypeptide in the present invention can significantly inhibit thrombus formation, can be used as a drug for the prevention or treatment of thrombotic diseases, and has clinical application value.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and more particularly to the use of a polypeptide in the prevention or treatment of thrombotic diseases. Background Art

[0002] Vascular embolism diseases (thrombotic diseases) are common diseases that seriously endanger human health, characterized by high incidence, high fatality rate and high disability rate. For example, myocardial infarction, cerebral infarction, pulmonary infarction caused by vascular embolism rank first among various causes of death. Anticoagulants are one of the main drugs for the prevention and treatment of thrombotic diseases. Although traditional anticoagulant drugs such as heparin and warfarin have good anticoagulant and antithrombotic effects, they all have deficiencies such as bleeding risk, thrombocytopenia, and the need for monitoring during use. Therefore, it is of great practical significance to further develop new anticoagulant and antithrombotic drugs with high efficiency and low side effects.

[0003] The coagulation system of humans and mammals is a process in which a series of coagulation factors are successively activated and finally fibrin is formed. Therefore, by inhibiting the corresponding coagulation factor targets, the activation of the coagulation system can be blocked, thereby preventing the formation of fibrin. In recent years, great attention has been paid to the development of inhibitor drugs targeting key coagulation factor targets in the coagulation process, such as factor Xa (fXa) and thrombin. For example, newly marketed factor Xa inhibitors (such as rivaroxaban) and thrombin inhibitors (such as dabigatran and hirudin) have reduced the bleeding risk (see the literature: "New Anticoagulants for the Prevention and Treatment of Venous Thromboembolism", Joo Hee Kim et al., Biomol Ther (Seoul), Vol. 25, No. 5, pp. 461-470, 2017). Recent studies have also shown that anticoagulant drugs targeting factor XIa (fXIa) have a lower bleeding risk than new drugs such as rivaroxaban and dabigatran (see the literature: "Emerging anticoagulant strategies", Fredenburgh JC et al., Blood, Vol. 129, No. 2, pp. 147-154, 2017). Although there is currently no new anticoagulant drug targeting fXIa entering clinical application, the research and development of inhibitors that can act on the fXIa target as new anticoagulant and antithrombotic drugs have recently received great attention. Summary of the Invention

[0004] The problem to be solved by the present invention is to provide a polypeptide drug with significant anticoagulant and antithrombotic effects for preventing or treating thrombotic diseases.

[0005] For preventing and treating thrombotic diseases, the present invention provides the pharmaceutical use of a polypeptide - NKI12, or its mutants, in thrombotic diseases. The amino acid sequence of NKI12 is: SEQ ID NO.1, or its mutants, such as the amino acid sequence: SEQ ID NO.2.

[0006] The clotting time experiment shows that NKI12 can significantly prolong the activated partial thromboplastin time (aPTT), and also has a certain effect on prolonging the prothrombin time (PT). The in vitro enzyme activity experiment shows that NKI12 mainly has a strong inhibitory effect on activated coagulation factor XI (fXIa), and also has a certain inhibitory effect on fXa.

[0007] The present invention has confirmed through animal experiments that NKI12 has an obvious effect of reducing the formation of various thrombi. Therefore, this polypeptide may be used as a drug for preventing or treating thrombotic diseases.

[0008] The present invention has confirmed through animal experiments that NKI12 has less impact on the normal hemostatic function of animals, indicating that the bleeding risk is relatively small when NKI12 is used as a drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The following drawings are only intended to illustrate and explain the present invention schematically and do not limit the scope of the present invention.

[0010] Figure 1 Shown is the diagram of the inhibitory effect of NKI12 on the activity of coagulation factors. Among them, the concentrations of activated coagulation factor XI (fXIa), Xa (fXa), IIa (fIIa), XIIa (fXIIa) and plasma kallikrein (PK) are all 1 nmol / L; the concentration of fVIIa in the activated coagulation factor VII - tissue factor complex (fVIIa / TF) is 5 nmol / L, and TF is 200 nmol / L. Except that the concentration of NKI12 inhibiting fVIIa / TF is 500 nmol / L, the concentration of NKI12 acting on other coagulation factors is 100 nmol / L. Inhibition rate = (V0 - V) / V0×100%, where V0 is the reaction rate of the control group and V is the reaction rate after adding NKI12. Each group is tested in 3 parallel experiments and the average value is taken. DETAILED DESCRIPTION OF THE INVENTION

[0011] In order to have a clearer understanding of the technical features, objectives and effects of the present invention, the specific embodiments of the present invention are now described. The following specific examples are only used to illustrate the present invention and do not limit the scope of the present invention.

[0012] Necator americanus is a blood-sucking parasite that parasitizes the human intestine. The inventor isolated the gene sequence of Necator americanus, obtained and confirmed through a series of experiments that a polypeptide of Necator americanus - NKI12 has good anticoagulant and antithrombotic effects, and has little effect on the hemostatic function of experimental animals. Therefore, NKI12 can be used as a drug for the prevention or treatment of thrombotic diseases, and has the characteristic of a relatively small bleeding risk.

[0013] Example 1 Anticoagulant activity of the polypeptide of the present invention

[0014] The polypeptides in this example, such as NKI12 (SEQ ID NO.1), or NKI12-1 (SEQ ID NO.2), were recombinantly prepared by the inventor using a prokaryotic expression system, with a purity greater than 95%. Specifically, the nucleic acid sequence encoding the target polypeptide was ligated into the prokaryotic expression vector pET32a-sumo. The recombinant plasmid with correct sequencing was transferred into Escherichia coli BL21(DE3). IPTG was used for induction expression. After the host bacteria were separated and ultrasonically disrupted, the expression product was purified by nickel affinity chromatography to obtain a fusion protein. The fusion partner was cleaved by SUMO protease, and the purified recombinant polypeptide NKI12 (rNKI12) was further obtained by affinity chromatography and ion exchange chromatography. The anticoagulant activity of the polypeptides in the present invention was observed by detecting the activated partial thromboplastin time (aPTT) and prothrombin time (PT).

[0015] Determination of activated partial thromboplastin time (aPTT): In a 96-well plate, 10 μl of the polypeptide of the present invention at a certain concentration (normal control group added with physiological saline) and 50 μl of normal human plasma were taken and mixed evenly. Then 20 μl of aPTT reagent (HYPHENBioMed, France) was added. After mixing, it was incubated at 37°C for 15 min. Then 20 μl of 0.05 mol / L CaCl 2 solution was added. The blood coagulation process was monitored with an enzyme-linked immunosorbent assay (ELISA) reader Elx808IU at absorbance A 630 , and the aPTT value was calculated. Each concentration was detected 3 times repeatedly.

[0016] Determination of prothrombin time (PT): In a 96-well plate, 10 μl of the polypeptide of the present invention at a certain concentration (normal control group added with physiological saline) and 45 μl of normal human plasma were added. After mixing, it was incubated at 37°C for 15 min. Then 45 μl of PT reagent (SIEMENS, Germany) was added. The blood coagulation process was monitored with an enzyme-linked immunosorbent assay (ELISA) reader Elx808IU (BioTek, United States) at absorbance A 630 , and the coagulation time was calculated, which was the PT value. Each concentration was detected 3 times repeatedly.

[0017] The experimental results are shown in Table 1: the aPTT and PT of the normal control group were 43.83±3.47 and 12.30±0.80 sec, respectively. The polypeptides in the present invention can significantly prolong aPTT. For example, NKI12 and NKI12-1 at 200 nmol / L can prolong the aPTT of normal human plasma to 147.0±6.7 and 143.8±5.9 sec, respectively, and the extension multiples are about 3.36 and 3.28 times, respectively. Although the polypeptides in the present invention have a certain effect of prolonging PT, the activity is relatively weak. For example, NKI12 and NKI12-1 at 200 nmol / L can prolong the PT of normal human plasma to 15.7±0.6 and 15.3±0.6 sec, respectively. The results show that NKI12 and NKI12-1 have strong anticoagulant effects, have a significant effect on prolonging aPTT, and have a weak activity on prolonging PT.

[0018] Table 1 Effects of the polypeptides of the present invention on aPTT and PT (n = 3)

[0019]

[0020] * Compared with the normal control group, P<0.01;

[0021] Example 2 Inhibitory effect of the polypeptides of the present invention on coagulation factors

[0022] The preparation method of the polypeptides in this example is the same as that in Example 1. The chromogenic substrate method was used to detect the effects of each anticoagulant peptide on coagulation factors. Human coagulation factor IIa, Xa, XIa, XIIa, and EGR-fXa were purchased from Haematologic Technologies Inc., USA. Human plasma kallikrein (PK) was a product of Enzyme Research Laboratories, USA. Recombinant fVIIa was a product of Novo-Nordisk (Denmark). rsTF was prepared by the research group of the inventor. The chromogenic substrate for coagulation factor Xa was S2765, the chromogenic substrates for coagulation factors VIIa and IIa were S2288, the chromogenic substrates for coagulation factors XIa and XIIa were S2366, and the chromogenic substrate for coagulation factor Xa was S2302. The substrates were all products of Chromogenix (Italy).

[0023] Take a 96-well enzyme-linked immunosorbent assay (ELISA) plate with a reaction system of 100 μl per well. 50 μL of thrombin (Factor IIa), Factor Xa, Factor XIa, Factor XIIa, and prekallikrein (PK) (each with a final concentration of 1 nmol / L) are respectively mixed with 10 μL of the corresponding different concentrations of the polypeptide of the present invention (or the PBS control group). (When observing the inhibitory effect on fVIIa / TF, use 5 nmol / L fVIIa + 200 nmol / L rsTF + 500 nmol / L EGR-fXa). After incubating at 25 °C for 15 min, add 40 μL of the chromogenic substrate (final concentration of 400 μmol / L). Use an Elx808IU (BioTek, United States) to scan and detect the enzyme reaction kinetics at absorbance A 405 and read the plate once every 14 seconds for 5 min to record the enzyme reaction rate and calculate the inhibition rate. The inhibition rate = (V0 - V) / V0 × 100%, where V0 is the reaction rate of the control group and V is the reaction rate after adding NKI12. Each group is tested in parallel 3 times and the average value is taken.

[0024] The experimental results show that NKI12 has a strong inhibitory effect on Factor XIa (fXIa). The polypeptide at a concentration of 100 nmol / L can inhibit approximately 90% of the activity of fXIa (1 nmol / L), 50% of the activity of fXa (1 nmol / L), and 10% of the activity of plasma prekallikrein PK (1 nmol / L), but has no obvious inhibitory effect on fVIIa / TF, fIIa, and fXIIa ( Figure 1 ), and the inhibitory effect of NKI12-1 on each coagulation factor is similar. The results indicate that NKI12 mainly has a strong inhibitory effect on fXIa.

[0025] Example 3 Effect of the polypeptide of the present invention on thrombus formation in rats

[0026] The method for preparing the polypeptide in this example is the same as that in Example 1. Take 40 SD rats (experimental animal certificate: SCXK (Yue) 2013-0008; SYXK (Yue) 2015-0147), weighing 180 - 220 g, and randomly divide them into a normal saline control group, a positive control group (low molecular weight heparin calcium, 260 U / kg, product of Guangdong Tianpu Biochemical Pharmaceutical Co., Ltd.), a low-dose group of NKI12 (100 μg·kg -1 ), a medium-dose group (200 μg·kg -1 ), and a high-dose group (400 μg·kg -1) Rats were anesthetized by intraperitoneal injection of 2% sodium pentobarbital, fixed in the supine position, and the trachea was intubated. The right common carotid artery and the left external jugular vein were surgically dissected. Inside a siliconized polyethylene tube with an inner diameter of 1.5 mm, a length of 22 cm, and filled with heparinized saline at 50 U / mL, a 4-0 surgical suture with a length of 5 cm was placed. One end of the polyethylene tube was inserted into the right common carotid artery, and the other end was inserted into the left external jugular vein. Then, the corresponding drugs were administered via the tail vein. 5 minutes after drug administration, the arterial clamp was opened, and the blood flowed from the right common carotid artery through the polyethylene tube back to the left external jugular vein. After 15 minutes of blood flow, the blood flow was interrupted, and the silk thread was quickly removed and weighed. The total weight minus the weight of the silk thread was the wet mass of the thrombus; Inhibition rate = (wet mass of thrombus in the normal saline control group - wet mass of thrombus in the drug administration group) / wet mass of thrombus in the normal saline control group × 100%.

[0027] The experimental results are shown in Table 2. Compared with the normal saline control group, all doses of NKI12 could significantly inhibit thrombus formation in rats (P < 0.01), and compared with low molecular weight heparin, the medium and high dose groups of NKI12 also had significant antithrombotic effects. The results indicate that NKI12 has a strong effect on inhibiting thrombus formation.

[0028] Table 2 Effects of the polypeptide of the present invention on arteriovenous bypass thrombosis in rats

[0029]

[0030] *Compared with the normal saline control group, P < 0.01

[0031] #Compared with the low molecular weight heparin group, P < 0.01

[0032] Example 4 Effects of the polypeptide of the present invention on thrombosis in the common carotid artery of rats

[0033] The preparation method of the polypeptide NKI12 in this example was the same as that in Example 1. 40 SD rats (animal experimental certificate: SCXK(Guangdong)2013 - 0008; SYXK(Guangdong)2015 - 0147), with a body weight of 180 - 220 g, were randomly divided into 5 groups, namely the sham operation group (except not using FeCl 3 , the other steps were the same as those in other groups), the model group, and the low, medium, and high dose groups of NKI12. After the rats were anesthetized by intraperitoneal injection of 2% sodium pentobarbital, an incision was made along the midline of the neck, and the right carotid artery was bluntly dissected for 1 cm in length. A sealing strip with a width of 0.6 cm was placed. 5 minutes after drug administration, a cotton ball soaked in 15% FeCl 3A filter paper (1.0 cm × 0.5 cm) of the solution was wrapped around the isolated common carotid artery segment for standby, and it was sealed with a sealing tape. After 15 min, the filter paper strip was removed. After 40 min, the blood vessels at both ends of the filter paper strip were ligated, the blood vessel segment wrapped by the filter paper strip was precisely cut, the remaining blood in the blood vessel was blotted dry with a clean filter paper, the wet weight of the blood vessel containing the thrombus was precisely weighed, and the blood vessel after removing the thrombus was weighed again. The difference between the two was the mass of the thrombus in the 0.5 cm long blood vessel segment. The sham operation group used normal saline instead of FeCl 3 The soaked filter paper strip. Inhibition rate = (wet weight of thrombus in the model group - wet weight of thrombus in the drug administration group) / wet weight of thrombus in the model group × 100%.

[0034] The experimental results are shown in Table 3. Compared with the model group, all doses of the NKI12 group could significantly inhibit the formation of thrombus in the common carotid artery of rats (P < 0.01), showing a dose-dependent relationship. The results indicate that NKI12 has a strong effect on anti-arterial thrombus formation.

[0035] Table 3 Effects of NKI12 on the formation of thrombus in the common carotid artery of rats

[0036]

[0037] * Compared with the model group, P < 0.01

[0038] Example 5 Experiment on the anti-venous thrombus formation of the polypeptide of the present invention in rats

[0039] The preparation method of the polypeptide NKI12 in this example was the same as that in Example 1. Fifty SD rats (animal experiment certificate: SCXK(Yue)2013 - 0008; SYXK(Yue)2015 - 0147), with a body weight of 180 - 220 g, were randomly divided into 5 groups, namely the sham operation group (except not using FeCl 3 except, the remaining steps were the same as those of other groups), the model group, and the low, medium, and high dose groups of NKI12. The rats were anesthetized by intraperitoneal injection of 2% sodium pentobarbital, the inferior vena cava was isolated by laparotomy, a silk thread was placed under the left renal vein for ligating the blood vessel, the drug was administered by tail vein injection, the inferior vena cava was ligated after 5 min to cause congestion, then the abdominal cavity was closed, and the abdominal cavity was opened again after 4 h. The blood vessel was clamped 2.0 cm below the ligation, and the longitudinal section was examined for thrombus formation. The thrombus formation ratio of each group was calculated based on the model group.

[0040] The experimental results are shown in Table 4. All groups of NKI12 could significantly inhibit the formation of thrombus in the inferior vena cava of rats (P < 0.01), showing a dose-dependent relationship. The results indicate that NKI12 has a strong effect on anti-venous thrombus formation.

[0041] Table 4 Effects of NKI12 on the formation of thrombus in the inferior vena cava of rats

[0042]

[0043] * Compared with the model group, P < 0.01

[0044] Example 6 Effect of the polypeptide of the present invention on the tail bleeding time of mice

[0045] The method for preparing the polypeptide in this example is the same as that in Example 1. Forty Balb / c mice (Animal Experiment Certificate: SCXK(Yue)2013 - 0008; SYXK(Yue)2015 - 0147), half male and half female, weighing 18 - 22 g, were randomly divided into 8 groups, with 5 mice in each group, namely: normal saline control group, low, medium, and high dose groups of recombinant NKI12 and NKI12 - 1, and low molecular weight heparin calcium group (product of Guangdong Tianpu Biochemical Pharmaceutical Co., Ltd.). The Balb / c mice were anesthetized by intraperitoneal injection of 2% pentobarbital sodium, and then the polypeptides, low molecular weight heparin calcium (375 U / kg), and normal saline of the present invention were respectively administered by tail vein injection. 2 minutes after tail vein administration, the mice's tails were cut at 0.5 cm from the tip and placed in physiological saline at 37°C, and the time from cutting to bleeding stop was recorded as the bleeding time, that is, the tail bleeding time, expressed as mean ± standard deviation represented.

[0046] The results are shown in Table 6. Compared with the low molecular weight heparin group, the tail bleeding time of each dose group of NKI12 and NKI12 - 1 was significantly reduced (P < 0.05); compared with the normal saline control group, the low and medium dose groups of NKI12 and each dose group of NKI12 - 1 had no obvious effect on the bleeding time. The results indicate that when the polypeptide of the present invention is used as a drug, the bleeding risk is relatively small.

[0047] Table 6 Effect of the polypeptide of the present invention on the tail bleeding time of mice

[0048]

[0049] * Compared with the low molecular weight heparin group, P < 0.05

[0050] # Compared with the blank control group, P > 0.05

[0051] The above - mentioned is only the illustrative specific embodiment of the present invention, and is not used to limit the scope of the present invention. Any equivalent changes, modifications, and combinations made by those skilled in the art without departing from the concept and principles of the present invention shall fall within the scope of protection of the present invention.

[0052]

[0053] Sequence Listing <110> Guangdong Medical University <120> Application of a polypeptide in preventing or treating thrombotic diseases <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 80 <212> PRT <213> Necator americanus <400> 1 Gly Gly Asn Gly Pro Glu Val Cys Leu Leu Glu Pro Asp Glu Gly Gln 1 5 10 15 Cys Arg Gly Ile Phe Arg Arg Trp Ala Trp Asn Pro Ala Glu Glu His 20 25 30 Cys Glu Pro Phe Glu Tyr Gly Gly Cys Gly Gly Asn Gly Asn Asn Phe 35 40 45 Met Thr Gln Lys Gln Cys Leu Asp Glu Cys Trp Asn Lys His Val Asp 50 55 60 Lys Glu His Trp Lys Glu Pro Glu Pro Arg Cys Ile Lys Tyr Ile Lys 65 70 75 80 <210> 2 <211> 51 <212> PRT <213> Necator americanus <400> 2 Cys Leu Leu Glu Pro Asp Glu Gly Gln Cys Arg Gly Ile Phe Arg Arg 1 5 10 15 Trp Ala Trp Asn Pro Ala Glu Glu His Cys Glu Pro Phe Glu Tyr Gly 20 25 30 Gly Cys Gly Gly Asn Gly Asn Asn Phe Met Thr Gln Lys Gln Cys Leu 35 40 45 Asp Glu Cys 50

Claims

1. Use of a polypeptide in the preparation of a medicament for preventing or treating thrombotic diseases, wherein the amino acid sequence of the polypeptide is as shown in SEQ ID NO.2.

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