APPLICATION OF ANTI-PLATELET THROMBOLYSIN IN PREPARING DRUG FOR TREATING ANEMIA

Abstract

A method of preparing a medicament for a treatment of anemia includes the step of using an antiplatelet thrombolysin in preparing the medicament, and the antiplatelet thrombolysin has an activity of improving the anemia. The antiplatelet thrombolysin consists of two peptide chains of α chain and β chain, in which the α chain amino acid sequence is shown in SEQ ID NO: 1, the β chain amino acid sequence is shown in SEQ ID NO: 2; or the antiplatelet thrombolysin is derived from the α chain amino acid sequence by substitution, deletion, or addition of one or more amino acids and has at least 95% identity with SEQ ID NO: 1; or the antiplatelet thrombolysin is derived from the β chain amino acid sequence by substitution, deletion, or addition of one or more amino acids and has at least 95% identity with SEQ ID NO: 2.

Claims

1. A method of preparing a medicament for a treatment of anemia, comprising the step of using an antiplatelet thrombolysin in preparing the medicament, wherein the antiplatelet thrombolysin consists of two peptide chains of α chain and β chain, and the antiplatelet thrombolysin has an activity of improving the anemia, wherein the amino acid sequence of the α chain is shown in SEQ ID NO: 1, the amino acid sequence of the β chain is shown in SEQ ID NO: 2; or the antiplatelet thrombolysin is derived from the amino acid sequence of the α chain by substitution, deletion, or addition of one or more amino acids and has at least 95% identity with SEQ ID NO: 1, or the antiplatelet thrombolysin is derived from the amino acid sequence of the β chain by substitution, deletion, or addition of one or more amino acids and has at least 95% identity with SEQ ID NO: 2.

2. The method according to claim 1, wherein the anemia is sickle cell anemia.

3. The method according to claim 1, wherein the medicament is a chemical drug or a biological preparation.

4. The method according to claim 1, wherein the medicament comprises a pharmaceutically acceptable excipient.

5. The method according to claim 1, wherein the medicament is an oral preparation or an injection.

6. The method according to claim 5, wherein the oral preparation is a tablet, a capsule, a pill, a granule, a dripping pill, a microcapsule or a pellet.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0024] FIG. 1 shows the interaction between neutrophils in a mouse model of sickle cell anemia;

[0025] FIG. 2 shows the interaction between platelets and neutrophils in a mouse model of sickle cell anemia;

[0026] FIG. 3 is a representative image of intravital microscopy, from left to right are BSA group, IgG group, antiplatelet thrombolysin group, antiPDI group, antiPDI+antiplatelet thrombolysin group, and the big arrow and the small arrow show blood flow and the rolling direction of neutrophils, respectively;

[0027] FIG. 4 shows the survival time of sickle cell anemia mice after administration.

DETAILED DESCRIPTION

[0028] The present invention provides use of antiplatelet thrombolysin in the preparation of a medicament for the treatment of anemia. Those skilled in the art can achieve it in view of this disclosure and appropriate improvement of the process parameters. In particular, it should be pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all deemed to be included in the present invention. The method and use of the present invention have been described through the preferred examples. It is obvious that those skilled in the art can make changes or appropriate modifications and combinations to the methods and uses described herein without departing from the content, spirit and scope of the present invention to achieve and apply the technology of the present invention.

[0029] The reagents used in the present invention are all common commercially available products, and all are available in the market.

[0030] The following examples further illustrate the present invention:

EXAMPLE 1

Anti-Platelet Thrombolysin Reduces Platelet-Neutrophil Aggregation in a Mouse Model of Sickle Cell Anemia Induced by TNF-α

[0031] Neutrophils (neutrophils) play a very important role in the non-specific cellular immune system of the blood. Activated neutrophils reach the diseased site through chemotaxis, and the bactericidal substances they carry will be released locally. Through their respective destructive effects, they will cause tissue damage and further lead to organ disorders. After neutrophils invade an organ, they will induce the accumulation of neutrophils in other important organs, resulting in multiple organ damage. At the same time, platelet selective receptors mediate the binding and adhesion of activated platelets and neutrophils, neutrophils aggregate, and accelerate tissue damage; on the other hand, platelets adhere to neutrophils, promote platelet aggregation, and mediate vascular occlusion. At the same time, the cell membrane of neutrophils can release tetraenoic acid. Under the action of enzymes, thromboxane and prostaglandins produced by tetraenoic acid have a significant effect on regulating the diameter and permeability of blood vessels, and can also cause inflammation and pain. The sickle red blood cells in the blood of sickle cell anemia are stiff and have poor deformability, and they are easy to block capillaries and cause local hypoxia and inflammation. The clinical manifestations are chronic hemolytic anemia, easy interference and recurrent pain, and ischemia leads to organ and tissue damage. The current routine treatment of sickle cell anemia is to prevent hypoxia, dehydration, and infection to relieve symptoms, reduce organ damage complications, promote hematopoiesis and prolong life.

[0032] By transplanting the bone marrow of Berkeley mice into 6-week-old WT mice that have been irradiated with a lethal dose, chimeric control and Berkeley mice were generated. Three months after transplantation, the chimera was confirmed by PCR and electrophoresis analysis. These chimeric Berkeley Mice are sickle cell anemia mice.

[0033] Sickle cell anemia model mouse platelets (2×107 cells/nil) and neutrophils (1×106 cells/ml) were taken, and labeled with DyLight488-anti-CD42C and Alexa Fluor647-anti-Ly-6G antibodies. Human neutrophils and platelets were respectively labeled with Alexa Fluor 488-anti-CD41 and FITC-anti-L-selectin antibodies. Neutrophils were induced with 20 ng/ml TNF-α for 5 minutes, and platelets were pre-incubated with control IgG (10 μg/ml) and anti PDI antibodies (protein disulfide bond isomerase inhibitor, 10 μg/ml), BSA (0.2 μg/ml), antiplatelet thrombolysin (0.2 μg/ml) and antiplatelet thrombolysin+antiPDI for 30 min respectively at room temperature, and then incubated with 0.025 U/ml thrombin at 37° C. for 5 minutes, and then incubated with 50 μM PPACK, stirred and mixed with platelets and neutrophils at 1000 rpm, fixed after 5 minutes and analyzed by flow cytometry.

[0034] During pathology, platelets adhere to neutrophils, promote platelet aggregation, mediate vascular occlusion, and aggravate pathophysiological reactions. The test results showed that 0.2 μg/ml of antiplatelet thrombolysin can reduce the aggregation of neutrophils in sickle cell anemia model mice (FIG. 1), and it can also reduce the combination of platelets and neutrophils (FIG. 2), thereby playing a role in inhibiting blood vessel damage.

[0035] In FIG. 1, the ordinate is the cell-cell interaction relative to the control group, and the abscissa is the experimental group from left to right: control IgG group, anti PDI group, BSA group, anfibatide group, IgG+BSA group, anti PDI+anfibatide group.

[0036] In FIG. 2, the ordinate is the interaction between platelets and neutrophils relative to the control group, and the abscissa is the experimental group from left to right: control IgG group, anti PDI group, BSA group, anfibatide group, IgG+BSA group, anti PDI+anfibatide group.

[0037] The experimental data was statistically analyzed by ANOVA and Tukey's test.

[0038] n=4, *P<0.05, **P<0.01, or ***P<0.001

EXAMPLE 2

Therapeutic Effect of Antiplatelet Thrombolysin on Mice with Sickle Cell Anemia

[0039] By transplanting the bone marrow of Berkeley mice into 6-week-old WT mice that have been irradiated with a lethal dose, chimeric control and Berkeley mice were generated. Three months after transplantation, the chimera was confirmed by PCR and electrophoresis analysis. These chimeric Berkeley Mice are sickle cell anemia mice. Successfully modeled mice were injected with 500 ng TNF-α through intraperitoneal injection to induce severe inflammation. After 3 hours of induction, single-blind administration was used. The administration groups included: IgG control (1.5 μg/g), antiPDI (BD34, 1.5 μg/g), BSA (25 ng/g), anfibatide (25 ng/g), antiPDI (1.5 μg/g)+anfibatide (25 ng/g).

[0040] After administration, the mice were observed by intravital microscope. During the study, the survival time of each mouse was recorded. The recording time started from the injection of TNFα and ended when the mice died or 6 h after injection of TNF-α. GraphPad Prism 7 software was used to analyze the data. Statistical analysis was performed by Student's t-test and ANOVA. n=8/group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0041] The results showed that, compared with the BSA control group, 25 ng/g antiplatelet thrombolysin eliminated the adhesion of platelets and neutrophils on the blood vessel wall of SCD mice by intravital microscope observation (FIG. 3). DyLight488-anti-CD42C and Alexa Fluor647-anti-Ly-6G antibody were used in the figure to label the platelets and neutrophils respectively. The large arrow indicates the direction of blood flow, and the small arrow indicates the rolling direction of neutrophils. The TNF-α stimulation and intravital microscopy surgery can cause acute vascular embolism, leading to the death of most SCD mice. The experimental observation period is the death of mice or 6 hours after the administration of TNFα.

[0042] Compared with the control groups, P=0.0249 for the comparison between BSA and anti-platelet thrombolysin group, P=0.028 for the comparison between BSA+IgG and BSA+anti-PDI group, P=0.0165 for the comparison between BSA+IgG and anti-platelet thrombolysin anti-PDI group. 50% survival time of mice treated with antiplatelet thrombolysin (Anfibatide) was 5.0 h, which was the longest survival time of mice in all administration groups (see FIG. 4), showing that antiplatelet thrombolysin has effective improving effect against sickle cell anemia in living bodies.