WO2022252280A1 - Use of free hemoglobin and derivative thereof against platelet aggregation - Google Patents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/41—Porphyrin- or corrin-ring-containing peptides
- A61K38/42—Haemoglobins; Myoglobins
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
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- the invention relates to the field of biomedicine, in particular to the application of free hemoglobin and its derivatives against platelet aggregation.
- VWF Von Willebrand factor
- endothelial cells which plays a key role in mediating platelet adhesion to the subendothelial membrane in the process of primary hemostasis.
- Inhibiting the combination of VWF and platelets is an important way to control the formation of arteriovenous and microthrombosis.
- the spatial structure and activity of VWF are regulated by the shear force of blood flow. Under normal circumstances, plasma VWF does not interact with platelets. Under the action of high shear force, the VWF molecule is stretched to expose the active A1 domain to bind with platelet GPIb, and then trigger aggregation reaction. When the combination of VWF and platelets is blocked, platelets cannot form aggregation and release coagulation signals, so the formation of thrombus will be inhibited.
- the first type is snake venom proteins, such as Agkisacucetin, etc., which inhibit the binding of VWF to platelets by competing with the GPIb receptor on platelets.
- the second type is DNA aptamer (Aptamer), such as ARC1779, this kind of DNA fragment blocks the binding of VWF and platelets by binding to VWF to form steric hindrance
- the third type is various antibodies or antibody derivatives, such as Caplacizumab, which is also passed through Combined with VWF or GPIb to achieve competitive inhibition.
- Hemoglobin is an iron-oxygen transport metalloprotein in the erythrocytes of almost all vertebrates. It is bound in erythrocytes under normal conditions, and will dissociate into plasma when it encounters hemolysis. Hemoglobin in red blood cells carries oxygen from the lungs to the rest of the body, where it releases oxygen, allowing aerobic respiration to provide energy to drive the organism's functions during metabolism. A healthy person has 12 to 20 grams of hemoglobin per 100 milliliters of blood. In mammals, hemoglobin makes up approximately 96% of the dry content (by weight) of red blood cells.
- hemoglobin In addition to being located inside the red blood cell, hemoglobin is also located on the outside of the red blood cell and its progenitor cells. Other hemoglobin-containing cells include A9 dopaminergic neurons in the substantia nigra, macrophages, alveolar cells, lung, retinal pigment epithelium, hepatocytes, mesangial cells in the kidney, endometrial cells, cervical cells, and vagina Epithelial Cells. In these tissues and cells, hemoglobin has the function of anti-oxidation and regulation of iron metabolism. Too much glucose in the blood attaches to hemoglobin and raises hemoglobin levels. The description of the function of hemoglobin is mainly focused on the transport of oxygen, and free hemoglobin has not been considered to be directly related to the coagulation process mediated by platelets/VWF.
- the object of the present invention is to provide an application of free hemoglobin and its derivatives against platelet aggregation.
- the present invention discloses a new application of free hemoglobin and its derivatives, and finds that it can selectively recognize and activate VWF in the activated state, but does not recognize the inactive VWF, and inhibits the binding of the activated VWF to platelets.
- the invention discloses the application of free hemoglobin or hemoglobin derivatives in the preparation of anti-platelet aggregation medicine.
- free hemoglobin or hemoglobin derivatives are free hemoglobin or hemoglobin derivatives in plasma that are not bound in cells.
- the drug against platelet aggregation is an anticoagulant drug.
- the diseases that anti-platelet aggregation drugs are used for prevention and/or treatment include thrombotic thrombocytopenic purpura (TTP), type 2B von Willebrand disease, immune thrombocytopenia (ITP), disseminated intravascular coagulation (DIC) and other platelet aggregation diseases.
- TTP thrombotic thrombocytopenic purpura
- ITP immune thrombocytopenia
- DIC disseminated intravascular coagulation
- other platelet aggregation diseases include thrombotic thrombocytopenic purpura (TTP), type 2B von Willebrand disease, immune thrombocytopenia (ITP), disseminated intravascular coagulation (DIC) and other platelet aggregation diseases.
- anti-platelet aggregation drugs are used to inhibit the binding of von Willebrand factor VWF to platelets.
- von Willebrand factor is von Willebrand factor in an activated state.
- the binding constant K d of hemoglobin binding to activated VWF is about 0.15 ⁇ M (0.96 mg/dL), which indicates that in the general hemolytic plasma free hemoglobin concentration range (45 to 200 mg/dL), after activation, VWF and Platelet binding can be inhibited.
- hemoglobin derivative is a hemoglobin mutant, preferably a recombinant amino acid mutant of hemoglobin designed to strengthen the interaction with von Willebrand factor.
- the dose of free hemoglobin or hemoglobin derivatives is 0.05-0.5 mg/mL.
- the dosage of free hemoglobin or hemoglobin derivatives is 0.01-0.4 mg/mL.
- the present invention has at least the following advantages:
- the invention discloses that free hemoglobin and its derivatives can selectively recognize activated VWF and inhibit its combination with platelets, thereby greatly reducing the risk of thrombotic diseases without causing loss of non-activated VWF. Compared with substances that inhibit the binding of VWF to platelets, it has outstanding advantages, which provides a direction for the development of new drugs in this field.
- Figure 1 is the result of the binding experiment between VWF and free hemoglobin
- Figure 2 is the results of platelet aggregation experiments in vitro of different experimental groups
- Figure 3 is the results of FeCl 3 induced thrombus experiments in different experimental groups.
- the free hemoglobin was adhered to the wells of the 96-well plate, and then blocked with 3% bovine serum albumin at room temperature for 2 hours for use.
- Activated VWF and non-activated VWF were added to the wells to which hemoglobin adhered in the form of gradient dilution from high concentrations, and incubated at room temperature for half an hour.
- the small wells were washed three times with phosphoric acid solution (1xPBS), and then the bound VWF protein was recognized with an anti-VWF antibody, and the VWF primary antibody was recognized with a secondary antibody, and finally the absorbance value of the chromogenic reagent at 405nm was used for quantitative analysis.
- the obtained results are shown in Figure 1.
- the abscissa in the figure represents the concentration of VWF. It can be seen from the figure that the binding constant K d of free hemoglobin binding to activated VWF is 0.15 ⁇ 0.06 ⁇ M, indicating that the binding of VWF and platelets after activation can be controlled. inhibition. However, the binding constant K d of free hemoglobin binding to non-activated VWF was above 10 ⁇ M, which indicated that non-activated VWF did not bind to free hemoglobin.
- the present invention also verifies the effect of free hemoglobin on platelet aggregation mediated by VWF in vitro, the steps are as follows:
- the platelets When activated VWF is mixed with washed platelets, the platelets will aggregate, changing their 600nm transmittance.
- the platelet aggregation curve is obtained by plotting the 600nm transmittance (y) and the mixing time (x).
- the activated VWF binds to platelets, leading to platelet activation and aggregation; under the interference of 0.05 mg/mL hemoglobin, the effect of platelet aggregation induced by VWF is significantly delayed, and VWF mediates
- the in vitro aggregation of platelets induced by VWF can be significantly inhibited; as the concentration of free hemoglobin increases, the degree of inhibition of platelet aggregation mediated by VWF also increases; and 0.4 mg/mL of free hemoglobin can completely inhibit the in vitro aggregation of platelets mediated by VWF .
- the present invention also verifies the thrombus-inhibiting effect of free hemoglobin through FeCl in vivo induced thrombus experiment, and the steps are as follows:
- Figure 3A and B are the fluorescent photos of the control group and the free hemoglobin group in the mouse carotid artery thrombosis experiment induced by FeCl3 respectively. It can be seen from the figure that when the free hemoglobin is injected into the mouse plasma to reach At 0.19mg/mL, the area of thrombus induced by FeCl 3 was significantly reduced, indicating that infusion of free hemoglobin into plasma can significantly reduce the area of thrombus.
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Abstract
Provided is antithrombotic activity of free hemoglobin or a derivative thereof, and also provided is a use of free hemoglobin or a hemoglobin derivative in preparation of an anti-platelet aggregation drug. The free hemoglobin and the derivative thereof can selectively identify a VWF in an activated state and inhibit its binding to platelets.
Description
本发明涉及生物医药领域，尤其涉及一种游离的血红蛋白及其衍生物抗血小板聚集的应用。The invention relates to the field of biomedicine, in particular to the application of free hemoglobin and its derivatives against platelet aggregation.
血管性血友病因子(VWF)是内皮细胞分泌的一种多聚体糖蛋白，在原发性止血过程中介导血小板粘附于内皮下膜来发挥关键作用。抑制VWF与血小板的结合是控制动静脉及微血栓形成的重要途径。VWF的空间结构和活性受血流剪切力调控，正常情况下，血浆VWF不与血小板相互作用。高剪切力作用下，VWF分子被拉伸暴露出活性的A1结构域与血小板GPIb结合、继而引发凝聚反应。当VWF与血小板结合被阻断后，血小板不能形成聚集和释放凝血信号，因此血栓的形成就会受到抑制。Von Willebrand factor (VWF) is a multimeric glycoprotein secreted by endothelial cells, which plays a key role in mediating platelet adhesion to the subendothelial membrane in the process of primary hemostasis. Inhibiting the combination of VWF and platelets is an important way to control the formation of arteriovenous and microthrombosis. The spatial structure and activity of VWF are regulated by the shear force of blood flow. Under normal circumstances, plasma VWF does not interact with platelets. Under the action of high shear force, the VWF molecule is stretched to expose the active A1 domain to bind with platelet GPIb, and then trigger aggregation reaction. When the combination of VWF and platelets is blocked, platelets cannot form aggregation and release coagulation signals, so the formation of thrombus will be inhibited.
已经公布的抑制VWF与血小板结合的物质主要有三类，第一类是蛇毒蛋白，例如Agkisacucetin等，他们通过竞争结合血小板的GPIb受体来抑制VWF与血小板的结合。第二类是DNA适体(Aptamer)，例如ARC1779，这类DNA片段通过结合VWF形成空间位阻来阻断VWF与血小板结合，第三类是各种抗体或抗体衍生物，例如Caplacizumab，也是通过结合VWF或者GPIb来实现竞争性抑制作用。上述三种物质虽然能保护血小板不结合VWF，但是他们都有一个共同的缺陷——即无法区分活化的VWF和非活化状态的VWF。这种对非活化VWF的无特异性结合会造成对血浆中正常循环的VWF蛋白的清除。因此，无一例外，这些药物在抑制血栓的同时带来严重的出血副作用。所以目前为止这些药物的适用范围都被严格限制在相对狭窄的领域。There are mainly three types of substances that have been published to inhibit the binding of VWF to platelets. The first type is snake venom proteins, such as Agkisacucetin, etc., which inhibit the binding of VWF to platelets by competing with the GPIb receptor on platelets. The second type is DNA aptamer (Aptamer), such as ARC1779, this kind of DNA fragment blocks the binding of VWF and platelets by binding to VWF to form steric hindrance, and the third type is various antibodies or antibody derivatives, such as Caplacizumab, which is also passed through Combined with VWF or GPIb to achieve competitive inhibition. Although the above three substances can protect platelets from binding to VWF, they all have a common defect—that is, they cannot distinguish activated VWF from non-activated VWF. This non-specific binding to inactive VWF results in the clearance of normally circulating VWF protein in the plasma. Therefore, without exception, these drugs bring serious bleeding side effects while inhibiting blood clots. Therefore, the scope of application of these drugs has been strictly limited to a relatively narrow field so far.
理想的抑制VWF与血小板结合的药物应当是有针对性的对活化之后的VWF进行阻断，对非活化的VWF不识别。这样就可以最大限度的保证VWF在血浆中的基本浓度，有效的攻击并削弱活化的VWF。但是目前为止还没有这样的药物出现。An ideal drug that inhibits the binding of VWF to platelets should specifically block activated VWF and not recognize inactive VWF. In this way, the basic concentration of VWF in plasma can be guaranteed to the greatest extent, and the activated VWF can be effectively attacked and weakened. But so far no such drug has appeared.
血红蛋白是几乎所有脊椎动物的红细胞中的含铁氧转运金属蛋白，正常情况下被束缚在红细胞中，遇到溶血状态时会游离到血浆当中。红细胞中的血红蛋白将氧气从肺中带到身体的其余部分，并在肺部释放氧气，允许有氧呼吸，以提供能量来推动生物体在新陈代谢过程中的功能。一个健康的人每100毫升血液中有12至20克血红蛋白。在哺乳动物中，血红蛋白约占红细胞干含量(按重量计)的96％。Hemoglobin is an iron-oxygen transport metalloprotein in the erythrocytes of almost all vertebrates. It is bound in erythrocytes under normal conditions, and will dissociate into plasma when it encounters hemolysis. Hemoglobin in red blood cells carries oxygen from the lungs to the rest of the body, where it releases oxygen, allowing aerobic respiration to provide energy to drive the organism's functions during metabolism. A healthy person has 12 to 20 grams of hemoglobin per 100 milliliters of blood. In mammals, hemoglobin makes up approximately 96% of the dry content (by weight) of red blood cells.
除了位于红细胞内部之外，血红蛋白也位于红细胞及其祖细胞的外部。其他含有血红蛋白的细胞包括黑质中的A9多巴胺能神经元，巨噬细胞，肺泡细胞，肺，视网膜色素上皮，肝细胞，肾脏中的肾小球膜细胞，子宫内膜细胞，宫颈细胞和阴道上皮细胞。在这些组织和细胞中，血红蛋白具有抗氧功能和调节铁代谢的功能。血液中的葡萄糖过多会附着在血红蛋白上，并升高血红蛋白的水平。人们对血红蛋白的功能描述主要集中在运输氧气方面，游离血红蛋白与血小板/VWF介导的凝血过程一直被认为没有直接关系。In addition to being located inside the red blood cell, hemoglobin is also located on the outside of the red blood cell and its progenitor cells. Other hemoglobin-containing cells include A9 dopaminergic neurons in the substantia nigra, macrophages, alveolar cells, lung, retinal pigment epithelium, hepatocytes, mesangial cells in the kidney, endometrial cells, cervical cells, and vagina Epithelial Cells. In these tissues and cells, hemoglobin has the function of anti-oxidation and regulation of iron metabolism. Too much glucose in the blood attaches to hemoglobin and raises hemoglobin levels. The description of the function of hemoglobin is mainly focused on the transport of oxygen, and free hemoglobin has not been considered to be directly related to the coagulation process mediated by platelets/VWF.
发明内容Contents of the invention
为解决上述技术问题，本发明的目的是提供一种游离的血红蛋白及其衍生物抗血小板聚集的应用，本发明公开了游离的血红蛋白及其衍生物的新用途，发现其能够选择性的识别活化状态的VWF，而不识别非活化的VWF，并抑制活化状态的VWF与血小板结合。In order to solve the above technical problems, the object of the present invention is to provide an application of free hemoglobin and its derivatives against platelet aggregation. The present invention discloses a new application of free hemoglobin and its derivatives, and finds that it can selectively recognize and activate VWF in the activated state, but does not recognize the inactive VWF, and inhibits the binding of the activated VWF to platelets.
本发明的技术方案如下：Technical scheme of the present invention is as follows:
本发明公开了游离的血红蛋白或血红蛋白衍生物在制备抗血小板聚集的药物中的应用。The invention discloses the application of free hemoglobin or hemoglobin derivatives in the preparation of anti-platelet aggregation medicine.
进一步地，游离的血红蛋白或血红蛋白衍生物为非拘束在细胞中的在血浆中游离的血红蛋白或血红蛋白衍生物。Further, free hemoglobin or hemoglobin derivatives are free hemoglobin or hemoglobin derivatives in plasma that are not bound in cells.
进一步地，抗血小板聚集的药物为抗凝血药。Further, the drug against platelet aggregation is an anticoagulant drug.
进一步地，抗血小板聚集的药物用于预防和/或治疗的疾病包括血栓性血小板减少性紫癜(TTP)、2B型血管性血友病、免疫性血小板减少症(ITP)、弥散性血管内凝血(DIC)等血小板聚集类疾病。Further, the diseases that anti-platelet aggregation drugs are used for prevention and/or treatment include thrombotic thrombocytopenic purpura (TTP), type 2B von Willebrand disease, immune thrombocytopenia (ITP), disseminated intravascular coagulation (DIC) and other platelet aggregation diseases.
进一步地，抗血小板聚集的药物用于抑制血管性血友病因子VWF与血小板的结合。Furthermore, anti-platelet aggregation drugs are used to inhibit the binding of von Willebrand factor VWF to platelets.
进一步地，血管性血友病因子为处于活化状态的血管性血友病因子。Further, von Willebrand factor is von Willebrand factor in an activated state.
进一步地，血红蛋白结合活化VWF的结合常数K
d是0.15μM(0.96mg/dL)左右，这个结合常数说明在一般的溶血性血浆自由血红蛋白浓度范围内(45到200mg/dL)，活化后VWF与血小板的结合可以被抑制。
Further, the binding constant K d of hemoglobin binding to activated VWF is about 0.15 μM (0.96 mg/dL), which indicates that in the general hemolytic plasma free hemoglobin concentration range (45 to 200 mg/dL), after activation, VWF and Platelet binding can be inhibited.
进一步地，血红蛋白衍生物为血红蛋白突变体，优选为重组的为加强与血管性血友病因子相互作用而设计的血红蛋白的氨基酸突变体。Further, the hemoglobin derivative is a hemoglobin mutant, preferably a recombinant amino acid mutant of hemoglobin designed to strengthen the interaction with von Willebrand factor.
进一步地，游离的血红蛋白或血红蛋白衍生物的使用剂量为0.05-0.5mg/mL。Further, the dose of free hemoglobin or hemoglobin derivatives is 0.05-0.5 mg/mL.
进一步地，游离的血红蛋白或血红蛋白衍生物的使用剂量为0.01-0.4mg/mL。Further, the dosage of free hemoglobin or hemoglobin derivatives is 0.01-0.4 mg/mL.
发明人对游离血红蛋白的分子机制研究发现，游离血红蛋白可结合到高活性的VWF的A1结构域从而抑制其与血小板结合，而游离血红蛋白并不结合低活性VWF的A1结构域。 且不同来源的游离血红蛋白，如牛、鼠和人的血红蛋白，均可以特异性结合活化之后的VWF，而不能结合非活化的VWF。The inventors studied the molecular mechanism of free hemoglobin and found that free hemoglobin can bind to the A1 domain of highly active VWF to inhibit its binding to platelets, while free hemoglobin does not bind to the A1 domain of low activity VWF. Moreover, free hemoglobin from different sources, such as bovine, mouse and human hemoglobin, can specifically bind to activated VWF, but cannot bind to inactive VWF.
借由上述方案，本发明至少具有以下优点：By means of the above solution, the present invention has at least the following advantages:
本发明公开了游离血红蛋白及其衍生物能够选择性识别活化状态的VWF，并抑制其与血小板结合，从而大幅降低血栓类疾病形成的风险，并且不会造成非活化VWF的损耗，与已经公布的抑制VWF与血小板结合的物质相比有突出的优势，为该领域的新药研发提供了方向。The invention discloses that free hemoglobin and its derivatives can selectively recognize activated VWF and inhibit its combination with platelets, thereby greatly reducing the risk of thrombotic diseases without causing loss of non-activated VWF. Compared with substances that inhibit the binding of VWF to platelets, it has outstanding advantages, which provides a direction for the development of new drugs in this field.
上述说明仅是本发明技术方案的概述，为了能够更清楚了解本发明的技术手段，并可依照说明书的内容予以实施，以下以本发明的较佳实施例并配合详细附图说明如后。The above description is only an overview of the technical solutions of the present invention. In order to understand the technical means of the present invention more clearly and implement them according to the contents of the description, the preferred embodiments of the present invention are described below with detailed drawings.
图1是VWF与游离血红蛋白结合实验结果；Figure 1 is the result of the binding experiment between VWF and free hemoglobin;
图2是不同实验组的体外血小板聚集实验结果；Figure 2 is the results of platelet aggregation experiments in vitro of different experimental groups;
图3是不同实验组的FeCl
3诱导血栓实验结果。
Figure 3 is the results of FeCl 3 induced thrombus experiments in different experimental groups.
下面结合实施例，对本发明的具体实施方式作进一步详细描述。以下实施例用于说明本发明，但不用来限制本发明的范围。Below in conjunction with the examples, the specific implementation of the present invention will be further described in detail. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.
实施例1Example 1
将游离血红蛋白粘附在96孔板小孔内，再用3％牛血清白蛋白室温封闭2小时备用。活化的VWF和非活化的VWF分别从高浓度以梯度稀释的方式加入血红蛋白粘附的孔内，室温培养半小时。用磷酸溶液(1xPBS)将小孔洗涤三次，然后用抗-VWF的抗体对结合的VWF蛋白质进行识别，再用二抗识别VWF一抗，最后用显色剂在405nm的吸收数值进行量化分析。The free hemoglobin was adhered to the wells of the 96-well plate, and then blocked with 3% bovine serum albumin at room temperature for 2 hours for use. Activated VWF and non-activated VWF were added to the wells to which hemoglobin adhered in the form of gradient dilution from high concentrations, and incubated at room temperature for half an hour. The small wells were washed three times with phosphoric acid solution (1xPBS), and then the bound VWF protein was recognized with an anti-VWF antibody, and the VWF primary antibody was recognized with a secondary antibody, and finally the absorbance value of the chromogenic reagent at 405nm was used for quantitative analysis.
所得结果如图1所示，图中横坐标代表VWF的浓度，从图中可看出，游离血红蛋白结合活化VWF的结合常数K
d为0.15±0.06μM，说明活化后VWF与血小板的结合可以被抑制。而游离血红蛋白结合非活化VWF的结合常数K
d为10μM以上，说明非活化的VWF与游离血红蛋白不发生结合。
The obtained results are shown in Figure 1. The abscissa in the figure represents the concentration of VWF. It can be seen from the figure that the binding constant K d of free hemoglobin binding to activated VWF is 0.15±0.06 μM, indicating that the binding of VWF and platelets after activation can be controlled. inhibition. However, the binding constant K d of free hemoglobin binding to non-activated VWF was above 10 μM, which indicated that non-activated VWF did not bind to free hemoglobin.
实施例2Example 2
本发明还验证了游离血红蛋白对体外VWF介导的血小板聚集的作用，步骤如下：The present invention also verifies the effect of free hemoglobin on platelet aggregation mediated by VWF in vitro, the steps are as follows:
将活化的VWF与洗涤血小板混合后，血小板会发生聚集，从而改变其600nm的透光度。 将600nm透光度(y)与混合时间(x)作图，则得到血小板聚集曲线。When activated VWF is mixed with washed platelets, the platelets will aggregate, changing their 600nm transmittance. The platelet aggregation curve is obtained by plotting the 600nm transmittance (y) and the mixing time (x).
如图2所示，当没有游离血红蛋白时，活化的VWF和血小板发生结合，进而导致血小板活化和聚集；在0.05mg/mL血红蛋白的干扰下，血小板受VWF诱导聚集的效应明显延后，VWF介导的血小板体外聚集可明显被抑制；随着游离血红蛋白浓度的升高，VWF介导的血小板体外聚集的抑制程度也增加；而0.4mg/mL的游离血红蛋白能完全抑制VWF介导的血小板体外聚集。As shown in Figure 2, when there is no free hemoglobin, the activated VWF binds to platelets, leading to platelet activation and aggregation; under the interference of 0.05 mg/mL hemoglobin, the effect of platelet aggregation induced by VWF is significantly delayed, and VWF mediates The in vitro aggregation of platelets induced by VWF can be significantly inhibited; as the concentration of free hemoglobin increases, the degree of inhibition of platelet aggregation mediated by VWF also increases; and 0.4 mg/mL of free hemoglobin can completely inhibit the in vitro aggregation of platelets mediated by VWF .
实施例3Example 3
本发明还通过体内FeCl
3诱导血栓实验对游离血红蛋白的抑制血栓效果进行验证，步骤如下：
The present invention also verifies the thrombus-inhibiting effect of free hemoglobin through FeCl in vivo induced thrombus experiment, and the steps are as follows:
用FeCl
3浸润的滤纸接触小鼠颈动脉可造成其内皮细胞损伤，从而出现血栓。将游离血红蛋白通过尾静脉注射后(终浓度0.19mg/mL)，用荧光显微镜观察其在FeCl
3诱导下的血栓形成。
Contacting mouse carotid arteries with FeCl 3 infiltrated filter paper can cause endothelial cell damage, resulting in thrombus. After the free hemoglobin was injected through the tail vein (final concentration 0.19 mg/mL), its thrombus formation induced by FeCl 3 was observed with a fluorescence microscope.
如图3所示，图3A、B分别是FeCl
3诱导的小鼠颈动脉血栓实验的对照组和游离血红蛋白组的荧光照片，从图中可看出，当向小鼠血浆中注入游离血红蛋白达到0.19mg/mL时，由FeCl
3诱导发生的血栓面积显著减少，说明输注游离血红蛋白到血浆中能显著减少血栓的面积。
As shown in Figure 3, Figure 3A and B are the fluorescent photos of the control group and the free hemoglobin group in the mouse carotid artery thrombosis experiment induced by FeCl3 respectively. It can be seen from the figure that when the free hemoglobin is injected into the mouse plasma to reach At 0.19mg/mL, the area of thrombus induced by FeCl 3 was significantly reduced, indicating that infusion of free hemoglobin into plasma can significantly reduce the area of thrombus.
以上所述仅是本发明的优选实施方式，并不用于限制本发明，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明技术原理的前提下，还可以做出若干改进和变型，这些改进和变型也应视为本发明的保护范围。The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention. It should be pointed out that for those of ordinary skill in the art, some improvements can be made without departing from the technical principle of the present invention. and modifications, these improvements and modifications should also be considered as the protection scope of the present invention.
Claims (9)
- 游离的血红蛋白或血红蛋白衍生物在制备抗血小板聚集的药物中的应用。The application of free hemoglobin or hemoglobin derivatives in the preparation of anti-platelet aggregation drugs.
- 根据权利要求1所述的应用，其特征在于：所述游离的血红蛋白或血红蛋白衍生物为非拘束在细胞中的在血浆中游离的血红蛋白或血红蛋白衍生物。The application according to claim 1, characterized in that: said free hemoglobin or hemoglobin derivative is free hemoglobin or hemoglobin derivative in plasma that is not bound in cells.
- 根据权利要求1所述的应用，其特征在于：所述抗血小板聚集的药物用于预防和/或治疗的疾病包括血栓性血小板减少性紫癜、2B型血管性血友病、免疫性血小板减少症和弥散性血管内凝血中的一种。The application according to claim 1, characterized in that: the diseases that the anti-platelet aggregation drugs are used for prevention and/or treatment include thrombotic thrombocytopenic purpura, type 2B von Willebrand disease, immune thrombocytopenia and disseminated intravascular coagulation.
- 根据权利要求1或3所述的应用，其特征在于：所述抗血小板聚集的药物用于抑制血管性血友病因子与血小板的结合。The use according to claim 1 or 3, characterized in that: the anti-platelet aggregation drug is used to inhibit the combination of von Willebrand factor and platelets.
- 根据权利要求4所述的应用，其特征在于：所述血管性血友病因子为处于活化状态的血管性血友病因子。The use according to claim 4, characterized in that: said von Willebrand factor is von Willebrand factor in an activated state.
- 根据权利要求1所述的应用，其特征在于：所述血红蛋白衍生物为血红蛋白突变体。The application according to claim 1, characterized in that: the hemoglobin derivative is a hemoglobin mutant.
- 根据权利要求1所述的应用，其特征在于：所述游离的血红蛋白或血红蛋白衍生物的使用剂量为0.05-0.5mg/mL。The application according to claim 1, characterized in that: the dosage of said free hemoglobin or hemoglobin derivative is 0.05-0.5mg/mL.
- 根据权利要求7所述的应用，其特征在于：所述游离的血红蛋白或血红蛋白衍生物的使用剂量为0.1-0.4mg/mL。The application according to claim 7, characterized in that: the dosage of the free hemoglobin or hemoglobin derivative is 0.1-0.4 mg/mL.
- 根据权利要求1所述的应用，其特征在于：所述抗血小板聚集的药物为抗凝血药。The application according to claim 1, characterized in that: the anti-platelet aggregation drug is an anticoagulant drug.
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| WO2018053427A1 (en) * | 2016-09-16 | 2018-03-22 | Duke University | Von willebrand factor (vwf)-targeting agents and methods of using the same |
| CN110624105A (en) * | 2019-09-24 | 2019-12-31 | 苏州大学 | Sequences of structurally sensitive polypeptide antigens of von Willebrand factor |
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| WO2018053427A1 (en) * | 2016-09-16 | 2018-03-22 | Duke University | Von willebrand factor (vwf)-targeting agents and methods of using the same |
| CN110624105A (en) * | 2019-09-24 | 2019-12-31 | 苏州大学 | Sequences of structurally sensitive polypeptide antigens of von Willebrand factor |
Non-Patent Citations (4)
| Title |
|---|
| ANNARAPU GOWTHAM KUMAR, RASHI SINGHAL , SHEETAL CHAWLA , AMRITA OJHA ,PRASENJIT GUCHHAIT: "HemoglobinMediated RegulationofPlateletFunctions", JOURNAL OF HEMATOLOGY & TRANSFUSION, vol. 4, no. 3, 23 September 2016 (2016-09-23), pages 1 - 7, XP093009331, ISSN: 2333-6684 * |
| CRUZ MIGUEL A.; GUCHHAIT PRASENJIT; BROWN RYANNE ASHLEY: "Extracellular Hemoglobin Increases the Binding of the Active Form of Von Willebrand Factor to Glycoprotein Ibα and Platelet Activation Under Shear Stress: Implications for Sickle Cell Disease and Other Hemolytic Disorders", BLOOD, vol. 118, no. 21, 18 November 2011 (2011-11-18), US , pages 2095, XP086622318, ISSN: 0006-4971, DOI: 10.1182/blood.V118.21.2095.2095 * |
| DA QI, TERUYA MIHO, GUCHHAIT PRASENJIT, TERUYA JUN, OLSON JOHN S, CRUZ MIGUEL A: "Free hemoglobin increases von Willebrand factor-mediated platelet adhesion in vitro: implications for circulatory devices.", BLOOD, vol. 126, no. 20, 12 November 2015 (2015-11-12), pages 2338 - 2341, XP093009328, DOI: 10.1182/blood-2015-05-648030 * |
| PAWLOSKI JOHN R., SWAMINATHAN RAJESH V., STAMLER JONATHAN S.: "Cell-Free and Erythrocytic S -Nitrosohemoglobin Inhibits Human Platelet Aggregation", CIRCULATION, AMERICAN HEART ASSOCIATION, vol. 97, no. 3, 27 January 1998 (1998-01-27), US , pages 263 - 267, XP093009325, ISSN: 0009-7322, DOI: 10.1161/01.CIR.97.3.263 * |
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