WO2008071081A1 - 低出血抗凝血融合蛋白的制备及其应用 - Google Patents

低出血抗凝血融合蛋白的制备及其应用 Download PDF

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WO2008071081A1
WO2008071081A1 PCT/CN2007/003526 CN2007003526W WO2008071081A1 WO 2008071081 A1 WO2008071081 A1 WO 2008071081A1 CN 2007003526 W CN2007003526 W CN 2007003526W WO 2008071081 A1 WO2008071081 A1 WO 2008071081A1
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recognized
coagulation factor
cleaved
oligopeptide
anticoagulant
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PCT/CN2007/003526
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WO2008071081A8 (zh
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Zuze Wu
Aiping Yu
Chuanling Zhang
Zhongxiong Tang
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Institute Of Radiation Medicine, Academy Of Military Medical Sciences, Pla
Beijing Sanly Sci-Tech Develop Inc. Ltd.
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Priority to JP2009540580A priority Critical patent/JP5345069B2/ja
Priority to US12/519,309 priority patent/US8101379B2/en
Priority to AT07845883T priority patent/ATE533789T1/de
Priority to CN2007800463406A priority patent/CN101668778B/zh
Priority to EP07845883A priority patent/EP2103630B1/en
Publication of WO2008071081A1 publication Critical patent/WO2008071081A1/zh
Publication of WO2008071081A8 publication Critical patent/WO2008071081A8/zh

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    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
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Definitions

  • the present invention belongs to the field of biotechnology and relates to a structure and a preparation method of a low-bleeding anticoagulant substance and its use in the prevention and treatment of thrombotic diseases and thrombosis-related diseases.
  • the present invention relates to a novel substance in which an anticoagulant substance is linked to an amino acid sequence which can be recognized and cleaved by several blood coagulation factors including thrombin, factor Xa (FXa), factor XIa (FXIa), and the like. , and the preparation and medicinal uses of these new substances.
  • Background technique is relates to a novel substance in which an anticoagulant substance is linked to an amino acid sequence which can be recognized and cleaved by several blood coagulation factors including thrombin, factor Xa (FXa), factor XIa (FXIa), and the like.
  • Cardiovascular and cerebrovascular diseases are the number one killers that threaten human health and life in recent years. Thrombosis is an important cause of many cardiovascular and cerebrovascular diseases. Anticoagulants are important drugs for preventing and treating thrombosis. The currently widely used anticoagulant is mainly heparin. An important disadvantage of heparin is thrombocytopenia (thrombocytopenia). The development of low molecular weight heparin now reduces the risk of heparin application, but does not overcome its shortcomings. Hirudin is a new anticoagulant that has been marketed in Europe in recent years.
  • thrombin It is a direct inhibitor of thrombin, but its strong inhibitory effect on thrombin can lead to blood coagulation related parameters such as bleeding time, APTX, TT and PT. It rises sharply and is associated with a risk of systemic or systemic bleeding.
  • the key to the guiding principle of the present invention is to conditionally release the anticoagulant activity of the anticoagulant, that is, under normal conditions, the substance has no anticoagulant activity.
  • the coagulation system When the coagulation system is activated, there is a possibility of thrombosis or thrombosis, the anticoagulant activity of the substance is released locally, creating an environment in which thrombus is not easily formed (ie, local anticoagulant activity occurs) to prevent thrombosis. Or the existing thrombus continues to grow, even dissolve the tiny emboli that has formed to achieve the function of preventing thrombosis. This overcomes the risk of systemic bleeding caused by anticoagulants such as heparin and hirudin.
  • hirudin is a single-chain polypeptide consisting of 65 to 66 amino acids. Its amino terminus can bind to the catalytic active site of thrombin and has anticoagulant activity. The carboxy terminus binds to the substrate recognition site of thrombin and has a strong specific affinity for thrombin.
  • a method for blocking the amino terminus of hirudin was designed to temporarily block the anticoagulant activity of hirudin.
  • the characteristics of the biochemical changes induced by the thrombus are used to restore the amino terminal blocked hirudin to the hirudin form, and to play a specific part in the thrombus that may or may have occurred. It is a new type of safe, effective anticoagulant that reduces the risk of systemic bleeding caused by hirudin.
  • the hirudin has been modified. Because the laboratory has been identified on the amino terminus of hirudin by an oligopeptide that can be recognized and cleaved by thrombin (the hirudin derivative is named TH) or the oligopeptide recognized and cleaved by coagulation factor Xa (the hirudin derivative is named It is FH) so that once the coagulation system is activated, the anticoagulant activity of hirudin is released, which acts as an anticoagulant and antithrombotic agent and reduces bleeding side effects. The results show that it has a certain effect, but the effect is still poor.
  • oligopeptides that can be recognized and lysed by several coagulation factors or other coagulation factors. It is hoped that the effect can be improved and the practical application can be achieved.
  • these oligopeptides can be efficiently recognized and cleaved by the corresponding coagulation factors when thrombosis occurs, and it is not currently speculated according to the relevant literature.
  • the laboratory has prepared two related proteins: 1 The oligopeptide of the hirudin is recognized and cleaved by thrombin and factor Xa, respectively.
  • the hirudin derivative is named GH; 2
  • the oligopeptide of the hirudin is linked to an oligopeptide which is recognized and cleaved by coagulation factor XI a and coagulation factor Xa, respectively, and the hirudin derivative is named EH.
  • the results showed that the two hirudin derivatives did not have anticoagulant activity under normal conditions in vitro and in vivo. Once the in vivo coagulation system was activated, the anticoagulant activity of hirudin could be efficiently released under the combined action of the above coagulation factors. , anticoagulant and antithrombotic effects.
  • An object of the present invention is to provide a substance which does not have anticoagulant activity itself, and which can locally release its anticoagulant activity in the presence of a thrombus when thrombus occurs or thrombus occurs, thereby preventing thrombosis.
  • the present inventors have now discovered that the sequence identified by coagulation factor XIa and coagulation factor Xa, or the sequence recognized by thrombin and coagulation factor Xa, respectively, can be linked to an anticoagulant substance to achieve closure of the anticoagulant activity of the anticoagulant substance, and Cleavage under certain conditions.
  • the derived anticoagulant substance has the following characteristics:
  • the anticoagulant substance such as the base end of hirudin is blocked by the sequence recognized by the above two coagulation factors, and has no anticoagulant activity in vitro and non-thrombus sites of the blood system, thereby avoiding or It reduces the side effects of systemic hemorrhage caused by anticoagulant substances such as hirudin; the derived anticoagulant is only required to release free anticoagulation only when thrombus occurs, under the action of the specific blood coagulation factor at the thrombus site.
  • the physical shield plays a role in the prevention and treatment of blood clots, so the side effects of systemic bleeding are significantly reduced.
  • the sequence recognized by the two coagulation factors respectively blocks the amino terminus of an anticoagulant substance such as hirudin, which can be cleaved by two coagulation factors in the body, and the effect is much better than the recognition sequence of a single coagulation factor.
  • Anticoagulant For example, the amino terminus of hirudin is blocked.
  • the first aspect of the present invention relates to an anticoagulant substance comprising an oligopeptide which is co-recognized and cleaved by coagulation factor XIa and coagulation factor Xa or an oligopeptide which is co-recognized and cleaved by thrombin and coagulation factor Xa.
  • a further aspect of the present invention relates to a method for producing an anticoagulant comprising an oligopeptide which is co-recognized and lysed by coagulation factor XIa and coagulation factor Xa or which is co-identified and cleaved by coagulation factor Xa, comprising a factor XIa
  • the nucleotide sequence corresponding to the co-recognition sequence of coagulation factor Xa or the sequence recognized by thrombin and coagulation factor Xa is linked to the anticoagulant protein gene, and these genes are then constructed into vectors suitable for expression, such as pBV220, pPIC9, pPIC91. [Sequence, these recombinant vectors containing the gene of interest are expressed in a suitable host, for example, in E. coli, yeast or animal cell systems to give new anticoagulant substances.
  • a further aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the above novel anticoagulant and a pharmaceutically acceptable carrier or excipient.
  • anticoagulant substance or "anticoagulant substance” means a substance capable of anticoagulation, such as hirudin, antithrombin I II, snake venom, or the like, or a mutant thereof. Or other substance having anticoagulant action, preferably hirudin or a mutant thereof.
  • the oligopeptide which is recognized by the factor Xa refers to the tripeptide sequence EPR (GluProArg) or the peptide containing the EPR, or the pentapeptide sequence GVYAR (GlyValTyrAlaArg) or the peptide containing GVYAR.
  • the anticoagulant substance of the present invention comprising an oligopeptide which is co-recognized and cleaved by coagulation factor XIa and coagulation factor Xa or which is co-identified and cleaved by coagulation factor Xa is preferably attached to the amino terminal of hirudin. EPR or GVYAR.
  • the anticoagulant substance of the present invention comprising an oligopeptide which is co-recognized and cleaved by coagulation factor XIa and coagulation factor Xa or which is co-identified and cleaved by coagulation factor Xa can be expressed in a suitable host system. Preferably expressed in E. coli or yeast.
  • Figure 1 shows the nucleotide sequences of EH and GH.
  • Figure 2 shows the amino acid sequences of EH and GH.
  • EH EPR-HV2
  • GH GVYAR-HV2
  • the restriction enzyme cleavage site Xho I and the coagulation factor XIa and the coagulation factor Xa co-recognition sequence EPR or the base sequence corresponding to the co-recognition sequence GVTAR of the coagulation factor Xa are introduced into the upstream of the hirudin (HV2) gene by PCR.
  • the EcoR I restriction site was introduced downstream of the HV2 gene, and the gene was ligated to the same digested PPIC9 plasmid to obtain recombinant plasmids pPIC9-EH and pPIC9-GH.
  • PIC9-EH and PPIC9-GH and pPIC9K were double-digested with BamH I and Sal I to obtain PPIC9K-EH and PPIC9K-GH.
  • Two recombinant plasmids were electrotransformed into the yeast genome and induced to express using methanol. The expressed product was isolated and purified to obtain the target proteins EH and GH.
  • GH, EH, FH and TH were cleaved with thrombin, FXa and FXIa, respectively, and the anticoagulant activity of the product was determined by fibrin clot method after lysis. The results are shown in Table 1.
  • Table 1 Analysis of anticoagulant activity of GH, EH, FH and TH before and after cleavage by thrombin, FXa and FXIa
  • FXIa 0 0 0 0 0 0 0 0 0 0 64 0 256 512
  • Table 1 GH can be recognized and cleaved by two clotting factors, thrombin and FXa, respectively.
  • EH can be recognized and lysed by FXa and FXIa clotting factors, respectively.
  • ⁇ ⁇
  • FH is only recognized and cleaved by FXa
  • TH is only recognized and cleaved by thrombin.
  • EH, GH, FH and TH can prolong the time of common carotid artery thrombosis in rats, and dose-dependent, indicating that these hirudin derivatives have anti-arterial Thrombosis function, and the effect is basically similar to each other, but it is very different in terms of bleeding side effects.
  • Table 3 and Table 4 that GH, EH, FH and TH have less influence on bleeding index than HV2. It shows that it is safer than HV2; and GH and EH have much less impact on the bleeding index than FH and TH, especially the bleeding time of GH is significantly lower than that of FH and TH group, indicating that GH and EH are reduced.
  • the side effects of bleeding are significantly better than FH or TH.

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Description

低出血抗凝血融合蛋白的制备及其应用 技术领域
本发明属于生物技术领域， 涉及一类低出血抗凝血物质的结 构和制备方法及其在防治血栓性疾病和血栓相关性疾病中的应 用。 具体而言， 本发明涉及抗凝血物质与能被数个凝血因子 （包 括凝血酶、 凝血因子 Xa ( FXa ) 、 凝血因子 XIa ( FXIa )等）识别 并裂解的氨基酸序列连接而成的新物质， 以及这些新物质的制备 和医药学用途。 背景技术
心脑血管疾病是近年来威胁人类健康和生命的头号杀手， 血 栓形成是许多心脑血管疾病的重要诱因， 抗凝剂是防治血栓形成 的重要药物。 目前临床上广泛应用的抗凝剂主要是肝素， 肝素的 一个重要缺点是引发血小板减少症 （thrombocytopenia ) 。 现在 又发展的低分子量肝素可以减轻肝素应用中的风险程度， 但不能 根本克服它的缺点。 水蛭素是近年来欧洲已经上市的一个新型抗 凝药物， 它是凝血酶的直接抑制剂，但它对凝血酶的强抑制作用， 可导致凝血相关参数， 如出血时间、 APTX、 TT和 PT等急剧升高， 从而伴有全身或系统性出血风险。
现有抗凝药物存在的上述问题已经越来越多的受到人们的关 注， 作为一个理想的抗凝药物， 在全身用药条件下， 应当具有明 确的抗凝抗栓效果， 但又不引起出血副作用， 使临.床用药安全性 提高。
为此， 本发明思想的指导原则的关键是使抗凝剂的抗凝活性 进行条件特异性释放， 即在正常条件下， 该类物质无抗凝活性， 而当凝血系统被激活， 有血栓形成可能或有血栓形成时， 该类物 质的抗凝活性才在该局部释放， 营造不易形成血栓的徵环境（即 在局部出现抗凝活性） ， 防止血栓形成或已有血栓的继续增长， 甚至溶解已形成的微小栓子以达到防治血栓形成的功能。 这就克 服了肝素、 水蛭素等抗凝剂用药引起系统性出血的风险。
例如水蛭素是由 65 ~ 66个氨基酸組成的单链多肽。其氨基端 可以与凝血酶催化活性位点结合， 具有抗凝活性， 羧基端与凝血 酶的底物识别位点结合， 对凝血酶有很强的特异性亲和作用。 本 文设计了一个封闭水蛭素氨基末端的措施， 以达到暂时封闭水蛭 素的抗凝活性的目的。 当体内凝血系统被激活发生血栓时， 利用 血栓发生过程中引发的生物化学变化的特点， 使氨基末端被封闭 的水蛭素重新回复为水蛭素原形， 在可能发生或已发生的血栓的 局部发挥特异性抗凝作用， 从而降低水蛭素引起系统性出血的危 险， 成为一类新型的、 安全的、 有效的抗凝剂。
在上述发明思想的指导下， 本文对水蛭素进行了修饰。 由于 本实验室曾在水蛭素的氨基末端连接上可被凝血酶识别并裂解的 寡肽（该水蛭素衍生物命名为 TH ) 或凝血因子 Xa识别并裂解的 寡肽（该水蛭素衍生物命名为 FH) , 以便一旦凝血系统被激活， 则 释放水蛭素的抗凝活性， 起到抗凝和抗栓作用， 且减少出血副作 用。 结果表明是有一定作用， 但是效果还差。 因此我们进而改变 思路， 考虑采取连接可被数个凝血因子或其它凝血因子识别并裂 解的寡肽进行修饰， 希望可以提高效果，达到具有实际应用的目 的。 当然这些寡肽在血栓发生时能否高效率地被相应的凝血因子 识别并裂解， 根据有关文献看来目前并无法推测。 本实验室就试 验制备了两个相关蛋白： ①水蛭素的氨基末端连接上可被凝血酶 和凝血因子 Xa 分别识别并裂解的寡肽， 该水蛭素衍生物命名为 GH; ②水蛭素的氨基末端连接上可被凝血因子 XI a和凝血因子 Xa 分别识别并裂解的寡肽， 该水蛭素衍生物命名为 EH。 结果表明该 两个水蛭素衍生物在体外和体内正常情况下不具有抗凝活性， 一 旦体内凝血系统被激活， 则在以上凝血因子的共同作用下， 可高 效地局部释放水蛭素的抗凝活性， 起到抗凝和抗栓作用。 当然， 在体内不存在血栓的部位， 则无抗凝活性， 故其出血副作用也显 著降低。 因此， 它不同于水蛭素、 肝素等抗凝剂， 是一类安全、 有效的抗凝抗栓剂。 从而， 此类具有低出血特征的抗凝剂对于防 治血栓形成的应用具有重要意义。
发明目的
本发明的目的在于提供一类本身无抗凝活性， 而当有血栓发 生倾向或血栓发生时， 可以在发生血栓局部释放其抗凝活性， 从 而防治血栓形成的物质。
发明内容
本发明现已发现，利用凝血因子 XIa和凝血因子 Xa分别识别 的序列或凝血酶和凝血因子 Xa 分别识别的序列连接抗凝血物质 可实现对此抗凝血物质抗凝活性的封闭， 并在一定条件下裂解。 衍生的抗凝血物质具有如下的特征： 抗凝血物质如水蛭素的 基 末端被上述两个凝血因子分别识别的序列封闭后， 在体外和血液 系统非血栓部位没有抗凝活性， 从而避免或减小了因抗凝血物质 如水蛭素引起的系统出血的副作用； 衍生的抗凝血物庸只有在血 栓发生时， 在血栓部位特有的凝血因子的作用下才局部释放出游 离的抗凝血物盾， 发挥防治血栓的作用， 因而全身出血副作用明 显降低。 而且， 由两个凝血因子分别识别的序列封闭抗凝血物质 如水蛭素的氨基末端， 在体内可以由两个凝血因子共同对其进行 裂解， 其效果大为优于由单个凝血因子的识别序列对抗凝血物质 如水蛭素的氨基末端进行封闭。 本发明基于上述特征的发现现已 完成。
因此， 本发明第一方面涉及含有被凝血因子 XIa和凝血因子 Xa共同识别并裂解的寡肽或被凝血酶和凝血因子 Xa共同识别并 裂解的寡肽的抗凝血物质。
本发明再一方面涉及含有被凝血因子 XIa和凝血因子 Xa共同 识别并裂解的寡肽或凝血酶和凝血因子 Xa 共同识别并裂解的寡 肽的抗凝血物质的制备方法， 包括将凝血因子 XIa和凝血因子 Xa 共同的识别序列或凝血酶和凝血因子 Xa 共同识别的序列所对应 的碱基序列与抗凝血蛋白基因进行连接， 然后这些基因构建至适 合表达的载体， 如 pBV220, pPIC9 , pPIC91 [等， 这些含有目的基 因的重组载体在适宜的宿主中表达， 例如在大肠杆菌， 酵母或动 物细胞体系中表达而得到新的抗凝血物质。
本发明再一方面涉及含有上述新的抗凝血物质及药用载体或 赋形剂的药物组合物。
根据本发明， 术语 "抗凝物质" 或 "抗凝血物质" 是指能够 抗凝血的物质， 如水蛭素、 抗凝血酶 I II、 蛇毒等， 或它们的突 变体。 或其它具有抗凝作用的物质， 优选水蛭素或其突变体。
根据本发明， 术语 "凝血因子 XIa和凝血因子 Xa共同识别的 连接肽或凝血酶和凝血因子 Xa共同识别的连接肽"或 "凝血因子 XIa和凝血因子 Xa共同识别的寡肽或凝血酶和凝血因子 Xa共同 识别的寡肽"是指三肽序列 EPR ( GluProArg )或含有 EPR的肽段， 或五肽序列 GVYAR (GlyValTyrAlaArg)或含有 GVYAR的肽段。
根据本发明， 本发明的含有被凝血因子 XIa 和凝血因子 Xa 共同识别并裂解的寡肽或凝血酶和凝血因子 Xa 共同识别并裂解 的寡肽的抗凝血物质优选在水蛭素氨基末端连接上 EPR或 GVYAR。 根据本发明，本发明的含有被凝血因子 XIa和凝血因子 Xa共 同识别并裂解的寡肽或凝血酶和凝血因子 Xa 共同识别并裂解的 寡肽的抗凝血物质可在适宜的宿主系统中表达， 优选在大肠杆菌 或酵母中表达。
附图说明
图 1表示 EH和 GH的核苷酸序列。
图 2表示 EH和 GH的氨基酸序列。
下面的实施例用来进一步说明本发明， 但并不意味着对发明 的任何限制。
实施例 EPR- HV2 ( EH )和 GVYAR- HV2 ( GH )的制备及其防治 血栓形成功能
一、 EH蛋白和 GH蛋白的制备
通过 PCR将限制性酶切位点 Xho I和凝血因子 XIa和凝血因 子 Xa共同识别序列 EPR或凝血酶和凝血因子 Xa共同识别序列 GVTAR对应的碱基序列引入水蛭素（HV2 )基因的上游， 在 HV2基 因下游引入 EcoR I酶切位点， 将该基因连接到相同酶切的 PPIC9 质粒上， 得到重组质粒 pPIC9- EH和 pPIC9- GH。 将 PIC9-EH和 PPIC9-GH和 pPIC9K用 BamH I和 Sal I双酶切后进行连接， 得到 PPIC9K-EH和 PPIC9K-GH。将两个重組质粒电转化重组到酵母基因 组， 利用甲醇进行诱导表达。 表达产物经分离纯化得到目的蛋白 EH和 GH。
二、 EH和 GH的生物活性
1.体外活性分析
GH、 EH、 FH和 TH分別用凝血酶、 FXa和 FXIa进行裂解， 裂 解后采用纤维蛋白凝块法测定产物的抗凝活性。 结果见表 1。 表 1 GH、 EH、 FH和 TH被凝血酶、 FXa和 FXIa裂解前后抗 凝活性分析
GH FH TH EH
group
Oh 3h 6h Oh 3h 6h Oh 3h 6h Oh 3h 6h thrombin 0 2 8 0 0 0 0 4 8 0 0 0
FXa 0 64 128 0 32 64 0 0 0 0 64 128
FXIa 0 0 0 0 0 0 0 0 64 0 256 512 由表 1可看出， GH可被凝血酶和 FXa两种凝血因子分别识别 并裂解， EH可以被 FXa和 FXIa两种凝血因子分别识别并裂解， ί
而 FH只被 FXa识别并裂解， TH只被凝血酶识别并裂解。
2. GH和 EH的抗栓作用和出血副作用分析
① GH和 EH在大鼠颈总动脉血栓模型中的抗栓作用和出血副 作用分析。 结果见表 2和表 3。
GH、 FH、 EH和 TH对大鼠颈总动脉血栓形成时间的影响
HV2 EH
組别 NS
2mg/kg 2mg/kg 4mg/kg
779. 30士 964. 10 ± 1083. 90 ± 血栓形成时间（S )
193. 47 179. 68* 227. 10"
-
HV2 GH
组别 NS
2mg/kg 2mg/kg 4mg/kg
1117. 00士 918. 10土
血栓形成时间（ S ) 1006. 7 ± 227. 37*
243. 95## 204. 16
HV2 FH
組别 NS
2mg/kg 2mg/kg 4mg/kg
779. 30士 988. 10 ± 血栓形成时间（ S ) 1096. 0 ± 255. 9*
193. 47 231. 16
血检形成时间 ( S ) 7= ^ 8= ― 每组动物 10只， 结果以 土 S表示。 HV2 -水蛭素， NS-生 理盐水
# P<0.05, 与 NS组相比 ## P<0.01， 与 NS组相比
EH、 GH、 FH和 ΊΉ对大鼠颈总动脉血栓模型血浆 TT的 影响
HV2 EH
纽別 NS
2mg/kg 2mg/kg 4mg/kg
ΤΤ变化百分数 100± 0 1005.62 ± 361.28**' ' 108.41 ±12.95 121.81± 13.68**
HV2 GH
纽別 NS
2mg/kg 2mg/kg 4mg/kg
ΤΤ变化百分数 100± 0 1005.62 ± 361.28**' 1 104.35 ± 17.21 114.94± 19.82**
HV2 FH
组別 NS
2mg/kg 2mg/kg 4mg/kg
ΤΤ变化百分数 100± 0 1005.62 ± 361.28*.*' 1 119.31± 16.32 149.66 ± 50.84*
HV2 TH
组別 NS
2mg/kg 2mg/kg 4mg/kg
ΤΤ变化百分数 100± 0 1005.62 ± 361.28**' ' 117.57±14.82 141.03 ± 54.31* 每组动物 10只， 结果按生理盐水组为 100%， 其它各組对生 理盐水组的变化百分数表示， 数据以 ±S表示。 HV2-水蛭素，
NS-生理盐水
* P<0.05, 与 NS组相比； * * P<0.01, 与 NS组相比 △ P<0.01, HV组与其它各组相比 ② GH、 EH、 TH和 FH在小鼠割尾出血模型中的出血副作用分 析。 结果见表 4。
EH、 GH、 FH和 TH对小鼠割尾出血时间的影响
HV2 EH
组别 rib
1. , 5mg/kg 1.5mg/kg 6mg/kg
203.89士 338.77 ± 出血时间 (S) 156.14土 125.08 1532. 5 ± 420. 74Δ
97.41 169.08*
HV2 GH
组别 M
1. , 5mg/kg 1.5mg/kg 6mg/kg
165.46 ± 304 ± 出血时间 ( s ) 156.14士 125- 08 1532. 5 ± 420.74Δ
142.42 178.99*'
HV2 FH
組别 S
1. , 5mg/kg 1.5mg/kg 6. Omg/kg
264.08士 569.14士 出血时间 ( s ) 156.14± 125.08 1532. 5 ± 420.74Δ
209.74 430.1*
HV2 TH
组别 NS
1. , mg/kg 1.5mg/kg 6mg/kg
241.42土 397.31士 出血时间 ( s ) 156.14± 125.08 1532. 5 ± 420.74Λ
194.49 286.12* 每组动物 15只， 结果以 士 S表示。
* P<0.05, 与 NS组相比； Δ P<0.01, HV2组与其它各组相 比；
# P<0.05， GH组与 FH组和 TH组相比
从表 2可看出， EH、 GH、 FH和 TH可延长大鼠颈总动脉血栓形成 时间， 且成剂量依赖关系， 说明这些水蛭素衍生物都具有抗动脉 血栓形成功能， 而且彼此效果基本类似， 但在出血副作用方面则 大不相同， 从表 3和表 4可看出， 与 HV2相比， GH、 EH、 FH和 TH对出 血指标的影响较小， 说明其比 HV2的安全性要高； 而其中 GH、 EH 比 FH、 TH对出血指标的影响更要小得多， 特别是 GH的出血时间更 明显低于 FH和 TH组， 说明 GH、 EH减低出血副作用方面要明显优于 FH或 TH。

Claims

1. 含有可被两种凝血因子共同识别并裂解的寡肽的抗凝血蛋 白。
2. 权利要求 1 的抗凝血蛋白， 包括水蛭素、 抗凝血酶 I I I、 蛇毒等， 或它们的突变体。
3. 权利要求 2的抗凝 jk蛋白， 优选水蛭素或其突变体。
4. 权利要求 1的含有被两种凝血因子共同识别并裂解的寡肽 的抗凝血蛋白， 其中凝血因子为凝血酶、 凝血因子 Xa、 凝血因子 XIa等。
5. 权利要求 1 的含有被两种凝血因子共同识别并裂解的寡肽 的抗凝血蛋白，其中寡肽为能被凝血因子 XIa和凝血因子 Xa共同 识别的氨基酸序列或凝血酶和凝血因子 Xa 共同识别的氨基酸序 列，或含有能被凝血因子 XIa和凝血因子 Xa共同识别的氨基酸序 列的寡肽或凝血酶和凝血因子 Xa共同识别的氨基酸序列的寡肽。
6. 权利要求 5的能被凝血因子 XIa和凝血因子 Xa共同识别 并裂解的氨基酸序列或凝血酶和凝血因子 Xa 共同识别并裂解的 氨基酸序列，为能被凝血因子 XIa和凝血因子 Xa共同识别并裂解 的氨基酸序列 EPR或含有 EPR的肽段， 或为能被凝血酶和凝血因 子 Xa共同识别并裂解的氨基酸序列 GVYAR或含有 GVYAR的肽段。
7. 权利要求 1的含有可被两种凝血因子共同识别并裂解的寡 肽的抗凝血蛋白， 包括在水蛭素 N末端连接 EPR或 GVYAR的抗凝 血蛋白 EH和 GH。
8. 制备含有被凝血因子 XIa和凝血因子 Xa共同识别并裂解 的寡肽或凝血酶和凝血因子 Xa 共同识别并裂解的寡肽的抗凝血 蛋白的方法， 其包括将编码 EPR或 GVYAR的碱基序列引入抗凝血 蛋白基因的上游，然后这些基因通过适合的表达载体在大肠杆菌， 酵.母或动物细胞等体系中表达。
9. 含有被凝血因子 XIa和凝血因子 Xa共同识别并裂解的寡肽 或凝血酶和凝血因子 Xa共同识别并裂解的寡肽的抗凝血蛋白及药 用载体或赋形剂的药物组合物。
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CN115572329A (zh) * 2021-06-21 2023-01-06 王大勇 一组活性增强代谢较慢的菲牛蛭基因重组水蛭素及其制备方法
CN115572329B (zh) * 2021-06-21 2024-02-06 王大勇 一组活性增强代谢较慢的菲牛蛭基因重组水蛭素及其制备方法

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