Novel Recombinant Anticoagulant Proteins

描述(申请人提供)：第一阶段：血管中的凝块形成(血栓形成)导致一系列严重的临床情况，如不稳定心绞痛、急性心肌梗死、缺血性中风、深静脉血栓形成、肺栓塞、弥漫性血管内凝血和外周血管疾病。在发达国家，它是导致死亡和残疾的主要原因。动脉和静脉血栓栓塞症的短期治疗和长期预防复发需要抗凝药物。目前可用的抗凝药物有各种局限性。需要更有效、更安全、更容易使用的抗凝剂。该应用的长期目标是开发高效的新型抗凝药物，以满足上述治疗需求。利用基因工程技术，EVAS治疗公司创造了一系列重组蛋白，以高亲和力结合阴离子磷脂，并有效地抑制膜相关凝血酶复合体。因此，这些抗凝蛋白是针对血栓的，非常有效，可以实现更好的抗血栓效果，同时降低全身出血副作用的风险。这一I期提案的主要目的是证明这些杂合蛋白在体外特异性定位于血栓形成细胞的能力，并在体内实现抗血栓作用。此外，还将进行小规模的大肠杆菌发酵和蛋白质生产过程，为体外和体内研究提供材料。这一第一阶段项目的具体目标是： 1.在体外建立融合蛋白与正常细胞和受干扰细胞的结合特异性。为了证明融合蛋白与血栓形成细胞的特异性结合，将研究融合蛋白与刺激的和静止的血小板和内皮细胞的不同结合。 2.验证融合蛋白的抗血栓作用，并在小鼠动脉血栓模型上完成量效关系研究。将使用小鼠动脉血栓形成模型来确定EVAs的抗凝血剂分子的抗血栓作用。 3.建立一种用于体外和体内研究的重组融合蛋白的生产工艺。将建立更高效的发酵和纯化工艺，为一期研究提供所需材料。该工艺也为重组蛋白的二期生产奠定了基础。

DESCRIPTION (provided by applicant): Phase I: Clot formation in the blood vessel (thrombosis) causes a broad range of serious clinical conditions, such as unstable angina, acute myocardial infarction, ischemic stroke, deep vein thrombosis, pulmonary embolism, disseminated intravascular coagulation, and peripheral vascular disease. It is a leading cause of death and disability in the developed countries. Anticoagulant drugs are needed for the short-term treatment of arterial and venous thromboembolic disorders and for the long-term prevention of recurrences. Currently available anticoagulant drugs have various limitations. More efficacious, safer, and easier to use anticoagulants are demanded. The long-term objective of this application is to develop highly effective new anticoagulant drugs to meet the above therapeutic needs. Using genetic engineering technology, EVAS Therapeutics has created a series of recombinant proteins that bind anionic phospholipids with high affinity and potently inhibit membrane-associated coagulation enzyme complexes. As a result, these anticoagulant proteins are site-targeted to thrombus, exceptionally potent, and may achieve superior antithrombotic efficacy with lower risk of systemic bleeding side effects. The main objective of this phase I proposal is to demonstrate the ability of these hybrid proteins to specifically localized on the thrombogenic cells in vitro and to achieve antithrombotic effect in vivo. In addition, small-scale E. coli fermentation and protein production processes will be carried out to supply materials for the in vitro and in vivo studies. The specific aims of this phase I project are to: 1. Establish the binding specificity of the fusion proteins to normal vs. perturbed cells in vitro. Differential binding of the fusion proteins to stimulated vs. resting platelets and endothelial cells will be studied in order to demonstrate the specific binding of the fusion proteins to thrombogenic cells. 2. Demonstrate the antithrombotic efficacies of the fusion proteins and complete the dose response study in a mouse arterial thrombosis model in vivo. A mouse arterial thrombosis model will be used to establish the antithrombotic efficacies of EVAS' anticoagulant molecules. 3. Develop a bioprocess to produce the recombinant fusion proteins for in vitro and in vivo studies. A more efficient fermentation and purification process will be established to provide the needed materials for Phase I research. This process will also lay a foundation for Phase II GLP production of the recombinant proteins.