HUMAN THROMBINS

正在生产人类α-凝血酶（约5g/年），用于详细说明 蛋白质结构、酶的专一性和生物活性的研究 这种中央生物调节丝氨酸蛋白酶在止血中的功能。 在 除了这种形式与高促凝血剂和所有其他 凝血酶归因的活性，具有最小凝血但高 合成基质活动正在准备， 蛋白水解裂解（例如，γ-凝血酶）或选择性化学品 修改（例如，硝化酪氨酸;氧化甲硫氨酸凝血酶）。 还制备了催化失活形式，其保留了 非共价蛋白结合或识别位点（例如，各种 亲和标记的;活性位点丝氨酸衍生的凝血酶）。 这些 修饰的凝血酶用于定位活性位点区域（例如， 在三维B链模型中），并将结合位点包围起来， 纤维蛋白（原）识别和大多数其它生物活性（例如， 血小板、成纤维细胞受体）。 正在检查结合相互作用 通过用固定化蛋白质进行固定相分配（例如， 各种凝血酶形式的非聚合纤维蛋白结合）。 唾液酸 凝血酶B链上单个碳水化合物部分的衍生化 不影响纤维蛋白（原）相互作用（例如，结合，凝血 活性）并且能够实现标记α-凝血酶的新方法（例如， 碳水化合物共轭荧光，放射性，生物素衍生物，或 免疫化学组）在凝血或受体加工过程中跟踪它 事件（例如，凝块截留的凝血酶）。 生物素或半抗原的复合 分别与抗生物素蛋白或抗体的缀合物不仅提供 凝血酶功能的相互作用探针， 纯化（例如，受体）。 酶联双抗体免疫分析 应该允许监视各种功能活动（例如，凝血酶 因子VIII Ag：C络合的增强），而稳定的对照 血浆应允许外部标准化和重新定义 凝血酶凝固单位（例如，血浆凝血酶原百分比）。 这样的方法 是多用途的设计，为我们自己的内部，各种协作， 许多非合作项目（例如，细胞蛋白质化学 生物学;临床血液学到癌症研究）， 来自我们的实验室（est。超过100万美元/年的人力 凝血酶）。 我们的研究将继续主要集中在纤维蛋白（原） α-凝血酶的识别位点。

Human Alpha-thrombin is being produced (approximately 5g/yr) for detailed studies on the protein structure, enzyme specificity and biological functions of this central, bioregulatory serine protease in hemostasis. In addition to this form with high procoagulant and all other thrombin-ascribed activities, forms with minimal clotting yet high synthetic-substrate activities are being prepared by either controlled proteolytic cleavage (e.g., Gamma-thrombin) or selective chemical modification (e.g., nitrated-tyrosine; oxidized-methionine thrombins). Catalytically inactivated forms are also being made which retain the noncovalent-protein binding or recognition site (e.g., various affinity-labeled; active-site serine derivatized thrombins). These modified thrombins are being used for mapping active-site regions (e.g., within the 3-dimensional B-chain model) and bracketing the binding site for fibrin(ogen) recognition and most other biological activities (e.g., platelet, fibroblast receptors). Binding interactions are being examined by stationary phase partitioning with immobilized proteins (e.g., nonpolymerized fibrin binding of various thrombin forms). Sialic acid derivatization of the single carbohydrate moiety on the thrombin B chain does not affect fibrin(ogen) interactions (e.g., binding, clotting activity) and enables new approaches for labeling Alpha-thrombin (e.g., carbohydrate conjugated fluorescent, radioactive, biotin-derivative, or immunochemical groups) to follow it during clotting or receptor processing events (e.g., clot entrapped thrombin). Complexing of biotin or hapten conjugates with avidin or antibodies, respectively, not only provides interactive probes of thrombin functions but also approaches for purification (e.g., receptors). Enzyme-linked double-antibody immunoassays should allow monitoring of various functional activities (e.g., thrombin enhancement of Factor VIII Ag:C complexing), whereas stabilized control plasma should permit external standardization and redefinition of the thrombin clotting unit (e.g., percent plasma prothrombin). Such approaches are multi-purpose designed for our own in-house, various collaborative, and numerous noncollaborative projects (e.g., Protein Chemistry to Cell Biology; Clinical Hematology to Cancer Research), which utilize materials originating from our laboratory (est. greater than $1-million/yr of human thrombin). Our research will continue to focus mainly on the fibrin(ogen) recognition site of Alpha-thrombin.