HUMAN THROMBINS

正在生产人α-凝血酶(约5g/年)，以获得详细的 蛋白质结构、酶特异性和生物学特性的研究 这种中心的、生物调节的丝氨酸蛋白酶在止血中的功能。在……里面 除了这种形式的高促凝血剂和所有其他 凝血酶活性，凝血最少但仍高的形式 合成底物的活性是由其中一个受控的 蛋白水解性裂解(例如，伽玛-凝血酶)或选择性化学物质 修饰(例如，硝化酪氨酸；氧化蛋氨酸凝血酶)。 催化灭活模板也在制造中，这种模板保留了 非共价-蛋白质结合或识别位点(例如，各种 亲和标记；活性部位丝氨酸衍生凝血酶)。这些 修饰的凝血酶正被用于绘制活性部位区域(例如， 在三维B链模型内)，并将结合位点括起来 纤维蛋白(原)识别和大多数其他生物活性(例如， 血小板、成纤维细胞受体)。正在检查绑定交互作用 通过固定化蛋白质的固定相分配(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链上单糖部分的衍生化 不影响纤维蛋白(原)相互作用(例如，结合、凝血 活性)并实现标记α-凝血酶的新方法(例如， 碳水化合物结合的荧光、放射性、生物素衍生物或 免疫化学组)在凝血或受体处理过程中跟随它 事件(例如，凝块包裹凝血酶)。生物素或半抗原的络合 分别与亲和素或抗体偶联，不仅提供 凝血酶功能的交互式探针，但也有方法 纯化(例如，受体)。酶联双抗体免疫测定法 应允许监测各种功能活动(例如凝血酶 因子VIII的Ag：C络合作用增强)，而稳定控制 血浆应该允许外部标准化和重新定义 凝血酶凝血单位(例如，血浆凝血酶原的百分比)。这样的方法 是为我们自己的内部、各种协作和 众多非协作项目(例如，从蛋白质化学到细胞 生物学；临床血液学到癌症研究)，利用材料 源自我们的实验室(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)上 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.