NEW HEPARIN-DEPENDENT PROTEASE INHIBITOR IN HUMAN PLASMA

在初步实验中，我发现了一种以前未被识别的 人血浆中的蛋白水解酶抑制剂。初步研究表明，这一点 抑制物是一种表观分子量为65,6000的蛋白质， 它通过形成非常稳定的1：1化学计量比络合物来灭活凝血酶 它在免疫学上与所有的 已知的血浆蛋白酶抑制剂。新型缓蚀剂的显著性能 它的作用明显是由肝素加速的。此外，在 肝素的浓度相对较高，这种抑制剂的竞争相当激烈 使用抗凝血酶III对凝血酶有效，尽管抗凝血酶III 之前被认为是唯一在数量上有意义的 血浆中凝血酶的抑制物。在此应用程序中，我建议执行 1)提纯足量的结构用缓蚀剂 学习。2)提高兔的抗体水平，建立一种特异性免疫分析方法 用于血浆中的新抑制物。3)测定部分NH_2-和 蛋白质的C00H-末端氨基酸序列。4)确定 纯化的抑制物对下列纯化的抑制物失活的能力 蛋白酶：凝血因子VIIa、IXa、XIIa(片段)、激肽释放酶、 纤溶酶和激活蛋白C)建立肝素依赖和 (4)中抑制蛋白水解酶的化学计量。6)详细执行 用动力学研究确定二级速率常数和/或 抑制剂-蛋白酶复合体在水溶液中的平衡解离常数 有无肝磷脂。7)分离粗肝素 通过与抑制剂结合的大小和亲和力制备，以及 比较每一组分激活新的抑制剂和 抗凝血酶III8)用14C标记肝素的高活性部分(S) 或~3H，并测定其离解常数和结合的化学计量比 新的抑制剂。9)确定新的抑制器是否经历 在与蛋白水解酶形成复合体的过程中有限的蛋白质分解。10)隔离 并对(9)中发现的蛋白水解物进行测序，以研究 缓蚀剂的反应部位的结构。

In preliminary experiments, I have identified a previously unrecognized protease inhibitor in human plasma. Initial studies indicate that this inhibitor is a protein with an apparent molecular weight of 65,6000, that it inactivates thrombin by forming a very stable 1:1 stoichiometric complex with the protease, and that it is immunologically distinct from all of the known plasma protease inhibitors. A striking property of the new inhibitor is that its action is markedly accelearted by heparin. In addition, at relatively high concentrations of heparin, this inhibitor competes quite effectively for thrombin with antithrombin III, although antithrombin III has previously been considered to be the only quantitatively significant inhibitor of thrombin in plasma. In this application, I propose to do the following: 1) Purify a sufficient quantity of the inhibitor for structural studies. 2) Raise antibodies in rabbits and develop a specific immunoassay for the new inhibitor in plasma. 3) Determine the partial NH2- and C00H-terminal amino acit sequences of the protein. 4) Determine the ability of the purified inhibitor to inactivate the following purified proteases: coagulation factors VIIa, IXa, XIIa (fragments), kallikrein, plasmin, and activated protein C. 5) Establish the heparin-dependence and stoichiometry of inhibition of the proteases in (4). 6) Perform detailed kinetic studies to determine the 2nd-order rate constant and/or the equilibrium dissociation constant of the inhibitor-protease complex in the presence and absence of heaprin. 7) Fractionate a crude heparin preparation by size and by affinity of binding to the inhibitor, and compare the ability of each fraction to activate both the new inhibitor and antithrombin III. 8) Label a highly active fraction(s) of heparin with 14C or 3H, and determine its dissociation constant and stoichiometry of binding to the new inhibitor. 9) Determine whether the new inhibitor undergoes limited proteolysis during complex formation with a protease. 10) Isolate and sequence the proteolytic gragments, if found, in (9) to investigate the structure of the reactive site of the inhibitor.