NMR OF BLOOD COAGULATION PROTEIN INHIBITORS

活化的Hageman因子(因子XII-a)是一种丝氨酸蛋白酶 参与凝血：激活凝血因子XI 凝血酶的形成，将血浆前激肽释放酶转化为激肽释放酶，以及 纤溶酶原对纤溶酶的促进作用，并增强其促凝血特性 因子VII.从南瓜(Cucurbita Maxima)中分离得到的胰酶抑制剂 种子，即CMTI-I、CMTI-III、CMTI-IV，每个种子都有一个分子 3 kD的重量和CMTI-V(MR 7 KD)是高度特异的 因子XII-a；其他具有中等序列的丝氨酸蛋白酶 与凝血因子XII-a的同源性(32%至37%)，如人纤溶酶、牛 凝乳酶和人凝血酶要么很弱，要么根本没有 被禁止了。CMTI-V最近被分离和鉴定：它 属于带有一个二硫键的马铃薯1抑制物家族，以及 它与较小的3kD蛋白没有任何序列同源性。 虽然维珍CMTI-I、CMTI-III和CMTI-V都抑制XII-a因子，但他们的 活性中心被水解(修饰)的形式不会。确定了 两种修饰前后缓蚀剂的溶液结构 二维核磁共振(2D)波谱将引领 为了更好地理解特异性抑制的机制 XII-a因子，可能导致新的治疗策略的开发 治疗血液凝血障碍。拟议工作的目标 是：1)继续序列特定的氢-1核磁共振指定 完整和修饰的CMTI-V及其二级结构鉴定 元素；2)演绎出处女和 利用核Overhaser效应衍生距离修正CMTI-V 和能量投射中的距离几何算法(DSPACE) 最小化程序(X-PLOR)；3)继续比较研究 将完整和活性中心的CMTI-I和CMTI-III通过多个- 量子质子探测的异核2D核磁共振波谱。

Activated Hageman factor (Factor XII-a) is a serine proteinase that is involved in blood coagulation: it activates Factor XI to bring about formation of thrombin, converts plasma prekallikrein to kallikrein, and plasminogen to plasmin, and increases the clot-promoting properties of Factor VII. Trypsin-inhibitors isolated from pumpkin (Cucurbita maxima) seeds, viz., CMTI-I, CMTI-III, CMTI-IV, each of which has a molecular weight of 3 Kd, and CMTI-V (Mr 7 Kd), are highly specific inhibitors of Factor XII-a; other serine proteases which bear moderate sequence homology (32 to 37%) to Factor XII-a, such as human plasmin, bovine chymotrypsin, and human thrombin are either weakly or not at all inhibited. CMTI-V has recently been isolated and characterized: it belongs to the Potato 1 inhibitor family with one disulfide bridge, and it does not bear any sequence homology to the smaller 3 Kd proteins. While virgin CMTI-I, CMTI-III, and CMTI-V all inhibit Factor XII-a, their reactive-site hydrolyzed (modified) forms do not. Determination of the solution structures of the intact and modified inhibitors by two- dimensional Nuclear Magnetic Resonance (2D NMR) spectroscopy will lead to a better understanding of the mechanism of specific inhibition of Factor XII-a and may lead to development of new therapeutic strategies for blood coagulation disorders. The objectives of the proposed work are: 1) to continue the sequence-specific hydrogen-1 NMR assignments of intact and modified CMTI-V and identify their secondary structural elements; 2) to deduce the three-dimensional structures of virgin and modified CMTI-V by the use of nuclear Overhauser effect-derived distances and a distance-geometry algorithm (DSPACE) in conjection with an energy minimization program (X-PLOR); and 3) to continue the comparative study of intact and reactive-site hydrolyzed CMTI-I and CMTI-III by multiple- quantum proton-detected heteronuclear 2D NMR spectroscopy.