Structure/Function Study of the Anticoagulant Protein S

血液凝固涉及钙依赖性大分子复合物在磷脂膜上的组装。血液凝固的倒数第二步是凝血酶原转化为凝血酶，然后负责将纤维蛋白原转化为纤维蛋白。 不受控制或过量的凝血酶生成导致血栓形成，这是发病率和死亡率的主要原因。 蛋白C抗凝剂途径是调节凝血酶形成的重要机制，由此活化的蛋白C（APC）与其辅因子蛋白S结合增强因子Va（FVa）和因子VIIIa（FVIIIa）的失活。 蛋白S是一种维生素K依赖性蛋白，具有APC依赖性和APC非依赖性抗凝活性。 蛋白S的APC非依赖性活性归因于其抑制“凝血酶原酶”和“tenase”促凝血复合物的能力。 本研究计划的主要目标是阐明蛋白S的N-末端结构域的结构和/或功能意义，包括：Gla结构域，凝血酶敏感区和第一表皮生长因子结构域。 首先，我们将在APC独立的抗凝机制中表征人蛋白S，PS（1-45）的Gla结构域。 PS（1-45）的化学合成及其钙结合、磷脂结合和功能性质的研究。 然后我们将评估该结构域抑制FVa和FX依赖性凝血酶原酶活性的能力。 最后，我们将使用NMR光谱法确定PS（1-45）在存在和不存在钙的情况下的三维结构。 我们相信，这两种结构之间的比较将确定的残基是关键的钙结合，从而参与钙依赖性构象扰动。 该提议的另一个主要目标涉及蛋白S的APC依赖性辅因子活性，其需要凝血酶敏感区（TSR）和第一表皮生长因子结构域（EGF 1）。首先，我们将表征重组表达的TSR-EGF 1（rhTSR-EGF 1）的结构和功能特性，以确保该蛋白质正确折叠。 同时，我们将均匀地15 N和13 C标记rhTSR-EGF 1，使我们能够使用异频NMR光谱确定TSR-EGF 1的三维结构。 然后，我们将使用荧光光谱、等温量热法和NMR光谱研究rhTSR-EGF 1与APC或FXa之间的蛋白质-蛋白质相互作用。 这种结构信息将确定参与复合物形成的关键表位，并提供有价值的信息，以了解参与调节血栓形成的机制，这是相当大的临床和基础科学的兴趣。 对这些机制的新见解可能会发现新的方法来干预病理性血栓形成。

Blood coagulation involves the assembly of calcium- dependent macromolecular complexes on phospholipid membranes. The penultimate step in blood coagulation is the conversion of prothrombin to thrombin, which is then responsible for the conversion of fibrinogen to fibrin. Uncontrolled or excessive thrombin generation leads to thrombosis, a major cause of morbidity and mortality. The protein C anticoagulant pathway is an essential mechanism for regulating thrombin formation, whereby activated protein C (APC) in association with its cofactor protein S enhances the inactivation of factor Va (FVa) and factor VIIIa (FVIIIa). Protein S, is a vitamin K-dependent protein that possesses both APC-dependent and APC-independent anticoagulant activity. The APC-independent activity of protein S has been ascribed to its ability to inhibit the "prothrombinase" and "tenase" procoagulant complexes. The major goals of this research proposal are to elucidate the structural and/or functional significance of the N-terminal domains of protein S including: the Gla domain, the thrombin sensitive region and the first epidermal growth factor domain. Initially, we will characterize the Gla domain of human protein S, PS(1-45), in the APC-independent mechanism of anticoagulation. PS (1-45) will be chemically synthesized and its calcium binding, phospholipid binding and functional properties investigated. We will then evaluate the ability of this domain to inhibit the FVa- and FX- dependent prothrombinase activity. Finally, we will determine the three-dimensional structure of PS(1-45) in the presence and absence of calcium using NMR spectroscopy. We believe that a comparison between these two structures will identify residues that are critical for calcium binding and thus involved in the calcium-dependent conformational perturbation. The other major goal of this proposal involves the APC-dependent cofactor activity of protein S, which requires the thrombin sensitive region (TSR) and first epidermal growth factor domain (EGF1). Initially, we will characterize the structural and functional properties of recombinantly expressed TSR-EGF1 (rhTSR-EGF1), to ensure this protein is properly folded. Simultaneously, we will uniformly 15N and 13C label rhTSR-EGF1 allowing us to determine the three-dimensional structure of TSR-EGF1 using heteronuclear NMR spectroscopy. We will then investigate the protein-protein interaction between rhTSR-EGF1 and APC or FXa using fluorescence spectroscopy, isothermal calorimetry and NMR spectroscopy. This structural information will identify the critical epitopes involved in complex formation and provide valuable information necessary to understand the mechanisms involved in regulating thrombus formation, which is of considerable clinical and basic science interest. New insights into these mechanisms may identify novel methods for the intervention in pathologic thrombus formation.