Structure/Function Study of the Anticoagulant Protein S

凝血包括在磷脂膜上组装钙依赖的大分子复合体。凝血的倒数第二步是凝血酶原转化为凝血酶，然后凝血酶负责纤维蛋白原转化为纤维蛋白。不受控制的或过量的凝血酶生成会导致血栓形成，这是发病率和死亡率的主要原因。蛋白C抗凝途径是调节凝血酶形成的重要机制，活化蛋白C与其辅因子S共同促进Va因子和VIIIa因子失活。S蛋白是一种依赖维生素K的蛋白，既具有抗凝活性又具有非抗凝活性。S蛋白的不依赖于碱性磷酸酶的活性被归因于其抑制“凝血酶原酶”和“肌张力酶”促凝剂复合体的能力。本研究的主要目的是阐明S蛋白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，我们将表征人蛋白S的GLA结构域，PS(1-45)，在抗凝不依赖于APC的机制中。将化学合成PS(1-45)，并研究其钙结合、磷脂结合和功能性质。然后我们将评估该结构域抑制FVA和FX依赖的凝血酶原酶活性的能力。最后，我们将利用核磁共振波谱确定PS(1-45)在有钙和无钙的情况下的三维结构。我们相信，通过对这两种结构的比较，将确定对钙结合至关重要的残基，从而参与钙依赖的构象扰动。这项建议的另一个主要目标涉及到S蛋白依赖于APC的辅因子活性，这需要凝血酶敏感区(TSR)和第一表皮生长因子结构域(EGF1)。首先，我们将鉴定重组表达的TSR-EGF1(rhTSR-EGF1)的结构和功能特性，以确保该蛋白正确折叠。同时，我们将统一地15N和13C标记rhTSR-EGF1，使我们能够使用异核核磁共振谱确定TSR-EGF1的三维结构。然后，我们将使用荧光光谱、等温量热法和核磁共振光谱研究重组人TSR-EGF1与APC或FXA之间的蛋白质-蛋白质相互作用。这些结构信息将识别参与复杂形成的关键表位，并提供必要的有价值的信息，以了解参与调节血栓形成的机制，这具有相当大的临床和基础科学兴趣。对这些机制的新见解可能会确定干预病理性血栓形成的新方法。

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.