Factor lXa-Factor Vllla complexes in blood coagulation

描述(由申请人提供)：将研究凝血因子IXa和凝血因子VIIIa结合在膜上的结构和功能，以及对体内凝血蛋白复合体形成重要的膜。所解决的关键问题是凝血因子IXa/凝血因子VIIIa复合体的结构，该复合体与膜结合的机制，以及微粒在体内作为凝血表面的作用。我们将利用多肽合成的凝血因子IX GLA结构域和毕赤酵母制备的凝血因子VIII的C2结构域，通过X射线结晶学和三维核磁共振技术建立凝血因子IX GLA结构域的三维结构：因子VIII C2结构域。对于核磁共振实验，将制备这些蛋白质的同位素富集型。我们将确定LysoPS：因子IX的结构，以定义区分其GLA结构域与膜表面结合的独特功能。为了了解凝血因子IXa/凝血因子VIIIa复合体的生理功能，我们将对小鼠微循环进行高速共聚焦和明场显微镜成像，以定位体内血栓形成过程中膜表面的凝血因子IXa：凝血因子VIIIa复合体。我们将在体内血栓形成过程中建立凝血因子IX GLA结构域与膜的结合，然后使用基于凝血因子X的荧光底物研究FixA：FVIIa复合体在体内血栓形成过程中膜上的组装和功能活性。我们将与血小板、内皮细胞以及来自白细胞、血小板和内皮细胞的微粒共定位凝血因子IXa/凝血因子VIIIa活性。我们假设，对凝血至关重要的表面反应，包括因子IXa/因子VIIIa复合体，需要血小板来源的微粒，而组织因子的传递则需要白细胞来源的微粒。我们将通过建立微粒子缺陷小鼠模型来确定微粒膜表面在体内凝血中的作用。这些小鼠将通过活体显微镜研究这些微粒缺陷小鼠的特征和血栓形成。为了确定支持血液凝固的特定膜表面，我们将通过注入合成微囊来挽救微粒缺陷小鼠的纤维蛋白形成，其中磷脂成分通过改变磷脂酰丝氨酸含量而变化。

DESCRIPTION (provided by applicant): The structure and function of Factor IXa and Factor VIIIa bound to membranes and the membranes important for blood coagulation protein complex formation in vivo will be studied. The critical problem addressed is the structure of the Factor IXa/Factor VIIIa complex, the mechanism by which this complex binds to membranes and the role of microparticles as the surface upon which blood coagulation takes place in vivo. We will establish the 3D structure of the Factor IX Gla domain:Factor VIII C2 domain by X-ray crystallography and by 3D NMR using the Factor IX Gla domain prepared by peptide synthesis and the C2 domain of Factor VIII prepared in Pichia pastoris. For the NMR experiments, isotopically enriched forms of these proteins will be prepared. We will determine the structure of lysoPS:Factor IX to define the unique features that distinguish its Gla domain binding to membrane surfaces. To understand the physiologic function of the Factor IXa/Factor VIIIa complex, we will perform high speed confocal and brightfield microscopy imaging of the mouse microcirculation to localize the Factor IXa:Factor VIIIa complex on membrane surfaces in vivo during thrombus formation. We will establish binding of the Factor IX Gla domain to membranes during thrombus development in vivo, then study the assembly and functional activity of the FIXa:FVIIIa complex on membranes in vivo during thrombus development using a fluorogenic substrate based upon Factor X. We will co-localize Factor IXa/Factor VIIIa activity with platelets, endothelial cells, and microparticles derived from leukocytes, platelets and endothelial cells. We hypothesize that the surface-based reactions that are critical for blood coagulation, including the Factor IXa/Factor VIIIa complex, require platelet-derived microparticles whereas the delivery of tissue factor requires leukocyte-derived microparticles. We will determine the role of microparticle membrane surfaces in blood coagulation in vivo by developing of microparticle-deficient mice models. These mice will be characterized and thrombus formation in these microparticle-deficient mice studied by intravital microscopy. To determine the specific membrane surfaces that support blood coagulation, we will rescue fibrin formation in microparticle-deficient mice by infusion of synthetic microvesicles in which the phospholipid composition is varied by altering the phosphatidylserine content.