Factor lXa-Factor Vllla complexes in blood coagulation

描述（由申请人提供）：将研究与膜结合的因子IXa和因子VIIIa的结构和功能以及对体内凝血蛋白复合物形成重要的膜。所解决的关键问题是因子IXa/因子VIIIa复合物的结构、该复合物与膜结合的机制以及微粒作为体内发生血液凝固的表面的作用。我们将使用通过肽合成制备的因子IX Gla结构域和在巴斯德毕赤酵母中制备的因子VIII的C2结构域，通过X射线晶体学和通过3D NMR建立因子IX Gla结构域的3D结构：因子VIII C2结构域。对于NMR实验，将制备这些蛋白质的同位素富集形式。我们将确定lysoPS的结构：因子IX以定义区分其Gla结构域与膜表面结合的独特特征。为了了解因子IXa/因子VIIIa复合物的生理功能，我们将对小鼠微循环进行高速共聚焦和明视野显微镜成像，以在血栓形成期间将因子IXa：因子VIIIa复合物定位在体内膜表面上。我们将在体内血栓形成过程中建立因子IX Gla结构域与膜的结合，然后使用基于因子X的荧光底物在体内血栓形成过程中研究FIXa：FVIIIa复合物在膜上的组装和功能活性。我们将使因子IXa/因子VIIIa活性与血小板、内皮细胞和源自白细胞、血小板和内皮细胞的微粒共定位。我们假设，基于表面的反应是血液凝固的关键，包括因子IXa/因子VIIIa复合物，需要血小板衍生的微粒，而组织因子的交付需要白细胞衍生的微粒。我们将通过开发微粒缺陷小鼠模型来确定微粒膜表面在体内血液凝固中的作用。将对这些小鼠进行表征，并通过活体显微镜研究这些微粒缺陷小鼠中的血栓形成。为了确定支持血液凝固的特定膜表面，我们将通过输注合成微泡来拯救微粒缺陷小鼠中的纤维蛋白形成，其中磷脂组成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.