Factor lXa-Factor Vllla complexes in blood coagulation

描述（由申请人提供）：将研究结合在膜上的因子IXa和因子VIIIa的结构和功能，以及体内凝血蛋白复合物形成的重要膜。关键问题是因子IXa/因子viia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血液凝固发生的表面的作用。我们将利用肽合成制备的Factor IX Gla结构域和毕赤酵母制备的Factor VIII的C2结构域，通过x射线晶体学和3D NMR建立因子IX Gla结构域的三维结构：因子VIII C2结构域。在核磁共振实验中，将制备这些蛋白质的同位素富集形式。我们将确定lysoPS:Factor IX的结构，以定义区分其与膜表面结合的玻璃结构域的独特特征。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场显微镜成像，以在血栓形成过程中定位因子IXa：因子VIIIa复合物在体内膜表面的位置。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的荧光底物研究在体内血栓形成过程中FIXa: fviii复合物在膜上的组装和功能活性。我们将因子IXa/因子viii ia活性与血小板、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。我们假设，对血液凝固至关重要的基于表面的反应，包括因子IXa/因子viii复合物，需要血小板衍生的微粒，而组织因子的递送则需要白细胞衍生的微粒。我们将通过开发微颗粒缺陷小鼠模型来确定微颗粒膜表面在体内血液凝固中的作用。这些小鼠将被表征，并通过活体显微镜研究这些微粒缺陷小鼠的血栓形成。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的微泡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改变。

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.