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.
| Arias-Salgado, Elena Garcia; Haj, Fawaz; Dubois, Christophe et al. (2005) PTP-1B is an essential positive regulator of platelet integrin signaling. J Cell Biol 170:837-45 |
