Phosphatidylserine exposure on the external leaflet of cell membranes is important for the assembly of the coagulation enzyme complexes tenase, the activator of factor X and prothrombinase, the activator of prothrombin on the membrane surface. There is increasing interest in the role of the membrane surfaces of microparticles in initiation of blood coagulation and assembly of coagulation enzyme complexes. A great deal is known about the assembly of the coagulation complexes in vitro; the identity of the important membrane surface(s) in vivo remains uncertain. The goals of these studies are to establish the source of procoagulant membrane surfaces, the temporal and spatial exposure of negatively charged phospholipids, and the site of prothrombinase assembly in vivo.
In Specific Aim 1 we will characterize microparticles generated in vitro from platelets, monocytes, neutrophils, and endothelial cells by size and by protein and phospholipid composition to identify a signature based on membrane protein composition to distinguish microparticles by cell source and subpopulation.
In Specific Aim 2 we will characterize microparticles isolated from human blood and compare them to microparticles generated in vitro. We anticipate that signatures that can identify the cell of origin of microparticle populations may be useful in studying human thrombotic and inflammatory states. We will also use the microparticle and cell signatures to identify these species in vivo in a thrombosis model in the mouse. Thus we will determine analogous signatures for mouse cells and microparticles.
In Specific Aim 3 we will determine the spatial and temporal exposure of aminophospholipids and assembly of prothrombinase in this model. To do these sorts of studies we have developed a unique imaging facility for acquisition of intravital microscopy data. The instrumentation is dedicated to intravital microscopy in living animals; acquires images at high speed (near video rate); has multiple fluorescence channels to allow observation of up to three fluorochromes simultaneously; has both widefield and confocal capabilities; is high resolution; permits 3D/volume reconstruction; provides digital image analysis.
