Monocytes/macrophages play key roles in inflammatory and atherosclerotic processes, which in several instances proceed at extravascular tissue sites and are characterized by a fibroproliferative response and fibrin deposition. Since thrombin is a potent effector of these responses, the overall hypothesis is that thrombin production at the monocyte/macrophage surface provides an important bioregulatory effector molecule at these sites. The objectives are to define the binding and kinetic parameters governing the molecular events which result in the expression of thrombin at the monocyte/macrophage surface and to begin to elucidate the cellular mechanisms which regulate the expression of a procoagulant phenotype. Particular emphasis is placed on how the monocyte/macrophage surface membrane regulates prothrombinase complex assembly and function through the expression of binding sites for the cofactor factor Va and the enzyme factor Xa, and the expression of membrane-bound proteases which activate the procofactor factor V to factor Va. Equilibrium binding studies using radiolabeled factors V, Va and Xa will be coupled to kinetic analyses of prothrombin activation to correlate binding and function. Proteolytic modification of factors V and Va will be detected using electrophoretic, autoradiographic techniques. Factor Xa binding to monocytes, which occurs independently of factor Va and generates factor IX( will be studied to determine how this interaction regulates factor Xa proteolytic activity and its participation in prothrombin and factor IX activation. The mechanisms by which cell activation and cell adherence to extracellular matrix protein components enhances these various binding events and proteolytic activities will be defined. In addition, the regions of the factor Va and factor Xa molecules which mediate their independent binding to monocytes will be determined using isolated protein fragments, synthetic peptides mimicking their known sequences and anti-peptide antibodies of inhibitory peptides. Finally, affinity chromatographic techniques will be used to isolate the monocyte/macrophage """"""""receptor"""""""" for factor Va. The overall goal of this project is to provide a detailed understanding of how thrombin generation is effected and regulated at the monocyte surface through a series of integrally related events. The formation of thrombin at the monocyte/macrophage surface appears to be pivotal to the physiological and pathophysiological functions these cells provide as they localize to vascular and extravascular tissue sites. The ability of monocytes/macrophages to participate in and regulate the molecular events leading to thrombin formation is an integral part of their physiological and pathophysiological roles in atherosclerosis, chronic inflammation and wound repair. Also, since monocytes interact with vascular endothelium, platelets and mesenchymal cells, and since all of these cells respond in a variety of ways to thrombin, it seems likely that the production of thrombin at the monocyte/macrophage membrane surface provides an important bioregulatory effector molecule at precise locations. This hypothesis is based on the observations that thrombin can be an important effector molecule in cell-cell signaling and in remodelling the extracellular environment, activities which are important in a variety of (patho)physiologic settings in which monocytes are known to participate. Our accomplishments included demonstrating that 1) like monocytes, pericytes derived from human brain microvessels, can activate and propagate the coagulant response through the extrinsic pathway (i.e. a tissue factor/factor VIIa complex followed by a prothrombinase complex), however, unlike monocytes the activities of the required enzyme complexes can be differentially regulated in response to agonist stimulation; 2) monocytes/macrophages sustain thrombin generation at their surface by protecting the cofactor factor Va from inactivation by activated protein C; and 3) proteolysis of factyor V by cathepsin G yields a cofactor with a reduced affinity for factor Xa, thus limiting thrombin generation. Our current plans are: 1) to complete the structure/function analyses and cofactor activities of factor Va cleaved by elastase and cathepsin G; 2) to define the activation pathway of factor IX by the tissue factor/factor VIIa complex assembled on monocytes, and 3) to define the mechanism by which monocytes inhibit factor Va inactivation by activated protein C.
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