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 begin to elucidate the cellular mechanisms which regulate the expression of a procoagulant phenotype. Our accomplishments included demonstrating that 1) factor VIIa bound to the integral membrane protein tissue factor, expressed on the monocyte membrane surface, exhibits different substrate specificity than factor VIIa bound to recombinant tissue factor relipidated with defined phospholipid vesicles, specifically concerning the inability of the cell-bound enzyme to recognize factor IXa as a substrate; 2) 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 and 3) monocytes alter factor Va cofactor activitiy through the expression of membrane-bound elastase and cathepsin G which produce a cofactor molecule with reduced affinity for factor Xa (=3-fold). 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 determine if the constitutive expression of tissue factor (such as that observed in our pericyte population) modulates its cofactor function in factor IX activation as compared to tissue factor induced on the monocyte membrane surface.
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