Thrombin is generated at the monocyte/macrophage surface through the assembly of the multicomponent coagulation enzyme prothrombinase, which consists of a membrane-bound, Ca++-dependent complex of factor Va and factor Xa. The primary objective of this proposal is to provide a quantitative understanding of the molecular and supramolecular events which result in the expression and regulation of thrombin generation at the monocyte/macrophage surface, particularly through studies of prothrombinase. Our studies will define both the kinetic and thermodynamic parameters governing prothrombinase complex assembly on monocytes/macrophages, and the central role which factor Va plays in mediating these binding interactions. Our goals include characterization of the ability of monocytes/macrophages to assemble an endogenous prothrombinase through synthesis, secretion and activation of factor V, factor X and prothrombin. We will determine how the activated protein C/protein S complex interacts with the monocyte/macrophage surface and other constituents of the prothrombinase complex to regulate its activity. Equilibrium binding techniques using radiolabeled proteins will be used to calculate the thermodynamic parameters and stoichiometries governing these various binding interactions. The formation of a functional enzymatic complex will be analyzed using kinetics of prothrombin activation. We will characterize other potential regulatory mechanisms which may modulate monocyte/macrophage prothrombinase activity. Protocols include: (1) to stimulate these cells with compounds known to enhance or suppress monocyte procoagulant and fibroproliferative activities, (2) to co-culture, and/or adhere monocytes to extracellular matrices, arterial and venous endothelial cells, fibroblasts and smooth muscle cells, (3) to determine if monocyte prothrombinase activity is expressed by discrete subpopulations, and (4) to determine the collaborative role played by lymphocyte subpopulations. The ability of monocytes to effect thrombin formation appears to be an integral part of their (patho)physiologic role in wound and tissue repair, chronic inflammation and atherosclerosis. The regulation of thrombin generation at the monocyte/macrophage surface may be pivotal to their related functions as they localize to vascular and extravascular tissue sites.
