The objectives of this project are to define and characterize reactions of the plasma proteinase inhibitor Alpha2-macroglobulin (Alpha2M) with proteinases and the interaction of the Alpha2M-proteinase complex with human peripheral blood monocytes.
The specific aims are as follows: 1) The reaction of Alpha2M with trypsin and thrombin will be examined to determine the functional relationship of the Alpha2M proteinase binding sites to each other. 2) The reactions at the thiolester site of Alpha2M and their effect on the conformation of Alpha2M will be characterized. 3) Studies in progress will be expanded to show that monocytes have a specific binding site for Alpha2M, amine-treated Alpha2M, or the Alpha2M-proteinase complex. 4) The number and affinity of binding sites on the monocyte will be determined. 5) The Alpha2M structural determinants recognized by the monocyte binding site will be defined. 6) The functional consequences of binding of Alpha2M to monocytes will be investigated to determine if binding and/or uptake constitute a control mechanism in either coagulation or fibrinolysis. The reactions of Alpha2M with proteinase and methylamine will be studied using intrinsic protein fluorescence and fluorescent probes to measure conformation changes. In parallel, the chemical state of the thiolester will be assayed with sulfhydryl reagents, by gel electrophoretic analyses and by incorporation of 14CH3NH2. Studies of the dissociability of radiolabelled thrombin and trypsin from Alpha2M will utilize anti-Alpha2M IgG coupled to agarose and macromolecular inhitibor-synthetic substrate assays. Binding of Alpha2M to human peripheral blood monocytes will be studied and the protein structural determinants for cell recognition identified by isolation of products of proteolytic digestion and chemical modification of Alpha2M. Functional effects of binding such as intracellular protein phosphorylation and calcium mobilization will be measured using 32PO4 and the calcium probe Quin-2. Cell culture techniques in conjunction with fibrin dissolution and plasma recalcification assays will be utilized to determine if Alpha2M binding causes development of procoagulant activity or secretion of plasminogen-activator. These studies are important for understanding the control of certain blood clotting and fibrinolytic activities, particularly as related to monocyte and macrophage function in inflammatory processes such as those accompanying infection, neoplasm, or the early stages of atherosclerotic plaque formation.
| Straight, D L; Jakoi, L; McKee, P A et al. (1988) Binding of alpha 2-macroglobulin-thrombin complexes and methylamine-treated alpha 2-macroglobulin to human blood monocytes. Biochemistry 27:2885-90 |
