Alzheimer's disease (AD) is an idiopathic progressive dementia affecting a large proportion of our increasingly aged population. Although the pathogenesis of AD is not completely understood, present data suggest that it probably involves the abnormal proteolytic cleavage of a large precursor protein to yield about 42 residue protein, referred to as amyloid Beta protein or A4, which subsequently polymerizes and forms the amyloid deposits characteristic of this disease. The precursor of the amyloid Beta-protein (i.e., amyloid Beta-protein precursor; APP) is encoded by at least three mRNAs that arise through alternative splicing of two exons. The two most abundantly expressed forms contain a 56 residue insert, which has significant homology with the Kunitz-type protease inhibitors (KPI). Platelet coagulation Factor Xla-inhibitor was recently identified to be a form of APP that contains the KPI domain. Presently, little information exists on the origin of APP in the platelet or its processing during the maturation of megakaryocytes/platelets. The long-term objective of the work described in this application is to provide a detailed understanding of the production and processing of APP associated with megakaryocytes/platelets. Immunologic probes will be developed against specific regions of the APP molecule and utilized in immunofluorescent and immunoelectron microscopic techniques to define the cellular distribution of APP in (i) intact platelets, (ii) platelets during adhesion/aggregation reactions, (iii) megakaryocytic cell lines, and (iv) freshly isolated and cultured bone marrow megakaryocytes as they mature. The forms of APP present in intact platelets, as well as the processing of APP that occurs during platelet adhesion/aggregation reactions, will be analyzed utilizing immunoblotting protocols and radioimmunoassays. These studies will form the framework for analyzing the location and forms of APP associated with abnormal platelets that are present in the blood of AD patients. The origin of platelet APP will then be defined by utilizing immunologic assays and metabolic labeling/immunoprecipitation experiments to analyze the production of APP during the differentiation of megakaryocytic cell lines and bone marrow-derived cells in vitro. These studies will be complemented by characterizing the protease activity(ies) responsible for the processing of megakaryocyte/platelet APP. Furthermore, the structural domain(s) on APP that are critical for its targeting into platelet alpha-granules will be defined by the use of expression vector/APP constructs in cell lines that exhibit a storage secretory pathway. These constructs will include deletion mutants of APP, site-directed mutants of APP, and hybrid APP molecules. Because platelets have been proposed as a peripheral model for neurons, it is expected that information on the mechanisms by which megakaryocytes/platelets process APP will likely provide an insight into the expression and cleavage of this protein by neurons.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL049563-01
Application #
3368689
Study Section
Special Emphasis Panel (ZHL1-CSR-J (01))
Project Start
1992-09-30
Project End
1997-09-29
Budget Start
1992-09-30
Budget End
1993-09-29
Support Year
1
Fiscal Year
1992
Total Cost
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Chuang, T L; Schleef, R R (1999) Identification of a nuclear targeting domain in the insertion between helices C and D in protease inhibitor-10. J Biol Chem 274:11194-8
Lang, I M; Moser, K M; Schleef, R R (1998) Elevated expression of urokinase-like plasminogen activator and plasminogen activator inhibitor type 1 during the vascular remodeling associated with pulmonary thromboembolism. Arterioscler Thromb Vasc Biol 18:808-15
Riewald, M; Chuang, T; Neubauer, A et al. (1998) Expression of bomapin, a novel human serpin, in normal/malignant hematopoiesis and in the monocytic cell lines THP-1 and AML-193. Blood 91:1256-62
Lang, I M; Barbas 3rd, C F; Schleef, R R (1996) Recombinant rabbit Fab with binding activity to type-1 plasminogen activator inhibitor derived from a phage-display library against human alpha-granules. Gene 172:295-8
Seiffert, D; Schleef, R R (1996) Two functionally distinct pools of vitronectin (Vn) in the blood circulation: identification of a heparin-binding competent population of Vn within platelet alpha-granules. Blood 88:552-60
Riewald, M; Morgenstern, K A; Schleef, R R (1996) Identification and characterization of the cytoplasmic antiproteinase (CAP) in human platelets. Evidence for the interaction of CAP with endogenous platelet proteins. J Biol Chem 271:7160-7
Lang, I M; Moser, K M; Schleef, R R (1996) Expression of Kunitz protease inhibitor--containing forms of amyloid beta-protein precursor within vascular thrombi. Circulation 94:2728-34
Lang, I M; Chuang, T L; Barbas 3rd, C F et al. (1996) Purification of storage granule protein-23. A novel protein identified by phage display technology and interaction with type I plasminogen activator inhibitor. J Biol Chem 271:30126-35
Lang, I M; Schleef, R R (1996) Calcium-dependent stabilization of type I plasminogen activator inhibitor within platelet alpha-granules. J Biol Chem 271:2754-61
Riewald, M; Schleef, R R (1996) Human cytoplasmic antiproteinase neutralizes rapidly and efficiently chymotrypsin and trypsin-like proteases utilizing distinct reactive site residues. J Biol Chem 271:14526-32

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