Intracellular Processing and Trafficking of Protease Inhibitors in Platelets

R.R. Schleef, J. Chang, I.M. Lang, L. Gombau, M. Riewald, C.F. Barbas^*

^* Department of Molecular Biology, TSRI

Platelets play a central role in cardiovascular diseases through the release and cell-surface expression of a variety of hemostatic proteins. During the formation of platelets within megakaryocytes in the bone marrow or after the release of platelets into the circulating blood, these molecules are deposited into storage organelles called -granules. Knowledge of the factors involved in the processing of proteins and their deposition into secretory granules will provide information on basic cellular activities and may reveal novel approaches for controlling the coagulation and fibrinolytic systems.

We have shown that coagulation and fibrinolytic protease inhibitors are packaged and stabilized within -granules in concert with a series of defined proteins in a calcium-dependent manner. To determine proteins that may participate in the targeting or storage of these potent inhibitors, we used filamentous bacteriophages to display proteins expressed by cells containing a regulated secretory pathway and enriched the proteins by using their affinity for different protease inhibitors. One novel cDNA clone that preferentially recognized solution-phase plasminogen activator inhibitor 1 and reacted positively with antibodies derived from a rabbit immunized with -granules was recombinately expressed, and the purified recombinant protein was used to generate polyclonal antibodies. These immunologic reagents were used to develop a purification protocol for a novel molecule that may be involved in the packaging of proteins into storage granules.

Because proteases are involved not only in the processing of proteins into storage granules but also in a number of basic cellular events, we have been analyzing the cytoplasmic molecules that control the activity of cell-associated proteases in the hematopoietic compartment. We have cloned a novel protease inhibitor that we call bomapin because its expression is restricted to hematopoietic cells within the bone marrow. To understand the expression of bomapin within the hematopoietic compartment, we examined RNA extracted from bone marrow or peripheral blood from healthy donors and from patients with leukemia. Bomapin mRNA was readily detected in normal bone marrow, at a level designated as medium. Bomapin expression in peripheral blood from healthy donors and from patients with chronic lymphocytic leukemia was low or undetectable. Blood from patients with chronic myeloid leukemia, chronic myelomonocytic leukemia, acute myeloid leukemia, and acute lymphocytic leukemia had low to medium levels of bomapin expression. In addition, one patient with acute monocytic leukemia had high levels of bomapin.

We extended these studies by analyzing the expression of bomapin in a series of tissue culture cell lines. Bomapin mRNA was detected in the monocytic THP-1 and AML-193 cell lines, and treatment of these cell lines with phorbol myristate acetate or TNF- reduced the bomapin mRNA levels over a 4-day period. Immunoblotting showed the presence of a 40-kD protein in the cytosol of THP-1 cells. Levels of bomapin antigen were correspondingly reduced after treatment with phorbol myristate acetate. Because phorbol myristate acetate and TNF- induce monocytic differentiation in THP-1 and AML-193 cells, these data suggest that bomapin may play a role in the regulation of protease activities, specifically during the early stages of cellular differentiation.