REGULATION OF PROTEASE NEXIN 1 ACTIVITY AND SECRETION

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Our goal is to elucidate mechanisms by which regulatory serine proteases are controlled at and near the cell surface by three protease nexins (PN-1, PN-2 and PN-3) which we identified in earlier studies. The PNs are protein protease inhibitors that are synthesized and released by a variety of cultured cells. They form covalent complexes with certain proteases in the extracellular environment; the complexes bind back to the cells and are rapidly internalized and degraded. With purified PN-1 and seven regulatory proteases it inactivates, we will measure second order association rate constants to provide a quantitative foundation for our studies. Then, based on our finding that the surface of fixed fibroblasts accelerates the reaction between PN-1 and thrombin, we will determine if the reactions between PN-1 and the other proteases are similarly accelerated. Some of the acceleration of the reaction between PN-1 and thrombin appears to involve cell surface/extracellular matrix glycosaminoglycans. We will check this by treating fibroblasts with glycosaminoglycan lyases, fixing the cells, and examining the effect of this on the ability of the cells to accelerate the reactions. In complimentary studies we will examine the effects of purified glycosaminoglycans, in the absence of cells, on the reactions between PN-1 and the proteases. About 50% of the acceleration of the PN-1 and thrombin reactions appears to involve the Mr=150,000 cell surface binding sites for thrombin. We will study the basis of this and determine if there are cell surface binding sites for the six other regulatory proteases that might similarly participate in their control. With PN-2 that we recently purified to homogeneity, we will screen for serine proteases it effectively inactivates, measure second order association rate constants with them, and begin studies on PN-2 along the lines of the above studies for PN-1. Finally, we will purify PN-3 from serum-free culture medium conditioned by fibroblasts.