Human neutrophil elastase (HNE) has long been linked to the pathology of a variety of inflammatory diseases and therefore is a potential target for therapeutic intervention. At least two other serine proteases, proteinase 3 (Pr3) and cathepsin G, are stored within the same neutrophil primary granules as HNE and are released from the cell at the same time at inflammatory sites. HNE and Pr3 are structurally and functionally very similar, and no substrate is currently available that is preferentially cleaved by Pr3 rather than HNE. Discrimination between these two proteases is the first step in elucidating their relative contributions to the development and spread of inflammatory diseases. Therefore, we have prepared new fluorescent peptidyl substrates derived from natural target proteins of the serpin family. This was done because serpins are rapidly cleaved within their reactive site loop whether they act as protease substrates or inhibitors. The hydrolysis of peptide substrates reflects the specificity of the parent serpin including those from ␣-1-protease inhibitor and monocyte neutrophil elastase inhibitor, two potent inhibitors of elastase and Pr3. More specific substrates for these proteases were derived from the reactive site loop of plasminogen activator inhibitor 1, proteinase inhibitors 6 and 9, and from the related viral cytokine response modifier A (CrmA). This improved specificity was obtained by using a cysteinyl residue at P1 for Pr3 and an Ile residue for HNE and because of occupation of protease S subsites. These substrates enabled us to quantify nanomolar concentrations of HNE and Pr3 that were free in solution or bound at the neutrophil surface. As membrane-bound proteases resist inhibition by endogenous inhibitors, measuring their activity at the surface of neutrophils may be a great help in understanding their role during inflammation. Human neutrophil elastase (HNE)1 and proteinase 3 (Pr3) are very closely related serine proteases that are stored in millimolar amounts within the primary granules of polymorphonuclear neutrophils (PMN) (1-3). The activation and degranulation of PMNs at an inflammatory site result in these proteases being translocated to the surface of the plasma membrane and/or released from the PMN (2, 4). Thus, they are involved in the proteolytic events associated with inflammation such as the lysis of extracellular matrix components, the control of cytokine activity, availability (5), platelet activation, and blood coagulation (6, 7).The contributions of HNE and Pr3 to the development of inflammatory diseases remain to be elucidated, mainly because they have very similar substrate specificities (1). Indeed, all of the synthetic substrates for Pr3 assayed to date are hydrolyzed faster by HNE (8, 9) so that it is virtually impossible to distinguish between the activities of these two proteases when they are both present in a biological fluid. However, Pr3 has some features that can help to distinguish it from HNE. It is not inhibited by secretory leukocyte protease inhibitor,...

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