Human Neutrophil Elastase Proteolytically Activates the Platelet Integrin αIIbβ3 through Cleavage of the Carboxyl Terminus of the αIIb Subunit Heavy Chain

Neutrophil elastase (NE) and cathepsin G are two serine proteinases released concomitantly by stimulated polymorphonuclear neutrophils. We previously demonstrated that while NE by itself does not activate human platelets, it strongly enhances the weak aggregation induced by a threshold concentration of cathepsin G (threshold of cathepsin G) (Renesto, P., and Chignard, M. (1993) Blood 82, 139-144). The aim of this study was to delineate the molecular mechanisms involved in this potentiation process. Two main pieces of data prompted us to focus on the activation of the platelet fibrinogen receptor, the αIIbβ3 integrin. First, previous studies have shown this integrin to be particularly prone to proteolytic regulation of its function. Second, we found that the potentiating activity of NE on the threshold of cathepsin G-induced platelet aggregation was strictly dependent on the presence of exogenous fibrinogen. Using flow cytometry analysis, NE was shown to trigger a time-dependent binding of PAC-1 and AP-5, two monoclonal antibodies specific for the activated and ligand-occupied conformers of αIIbβ3. Furthermore, the potentiated aggregation was shown to result from an increased capacity of platelets to bind fibrinogen. Indeed, the combination of NE and threshold of cathepsin G increased the binding of PAC-1 ≈5.5-fold over basal values measured on nontreated platelets, whereas this binding raised only by ≈3-fold in threshold of cathepsin G-stimulated platelets (p < 0.05). By contrast, phosphatidic acid accumulation, pleckstrin phosphorylation, and calcium mobilization produced by the combination of NE and threshold of cathepsin G were not significantly different from those measured with threshold of cathepsin G alone (p > 0.05), indicating that the phospholipase C/protein kinase C pathway is not involved in the potentiation of aggregation. The foregoing data, as well as the requirement of catalytically active NE to trigger αIIbβ3 activation and potentiate threshold of cathepsin G-initiated platelet aggregation, led us to examine whether the structure of this integrin was affected by NE. Immunoblot and flow cytometry analysis revealed a limited proteolysis of the carboxyl terminus of the αIIb subunit heavy chain (αIIbH), as judged by the disappearance of the epitope for the monoclonal antibody PMI-1. Mass spectrometry studies performed on a synthetic peptide mapping over the cleavage domain of αIIbH predicted the site of proteolysis as located between Val837 and Asp838. Treatment by NE of ATP-depleted platelets or Chinese hamster ovary cells expressing human recombinant αIIbβ3 clearly established that activation of the integrin was independent of signal transduction events and was concomitant with the proteolysis of αIIbH. In support of this latter observation, a close correlation was observed between the kinetics of proteolysis of αIIbH on platelets and that of expression of the ligand binding activity of αIIbβ3 (r2 = 0.902, p ≤ 0.005). However, only a subpopulation (≈25%) of the proteolyzed αIIbβ3 appeared to fully express the ligand binding capacity. Altogether, these results demonstrate that NE up-regulates the fibrinogen binding activity of αIIbβ3 through a restricted proteolysis of the αIIb subunit, and that this process is relevant for the potentiation of platelet aggregation.