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

Abstract

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 ␣ IIb ␤ 3 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 ligandoccupied conformers of ␣ IIb ␤ 3 . 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 Ϸ5.5-fold over basal values measured on nontreated platelets, whereas this binding raised only by Ϸ3- Thrombosis and inflammation are processes which result from complex relationships between various vascular cell types, i.e. endothelial cells, leukocytes, and platelets (1, 2). As part of such a cell cooperation network, polymorphonuclear neutrophils contribute to vessel injury not only by their own, but also through interactions with platelets. Thus, neutrophils are found admixed with platelets in the core of vascular occlusions in several experimental models (1, 3), and more importantly, a neutrophil-dependent platelet deposition has been described in arterial injuries (4 -6). Neutrophil-mediated platelet activation can be demonstrated in vitro by adding specific neutrophil agonists such as the formyl-Met-Leu-Phe (fMLP) peptide, tumor necrosis factor-␣, or interleukin-8 to autologous neutrophil-platelet mixed suspensions (7-10). Cathepsin G, a serine proteinase stored in the azurophilic granules of neutrophils and released upon their stimulation, has been established as the major mediator of this cell-to-cell interaction (11-13). Acting similarly to ␣-thrombin, another serine proteinase agonist of platelets, cathepsin G-induced platelet activation results in massive exocytosis and aggregation reactions. The

Membrane-proximal α/β Stalk Interactions Differentially Regulate Integrin Activation

“…Prevention of this endoproteolytic cleavage by mutagenesis abrogated ␣6␤1 activation by phorbol myristate acetate (31). Neutrophil elastase cleaves ␣IIb between Val 837 and Asp 838 , close to the endoproteolytic cleavage site in the calf-2 domain (32). Although the elastase does not, by itself, induce platelet aggregation, it greatly potentiates ␣IIb␤3 activation by cathepsin G and Mn 2ϩ (17,33).…”

Regulation of P-selectin binding to the neutrophil P-selectin counter-receptor P-selectin glycoprotein ligand-1 by neutrophil elastase and cathepsin G

“…However, when neutrophils were mixed with platelets under conditions that induced degranulation, and in the presence of TRAP to activate platelets and cause P-selectin surface expression, we saw evidence of proteolysis of only PSGL-1. Several reports have demonstrated an ability of cathepsin G [40][41][42] and elastase 42,43 to act on platelets in washed platelet systems in a rapid proteolytic fashion; however, platelet P-selectin has not previously been shown to be a substrate for cathepsin G or elastase. Results presented here suggest that in purified systems, cathepsin G and elastase can both use PSGL-1 and P-selectin as substrates.…”

Agonist-specific Structural Rearrangements of Integrin αIIbβ3

“…In this respect, it is remarkable that, although the M3 epitope is always exposed, the epitope for PMI-1, an antibody directed against the very same end of the C terminus of ␣ IIbH , is fully exposed only after platelet aggregation and fibrinogen binding, after RGD peptide binding, or when the divalent ion concentration is Ͻ10 M (1). Similarly, selective removal of the C-terminal end of ␣ IIbH by ␣-chymotrypsin or neutrophil elastase seems to be related to the activation of platelets and fibrinogen binding (46).…”