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(Received for publication, November 14, 1996, and in revised form, January 16, 1997)
From the 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
Volume 272, Number 17,
Issue of April 25, 1997
pp. 11636-11647
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
IIb
3 through Cleavage of the
Carboxyl Terminus of the IIb Subunit Heavy Chain
INVOLVEMENT IN THE POTENTIATION OF PLATELET AGGREGATION
,
,
,
,
,
and
Unité de Pharmacologie Cellulaire,
Laboratoire de Spectrométrie de Masse
Bioorganique, Laboratoire Franco-Luxembourgeois de Recherche
Biomédicale, Centre Universitaire, Grand-Duchy, Luxembourg
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
ligand-occupied conformers of IIb3.
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
IIb3 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 IIb subunit heavy chain
(IIbH), 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
IIbH 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 IIb3 clearly established that
activation of the integrin was independent of signal transduction events and was concomitant with the proteolysis of IIbH.
In support of this latter observation, a close correlation was observed
between the kinetics of proteolysis of IIbH on platelets
and that of expression of the ligand binding activity of
IIb3 (r2 = 0.902, p 
0.005). However, only a subpopulation (25%)
of the proteolyzed IIb3 appeared to fully
express the ligand binding capacity. Altogether, these results
demonstrate that NE up-regulates the fibrinogen binding activity of
IIb3 through a restricted proteolysis of
the IIb subunit, and that this process is relevant for
the potentiation of platelet aggregation.
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