Blood, Vol. 93 No. 11 (June 1), 1999:
pp. 4019-4020
CORRESPONDENCE
Abciximab Binding to Glycoprotein IIb-IIIa and Protein Tyrosine
Phosphorylation in Human Platelets
 |
LETTER |
To the Editor:
We read with interest the report of Peter et al1 in which
the authors showed that the binding of abciximab (c7E3 Fab fragments), a new generation anti-thrombotic drug2,3 induced
conformation changes in glycoprotein (GP) IIb-IIIa. According to these
authors, these changes were such that when abciximab dissociated from
the complex, a process facilitated in their experimentation by a
large-scale dilution of the platelet suspensions, fibrinogen was able
to bind without the normal requirement for platelet stimulation by an agonist such as adenosine diphosphate (ADP) or thrombin. It is now well
known that in circulating platelets the level of protein tyrosine
phosphorylation is in a basal state, and that platelet stimulation by
ADP, thrombin, or collagen leads to a rapid increase in the tyrosine
phosphorylation of a number of cytoplasmic proteins.4-6 Such increases occur not only as a result of the interaction of the
primary agonist with its receptor, but also following fibrinogen binding to GP IIb-IIIa complexes and during platelet aggregation. Tyrosine phosphorylation as a result of integrin occupancy has become
an often-cited example of `outside-in' signaling.7
We were interested in establishing whether abciximab binding itself led
to increased tyrosine protein phosphorylation in platelets and whether
the drug inhibited the tyrosine phosphorylation response induced after
primary receptor activation. Washed platelets prepared as described
elsewhere,8 were suspended at 2.5 × 108/mL in
HEPES-buffered Tyrode's buffer, pH 7.4, containing 2 mmol/L Ca2+,8 and incubated for 30 minutes at 37°C
without stirring (except for an initial mixing) in the presence or
absence of 10 µg/mL abciximab (Lilly-France, Saint-Cloud,
France). That the surface pool of GP IIb-IIIa receptors is
saturated by abciximab under these conditions has already been
established in our laboratory.8 The suspensions were then
incubated in a platelet aggregometer (PAP-4 model; Biodata Corp, Paris,
France), with stirring in the presence or absence of 12.5 or 25 µmol/L thrombin receptor activating peptide (TRAP-14 mer; Neosystem,
Strasbourg, France). The aggregometer tracings were recorded. TRAP-14
mer-induced platelet aggregation was inhibited by abciximab, although a
small residual aggregation associated with a 10% to 20% light
transmission change persisted due to the exposure of unblocked
complexes from the internal pool.8,9 Samples were obtained
at 30 seconds, 1 minute, and 5 minutes, and immediately solubilized in
a buffer containing sodium dodecyl sulfate (SDS) and a cocktail of
inhibitors of proteases and tyrosine phosphatases as reported
previously from our laboratory.10 Proteins were separated
by electrophoresis on 7% to 12% gradient gels and transferred to
nitrocellulose membrane with phosphorylated tyrosine residues revealed
using the monoclonal antibody PY99 (0.1 µg/mL; Santa Cruz
Biotechnology, Santa Cruz, CA) and a chemiluminescence procedure.10
Significantly, at no time point did the protein tyrosine
phosphorylation profiles of unstimulated platelets incubated with abciximab differ from those incubated without this antithrombotic drug.
Typical results are shown in Fig 1; these
represent experiments performed on platelets from three donors. As
expected, TRAP-14 mer-induced platelet aggregation was associated with
markedly increased levels of protein tyrosine phosphorylation.
Increased labeling of bands at 60 (probably pp60c-src) and 31 kD was unaffected by the presence of c7E3 Fab, and the
increased phosphorylation of these proteins is probably a direct result
of activation through the PAR-1 receptor. Abciximab inhibited to
varying extents the TRAP-induced phosphorylation of bands of 130, 116/120, 100/105, 77/80, 64, and 42 kD. Allowing for small differences
in protein migration due to different experimental conditions (and
taking into account the increased sensitivity of PY99 with respect to previously used antibodies), our results are similar to those obtained
by Rosa et al5 for thrombin-activated platelets of patients
with type I and type II Glanzmann thrombasthenia. These authors
additionally showed that RGDS peptide and the intact anti-GP IIb-IIIa
monoclonal antibody 10E5 also selectively blocked
tyrosine-phosphorylation induced after GP IIb-IIIa engagement. Thus,
c7E3 Fab fragments, as shown in our study, exert inhibitory effects on
agonist-induced tyrosine phosphorylation probably linked to their
inhibition of the platelet aggregation response.
Furthermore, our results show that any conformation changes induced
within the complex by abciximab do not lead to outside-in signaling
linked to tyrosine phosphorylation. Therefore, abciximab does not mimic
fibrinogen, further suggesting that cross-linking or immobilization of
the occupied receptors is a necessary additional step for outside-in
signaling by the integrin.7 It is also pertinent to
emphasize that the trafficking of c7E3 Fab into and out of the platelet
as recently shown by us8 is unlikely to be driven by an
upregulation of protein tyrosine phosphorylation.
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| Fig 1.
Washed platelets were preincubated with or without
abciximab (10 µg/mL) for 30 minutes before being incubated with two
concentrations of TRAP-14 mer under aggregating conditions. Samples
were taken at the peak of the aggregation (2 minutes) and platelet
proteins were separated by SDS-polyacrylamide gel electrophoresis
before the detection of tyrosine-phosphate groups by Western blotting
using the monoclonal antibody PY99.
|
|
Eric Cazes
Paquita Nurden
Alan T. Nurden
UMR 5533 CNRS
Hôpital Cardiologique
Pessac, France
| |
REFERENCES |
1.
Peter K, Schwartz M, Ylänne J, Kohler B, Moser M, Nordt T, Salbach P, Kübler W, Bode W:
Induction of fibrinogen binding and platelet aggregation as a potential intrinsic property of various glycoprotein IIb/IIIa (
IIb
3) inhibitors.
Blood
92:3240, 1998[Abstract/Free Full Text]
2.
Coller BS:
Platelet GP IIb/IIIa antagonists: The first anti-integrin receptor therapeutics.
J Clin Invest
100:57, 1997 (suppl)
3.
Mascelli MA, Lance ET, Damaraju L, Wagner CL, Weisman HF, Jordan RE:
Pharmacodynamic profile of short-term abciximab treatment demonstrates prolonged platelet inhibition with gradual recovery from GP IIb/IIIa receptor blockade.
Circulation
97:1680, 1998[Abstract/Free Full Text]
4.
Levy-Toledano S, Maclouf J, Rosa J-P, Gallet C, Vallès G, Nurden P, Nurden AT:
Abnormal tyrosine phosphorylation linked to a defective interaction between ADP and its receptor on platelets.
Thromb Haemost
80:463, 1998[Medline]
[Order article via Infotrieve]
5.
Rosa J-P, Artçanuthurry V, Grelac F, Maclouf J, Caen JP, Lévy-Toledano S:
Reassessment of protein tyrosine phosphorylation in thrombasthenic platelets: Evidence that phosphorylation of cortactin and a 64-kD protein is dependent on thrombin activation and integrin IIb3.
Blood
89:4385, 1997[Abstract/Free Full Text]
6.
Ezumi Y, Shindoh K, Tsuji M, Takayama H:
Physical and functional association of the Src family kinases Fyn and Lyn with the collagen receptor glycoprotein VI-Fc receptor 
-chain complex on human platelets.
J Exp Med
188:267, 1998[Abstract/Free Full Text]
7.
Shattil SJ, Kashiwagi H, Pampori N:
Integrin signalling: The platelet paradigm.
Blood
91:2645, 1998[Free Full Text]
8.
Nurden P, Poujol C, Durrieu-Jais C, Winckler J, Combrie R, Macchi L, Bihour C, Wagner C, Jordan R, Nurden AT:
Labeling of the internal pool of GP IIb-IIIa in platelets of patients receiving c7E3 Fab fragments (abciximab): Flow and endocytic mechanisms contribute to the transport.
Blood
93:1622, 1999[Abstract/Free Full Text]
9.
Bihour C, Durrieu-Jais C, Macchi L, Poujol C, Coste P, Besse P, Nurden P, Nurden AT:
Expression of platelet activation and the interpatient variation in response to abciximab.
Arterioscler Thromb Vasc Biol
19:212, 1999[Abstract/Free Full Text]
10.
Pasquet J-M, Dachary-Prigent J, Nurden AT:
Microvesicle release is associated with extensive protein tyrosine dephosphorylation in platelets stimulated by A23187 or a mixture of thrombin and collagen.
Biochem J
333:591, 1998
