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Blood, Vol. 93 No. 5 (March 1), 1999:
pp. 1622-1633
By
From UMR 5533 CNRS, IFR Coeur-Vaisseaux-Thrombose and Unité des
Soins Intensifs, Hôpital Cardiologique, Pessac, France; and
Centocor, Malvern, PA.
Abciximab is a new antiplatelet therapeutic in ischemic
cardiovascular disease. The drug, chimeric Fab fragments of a murine monoclonal antibody (MoAb) (c7E3), blocks GP IIb-IIIa function. However, its capacity to reach all receptor pools in platelets is
unknown. Electron microscopy and immunogold labeling were used to
localize abciximab in platelets of patients receiving the drug for up
to 24 hours. Studies on frozen-thin sections showed that c7E3 Fab, in
addition to the surface pool, also labeled the surface-connected canalicular system (SCCS) and
GP IIb-IIIa COMPLEXES (INTEGRIN
The first major trial using abciximab was the EPIC study, which showed
that it can reduce by 35% the major complications of PTCA at the
primary end point of 30 days.12 These results were obtained
using a regimen in which a bolus of 0.25 µg/kg was followed by an
infusion of 10 µg/min for 12 hours. In the EPIC study, platelet aggregation induced by ADP was totally inhibited during the perfusion. The lack of platelet aggregation in patients receiving abciximab can be
compared with that observed in the inherited disorder, Glanzmann's
thrombasthenia.13 Nevertheless, if there are similarities in the response of platelets to ADP, the response to thrombin is
different. Whereas in thrombasthenia the platelets do not aggregate to
thrombin, Kleimann et al14 showed that following
administration of a bolus of c7E3, platelets of patients with ischemic
coronary disease continue to show a residual aggregation response to
thrombin, thereby implying the continued presence of unblocked
receptors. Recently, we reported similar results for platelets of
patients receiving a bolus of c7E3 Fab followed by an
infusion.15 An apparently internal pool of GP IIb-IIIa free
of abciximab was detected using a competitive MoAb, AP-2, and the
surface expression of this pool was shown after thrombin
stimulation.15
Our current study investigated the mechanisms whereby abciximab gains
access to the different pools of GP IIb-IIIa in platelets. To do this,
we first used electron microscopy (EM) and immunogold labeling to
detect c7E3 Fab on frozen ultrathin sections of platelets isolated from
patients undergoing antithrombotic therapy. The patients in question
received abciximab for 18 to 24 hours before prescheduled PTCA under
the conditions defined in the CAPTURE protocol.15,16 Using
a previously characterized rabbit antibody monospecific for c7E3 Fab
fragments,17,18 we observed that abciximab not only labeled
the surface membrane but also penetrated within the SCCS and was
detected on the membranes of some Patients and the Abciximab Infusion Protocol
Platelet Preparations
Platelet Aggregation
Short-Duration Incubations of Abciximab With Platelets In Vitro Blood from normal subjects or from a patient with type I Glanzmann's thrombasthenia (see George et al,21 patient no. 9) with a homozygous mutation 1063G'A in exon 12 of the gene coding for GP IIb giving rise to a E355K mutation22 was taken into ACD-A anticoagulant and washed platelets prepared as described above. The platelets suspended at 250,000/µL in HBMT were incubated with abciximab (ReoPro; Centocor, Malvern, PA) at 10 µg/mL for times ranging between 1 and 60 minutes. Platelets were then fixed directly.Electron Microscopy Sample preparation. Washed platelets from patients receiving abciximab or platelets from subjects incubated with abciximab in vitro were fixed in the presence of a mixture of 1% (wt/vol) paraformaldehyde (PFA; Sigma) and 0.1% (wt/vol) glutaraldehyde (Fluka AG, Buchs, Switzerland) or with 1.25% (wt/vol) glutaraldehyde in 0.1 mol/L phosphate buffer, pH 7.2, for 30 minutes at room temperature. Fixed platelets were washed 3× in phosphate-buffered saline (PBS), pH 7.2.23 The pellet was subdivided into small blocks that were cryoprotected with 2.3 mol/L sucrose before being frozen in liquid propane using the Reichert KF 80 freezing system (Leica, Vienna, Austria). Preparation of ultrathin sections and immunogold staining. Procedures were basically as previously described by us.23,24 In brief, ultrathin sections of platelets were cut with a Reichert-Jung Ultracut E ultramicrotome equipped with a FC 4E cryokit attachment (Reichert-Jung, Vienna, Austria) and placed on collodion-coated nickel grids. In subsequent steps, the grids were transferred onto drops containing antibodies or washing buffer as described below. Rabbit antibody prepared against c7E3 was used at a dilution of 1/100 of affinity-purified IgG (a gift from Centocor).17,18 Anti-clathrin heavy-chain MoAb (mouse IgG1 purchased from MedGene Science, Pantin, France) was used at 5 µg/mL. All dilutions were in PBS, pH 7.2, containing 0.1% (wt/vol) BSA (Sigma). The grids were kept floating on the antibody-containing solution for 1 hour at room temperature. After 3 washes, bound antibody was revealed by incubating for 1 hour with gold-labeled anti-rabbit or anti-mouse IgG (1/100 dilution of Auroprobe EM GAR or GAM G5 or G10 [Amersham, Les Ulis, France]). After 3 more washes, the cryosections were stained by uranyl acetate and embedded in a thin film of methylcellulose before being observed at 80 kV in a Jeol JEM-1010 transmission electron microscope (Jeol, Croissy-sur-Seine, France).24 In the double-labeling experiments, sections were serially incubated with rabbit anti-c7E3, GAR G5, murine anti-clathrin MoAb and GAM G10 with three washings between the addition of each new antibody. Controls involved the substitution of primary antibodies with nonimmune rabbit or mouse IgG during the labeling of the sections. Preliminary experiments established that GAM G10 failed to detect bound abciximab and did not cross-react with rabbit IgG. In studies on platelets from the patients, control sections were also prepared from platelets taken from each patient before their receiving abciximab. Sections of platelets from the patient with Glanzmann's thrombasthenia were tested with the anti-c7E3 antibody only. Computer analysis.
The distribution of gold particles revealing bound abciximab was
evaluated for patients 1, 2, and 3 on platelet sections photographed using a Sony XC 077C camera (Sony, France, Paris) digitalized in 640 × 480 pixels coded in 256 levels of gray. Image analysis was
performed using a program constructed by C. Allasia (Faculté de
Médecine, Université de la Mediterranée, Marseille,
France) and installed on an IBM PC Pentium 166 computer equipped with Image Series 640 cards (Matrox France, Paris) driven by the IPS program
(Unilog, Grenoble, France). Gold particles located on the surface
membrane or within the internal compartment of platelets (SCCS + Flow Cytometry Platelets of patients receiving c7E3. Samples of washed platelets (108/mL) were fixed with 2% (vol/vol) PFA for 30 minutes at room temperature. The samples were washed 3× in PBS, pH 7.2, containing 0.1% (wt/vol) BSA and then incubated for 30 minutes at room temperature with 100 µL of fluorescein isothiocyanate (FITC)-conjugated F(ab')2 fragments of a goat antibody monospecific for human IgG F(ab')2 (Jackson ImmunoResearch, West Grove, PA) diluted 1/100 in HBMT. Preliminary studies had confirmed that the murine sequences were largely inaccessible to the anti-mouse IgG antibodies in flow cytometry when c7E3 Fab was bound to platelets.15,19 Control measurements were made using FITC-labeled F(ab')2 fragments of a goat antibody monospecific for the human IgG Fc fragment (Jackson ImmunoResearch). The platelet suspension was then diluted with 750 µL HBMT before analysis using a Becton Dickinson FACScan flow cytometer (Becton Dickinson, Le Pont de Claix, France). The fluorescence intensity was determined for the platelet population identified by gating on light scattering parameters (forward scatter versus side scatter).20,23 Ten thousand cells were analyzed for each sample and the data were registered as log fluorescence. Mean fluorescence intensities (MFI) as a measure of antibody binding were expressed using the LYSYS II conversion software in an arithmetic mode. Short-duration incubations of abciximab with platelets in vitro. Unblocked GP IIb-IIIa complexes were located using AP-2 (a gift from Dr Thomas Kunicki, Scripp's Research Institute, La Jolla, CA), a murine monoclonal antibody that we have shown to be competitive with c7E3 Fab for GP IIb-IIIa.15,19 Isolated platelets were incubated or not with 10 µg/mL c7E3 Fab for 30 minutes as described above. After being washed twice, samples were then incubated in the presence or absence of 0.5 U/mL thrombin for 10 minutes. Samples were immediately fixed with PFA as described above. Aliquots of fixed platelets (10 µL at a concentration of 100,000/µL) were added to polystyrene tubes containing 100 µL HBMT and 7 µg/mL AP-2. Controls were performed in the absence of primary antibody or in the presence of purified myeloma mouse IgG (Sigma). All samples were incubated for 15 minutes at room temperature without stirring. Then 10 µL of a predetermined optimal concentration of FITC-conjugated affinity-purified F(ab')2 fragments of sheep antibody to mouse IgG (Silenus, Eurobio, Les Ulis, France) were added. The tubes were left in the dark at room temperature for an additional 15 minutes before analysis with the FACScan as described above. Statistical analysis. Data were analyzed using the Student's t-test for unpaired data. A value of P < .05 was considered to be statistically significant.
Studies on Platelets Obtained From Patients Receiving Abciximab Platelet function testing.
The effects of abciximab on platelet aggregation induced by ADP and by
TRAP-14 mer were studied for each patient at the sampling times shown
in Fig 1. A greater than 80% inhibition of ADP-induced platelet
aggregation was obtained for each patient during the infusion and was
seen as early as 3 hours after the onset of therapy for the three
patients tested at this time point. This consistency allowed us to
group the results for the patients together
(Fig 2). The recovery of the aggregation
response had begun within 24 hours after the infusion was stopped, and
was extensive after 48 hours, although the difference remained
statistically significant compared with the pretreatment value. With
TRAP-14 mer, which mimics thrombin and induces a secretion-dependent
aggregation in platelet-rich plasma, the inhibition was more modest
during the infusion, and residual aggregation of at least 50% of
initial levels was observed for each patient. The aggregation response to TRAP-14 mer was no longer statistically lower 48 hours after the
infusion had ended. The results with TRAP-14 mer were compatible with
the presence of a pool of GP IIb-IIIa complexes inaccessible to
abciximab even after a combined therapy of bolus and infusion.
Localization of abciximab on platelet sections by immunoelectron
microscopy.
Initial controls performed using platelets isolated from each patient
before therapy showed that the rabbit anti-c7E3 antibody did not label
the cryosections in the absence of abciximab (data not shown). For
platelets taken from the patients during the abcximab infusion, we
observed a regular labeling of the platelet surface, which was often
dense (Fig 3). In Fig 3A, the sample was
taken 3 hours after the onset of the infusion to patient 1. The
illustration is typical of the results obtained for each of the three
patients tested at this early time point. Significantly, labeling can
already be seen inside the platelet, where it was localized both to the SCCS and to the luminal surface of the membranes of some but not all
Computer analysis.
The number of gold particles was quantified on sections of a minimum of
10 platelets for each sample taken during and after abciximab infusion
for patients 1, 2, and 3 (Table 1). A
computer program also compared the distribution of gold particles
between the surface and internal membrane systems. During the infusion, the mean gold particle density on the surface of platelets of each
patient was consistently high. Particles associated with internal
membrane systems represented 24%, 28%, and 31% of the total number
counted for each patient. These values changed little in the 48 hours
that followed the end of the infusion, although there was slightly more
intersubject variation. After 48 hours, labeling both at the surface
and within the platelet had decreased, and although the percentage of
gold particles inside the platelet was marginally greater, these
measurements confirmed that there was not a progressive and
unidirectional accumulation of abciximab within the platelets.
Flow cytometry.
As a supplement to the EM studies, we used flow cytometry to evaluate
the levels of c7E3 Fab bound to the platelet surface. A rapid and large
increase in the MFI was observed during the infusion of abciximab. The
grouped MFI values during the infusion (266.4 ± 74.3; n = 7) were
the maximum obtained; levels remained high at 48 hours (218.3 ± 90.8, P = .031; n = 6) but fell progressively at time points
greater than 48 hours (162 ± 20.8, P = .024; n = 4).
Interestingly, a single peak of fluorescence was always observed on the
histograms, showing that new platelets produced after the end of the
abciximab infusion possessed c7E3 Fab, seemingly confirming an exchange
between platelets via the plasma pool as suggested by Christopoulos et
al.17,18 The positivity was not related to the binding of
plasma immunoglobulins, either to prebound c7E3 or to exposed platelet
neoantigens, as the binding of FITC-labeled anti-human Fc fragments
antibody remained negative.
Short-Duration Incubations of Platelets With Abciximab In Vitro
Immunogold staining for abciximab.
Platelets from normal subjects were incubated with 10 µg/mL c7E3 Fab
for periods of 1 minute, 5 minutes, or 1 hour. The results showed that
some abciximab was already seen inside the cell after a 1-minute
incubation but that labeling was greater after 1 hour. Figure 5A illustrates a platelet incubated
for 1 minute with c7E3 Fab. In Fig 5B, normal platelets have been
incubated for 1 hour with c7E3; a long, thin channel of SCCS located
parallel to the plasma membrane is strongly labeled; and gold beads
were associated with some
Colocalization with clathrin.
Because translocation of bound ligands from the surface to the internal
compartment is classically believed to involve the formation of
endocytic vesicles containing clathrin, we performed double staining of
sections with the rabbit anti-c7E3 antibody and a murine MoAb to
clathrin. As shown in Fig 7, colocalization was observed (A) in vesicular structures within the platelets and (B
and C) within
Flow cytometry.
To analyze the free pool of GP IIb-IIIa, we have used AP-2, a murine
MoAb that is competitive for c7E3 as described
previously.15,19 Incubation of normal platelets for 30 minutes with 10 µg/mL of c7E3 Fab in vitro resulted in a severe
reduction in the binding of AP-2 to the surface of unstimulated
platelets. We found that the MFI after incubation was 9.20 ± 4.3%
of the initial values. After stimulation of untreated platelets with
thrombin 0.5 U/mL for 10 minutes, the MFI for AP-2 binding was 202 ± 20.48% (n = 5) of that seen for unstimulated platelets. After
incubation with c7E3, the MFI after thrombin treatment was 33.80 ± 10.91% (n = 5) of the value obtained in the absence of drug. The
results, illustrated in Fig 8, therefore
confirm that an appreciable pool of unblocked GP IIb-IIIa appeared on
the platelet surface after thrombin stimulation and c7E3 incubation.
The conclusion from these experiments is that only part of the internal
pool, which can represent about half of the total pool of GP IIb-IIIa,
was in contact with abciximab at any one time. Quantitatively, the values obtained in flow cytometry in the in vitro experiments were very
close to the results found for patients receiving c7E3 and where
antibody distribution was assessed by computer analysis of labeled
electron micrographs.
In this study, we have localized c7E3 Fab fragments within the
different membrane compartments of platelets using two model systems:
(1) platelets from patients receiving abciximab for ~24 hours during
antithrombotic therapy and (2) control platelets given a short-duration
challenge with abciximab in vitro. In view of their small size,
monovalent nature, and therapeutic use, c7E3 Fab are interesting probes
for following GP IIb-IIIa movements in platelets. The initial detection
of c7E3 Fab within the SCCS and on the membrane of some Submitted February 24, 1998; accepted October 22, 1998.
Supported by the CNRS, Université de Bordeaux II (DRED), the
Ministère de l'Enseignement Supérieur et de la Recherche
(ACC-SV No. 9), and Lilly-France. C.P. received doctoral grants from
the GEHT (Synthelabo) and the Société Française
d'Hématologie.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Paquita Nurden, MD, UMR 5533 CNRS, Hôpital Cardiologique, 33604 Pessac, France; e-mail:
Paquita.Nurden{at}cnrshl.u-bordeaux2.fr.
1.
Wencel-Drake JD, Plow EF, Kunicki TJ, Woods VL, Keller DM, Ginsberg MH:
Localization of internal pools of membrane glycoproteins involved in platelet adhesive responses.
Am J Pathol
124:324, 1986[Abstract]
2.
Cramer ER, Savidge GF, Vainchenker W, Berndt MC, Pidard D, Caen JP, Masse J-M, Breton-Gorius J:
Alpha-granule pool of glycoprotein IIb-IIIa in normal and pathologic platelets and megakaryocytes.
Blood
75:1220, 1990
3.
Heilmann E, Hourdillé P, Pruvost A, Paponneau A, Nurden AT:
Thrombin-induced platelet aggregates have a dynamic structure: Time-dependent redistribution of GP IIb-IIIa complexes and secreted adhesive proteins.
Arterioscler Thromb
11:704, 1991
4.
Suzuki H, Kaneko T, Sakamoto T, Nakagawa M, Miyamoto T, Yamada M, Tanoue K:
Redistribution of
5.
Nurden AT, Nurden P:
A review of the role of platelet membrane glycoproteins in the platelet-vessel wall interaction.
Baillieres Clin Haematol
6:653, 1993[Medline]
[Order article via Infotrieve]
6.
Nurden P:
Bidirectional trafficking of membrane glycoproteins following platelet activation in suspension.
Thromb Haemost
78:1305, 1997[Medline]
[Order article via Infotrieve]
7.
Coller BS:
Platelet GPIIb/IIIa antagonists: The first anti-integrin receptor therapeutics.
J Clin Invest
7:1467, 1997
8.
Tcheng JE, Ellis SG, George BS, Kereiakes J, Kleiman NS, Talley JD, Wzang AL, Weisman HD, Califf M, Topol EJ:
Pharmacodynamics of chimeric glycoprotein IIb/IIIa integrin antiplatelet antibody Fab 7E3 in high-risk coronary angioplasty.
Circulation
90:1757, 1994
9.
Schulman SP, Goldschmidt-Clermont PJ, Topol EJ, Califf RM, Navetta FI, Willerson JT, Chandra NC, Guerci AD, Ferguson JJ, Harrington RA, Lincoff AM, Yakubov SJ, Bray PF, Bahr RD, Wolfe CL, Yock PG, Anderson HV, Nygaard TW, Mason SJ, Effron MB, Fatterpacker A, Raskin S, Smith J, Brashear L, Gottdiener P, du Mee C, Kitt MM, Gerstenblith G:
Effects of integrilin, a platelet glycoprotein IIb/IIIa receptor antagonist, in unstable angina. A randomized multicenter trial.
Circulation
94:2083, 1996
10.
Théroux P, Kouz S, Roy L, Knudtson ML, Diodati JG, Marquis J-F, Nasmith J, Fung AY, Boudreault J-R, Delage F, Dupuis R, Kells C, Bokslag M, Steiner B, Rapold HJ, on behalf of the investigators:
Platelet membrane receptor glycoprotein IIb/IIIa antagonism in unstable angina. The Canadian Lamifiban study.
Circulation
94:899, 1996
11.
Tcheng JE:
Glycoprotein IIb/IIIa receptor inhibitors: Putting the EPIC, IMPACT II, RESTORE, and EPILOG trials into perspective.
Am J Cardiol
78:35, 1996[Medline]
[Order article via Infotrieve]
12.
The EPIC Investigators:
Use of a monoclonal antibody directed against the platelet glycoprotein IIb-IIIa receptor in high-risk coronary angioplasty.
N Engl J Med
330:956, 1994
13.
George JN, Nurden AT, Phillips DR:
Molecular defects in the interaction of platelets with the vessel wall.
N Engl J Med
311:1094, 1984
14.
Kleimann NS, Raizner AE, Jordan R, Wang AL, Norton D, Mace KF, Joshi A, Coller BS, Weisman HF:
Differential inhibition of platelet aggregation induced by adenosine diphosphate or a thrombin receptor-activating peptide in patients treated with bolus chimeric 7E3 Fab: Implications for inhibition of the internal pool of GP IIb-IIIa receptors.
J Am Coll Cardiol
26:1665, 1995[Abstract]
15. Bihour C, Durrieu-Jaïs 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 (in press)
16.
The Capture Investigators:
Randomised placebo-controlled trial of abciximab before and during coronary intervention in refractory unstable angina: The CAPTURE study.
Lancet
349:1429, 1997[Medline]
[Order article via Infotrieve]
17.
Christopoulos C:
Platelet surface IgG in patients receiving infusions of Fab fragments of a chimaeric monoclonal antibody to glycoprotein IIb-IIIa.
Clin Exp Immunol
98:6, 1994[Medline]
[Order article via Infotrieve]
18.
Christopoulos C, Mackie I, Lahiri A, Machin S:
Flow cytometric observations on the in vivo use of Fab fragments of a chimaeric monoclonal antibody to platelet glycoprotein IIb-IIIa.
Blood Coagul Fibrinolysis
4:729, 1993[Medline]
[Order article via Infotrieve]
19.
Macchi L, Clofent-Sanchez G, Marit G, Bihour C, Durrieu-Jais C, Besse P, Nurden P, Nurden AT:
PAICA: A new method for characterizing platelet-associated antibodies
20.
Humbert M, Nurden P, Bihour C, Pasquet J-M, Winckler J, Heilmann E, Savi P, Herbert J-M, Kunicki TJ, Nurden AT:
Ultrastructural studies of platelet aggregates from human subjects receiving clopidogrel and from a patient with an inherited defect of an ADP-dependent pathway of platelet activation.
Arterioscler Thromb Vasc Biol
16:1532, 1996
21.
George JN, Caen JP, Nurden AT:
Glanzmann's thrombasthenia: The spectrum of clinical disease.
Blood
75:1383, 1990
22.
Bourre F, Peyruchaud O, Bray P, Combrié R, Nurden P, Nurden AT:
A point mutation in the gene for platelets GP IIb leads to a substitution in a highly conserved amino acid located between the second and the third Ca++-binding domain.
Blood
86:452a, 1995 (abstr)
23.
Nurden P, Humbert M, Piotrowicz RS, Bihour C, Poujol C, Nurden AT, Kunicki TJ:
The determination of ligand-occupied
24.
Poujol C, Nurden AT, Nurden P:
Ultrastructural analysis of the vitronectin receptor (
25.
Zucker-Franklin D:
Endocytosis by human platelets: Metabolic and freeze-fracture studies.
J Cell Biol
91:706, 1981
26.
White JG, Clawson CC:
Effects of large latex particle uptake of the surface connected canalicular system of blood platelets: A freeze-fracture and cytochemical study.
Ultrastruct Pathol
2:277, 1981[Medline]
[Order article via Infotrieve]
27.
Isenberg WM, Bainton DF, Newman PJ:
Monoclonal antibodies bound to subunits of the integrin GP IIb-IIIa are internalized and interfere with filopodia formation and platelet aggregation.
Blood
76:1564, 1990
28.
Stenberg PE, Shuman MA, Levine SP, Bainton DF:
Redistribution of alpha-granules and their contents in thrombin-stimulated platelets.
J Cell Biol
98:748, 1984
29.
Escolar G, White JG:
The platelet open canalicular system: A final common pathway.
Blood Cells
17:467, 1991[Medline]
[Order article via Infotrieve]
30.
Yeager M:
Structure of cardiac gap junction intercellular channels.
J Struct Biol
121:231, 1998[Medline]
[Order article via Infotrieve]
31.
Baron V, Alengrin F, Van Obberghen E:
Dynamin associates with Src-homology collagen (Shc) and becomes tyrosine phosphorylated in response to insulin.
Endocrinology
139:3034, 1998
32.
Escolar G, Leistikow E, White JG:
The fate of the open canalicular system in surface and suspension-activated platelets.
Blood
74:1983, 1989
33.
Wencel-Drake JD, Boudignon-Proudhon C, Dieter MG, Criss AB, Parise LV:
Internalization of bound fibrinogen modulates platelet aggregation.
Blood
87:602, 1996
34.
Behnke O:
Coated pits and vesicles transfer plasma components to platelet granules.
Thromb Haemost
62:718, 1989[Medline]
[Order article via Infotrieve]
35.
Behnke O:
Degrading and non-degrading pathways in fluid-phase (non-adsorptive) endocytosis in human blood platelets.
J Submicrosc Cytol Pathol
24:169, 1992[Medline]
[Order article via Infotrieve]
36.
Klinger MHF, Klüter H:
Immunocytochemical colocalization of adhesive proteins with clathrin in human blood platelets: Further evidence for coated vesicle-mediated transport of von Willebrand factor, fibrinogen and fibronectin.
Cell Tissue Res
279:453, 1995[Medline]
[Order article via Infotrieve]
37.
Stenberg PE, Pestina TI, Barrie RJ, Jackson CW:
The Src family kinases, Fgr, Fyn, Lck, and Lyn colocalize with coated membranes in platelets.
Blood
89:2384, 1997
38.
George JN:
Platelet immunoglobulin G: Its significance for the evaluation of thrombocytopenia and for understanding the origin of
39.
Santoso S, Zimmerman U, Neppert J, Mueller-Eckhardt C:
Receptor patching and capping of platelet membranes induced by monoclonal antibodies.
Blood
67:343, 1986
40.
Morgenstern E, Ruf A, Patscheke H:
Transport of anti-glycoprotein IIb/IIIa-antibodies into the alpha-granules of unstimulated human blood platelets.
Thromb Haemost
67:121, 1992[Medline]
[Order article via Infotrieve]
41.
Handagama P, Bainton D, Jacques Y, Conn MT, Lazarus RA, Shuman MA:
Kistrin, an integrin antagonist, blocks endocytosis of fibrinogen into guinea pig megakaryocyte and platelet
42.
Wencel-Drake JD, Frelinger AL III, Dieter MG, Lam SC-T:
Arg-Gly-Asp-dependent occupancy of GP IIb/IIIa by applagin: Evidence for internalization and cycling of a platelet integrin.
Blood
81:62, 1993
43.
Harrison P, Wilbourn B, Cramer E, Faint R, Mackie IJ, Bhattacharya S, Lahiri A, Tensa D, Machin SJ, Savidge G:
The influence of therapeutic blocking of GP IIb/IIIa on platelet
44.
Raub TJ, Denny JB, Roberts RM:
Cell surface glycoproteins of CHO cells. I. Internalization and rapid recycling.
Exp Cell Res
165:73, 1992
45.
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
46.
Simmon ML, Jan de Boer M, van den Brand MJBM, van Miltenburg AJM, Hoorntje JCA, Heyn drickx GR, van der Wieken LR, De Bono D, Rutsch W, Schaible TF, Weisman HF, Klootwijk P, Nijssen KM, Stibbe J, de Feyter PJ, and the European Cooperative Study Group:
Randomized trial of a GPIIb/IIIa platelet receptor blocker in refractory unstable angina.
Circulation
89:596, 1994
47.
Ferguson JJ:
Epilog and Capture trials halted because of positive interim results.
Circulation
93:637, 1996
48.
Woods VL, Wolff LE, Keller DM:
Resting platelets contain a substantially centrally located pool of glycoprotein IIb/IIIa complex which may be accessible to some but not other extracellular proteins.
J Biol Chem
261:15242, 1986
49.
Niiya K, Hodson E, Bader R, Byers-Ward V, Koziol JA, Plow EF, Ruggeri ZM:
Increased surface expression of the membrane glycoprotein IIb/IIIa complex induced by platelet activation. Relationship to the binding of fibrinogen and platelet aggregation.
Blood
70:475, 1987
50.
Wagner CL, Mascelli MA, Neblock DS, Weisman HF, Coller BS, Jordan RJ:
Analysis of GP IIb/IIIa receptor number by quantification of 7E3 binding to human platelets.
Blood
88:907, 1996
51.
The EPILOG Investigators:
Platelet glycoprotein IIb/IIIa receptor blockade and low dose heparin during percutaneous coronary revascularization.
N Engl J Med
24:1689, 1997
52.
Müller TH, Weisenberger H, Brickl R, Narjes H, Himmelsbach F, Krause J:
Profound and sustained inhibition of platelet aggregation by Fradafiban, a nonpeptide platelet glycoprotein IIb/IIIa antagonist, and its orally active prodrug, Lefradafiban, in men.
Circulation
96:1130, 1997
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Top
Abstract
Introduction
-granules. Analysis of gold particle distribution showed that intraplatelet labeling was not accumulative and in equilibrium with the surface pool. After short-term incubations of platelets with c7E3 Fab in vitro, gold particles were often seen in
lines within thin elements of the SCCS, some of which appeared in
contact with 
IIb
3), are not only
constituents of the surface membrane of platelets, they are also widely
present in internal membrane compartments, including those of the
surface-connected canalicular system (SCCS) and the
Its application to study of idiopathic thrombocytopenic purpura and to the detection of platelet-bound c7E3.
Thromb Haemost
76:1020, 1996
