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IMMUNOBIOLOGY
From the Department of Molecular Oncology, General
Surgery, University of Witten-Herdecke, Wuppertal, Germany; and
Department of Clinical Immunology, Hannover Medical School, Hannover,
Germany.
The pathogenic effects of antiplatelet antibodies were investigated
in mice. Monoclonal antibodies (mAbs) of different immunoglobulin G
subclass directed against mouse GPIIbIIIa, GPIIIa, GPIb Autoimmune thrombocytopenic purpura (ITP) is an
autoimmune bleeding disease in which autoantibodies are directed
against the individual's own platelets, resulting in increased
platelet destruction and frequently severe bleeding.1 It
is well recognized that the vast majority of antiplatelet antibodies in
patients with ITP are directed against the 2 most prominent membrane
glycoprotein (GP) receptors GPIIbIIIa
( (NZW × BXSB)F1 mice, which develop systemic autoimmunity
including progressive thrombocytopenia, have served as a model for ITP,
as antiplatelet antibodies are detectable in those mice.12 The antigens recognized by these antibodies, however, have not been
identified, making it difficult to determine the importance of the
antigenic specificity of antiplatelet antibodies in the development of
ITP. This may be of considerable importance, as platelet functions
could be influenced by certain antibodies resulting in
(antigen-specific) mechanisms of platelet destruction and, possibly,
further inflammatory responses. We recently confirmed this hypothesis
when we reported that the first defined monoclonal antibody (mAb)
against mouse GPIIbIIIa [MWReg30, rat immunoglobulin G1 (IgG1)]
specifically induced strong thrombocytopenia and acute systemic
reactions in mice.13
Systematic in vivo studies on the relevance of the antigenic
specificity of antiplatelet antibodies for their pathogenic effects have not been performed to date, because defined antibodies suited for
in vivo studies were not available. Thus, it is presently not clear
which parameters determine the pathogenic effects of antiplatelet
antibodies: (1) antigenic specificity, (2) epitope-specific effects
leading to platelet agglutination/aggregation, (3) abundance of the
antigen on the membrane, or (4) IgG subclass of the antibody. To
address these issues, we generated panels of novel mAbs of different
isotype subclasses directed against nonoverlapping epitopes on mouse
GPIIbIIIa, GPIb-IX, and GPV and examined their pathogenic effects in vivo.
Here we demonstrate Fc-dependent and -independent mechanisms of
platelet destruction and provide evidence that the antigenic specificity of antiplatelet antibodies strictly determines their pathogenic potential. Furthermore, we show that antibodies directed against conformational epitopes on GPIIbIIIa induce previously unrecognized inflammatory responses that seem to be the major cause of
the bleeding problems in those individuals.
Animals
Reagents
Antibodies The rat antimouse P-selectin mAb RB40.34 was kindly provided by D. Vestweber (Münster, Germany). Polyclonal rabbit antibodies to human fibrinogen and vWF were purchased from DAKO and were modified in our laboratories. Rabbit anti-fluorescein isothiocyanate (FITC)-HRP, rabbit anti-rat immunoglobulin-FITC were purchased from DAKO. All other antibodies were generated, produced, and modified in our laboratories.Platelet preparation and counting Mice were bled under ether anesthesia from the retro-orbital plexus. Blood was collected in a tube containing 10% (v/v) 0.1 mol/L sodium citrate or 7.5 U/mL heparin, and platelet-rich plasma was obtained by centrifugation at 300g for 10 minutes at room temperature (RT). The platelets were washed twice with phosphate-buffered saline (PBS) by centrifugation at 1300g for 10 minutes and used immediately. Isolated platelets did not show any signs of activation as shown by flow cytometry (staining for P-selectin and surface-expressed fibrinogen). For determination of platelet counts, blood (20 µL) was obtained from the retro-orbital plexus of anesthetized mice by using siliconized microcapillaries and immediately diluted 1:100 in Unopette kits (Becton Dickinson, Heidelberg, Germany). The diluted blood sample was allowed to settle for 20 minutes in an Improved Neubauer hemocytometer (Superior, Bad Mergentheim, Germany), and platelets were counted under a phase contrast microscope at ×400 magnification.Production of mAbs Female Wistar rats, 6 to 8 weeks of age, were immunized repeatedly with mouse platelets or with purified antigens. The rat spleen cells were then fused with mouse myeloma cells (Ag8.653), and hybridomas were selected in HAT medium. Hybridomas secreting mAbs directed against platelet receptors were identified by flow cytometry. Briefly, a 1:1 mixture of resting and thrombin-activated platelets (106) was incubated with 100 µL supernatant for 30 minutes at RT, washed with PBS (1300g, 10 minutes), and stained with FITC-labeled rabbit anti-rat immunoglobulin (DAKO) for 15 minutes. Samples were analyzed on a FACScan (Becton Dickinson) in the set-up mode. Platelets were gated by FSC/SSC characteristics. Positive hybridomas were subcloned twice before large-scale production. Isotype subclasses were determined by enzyme-linked immunosorbent assay with alkaline phosphatase-conjugated isotype-specific antibodies (Pharmingen, Hamburg, Germany).Modification of antibodies Affinity-purified antibodies were fluoresceinated to a fluorescein-to-protein ratio of approximately 3:1 by standard methods with FITC (Sigma) and separated from free FITC by gel filtration on a PD-10 column (Pharmacia, Uppsala, Sweden). F(ab)2 fragments were generated by 24-hour incubation of 10 mg/mL mAb with immobilized pepsin (Pierce). After peptic digest, the preparations were applied to an immobilized protein A column followed by an immobilized protein G column (Pharmacia) to remove Fc fragments and any undigested IgG. The purity of the F(ab)2 fragments was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining of the gel.Immunoprecipitation and immunoblotting Immunoprecipitation was performed as described previously.14 Briefly, 108 washed platelets were surface labeled with EZ-Link sulfo-NHS-LC-biotin (Pierce; 100 µg/mL in PBS) and subsequently solubilized in 1 mL lysis buffer (Tris-buffered saline containing 20 mmol/L Tris/HCl, pH 8, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride, 2 µg/mL aprotinin, 0.5 µg/mL leupeptin, and 0.5% Nonidet P-40; all from Boehringer Mannheim). Cell debris was removed by centrifugation (15 000g, 10 minutes). After preclearing (8 hours), 10 µg mAb was added together with 25 µL protein G-Sepharose (Pharmacia), and precipitation took place overnight at 4°C. Samples were separated on a 9% to 15% gradient SDS-PAGE along with a molecular weight marker and then transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was incubated with streptavidin-HRP (1 µg/mL; DAKO) for 1 hour after blocking. After extensive washing, biotinylated proteins were visualized by echochemiluminescence (ECL; Amersham).For immunoblotting, platelets were not surface labeled. After lysis, the whole-cell extract was run on a SDS-PAGE gel and transferred to a PVDF membrane. The membrane was first incubated with 5 µg/mL FITC-labeled primary antibody followed by rabbit anti-FITC-HRP (1 µg/mL). Proteins were visualized by ECL. Flow cytometry Freshly isolated platelets were washed twice with PBS and then resuspended in platelet buffer (20 mmol/L Tris-HCl pH 7, 0.9% NaCl, 1 mmol/L CaCl2) at a concentration of 4 × 104/µL. Samples of 25 µL were preincubated with 50 µg/mL unlabeled mAb for 30 minutes at RT, followed by addition of saturating amounts of FITC-labeled antibodies. After 15 minutes incubation at RT, the samples were analyzed on a FACScan. Platelets were gated by FSC/SSC characteristics. For analysis of platelets from antibody-treated mice, whole blood was diluted 1:20 in platelet buffer and stained with the indicated fluorophore-labeled mAbs for 10 minutes at RT.In vivo experiments Ether-anesthetized mice received the indicated amounts of antibody intravenously. For the hypothermia measurement, body temperature was measured at the indicated times with a rectal probe. For the blockage of platelet-activating factor (PAF) receptors, mice received the indicated amounts of WEB2170 BS (in 200 µL sterile PBS) intravenously.
Characterization of antiplatelet antibodies To investigate the mechanisms of immune thrombocytopenia, mAbs of different IgG subclass against different epitopes on the dominant surface receptors on mouse platelets were generated GPIIbIIIa and
GPIb-IX-V. The anti-GPIb-IX mAbs (p0p1-5) have been described recently.15 P0p3-5 are directed against different epitopes
on the glycocalicin (GC) portion of GPIb , whereas p0p1,2 recognize epitopes on the GPIb-IX complex distinct from the GC portion of GPIb
(furthermore referred to as GPIb-IX). P0p6 (IgG2b) is also directed
against GPIb-IX (not shown). The mAbs against epitopes on GPIIbIIIa
were generated as described.13 JON1-3 precipitated the
platelet GPIIbIIIa complex (Figure 1A)
but not integrin V 3 from
surface-biotinylated bEnd.3 endothelioma cells (not shown), demonstrating that these mAbs are not directed against the GPIIIa subunit. Immunohistochemical staining of acetone-fixed frozen sections
of various organs (lung, liver, spleen, kidney, heart, and intestine)
confirmed earlier studies in which antibodies against GPIb, GPIb-IX
(p0p-mAbs), or GPIIbIIIa (JON-mAbs, MWReg30) exclusively bound to
platelets/megakaryocytes13,15 (not shown). In contrast, EDL1-3 recognized the GPIIIa chain in a Western blot analysis under
nonreducing conditions (Figure 1B) and precipitated integrin V3 from surface-biotinylated bEnd.3
endothelioma cells (not shown). The anti-GPV mAbs (DOM mAbs) were
obtained from fusions of spleen cells from rats immunized with affinity
purified surface-cross-linked GPIb-IX-V complexes and identified by
flow cytometric and biochemical methods (W.B. et al, manuscript in
preparation). DOM1-3 precipitated an approximate 80-kD protein from
platelet lysates (Figure 1C) and an approximate 68-kD band from the
supernatant of thrombin-activated platelets (not shown). Three
different mAbs against CD31 (PECAM; KIR1-3, Figure 1C) were used as
controls in the current study, because this platelet antigen is not a
common target for autoimmune antibodies in patients with ITP. Flow
cytometric preincubation experiments demonstrated that mAbs directed
against identical antigens did not block each other's binding (not
shown). None of the mAbs used in the current study induced platelet
activation (surface expression of P-selectin or fibrinogen). A summary
of all mAbs is shown in Table 1.
Intensity of thrombocytopenia depends on the antigenic specificity but not on the IgG subclass of the injected antibody To examine the pathogenic effects of the mAbs in vivo, mice received 100 µg purified mAb intravenously, and platelet counts were monitored for 6 days at the indicated time intervals. As shown in Figure 2A, most mAbs induced thrombocytopenia, but intensity and time course differed significantly between the mAb panels. Injection of JON mAbs (anti-GPIIbIIIa) of either IgG subclass resulted in a drop of platelet count to below 15% of control within 1 hour that further decreased to less than 3% at 24 hours and remained at this level for 3-4 days. Comparable results were obtained with the anti-GC mAbs p0p3-5, confirming earlier results.15 After 24 hours, intestinal hemorrhages and subcutaneous bleeding were evident in anti-GPIIbIIIa-treated mice (not shown). Consequently, hematocrits in those animals decreased and reached a minimum on day 3-4 (Figure 2B). Although anti-GPIb
antibodies displayed virtually identical cytotoxic effects as
anti-GPIIbIIIa antibodies, they induced neither subcutaneous nor
intestinal hemorrhages. As a result, the hematocrits were not
significantly affected (Figure 2B). In contrast to anti-GPIIbIIIa and
anti-GPIb antibodies, injection of mAbs directed against GPIIIa,
GPV, or GPIb-IX of either isotype resulted in delayed platelet clearing
and a maximum drop of platelet counts to approximately 50% of control
for some mAbs after 24 hours and in increasing counts after 48 hours.
The anti-CD31 mAbs had virtually no effect on platelet counts. Bleeding or decreased hematocrits were not detectable in any of these mice (Figure 2B).
Phenotypes of circulating platelets in antiplatelet-treated mice In all mice treated with antibodies against GPIb-IX, GPIIIa, GPV, or CD31, more than 99% of the circulating platelets were opsonized with the respective mAb 6 hours after injection as demonstrated by flow cytometric detection of surface-bound rat IgG. Furthermore, the surface expression of GPIIbIIIa, GPIb, GPV, and CD9
was unchanged compared with control platelets (Table
2). In contrast, the remaining platelets
circulating in mice on injection of anti-GPIb or anti-GPIIbIIIa antibodies displayed different phenotypes. While p0p3-5 had induced shedding of GPIb on some but not all remaining platelets, injection of JON1-3 had resulted in a more than 90% loss of surface-expressed GPIIbIIIa (Figure 3A). These
GPIIbIIIa-negative platelets, however, expressed virtually normal
amounts of GPIb, GPIb-IX, and GPV, whereas the CD9 levels were
decreased (Table 2). The internal pools of GPIIbIIIa had not been
affected, as shown by the translocation of these receptors (along with
P-selectin) to the surface on thrombin activation (Figure 3B). Western
blot analysis of a whole cell lysate of these platelets confirmed that
the total amount of GPIIIa per platelet was reduced to approximately
half of normal (not shown). The membrane GP composition on the
circulating platelets in both anti-GPIb- and anti-GPIIbIIIa-treated
mice remained unchanged as long as they were detectable in the
circulation (approximately 7 days).
All anti-GPIIbIIIa mAbs induce PAF-dependent acute systemic reactions A bolus injection of either JON1-3 (n = 6 per group) induced severe hypothermia in mice. This condition was accompanied by peripheral vasodilation and uncoordinated movements as described to occur on injection of MWReg30.13 In contrast, injection of EDL-, p0p-, DOM-, or KIR-mAbs (n = 6 for each mAb) induced neither hypothermia nor any other signs of acute reactions (Figure 4A). PAF was suspected to be a critical mediator of the observed pathology, as this potent inflammatory phospholipid is known to induce similar effects in mice.16 To test this hypothesis, mice were treated with different doses (1 mg/kg, 3mg/kg, or 10 mg/kg) of the potent PAF receptor antagonist WEB2170 BS17 60 minutes before injection of anti-GPIIbIIIa (JON1-3). Pretreatment with 1mg/kg (as with the other doses) WEB2170 BS abolished hypothermia induced by either mAb (Figure 4B). The body temperature was monitored for 6 hours, and blood was subsequently drawn from the retro-orbital plexus for determination of platelet counts. WEB2170 BS did not prevent JON-induced thrombocytopenia. In contrast, platelet counts were even lower in WEB2170 BS-pretreated mice (Figure 4C). Thus, PAF is a mediator of anti-GPIIbIIIa-induced systemic reactions, but it is clearly not involved in the development of thrombocytopenia. Importantly, the WEB2170 BS-treated mice, like the control mice, developed subcutaneous bleeding and significantly decreased hematocrits, indicating that PAF was not responsible for these complications (not shown).
Importance of the Fc part of anti-GPIIbIIIa and
anti-GPIb were generated. Only IgG2a and IgG2b antibodies were used
as F(ab)2 fragments because rat IgG1 was resistant against
pepsin digest. Therefore, further antibodies against GPIIbIIIa (JON/A
and JON9) and GPIb (p0p8, p0p11) were used in this part of the
study. The F(ab)2 fragments of anti-GPIIbIIIa mAbs (JON1,
JON2, JON/A, and JON9) neither induced hypothermia (Figure
5A), peripheral vasodilation, nor
significant bleeding (n = 6 per group) and only had mild effects on
platelet counts (Figure 5B). Membrane-bound F(ab)2
fragments were demonstrated by staining platelets ex vivo with rabbit
anti-rat IgG-FITC. The surface levels of GPIIbIIIa on these platelets
remained virtually unchanged for at least 3 days (not shown). Thus,
anti-GPIIbIIIa-induced hypothermia, thrombocytopenia, loss of
GPIIbIIIa, and the resulting bleeding problems depended on the Fc part
of the mAbs. In contrast, F(ab)2 fragments of anti-GPIb
mAbs (p0p3, p0p4, p0p8, and p0p11) induced thrombocytopenia with the
same intensity as the intact antibodies, confirming earlier
results15 and demonstrating that anti-GPIb-mediated
thrombocytopenia is Fc independent. To compare the cytotoxic effects of
anti-GPIIbIIIa and anti-GPIb antibodies, we performed dose-response
studies with intact IgG and F(ab)2 fragments of JON1, JON2,
p0p3, and p0p4. As shown in Figure 5C, low doses (3 and 10 µg) of
intact anti-GPIb mAbs had slightly stronger effects on platelet
counts than anti-GPIIbIIIa mAbs, whereas injection of higher doses
resulted in a more than 90% drop of platelet counts in all mice. The
F(ab)2 fragments of p0p3 and p0p4 induced thrombocytopenia
at similar doses as the intact antibodies, whereas the
F(ab)2 fragments of JON1 and JON2 had no significant
effects on platelets counts.
Repeated injections of low doses of anti-GPIIbIIIa antibodies induce severe bleeding A bolus injection of antiplatelet antibody does not reflect the situation in patients in which ITP is often a chronic disease, suggesting that antiplatelet antibodies reach the circulation continuously. To mimic this situation, mice were repeatedly injected with low amounts of anti-GPIIbIIIa or anti-GPIb antibodies
(7 × 7.5 µg intraperitoneally within 8 hours). The acute systemic responses seen on a bolus injection of more than 10 µg anti-GPIIbIIIa were not detectable in these mice, confirming that this process requires a threshold dose of antibody to occur.13 The
number of circulating platelets, however, decreased to less than 3% of normal within 24 hours in all mice (Figure
6A), and the remaining cells displayed
the same phenotype as those found in bolus-injected mice (see above).
All JON-treated mice developed marked intestinal and subcutaneous
hemorrhages and markedly decreased hematocrits, whereas these effects
were not observed in p0p-treated mice (Figure 6B-C). As with the bolus
injection, WEB2170 BS had no protective effect on thrombocytopenia and
bleeding (not shown).
In this study, we investigated the pathogenic effects of
antibodies directed against different epitopes on the major platelet antigens GPIIbIIIa and GPIb-IX-V systematically for the first time. It
is well recognized that the majority of autoimmune antiplatelet antibodies in patients with ITP are directed against epitopes on
GPIIbIIIa and/or GPIb-IX-V.2 However, in most patients
with ITP, autoantibodies are directed against different antigens,
making it difficult to determine potentially existing antigen-specific mechanisms of platelet destruction. Our results strongly suggest that
antibodies directed against GPIIbIIIa and GPIb In contrast, all antibodies against the GPIIbIIIa complex induced
thrombocytopenia with similar efficiency as anti-GPIb The anti-GPIIbIIIa-induced acute reactions only occurred when antibody
amounts of more than 10 µg were given as a bolus injection (which
does not reflect the situation in patients with ITP). Probably, lower
amounts of antibody also induced PAF production, but the concentrations
may not have been high enough to affect the systemic circulation. The
very short half-life of PAF in vivo may explain why the critical
concentration was not reached by repeated injections of anti-GPIIbIIIa
antibodies. The source of PAF could not be identified in the present
study, but it seems likely that Fc We found that PAF itself is not involved in the development of
thrombocytopenia and the increased bleeding tendency in
anti-GPIIbIIIa-treated mice (Figure 4). This finding confirms results
from studies in patients with ITP in which PAF-receptor antagonists had
no beneficial effects.28 However, the lack of circulating
platelets alone cannot account for the marked blood loss as no such
effect was observed in anti-GPIb Taken together, the results presented here provide the first evidence
that antibodies directed against GPIb
The authors would like to thank N. Huss for critically reading the manuscript and U. Barnfred for constant support.
Submitted January 27, 2000; accepted June 7, 2000.
Supported in part by grant Ni 556/2-1 (to B.N. and J.E.G.) from the Deutsche Forschungsgemeinschaft and the Bayer ag, Germany.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Bernhard Nieswandt, IMMI, Klinikum Wuppertal, Universität Witten-Herdecke, Heusnerstr. 40, D-42283 Wuppertal, Germany; e-mail: nieswand{at}klinikum-wuppertal.de.
1. Semple JW, Lazarus AH, Freedman J. The cellular immunology associated with autoimmune thrombocytopenic purpura: an update. Transfus Sci. 1998;19:245-251[Medline] [Order article via Infotrieve]. 2. Beardsley DS, Ertem M. Platelet autoantibodies in immune thrombocytopenic purpura. Transfus Sci. 1998;19:237-244[Medline] [Order article via Infotrieve]. 3. Macchi L, Nurden P, Marit G, et al. Autoimmune thrombocytopenic purpura (AITP) and acquired thrombasthenia due to autoantibodies to GP IIb-IIIa in a patient with an unusual platelet membrane glycoprotein composition. Am J Hematol. 1998;57:164-175[Medline] [Order article via Infotrieve].
4.
Niessner H, Clemetson KJ, Panzer S, Mueller-Eckhardt C, Santoso S, Bettelheim P.
Acquired thrombasthenia due to GPIIb/IIIa-specific platelet autoantibodies.
Blood.
1986;68:571-576 5. Kubota T, Tanoue K, Murohashi I, et al. Autoantibody against platelet glycoprotein IIb/IIIa in a patient with non-Hodgkin's lymphoma. Thromb Res. 1989;53:379-386[Medline] [Order article via Infotrieve]. 6. Meyer M, Kirchmaier CM, Schirmer A, Spangenberg P, Strohl C, Breddin K. Acquired disorder of platelet function associated with autoantibodies against membrane glycoprotein IIb-IIIa complex-1: glycoprotein analysis. Thromb Haemost. 1991;65:491-496[Medline] [Order article via Infotrieve]. 7. Balduini CL, Bertolino G, Noris P, et al. Defect of platelet aggregation and adhesion induced by autoantibodies against platelet glycoprotein IIIa. Thromb Haemost. 1992;68:208-213[Medline] [Order article via Infotrieve]. 8. Spangenberg P, Kirchmaier CM, Schirmer A, Meyer M, Breddin HK. Functional studies on platelets of a patient with an acquired disorder of platelet function associated with autoantibodies against membrane glycoprotein IIB/IIIA complex. Thromb Res. 1993;69:435-442[Medline] [Order article via Infotrieve]. 9. Garvey B. Management of chronic autoimmune thrombocytopenic purpura (ITP) in adults. Transfus Sci. 1998;19:269-277[Medline] [Order article via Infotrieve]. 10. George JN. Management of chronic refractory idiopathic thrombocytopenic purpura. In: George JN,Ballem PJ,Burstein SA, eds. Platelets (education booklet). Washington, DC: American Society of Hematology; 1994:77-79.
11.
George JN, Woolf SH, Raskob GE, et al.
Idiopathic thrombocytopenic purpura: a practice guideline developed by explicit methods for the American Society of Hematology.
Blood.
1996;88:3-40
12.
Mizutani H, Engelman RW, Kurata Y, Ikehara S, Good RA.
Development and characterization of monoclonal antiplatelet autoantibodies from autoimmune thrombocytopenic purpura-prone (NZW × BXSB)F1 mice.
Blood.
1993;82:837-844
13.
Nieswandt B, Echtenacher B, Wachs FP, et al.
Acute systemic reaction and lung alterations induced by an antiplatelet integrin gpIIb/IIIa antibody in mice.
Blood.
1999;94:684-693
14.
Rehli M, Krause SW, Kreutz M, Andreesen R.
Carboxypeptidase M is identical to the MAX.1 antigen and its expression is associated with monocyte to macrophage differentiation.
J Biol Chem.
1995;270:15644-15649
15.
Bergmeier W, Rackebrandt K, Zirngibl H, Nieswandt B.
Structural and functional characterization of the mouse von Willebrand factor receptor complex GPIb-IX with novel monoclonal antibodies.
Blood.
2000;95:886-895 16. Sun XM, Hsueh W. Platelet-activating factor produces shock, in vivo complement activation, and tissue injury in mice. J Immunol. 1991;147:509-514[Abstract]. 17. Libert C, Van Molle W, Brouckaert P, Fiers W. Platelet-activating factor is a mediator in tumor necrosis factor/galactosamine-induced lethality. J Inflamm. 1995;46:139-143[Medline] [Order article via Infotrieve].
18.
Cadroy Y, Hanson SR, Kelly AB, et al.
Relative antithrombotic effects of monoclonal antibodies targeting different platelet glycoprotein-adhesive molecule interactions in nonhuman primates.
Blood.
1994;83:3218-3224 19. Lazarus AH, Freedman J, Semple JW. Intravenous immunoglobulin and anti-D in idiopathic thrombocytopenic purpura (ITP): mechanisms of action. Transfus Sci. 1998;19:289-294[Medline] [Order article via Infotrieve].
20.
Woods VLJ, Kurata Y, Montgomery RR, et al.
Autoantibodies against platelet glycoprotein Ib in patients with chronic immune thrombocytopenic purpura.
Blood.
1984;64:156-160
21.
Woods VLJ, Oh EH, Mason D, McMillan R.
Autoantibodies against the platelet glycoprotein IIb/IIIa complex in patients with chronic ITP.
Blood.
1984;63:368-375
22.
Fujisawa K, O'Toole TE, Tani P, et al.
Autoantibodies to the presumptive cytoplasmic domain of platelet glycoprotein IIIa in patients with chronic immune thrombocytopenic purpura.
Blood.
1991;77:2207-2213 23. Bowditch RD, Tani P, McMillan R. Reactivity of autoantibodies from chronic ITP patients with recombinant glycoprotein IIIa peptides. Br J Haematol. 1995;91:178-184[Medline] [Order article via Infotrieve]. 24. Ravetch JV. Fc receptors. Curr Opin Immunol. 1997;9:121-125[Medline] [Order article via Infotrieve]. 25. Hazenbos WL, Gessner JE, Hofhuis FM, et al. Impaired IgG-dependent anaphylaxis and Arthus reaction in Fc gamma RIII (CD16) deficient mice. Immunity. 1996;5:181-188[Medline] [Order article via Infotrieve].
26.
Gemmell CH, Sefton MV, Yeo EL.
Platelet-derived microparticle formation involves glycoprotein IIb-IIIa: inhibition by RGDS and a Glanzmann's thrombasthenia defect.
J Biol Chem.
1993;268:14586-14589 27. Indig FE, Diaz-Gonzalez F, Ginsberg MH. Analysis of the tetraspanin CD9-integrin alphaIIbbeta3 (GPIIb-IIIa) complex in platelet membranes and transfected cells. Biochem J. 1997;327:291-298. 28. Giers G, Janzarik H, Kempe ER, Mueller-Eckhardt C. Failure of the platelet-activating factor antagonist WEB 2086 BS for treatment of chronic autoimmune thrombocytopenia. Blut. 1990;61:21-24[Medline] [Order article via Infotrieve].
© 2000 by The American Society of Hematology.
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B. D. Elzey, N. W. Schmidt, S. A. Crist, T. P. Kresowik, J. T. Harty, B. Nieswandt, and T. L. Ratliff Platelet-derived CD154 enables T-cell priming and protection against Listeria monocytogenes challenge Blood, April 1, 2008; 111(7): 3684 - 3691. [Abstract] [Full Text] [PDF]
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 K. Venkataraman, Y.-M. Lee, J. Michaud, S. Thangada, Y. Ai, H. L. Bonkovsky, N. S. Parikh, C. Habrukowich, and T. Hla Vascular Endothelium As a Contributor of Plasma Sphingosine 1-Phosphate Circ. Res., March 28, 2008; 102(6): 669 - 676. [Abstract] [Full Text] [PDF]
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S. H. Slofstra, M. F. Bijlsma, A. P. Groot, P. H. Reitsma, T. Lindhout, H. ten Cate, and C. A. Spek Protease-activated receptor-4 inhibition protects from multiorgan failure in a murine model of systemic inflammation Blood, November 1, 2007; 110(9): 3176 - 3182. [Abstract] [Full Text] [PDF]
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T. S. Dhanjal, C. Pendaries, E. A. Ross, M. K. Larson, M. B. Protty, C. D. Buckley, and S. P. Watson A novel role for PECAM-1 in megakaryocytokinesis and recovery of platelet counts in thrombocytopenic mice Blood, May 15, 2007; 109(10): 4237 - 4244. [Abstract] [Full Text] [PDF]
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 C. Kleinschnitz, M. Pozgajova, M. Pham, M. Bendszus, B. Nieswandt, and G. Stoll Targeting Platelets in Acute Experimental Stroke: Impact of Glycoprotein Ib, VI, and IIb/IIIa Blockade on Infarct Size, Functional Outcome, and Intracranial Bleeding Circulation, May 1, 2007; 115(17): 2323 - 2330. [Abstract] [Full Text] [PDF]
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R. S. Go, K. L. Johnston, and K. C. Bruden The association between platelet autoantibody specificity and response to intravenous immunoglobulin G in the treatment of patients with immune thrombocytopenia Haematologica, February 1, 2007; 92(2): 283 - 284. [Abstract] [Full Text] [PDF]
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 Z. Li, R. E. Rumbaut, A. R. Burns, and C. W. Smith Platelet Response to Corneal Abrasion Is Necessary for Acute Inflammation and Efficient Re-epithelialization Invest. Ophthalmol. Vis. Sci., November 1, 2006; 47(11): 4794 - 4802. [Abstract] [Full Text] [PDF]
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M. L. Webster, E. Sayeh, M. Crow, P. Chen, B. Nieswandt, J. Freedman, and H. Ni Relative efficacy of intravenous immunoglobulin G in ameliorating thrombocytopenia induced by antiplatelet GPIIbIIIa versus GPIb{alpha} antibodies Blood, August 1, 2006; 108(3): 943 - 946. [Abstract] [Full Text] [PDF]
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 V. Schulte, H. P. Reusch, M. Pozgajova, D. Varga-Szabo, C. Gachet, and B. Nieswandt Two-Phase Antithrombotic Protection After Anti-Glycoprotein VI Treatment in Mice Arterioscler. Thromb. Vasc. Biol., July 1, 2006; 26(7): 1640 - 1647. [Abstract] [Full Text] [PDF]
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 J. Kisucka, C. E. Butterfield, D. G. Duda, S. C. Eichenberger, S. Saffaripour, J. Ware, Z. M. Ruggeri, R. K. Jain, J. Folkman, and D. D. Wagner Platelets and platelet adhesion support angiogenesis while preventing excessive hemorrhage PNAS, January 24, 2006; 103(4): 855 - 860. [Abstract] [Full Text] [PDF]
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 T. Rabie, A. Strehl, A. Ludwig, and B. Nieswandt Evidence for a Role of ADAM17 (TACE) in the Regulation of Platelet Glycoprotein V J. Biol. Chem., April 15, 2005; 280(15): 14462 - 14468. [Abstract] [Full Text] [PDF]
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A. Musaji, F. Cormont, G. Thirion, C. L. Cambiaso, and J.-P. Coutelier Exacerbation of autoantibody-mediated thrombocytopenic purpura by infection with mouse viruses Blood, October 1, 2004; 104(7): 2102 - 2106. [Abstract] [Full Text] [PDF]
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S. Gruner, M. Prostredna, V. Schulte, T. Krieg, B. Eckes, C. Brakebusch, and B. Nieswandt Multiple integrin-ligand interactions synergize in shear-resistant platelet adhesion at sites of arterial injury in vivo Blood, December 1, 2003; 102(12): 4021 - 4027. [Abstract] [Full Text] [PDF]
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W. Bergmeier, P. C. Burger, C. L. Piffath, K. M. Hoffmeister, J. H. Hartwig, B. Nieswandt, and D. D. Wagner Metalloproteinase inhibitors improve the recovery and hemostatic function of in vitro-aged or -injured mouse platelets Blood, December 1, 2003; 102(12): 4229 - 4235. [Abstract] [Full Text] [PDF]
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 B. Nieswandt, W. Bergmeier, V. Schulte, T. Takai, U. Baumann, R. E. Schmidt, H. Zirngibl, W. Bloch, and J. E. Gessner Targeting of platelet integrin {alpha}IIb{beta}3 determines systemic reaction and bleeding in murine thrombocytopenia regulated by activating and inhibitory Fc{gamma}R Int. Immunol., March 1, 2003; 15(3): 341 - 349. [Abstract] [Full Text] [PDF]
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S. Massberg, M. Gawaz, S. Gruner, V. Schulte, I. Konrad, D. Zohlnhofer, U. Heinzmann, and B. Nieswandt A Crucial Role of Glycoprotein VI for Platelet Recruitment to the Injured Arterial Wall In Vivo J. Exp. Med., January 6, 2003; 197(1): 41 - 49. [Abstract] [Full Text] [PDF]
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Y. Kaluzhny, G. Yu, S. Sun, P. A. Toselli, B. Nieswandt, C. W. Jackson, and K. Ravid BclxL overexpression in megakaryocytes leads to impaired platelet fragmentation Blood, August 13, 2002; 100(5): 1670 - 1678. [Abstract] [Full Text] [PDF]
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O. Holtkotter, B. Nieswandt, N. Smyth, W. Muller, M. Hafner, V. Schulte, T. Krieg, and B. Eckes Integrin alpha 2-Deficient Mice Develop Normally, Are Fertile, but Display Partially Defective Platelet Interaction with Collagen J. Biol. Chem., March 22, 2002; 277(13): 10789 - 10794. [Abstract] [Full Text] [PDF]
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M.-P. Gratacap, B. Payrastre, B. Nieswandt, and S. Offermanns Differential Regulation of Rho and Rac through Heterotrimeric G-proteins and Cyclic Nucleotides J. Biol. Chem., December 14, 2001; 276(51): 47906 - 47913. [Abstract] [Full Text] [PDF]
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S. Kosugi, Y. Tomiyama, S. Honda, H. Kato, T. Kiyoi, H. Kashiwagi, Y. Kurata, and Y. Matsuzawa Platelet-associated anti-GPIIb-IIIa autoantibodies in chronic immune thrombocytopenic purpura recognizing epitopes close to the ligand-binding site of glycoprotein (GP) IIb Blood, September 15, 2001; 98(6): 1819 - 1827. [Abstract] [Full Text] [PDF]
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S. Moog, P. Mangin, N. Lenain, C. Strassel, C. Ravanat, S. Schuhler, M. Freund, M. Santer, M. Kahn, B. Nieswandt, et al. Platelet glycoprotein V binds to collagen and participates in platelet adhesion and aggregation Blood, August 15, 2001; 98(4): 1038 - 1046. [Abstract] [Full Text] [PDF]
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J. L. Teeling, T. Jansen-Hendriks, T. W. Kuijpers, M. de Haas, J. G. J. van de Winkel, C. E. Hack, and W. K. Bleeker Therapeutic efficacy of intravenous immunoglobulin preparations depends on the immunoglobulin G dimers: studies in experimental immune thrombocytopenia Blood, August 15, 2001; 98(4): 1095 - 1099. [Abstract] [Full Text] [PDF]
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 R. J. Hansen and J. P. Balthasar Pharmacokinetics, Pharmacodynamics, and Platelet Binding of an Anti-Glycoprotein IIb/IIIa Monoclonal Antibody (7E3) in the Rat: A Quantitative Rat Model of Immune Thrombocytopenic Purpura J. Pharmacol. Exp. Ther., July 1, 2001; 298(1): 165 - 171. [Abstract] [Full Text]
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B. Nieswandt, W. Bergmeier, A. Eckly, V. Schulte, P. Ohlmann, J.-P. Cazenave, H. Zirngibl, S. Offermanns, and C. Gachet Evidence for cross-talk between glycoprotein VI and Gi-coupled receptors during collagen-induced platelet aggregation Blood, June 15, 2001; 97(12): 3829 - 3835. [Abstract] [Full Text] [PDF]
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B. Nieswandt, V. Schulte, W. Bergmeier, R. Mokhtari-Nejad, K. Rackebrandt, J.-P. Cazenave, P. Ohlmann, C. Gachet, and H. Zirngibl Long-term Antithrombotic Protection by In Vivo Depletion of Platelet Glycoprotein VI in Mice J. Exp. Med., February 12, 2001; 193(4): 459 - 470. [Abstract] [Full Text] [PDF]
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W. Bergmeier, D. Bouvard, J. A. Eble, R. Mokhtari-Nejad, V. Schulte, H. Zirngibl, C. Brakebusch, R. Fassler, and B. Nieswandt Rhodocytin (Aggretin) Activates Platelets Lacking alpha 2beta 1 Integrin, Glycoprotein VI, and the Ligand-binding Domain of Glycoprotein Ibalpha J. Biol. Chem., June 29, 2001; 276(27): 25121 - 25126. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
: IgG1; 
:
IgG2a; 
: IgG2b). (B) Hematocrits were determined at the
indicated times.
R)-bearing cells in the RES are not
involved in anti-GPIb