|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
Blood, Vol. 95 No. 10 (May 15), 2000:
pp. 3168-3175
IMMUNOBIOLOGY
From the Department of Immunology, Institute for Cell Biology, and
Department of Transfusion Medicine, University Hospital,
Tübingen, Germany.
The origin and the function of HLA class I molecules present on the
surface of human platelets are still unclear. In particular, it is
controversial which fraction of these class I molecules represents
integral membrane components derived from the megakaryocyte-platelet lineage versus soluble plasma HLA molecules acquired by adsorption. Results of the present study show that HLA-A2 ligands isolated from
platelets possess the same peptide motif as described for HLA-A2-associated peptides obtained from nucleated cells. Sequencing of
these platelet-derived peptides reveals that they originate mainly from
ubiquitously expressed proteins also present in the megakaryocyte-platelet lineage. Moreover, one of these peptides derives
from the GPIX protein, which is specifically expressed by platelets and
their precursors. Platelet HLA molecules are unstable in vitro at
37°C, but can be partially stabilized by addition of exogenous
Class I molecules of the major histocompatibility
complex (MHC) are heterodimeric glycoproteins expressed at the
cytoplasmic membrane of almost all cells. They consist of a polymorphic
heavy chain of 45 kd noncovalently linked to
Platelets express HLA molecules to a lesser extent than peripheral
mononuclear cells (PBMCs); the number of HLA molecules at their surface
has been reported to vary between 50,000 and 100,000.2
Taking into account the number of platelets, it can be estimated that
approximately two thirds of the HLA molecules from blood are carried by
thrombocytes. The origin of platelet-surface class I molecules has been
discussed intensively. Different groups have shown that HLA-A and -B
antigens of platelets are integral membrane constituents with similar
biochemical properties to HLA molecules obtained from nucleated
cells.3,4 Other experiments, based on chloroquine treatment
or coincubation of platelets with allogenic plasma, suggest that most
HLA molecules do not have an endogenous source but are adsorbed from
the plasma.5,6 Taken together, these data argue for
coexpression of integral membrane and adsorbed HLA molecules on
platelets, although the contribution of these 2 types is
controversial.7,8
The nature of the peptides that are possibly bound to platelet HLA
molecules has not been investigated, and the exact function of
thrombocyte class I molecules is still unknown. However, it has been
proposed that they are implicated in alloimmunization following
multiple platelet transfusions. Production of anti-HLA class I
alloantibodies has been reported in about half the patients receiving
nonleukocyte-depleted thrombocyte concentrates, and refractoriness to
platelet transfusion occurs secondarily.9-13 Numerous
studies have shown that depletion of contaminant leukocytes from
platelet products efficiently decreases the risk of alloimmunization (< 20%) and refractoriness (< 3%).10,14,15 However,
previous pregnancies increase the risk of later immunization, even if
leukocyte-depleted platelet concentrates are used.16
Successful treatment of platelet refractoriness has recently
been obtained by transfusing platelets matched for HLA or by using
acid-stripped platelets.11,17 All these in vivo data
suggest that HLA molecules present on contaminating leukocytes rather
than on platelets induce primary anti-HLA alloimmunization after
transfusion, although platelets may be responsible for a reactivation
of secondary response in preimmunized patients. It was then
hypothesized that contaminating HLA class II antigen-presenting cells
(APC) present in the thrombocyte concentrates are responsible for
initiating T-cell immunity and further production of alloantibodies. In
contrast to these studies, several reports in mouse models showed that
repeated leukocyte-depleted platelet transfusions induced
CD4+ and CD8+ T-lymphocyte activation and
alloantibody formation, presumably by a cross-presentation system
involving responder APC.18,19 It was also reported recently
that extreme leukocyte reduction can re-enhance in vivo allogenic
platelet immunity in mice.20 Thus, it remains controversial
whether class I molecules from platelets are efficient in inducing an
in vivo immune response.
In reexamining this issue, we have studied the major functional
features of thrombocyte HLA class I molecules. We could show that
peptides extracted from platelet HLA-A2 molecules follow the rules of
the peptide motif described for this allele. We also observed that HLA
molecules are unstable when thrombocytes are incubated at
37°C, but can be partially stabilized when exogenous Platelet preparations
Extraction of HLA-A2 molecules from platelets and analysis of
peptidic ligands
Analysis of platelet HLA surface expression and peptide stabilization assays HLA-A2+ platelets (100 × 106) were incubated at 37°C in citrate buffer, pH 6.2, for different periods of time. For stripping of HLA surface molecules, a citric acid solution (pH 3.0) was prepared by mixing equal volumes of 0.263 mol/L citric acid and 0.123 mol/L Na2HPO4, as already described.23 Platelets were pelleted, gently resuspended in 500 µL of this solution, incubated at room temperature for 30 seconds under agitation, and washed twice in PBS. Peptide stabilization assays were performed as previously described,24 in citrate buffer, pH 6.2, at 37°C and 5% CO2 in the presence of human 2-microglobulin at 3 µg/mL (Biotrend, Cologne, Germany) and peptides at 100 µg/mL. Aliquots of platelets were collected at different time points and stained with MoAbs.
Synthetic peptides Peptides were synthesized in an automated peptide synthesizer 432A (Applied Biosystems). Purity was always more than 80%. Mat 58-66 (GILGFVFTL, Influenza A matrix protein) and gp190 367-375 (SLFTDPLEL, gp190 from Plasmodium falciparum) are strong HLA-A*0201 binders.1 Peptides CH10 68-77 (VGDIILMDKY, CH10 protein from Chlamydia pneumoniae), TYBO 11-19 (ASFDKAKLK, Thymosine10, natural ligand of HLA-A11), and Ras 8-16 (VVGAGGVGK, K-Ras,
HLA-A11 binder) were used as negative controls.1
Antibodies and staining for fluorescence-activated cell sorter (FACS) analysis W6/32 (anti-HLA class I monomorphic, IgG2a), BB7.2 (anti-HLA-A2, IgG2b), and B1.23.2 (anti-HLA-B and -C alleles, IgG2b) were used as purified MoAbs or as hybridoma culture supernatants. Optimal concentrations for staining had been previously determined. Background control stainings were evaluated with a mouse IgG2b isotype control (Coulter Immunotech, Hamburg, Germany) or OKT8 MoAb (anti-CD8, IgG2a); alternatively, fluorescein isothiocyanate (FITC)-coupled goat-antimouse IgG (GAM-FITC, Dianova, Hamburg, Germany) alone was used. There was no significant difference in the background fluorescence using these 3 methods. CD41-FITC MoAb, specific for the GpIIb protein expressed exclusively by megakaryocytes and platelets, was purchased from Coulter Immunotech.In vitro allostimulation of PBMCs and cytotoxic assays The fresh PBMCs were obtained by Ficoll-Hypaque gradient centrifugation (Ficoll-Paque, Pharmacia, Oslo, Norway) of buffy coats from HLA-A2 and HLA-A2+ typed healthy
donors (Blood Bank, Tübingen, Germany).
HLA-A2 peptide motif and identification of natural HLA class I ligands from platelets To determine whether HLA class I molecules associated to platelets contain peptides and to analyze these peptides, we performed HLA-A2 ligand extractions using platelet pool concentrates. An RP-HPLC profile obtained from one of these peptide extractions is presented in Figure 1. This profile is very similar to those usually obtained from different cell types. In particular, peptides eluted between 26 minutes and 52 minutes, showing similar hydrophobicity properties to HLA-A2 peptide ligands from other cells.1,21,25 Peptide fractions were pooled and sequenced, and the results are given in Table 1. Position 2 showed a dominant preference for Leu residue, Met also being strongly represented. At position 9, Leu and Val were found almost exclusively. Thus, amino acids in positions 2 and 9 appear to be anchor residues. Moreover, the dominant residue in position 6 was Ile. Although some differences were observed for auxiliary anchor (position 6) or preferred residues, the motif found here is very similar to the classical HLA-A*0201 motif.21 Fifteen individual peptides could be identified, 12 nonamers, 1 octamer and 1 decamer, and 1 peptide longer than 11 amino acids (this peptide could not be entirely sequenced). Such length variability has been well documented for most of the natural HLA ligands.1 Among the 15 peptides, 12 were classified as HLA-A2 natural ligands; 9 of them contain 2 anchor residues at positions 2 and 9-10, and for 3 of them, 1 of the 2 anchors was present. Ile at position 6 was found in 4 peptides. Peptides YLLPAIVHI, derived from the p68 RNA helicase, and ILMEHIHKL, derived from the 60S ribosomal protein L19 were previously isolated from HLA-A*0201 and HLA-A*0214 peptide extracts, respectively.25,26 Most of the HLA-A2 ligands identified here originate from ubiquitously expressed cytosolic or nuclear proteins, and thus are not specific for the megakaryocyte-platelet lineage. To control that extracted peptides derived from platelets and not from contaminating leukocytes, several peptide extractions were also performed using the leukocyte-rich fraction obtained after density gradient separation of platelet concentrates; for the experiment with the highest amount of platelets, the amount of HLA-A2-associated peptides found in the leukocyte-rich fraction was only 5% of that obtained with the separated platelet fraction, as detected by Edman degradation (data not shown). Thus, even if the gradient-separated platelets still contain some leukocytes, these should account for less than 5% of the total peptides detected. Moreover, peptide FLLWATAEA derives from the signal sequence of the GPIX protein, which has been shown to be exclusively expressed by mature megakaryocytes undergoing fragmentation and by platelets. This peptide could be sequenced from 3 of 4 extraction experiments, and each time appears as the dominant peptide, still more abundant than the main HLA-A*0201 ligand YLLPAIVHI. We concluded that it is most likely prominent among platelet HLA-A2 ligands. We also confirmed that synthetic FLLWATAEA peptide binds to HLA-A*0201 molecules using an in vitro stabilization assay with TAP-deficient T2 cells (data not shown). Three other peptides derived from the fibrinogen and chains were identified. As seen in
Table 1, their amino acid sequence did not fit the HLA-A*0201 motif or, indeed, any other HLA-A2 motif known. These peptides may derive from
contaminating fibrinogen present during the HLA extraction procedure
and may not be relevant for HLA binding. However, because nonmotif
peptides have been occasionally described as MHC ligands, we do not
want to exclude this possibility. Peptides 3 to 6 amino acids in length
were also found occasionally, but because such short peptides did not
bind to HLA-A*0201 in vitro (data not shown), they might constitute
short fragments generated from longer peptides during peptide
preparation. Taken together, our results show that platelet HLA-A2
molecules bind peptides derived from intracellular proteins. The
peptide motif and properties of these peptides are very similar to
those described for HLA-A*0201 ligands (HLA-A*0201 accounts for about
96% of all HLA-A2 alleles present in the Caucasian population).
Moreover, one of the peptides identified derives from the GPIX protein,
which is specifically expressed in the megakaryocyte-platelet lineage.
Platelets lose HLA surface expression after incubation at 37°C To examine the stability of thrombocyte surface HLA molecules, HLA-A2+ platelets were incubated at 37°C in citrate buffer pH 6.2, and HLA expression measured at different time points. Figure 2 shows a representative result from 5 experiments. HLA-A2+ freshly isolated platelets were stained with the conformation-dependent W6/32 (pan-anti-HLA class I molecules) and BB7.2 (HLA-A2 specific) MoAbs. This staining is weaker than that observed with PBMCs, which is in agreement with a previous report.2 After 24 hours of incubation (day 1), HLA surface expression was strongly reduced, with 34% and 20% of initial fluorescence remaining after staining with W6/32 and BB7.2, respectively. Further decrease of fluorescence was observed at day 2. Finally, after 4 days of incubation, the majority of integral HLA molecules had disappeared from the platelet surface, because less than 15% of initial surface expression was still visible. Interestingly, addition of 10% FCS to the medium could partially retain the staining with MoAbs, probably by stabilization of HLA complexes by exogenous bovine2-microglobulin (data not shown). Finally,
expression of GpIIb, as assessed by CD41 MoAb, remains stable even
after 4 days at 37°C, suggesting that platelet membrane integrity
was not affected by the assay conditions. These results demonstrate
that class I 2-microglobulin-peptide complexes are
unstable at the platelet surface and are rapidly dissociated after in
vitro incubation at 37°C.
Stabilization of platelet HLA-A2 molecules by exogenous HLA-A2 binding peptides Because we observed that properly folded HLA molecules disappear rapidly from the platelet surface after incubation at 37°C, we tested whether this dissociation could be compensated by addition of exogenous2-microglobulin and peptide to the medium.
HLA-A2+ platelets were incubated at 37°C for 24 hours,
in medium alone, or in the presence of 2-microglobulin
and different HLA class I binding peptides. HLA expression was then
analyzed. Staining of fresh platelets before incubation at 37°C was
used as control. Results of a representative experiment from 3 are
shown in Table 2. After 24 hours of
incubation at 37°C, HLA surface expression was strongly reduced, as
already observed in Figure 2. This was detected after staining with
W6/32 and BB7.2 MoAbs, as well as with B1.23.2 MoAb (anti-HLA-B and -C
alleles). Addition of Mat 58-66 (HLA-A*0201 epitope) and gp190 367-375 (strong HLA-A2 binder) peptides was able to significantly retain
staining of HLA-A2 molecules at the platelet surface. The effect
observed with the gp190 peptide was stronger than with the Mat peptide,
and about 70% of initial surface expression remained after 24 hours,
as compared to 50% without peptide. This could be observed using W6/32
and BB7.2 MoAbs, but not B1.23.2 MoAb, showing that stabilization of
surface HLA molecules occurred on specific binding of Mat and gp190
peptides to HLA-A2. Two other peptides, CH10 68-77 and TYBO 11-19, which do not bind to HLA-A2, could not induce any HLA stabilization, as
assessed with the different MoAbs tested. Finally, as already seen in
Figure 2, CD41 expression was almost stable after 24 hours and remained
unchanged after incubation with the different peptides.
In vitro allostimulation experiments
To investigate the origin and function of platelet MHC class I
molecules, we studied platelet-derived peptides presented by the common
HLA-A*0201 allelic product. We found that HLA-A2 molecules isolated
from platelets are able to bind peptides of 8 to 10 amino acids, and
that the binding motif of these peptides is very similar to the
HLA-A*0201 motif described (HLA-A*0201 counts for 96% of HLA-A2
alleles of the Caucasian population).1,21 The classical Leu
at position 2 and Leu or Val at position 8 to 10 as anchoring residues
were present in 11 of 12 and 10 of 12 identified ligands, respectively
(Table 1). Ile as auxiliary anchor residue at position 6, and other
preferred residues identified in our study, have also been described
for HLA-A*0201 ligands or T cell epitopes.1 Of 12 individual peptides that could be sequenced, 2 have already been found
in other cell extracts,25,26 confirming that platelet HLA-A2 molecules bind the same kind of peptides as those from other
cells. When the protein source of the peptides could be established, it
was evident that most of these proteins are ubiquitously expressed
(Table 1). One peptide was derived from the human actin cytoskeleton-associated protein talin, which has been shown to be
highly expressed in thrombocytes, accounting for 3% to 8% of whole
platelet proteins. Another abundant peptide was derived from the signal
sequence of the GPIX glycoprotein, which is specifically expressed in
the megakaryocyte-platelet lineage and has not been found in any
other cell. These results demonstrate that HLA-A2-bound peptides
from platelets are derived from endogenously processed proteins that
are expressed at the megakaryocyte stage. Although we cannot exclude
that a number of HLA molecules present at the platelet surface are
adsorbed from the serum, our data indicate that the majority of these
HLA molecules are of endogenous origin, as previously
suggested.27 The stage of platelet development at which
peptides to be presented by HLA molecules are generated is not known.
Because platelets are anucleated cytoplasmic fragments of
megakaryocytes with a short life span, it is generally thought that
synthesis of proteins occurs at the megakaryocyte stage. However,
endoplasmic reticulum and ribosomes have been identified in
platelets.28 Protein biosynthesis, including HLA class I molecules, and presence of a functional proteasomal complex have also
been reported.28-30 Thus, 2 scenarios are possible: (1) HLA molecules are synthesized and loaded with peptides at the nucleated megakaryocyte stage, or (2) remaining de novo synthesis of HLA molecules from platelet messenger RNA (mRNA) and components of the
processing machinery allow the entire assembly of peptide-loaded HLA
molecules at the thrombocyte stage.
We wish to thank D. Wernet, M. Schnaidt, and S. Pascolo for critically
reading the manuscript; G. Mayer and H. Northoff from the
Tübingen Blood Bank and A. König from the Stuttgart Blood Center for providing the platelet concentrates and buffy coats; L. Yakes for editorial help; and P. Hrsti Submitted May 24, 1999; accepted January 11, 2000.
Supported by a Marie Curie Fellowship awarded to C.G. (CT-96-1298) by
the Deutsche Forschungsgemeinschaft (Leibniz Programm Ra 369/4-1), and
by the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie.
Reprints: Cécile Gouttefangeas, Department of Immunology,
Institute for Cell Biology, Auf der Morgenstelle 15, 72076 Tübingen, 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.
1.
Rammensee HG, Bachmann J, Stevanovi
2.
Kao KJ, Cook DJ, Scornik JC.
Quantitative analysis of platelet surface HLA by W6/32 anti-HLA monoclonal antibody.
Blood.
1986;68:627-632
3.
Gockerman JP, Jacob W.
Purification and characterization of papain-solubilized HLA antigens from human platelets.
Blood.
1979;53:838-850
4.
Trägårdh L, Klareskog L, Curman B, Rask L, Peterson PA.
Isolation and properties of detergent-solubilized HLA antigens obtained from platelets.
Scand J Immunol.
1979;9:303-314[Medline]
[Order article via Infotrieve].
5.
Lalezari P, Driscoll AM.
Ability of thrombocytes to acquire HLA specificity from plasma.
Blood.
1982;59:167-170
6.
Kao KJ, Scornik JC, Riley WJ, McQueen CF.
Association between HLA phenotype and HLA concentration in plasma or platelets.
Hum Immunol.
1988;21:115-124[Medline]
[Order article via Infotrieve].
7.
Kao KJ.
Selective elution of HLA antigens and
8.
Santoso S, Mueller-Eckhardt G, Santoso S, Kiefel V, Mueller-Eckhardt C.
HLA antigens on platelet membranes.
Vox Sang.
1986;51:327-333[Medline]
[Order article via Infotrieve].
9.
Murphy MF, Metcalfe P, Thomas H, et al.
Use of leucocyte-poor blood components and HLA-matched-platelet donors to prevent HLA alloimmunization.
Br J Haematol.
1986;62:529-534[Medline]
[Order article via Infotrieve].
10.
Vamvakas EC.
Meta-analysis of randomized controlled trials of the efficacy of white cell reduction in preventing HLA-alloimmunization and refractoriness to random-donor platelet transfusions.
Transfus Med Rev.
1998;2:258-270.
11.
Kekomäki S, Volin L, Koistinen P, et al.
Successful treatment of platelet transfusion refractoriness: the use of platelet transfusions matched for both human leucocyte antigens (HLA) and human platelet alloantigens (HPA) in alloimmunized patients with leukaemia.
Eur J Haematol.
1998;60:112-118[Medline]
[Order article via Infotrieve].
12.
Schnaidt M, Northoff H, Wernet D.
Frequency and specificity of platelet-specific alloantibodies in HLA-immunized haematologic-oncologic patients.
Transfus Med.
1996;6:111-114[Medline]
[Order article via Infotrieve].
13.
Novotny VMJ, van Doorn R, Witvliet MD, Claas FHJ, Brand A.
Occurrence of allogenic HLA and non-HLA antibodies after transfusion of prestorage filtered platelets and red blood cells: a prospective study.
Blood.
1995;85:1736-1741
14.
Claas FHJ, Smeenk RJT, Schmidt R, van Steenbrugge GJ, Eernisse JG.
Alloimmunization against MHC-antigens after platelet transfusion is due to contaminating leucocytes in platelet suspensions.
Exp Haematol.
1981;9:84-89[Medline]
[Order article via Infotrieve].
15.
Eernisse JG, Brand A.
Prevention of platelet refractoriness due to HLA antibodies by administration of leukocyte-poor blood components.
Exp Haematol.
1981;9:77-83[Medline]
[Order article via Infotrieve].
16.
Ten Haaft MA, Van den Berg-Loonen PM, Van Rhenen DJ.
Prevention of primary HLA class I allo-immunization with leucocyte-poor blood components produced without the use of platelet filters.
Vox Sang.
1992;63:257-261[Medline]
[Order article via Infotrieve].
17.
Castro E, Muncunill J, Barea L, Gonzãlez R, Fernãndez-Villalta MJ.
Acid elution of platelet HLA-class I antigens in the treatment of a refractory patient.
Br J Haematol.
1998;100:245-246[Medline]
[Order article via Infotrieve].
18.
Semple JW, Speck ER, Milev YP, Blanchette V, Freedman J.
Indirect allorecognition of platelets by T helper cells during platelet transfusions correlates with anti-major histocompatibility complex antibody and cytotoxic. T lymphocyte formation.
Blood.
1995;86:805-812
19.
Bang A, Speck ER, Blanchette VS, Freedman J, Semple JW.
Recipient humoral immunity against leukoreduced allogenic platelets is suppressed by aminoguanidine, a selective inhibitor of inducible nitric oxide synthase.
Blood.
1996;88:2959-2966
20.
Semple JW, Speck ER, Cosgrave D, Lazarus AH, Blanchette VS, Freedman J.
Extreme leukoreduction of major histocompatibility complex class II positive B cells enhances allogenic platelet immunity.
Blood.
1999;93:713-720
21.
Falk K, Rötzschke O, Stevanovi
22.
Seeger FH, Arnold D, Dumrese T, et al.
The HLA-B*1516 motif demonstrates HLA-B-specific P2 pocket characteristics.
Immunogenetics.
1998;48:156-160[Medline]
[Order article via Infotrieve].
23.
Sugawara S, Abo T, Kumagai K.
A simple method to eliminate the antigenicity of surface class I MHC molecules from the membrane of viable cells by acid treatment at pH 3.
J Immunol Methods.
1987;100:83-90[Medline]
[Order article via Infotrieve].
24.
Gouttefangeas C, Demeur C, Connan F, Balayn N, Choppin J, Tilkin-Mariamé AF.
Differential binding to frequent HLA-A alleles of p21 RAS derived peptides bearing oncogenic substitutions at position 12 or 13.
Hum Immunol.
1997;55:117-126[Medline]
[Order article via Infotrieve].
25.
Hunt DF, Henderson RA, Shabanowitz J, et al.
Characterization of peptides bound to the class I MHC molecule HLA-A2.1 by mass spectrometry.
Science.
1992;255:1261-1263
26.
Barouch D, Friede T, Stevanovi
27.
Neumüller J, Tohidast-Akrad M, Fisher M, Mayr WR.
Influence of chloroquine or acid treatment of human platelets on the antigenicity of HLA and the "thrombocyte-specific" glycoproteins Ia/IIa, IIb, and IIb/IIIa.
Vox Sang.
1993;65:223-231[Medline]
[Order article via Infotrieve].
28.
Kieffer N, Guichard J, Farcet JP, Vainchenker W, Breton-Gorius J.
Biosynthesis of major platelet proteins in human blood platelets.
Eur J Biochem.
1987;164:189-195[Medline]
[Order article via Infotrieve].
29.
Santoso S, Kalb R, Kiefel V, Mueller-Eckhardt C.
The presence of messenger RNA for HLA class I in human platelets and its capability for protein biosynthesis.
Br J Haematol.
1993;84:451-456[Medline]
[Order article via Infotrieve].
30.
Yukawa M, Sakon M, Kambayashi JI, et al.
Proteasome and its novel endogeneous activator in human platelets.
Biochem Biophys Res Commun.
1991;178:256-262[Medline]
[Order article via Infotrieve].
31.
Kao KJ.
Stability of platelet and plasma HLA concentrations in healthy adults or random-donor platelet concentrates.
Transfusion.
1989;29:328-331[Medline]
[Order article via Infotrieve].
32.
Kurata Y, Oshida M, Take H, et al.
New approach to eliminate HLA class I antigens from platelet surface without cell damage: acid-treatment at pH 3.0.
Vox Sang.
1989;57:199-204[Medline]
[Order article via Infotrieve].
33.
Pereira J, Cretney C, Aster RH.
Variation of class I HLA antigen expression among platelet density cohorts: a possible index of platelet age?
Blood.
1988;71:516-519
34.
Goeken NE.
Human suppressor cell induction in vitro: preferential activation by class I MHC antigen.
J Immunol.
1984;132:2291-2299[Abstract].
35.
Henn V, Slupsky JR, Gräfe M, et al.
CD40 ligand on activated platelets triggers an inflammatory reaction of endothelial cells.
Nature.
1998;391:591-594[Medline]
[Order article via Infotrieve].
36.
Crow AR, Freedman J, Hannach B, Blanchette V, Lazarus AH.
Antibody-mediated inhibition of the human alloimmune response to platelet transfusion in Hu-PBL-SCID mice.
Br J Haematol.
1999;104:919-924[Medline]
[Order article via Infotrieve].
37.
Barry OP, Praticò D, Savani RC, Fitzgerald GA.
Modulation of monocyte-endothelial cell interactions by platelet microparticles.
J Clin Invest.
1998;102:136-144[Medline]
[Order article via Infotrieve].
38.
Elstad MR, McIntyre TM, Prescott SM, Zimmerman GA.
The interaction of leukocytes with platelets in blood coagulation.
Curr Opin Hematol.
1995;2:47-54[Medline]
[Order article via Infotrieve].
39.
Weyrich AS, Elstad MR, McEver RP, et al.
Activated platelets signal chemokine synthesis by human monocytes.
J Clin Invest.
1996;97:1525-1534[Medline]
[Order article via Infotrieve].
40.
Neumann FJ, Marx N, Gawaz M, et al.
Induction of cytokine expression in leukocytes by binding of thrombin-stimulated platelets.
Circulation.
1997;95:2387-2394
41.
Aiura K, Clark BD, Dinarello CA, et al.
Interaction with autologous platelets multiplies interleukin-1 and tumor necrosis factor production in mononuclear cells.
J Infect Dis.
1997;175:123-129[Medline]
[Order article via Infotrieve].
42.
Diacovo TG, Catalina MD, Siegelman MH, von Andrian UH.
Circulating activated platelets reconstitute lymphocyte homing and immunity in L-selectin-deficient mice.
J Exp Med.
1998;187:197-204
43.
Joseph M, Auriault C, Capron A, Vorng H, Viens P.
A new function for platelets: IgE-dependent killing of schistosomes.
Nature.
1983;303:810-812[Medline]
[Order article via Infotrieve].
44.
Pancré V, Joseph M, Mazingue C, Wietzerbin J, Capron A, Auriault C.
Induction of platelet cytotoxic functions by lymphokines: role of interferon-
45.
Damonneville M, Wietzerbin J, Pancré V, et al.
Recombinant tumor necrosis factors mediate platelet cytotoxicity to Schistosoma mansoni larvae.
J Immunol.
1988;140:3962-3965[Abstract].
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2000 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
, and
Top
Abstract
Introduction
20°C. For
peptide stabilization and allostimulation experiments, fresh
thrombocyte concentrates from HLA-A2-typed donors or platelets obtained
from HLA-A2+ buffy coats after Ficoll-Hypaque density
gradient were used. Platelets were centrifuged at 800g for 10 minutes, washed twice, resuspended in CPD citrate buffer, pH 6.2, consisting of 0.327 g sodium citrate H2O, 2.63 g natrium
citrate, 0.251 g natrium dihydrogenophosphate 2H2O, 2.55 g
dextrose H2O in 100 mL water for injection (Baxter
Healthcare, Maurepas, France), and counted. Leukocyte contamination was
registered by microscope and was always less than
0.2 × 106/mL. When needed, platelets were stored at
4°C.
.
