Blood, Vol. 94 No. 9 (November 1), 1999:
pp. 3271-3273
CORRESPONDENCE
Glycoprotein IIb/IIIa Expression on Hematopoietic Stem
Cells: Constitutive Expression or Platelet Adhesion?
 |
LETTER |
To the Editor:
The platelet glycoprotein (GP) IIb/IIIa is an important integrin
(
IIb
3) involved in cell-substratum adhesion, which plays a key
role in the function of megakaryocytes and platelets. This complex
could also play a relevant role in hematopoiesis if expressed in more
primitive multilineage hematopoietic progenitor cells. Thus, although
an extensive search has been made to identify the expression of this
integrin on hematopoietic progenitors, there are conflicting data about
the more primitive hematopoietic stem cell (HSC) capable of expressing
the platelet GP IIb/IIIa. Antisera against purified GPs IIb and IIIa
inhibit colony-forming unit (CFU)-mix in some
experiments,1,2 but not in others.3 Moreover, the GP IIb/IIIa mRNA has been detected in human
CD34+-enriched cell populations.4 Transgenic
mice with conditional toxigene suggest that the GP IIb promoter was
active in primitive hematopoietic progenitor cells with myeloid,
erythroid, and megakaryocytic potential, but decreased progressively as
differentiation proceeded toward the erythroid and myeloid
lineages.5,6 However, cell surface expression of GP
IIb/IIIa in HSC had not been fully demonstrated. Murray et
al7 suggest such expression, reporting that the GP IIb/IIIa+ cell population is also positive for the human
HSC marker CD34+. The recent report of Ody et
al8 is the first evidence of GP IIb/IIIa expression in HSC.
By flow cytometry, these authors sorted GP IIb/IIIa+ cells
from avian intraaortic clusters and from embryonic and adult bone
marrow. Then, by means of clonogenic assays for the multilineage
potential of HSC, they showed that the sorted cells were able to
differentiate into myeloid, erythroid, and thrombocytic lineages. Thus,
they suggest the attractive idea that GP IIb/IIIa is expressed in
primitive HSC and, therefore, this integrin should not be considered as
a specific marker of the megakaryocytic lineage, but it could be a
useful tool for the characterization and localization of hematopoietic progenitors.
However, some points should be considered with caution when analyzing
these results. Firstly, we have observed that a percentage of human
primitive multilineage hematopoietic cells (CD34+,
c-Kit+) from distinct sources (human cord blood, bone
marrow, and recombinant human granulocyte colony-stimulating factor
[rhG-CSF] mobilized peripheral blood from healthy subjects) are also
positive for platelet markers. Between 10% and 20% of
CD34+/c-kit+ cells with light scatter
characteristics of true progenitor cells (after convenient exclusion of
those CD34+ cells also positive for monocytic antigens)
from these sources do present a high expression of GP IIIa (CD61), GP
Ib (CD42b), GP Ia (CD49b), and P-selectin (CD62).
Figure 1 shows a representative example of
c-kit, CD34, and platelet-marker (CD42b and CD61) expression on a
sample from an rhG-CSF-mobilized donor leukapheresis.
Furthermore, we have also observed that the expression of these
antigens decreased when samples were washed with EDTA before monoclonal
antibody incubation. Therefore, our results cannot be explained by the
constitutive expression of these platelet markers in HSC. More likely,
these data suggest that the expression of these platelet-associated antigens might be the consequence of platelet adherence to progenitor cell surface. In fact, other authors have reported the in vitro P-selectin-dependent binding of platelet to HSC.9,10
Moreover, the recently described expression of the P-selectin
glycoprotein-1 (PSGP-1), the putative counterreceptor for P-selectin,
on CD34+ progenitor cells does also support platelet
adhesion to HSC.11 According with these data, sorting of GP
IIb/IIIa+ cells could achieve a positive selection of HSC
bound to platelets. Therefore, the culture of these selected cells in
different hematopoietic differentiation medium could easily produce a
high number of colonies of distinct lineages. The
observations suggested by Ody et al could have a fundamental relevance,
but we believe it could be necessary to discard the adhesion of
platelets to the sorted GP IIb/IIIa+ cells. The use of
specific platelet markers, such as GP Ib, in the sorting assays,
and/or the determination of platelet-antigen expression in the sorted
GP IIb/IIIa+ population should be performed to clarify this
point.
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| Fig 1.
c-Kit, CD42b, (GP Ib ), and CD61 (GP IIIa) expression
on human leukapheresis product after mobilization with rhG-CSF.
CD34+/CD14 low side scatter cells are
shown in red. Fifty thousand events were acquired.
|
|
J. Gómez-Espuch
J. Corral
R. González-Conejero
F. Ortuño
J.M. Moraleda
V. Vicente
Hematology and Clinical Oncology
Hospital General
Universitario
Centro Regional de Hemodonación
Murcia,
Spain
| |
REFERENCES |
1.
Berridge MV, Ralph SJ, Tan AS:
Cell-lineage antigens of stem cell-megakaryocyte-platelet lineage are associated with the platelet IIb-IIIa glycoprotein complex.
Blood
66:76, 1985[Abstract/Free Full Text]
2.
Fraser JK, Leahy MF, Berridge MV:
Expression of antigens of the platelet glycoprotein IIb-IIIa complex on human hematopoietic stem cells.
Blood
68:762, 1986[Abstract/Free Full Text]
3.
Levene RB, Lamaziere J-MD, Broxmeyer HE, Lu L, Rabellino EM:
Human megakaryocytic V. Changes in the phenotypic profile of differentiating megakaryocytes.
J Exp Med
161:457, 1985[Abstract/Free Full Text]
4.
Molla A, Andrieux A, Chapel A, Schweitzer A, Berthier R, Marguerie G:
Lack of transcription and expression of the IIB integrin in human early hematopoietic stem cells.
Br J Haematol
82:635, 1992[Medline]
[Order article via Infotrieve]
5.
Tronik-Le Roux D, Roullot V, Schweitzer A, Berthier R, Marguerie G:
Suppression of erythro-megakaryocytopoiesis and the induction of reversible thrombocytopenia in mice transgenic for the thymidine kinase gene targeted by the platelet glycoprotein IIb promoter.
J Exp Med
181:2141, 1995[Abstract/Free Full Text]
6.
Tropel P, Roullot V, Vernet M, Poujol C, Pointu H, Nurden P, Marguerie G, Tronik-Le Roux D:
A 2.7-kb portion of the 5' flanking region of the murine glycoprotein IIb gene is transcriptionally active in primitive hematopoietic progenitor cells.
Blood
90:2995, 1997[Abstract/Free Full Text]
7.
Murray LJ, Mandich D, Bruno E, Di Giusto RK, Fu W-C, Sutherkand DR, Hoffman R, Tsukamoto A:
Fetal bone marrow CD34+ CD41+ cells are enriched for multipotent hematopoietic progenitors, but not for pluripotent stem cells.
Exp Hematol
24:236, 1996[Medline]
[Order article via Infotrieve]
8.
Ody C, Vaigot P, Quéré P, Imhof BA, Corbel C:
Glycoprotein IIb-IIIa is expressed on avian multilineage hematopoietic progenitor cells.
Blood
93:2898, 1999[Abstract/Free Full Text]
9.
Zannettino AC, Berndt MC, Butcher C, Butcher EC, Vadas MA, Simmons PJ:
Primitive human hematopoietic progenitors adhere to P-Selectin (CD62P).
Blood
85:3466, 1995[Abstract/Free Full Text]
10.
Laszik Z, Jansen PJ, Cummings RD, Tedder T, McEver RP, Moore KL:
P-Selectin glycoprotein ligand-1 is broadly expressed in cells of myeloid, lymphoid, and dendritic lineage and in some nonhematopoietic cells.
Blood
88:3010, 1996[Abstract/Free Full Text]
11.
Yang J, Furie BC, Furie C:
The biology of P-Selectin Glycoprotein ligand-1: Its role as a selectin counterreceptor in leukocyte-endothelial and leukocyte-platelet interaction.
Thromb Haemost
81:1, 1999[Medline]
[Order article via Infotrieve]
Response
According to the authors, the immunoreactivity of hematopoietic stem
cells could be caused by contaminating adherent platelets. Thus, they
question our recently published data.1
Platelets do not exist in birds, and thrombocytes are the functional
equivalents to mammalian platelets. These are large nucleated cells
that express GPIIbIIIa at a high level (Fig 3 in ref 1). In the
c-kit/GPIIbIIIa double-positive bone marrow population selected by
cytofluorimetric cell sorting (Fig 4 in ref 1), thrombocytes were
excluded by the criteria of their high level of GPIIbIIIa expression.
The size of the thromboblasts (thrombocyte precursors) is similar to
the size of hematopoietic progenitors in the selected population, and
doublets or larger aggregates were excluded in the sorting procedure by
the choice of the forward and side scatter window. Therefore, the
thromboblasts included in the sorted progenitor population were present
as single cells and not bound to hematopoietic progenitors.
Furthermore, the potentialities of the double-positive cells were
tested in vitro and in vivo and these cells were able to differentiate
into all hematopoietic lineages. In conclusion, we showed that
c-kit+ multipotential hematopoietic progenitors coexpress GPIIbIIIa.
Nevertheless, in mammals, considering the small size of the platelets,
their interaction with hematopoietic cells is more difficult to exclude
by flow cytometry. The fluorescence-activated cell sorting (FACS)
profiles from Vicente et al using G-CSF-mobilized peripheral blood
progenitors show that the c-kit+ population contains cells
expressing megakaryocytic markers in the light scatter window
characteristic of progenitor cells. These cells could be contaminating
platelets adhering to progenitor cells or megakaryocytic precursors
expressing lineage markers along with c-kit. By analogy with chicken
adult bone marrow staining with c-kit and GPIIbIIIa monoclonal
antibodies, the CD61hi cell population may contain adherent
platelets because in the chicken, GPIIbIIIahi cells are
exclusively composed of thrombocytes (Fig 3 in ref 1). In contrast, the
CD61lo population could contain megakaryocytic precursors
and multipotential hematopoietic progenitors as it is for the chicken
(Fig 3 in ref 1). From the experiments performed with the chicken, we
showed that the multilineage hematopoietic progenitors were exclusively located in the GPIIbIIIalo c-kit+ population.
Considering the in vivo data in mice2,3 showing that
impaired GPIIb expression prevents normal hematopoiesis, we think that
GPIIbIIIa expression by multilineage hematopoietic progenitor cells is
a general feature and that it plays a functional role in hematopoiesis.
Although it is obviously not the case in the chicken, our data cannot
rule out adhesion of platelets to mammalian hematopoietic progenitors.
This idea is still intriguing, but the demonstration would require a
careful and thorough analysis that is not provided by the current comments.
Christiane Ody
Beat A. Imhof
Department
of Pathology
Centre Medical Universitaire
Geneva,
Switzerland
Catherine Corbel
Institut
d'Embryologie
CNRS UPR 9064
Nogent-sur-Marne, France
| |
REFERENCES |
1.
Ody C, Vaigot P, Quere P, Imhof BA, Corbel C:
Glycoprotein IIb-IIIa is expressed on avian multilineage hematopoietic progenitor cells.
Blood
93:2898, 1999
2.
Tronik-Le Roux D, Roullot V, Schweitzer A, Berthier R, Marguerie G:
Suppression of erythro-megakaryocytopoiesis and the induction of reversible thrombocytopenia in mice transgenic for the thymidine kinase gene targeted by the platelet glycoprotein alpha IIb promoter [published erratum appears in J Exp Med 182:1177, 1995].
J Exp Med
181:2141, 1995
3.
Tropel P, Roullot V, Vernet M, Poujol C, Pointu H, Nurden P, Marguerie G, Tronik-Le Roux D:
A 2,7-kb portion of the 5' flanking region of the murine glycoprotein alphaIIb gene is transcriptionally active in primitive hematopoietic progenitor cells.
Blood
90:2995, 1997
