The value of flow cytometric analysis of platelet glycoprotein expression of CD34+ cells measured under conditions that prevent P-selectin-mediated binding of platelets.
In the present study, we show by adhesion assays and ultrastructural studies that platelets can bind to CD34+ cells from human blood and bone marrow and that this interaction interferes with the accurate detection of endogenously expressed platelet glycoproteins (GPs). The interaction between these cells was found to be reversible, dependent on divalent cations, and mediated by P-selectin. Enzymatic characterization showed the involvement of sialic acid residues, protein(s). The demonstration of mRNA for the P-selectin glycoprotein ligand 1 (PSGL-1) in the CD34+ cells by polymerase chain reaction (PCR) analysis suggests that this molecule is present in these cells. Under conditions that prevent platelet adhesion, a small but distinct subpopulation of CD34+ cells diffusely expressed the platelet GPIIb/IIIa complex. These cells were visualized by immunochemical studies. Furthermore, synthesis of mRNA for GPIIb and GPIIIa by CD34+ cells was shown using PCR analysis. The semiquantitative PCR results show relatively higher amounts of GPIIb mRNA than of PF4 mRNA in CD34+CD41+ cells in comparison with this ratio in platelets. This finding is a strong indication that the PCR results are not caused by contaminating adhering platelets. MoAbs against GPIa GPIb alpha, GPV, P-selectin, and the alpha-chain of the vitronectin receptor did not react with CD34+ cells. The number of CD34+ cells expressing GPIIb/IIIa present in peripheral blood stem cell (PBSC) transplants was determined and was correlated with platelet recovery after intensive chemotherapy in 27 patients. The number of CD34+CD41+ cells correlated significantly better with the time of platelet recovery after PBSC transplantation (r = .83, P = .04) than did the total number of CD34+ cells (r = .55). Statistical analysis produced a threshold value for rapid platelet recovery of 0.34 x 10(6) CD34+CD41+ cells/kg. This study suggests that if performed in the presence of EDTA the flow cytometric measurement of GPIIb/IIIa on CD34+ cells provides the most accurate indication of the platelet reconstitutive capacity of the PBSC transplant.
Dercksen
M W
MW
European Cancer Centre, Amsterdam, The Netherlands.
Weimar
I S
IS
Richel
D J
DJ
Breton-Gorius
J
J
Vainchenker
W
W
Slaper-Cortenbach
C M
CM
Pinedo
H M
HM
von dem Borne
A E
AE
Gerritsen
W R
WR
van der Schoot
C E
CE
D018952:Antigens, CD34 / Q000032:analysis*; D000943:Antigens, Differentiation / Q000032:analysis*; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology / Q000627:therapeutic use; D016477:Artifacts; D001483:Base Sequence; D001773:Blood Cells; D001853:Bone Marrow / Q000473:pathology; D002454:Cell Differentiation; D004492:Edetic Acid / Q000494:pharmacology; D000069585:Filgrastim; D005434:Flow Cytometry; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology; D006331:Heart Diseases / Q000473:pathology / Q000601:surgery; D006410:Hematopoiesis / Q000187:drug effects; D018380:Hematopoietic Stem Cell Transplantation; D006801:Humans; D016130:Immunophenotyping; D008533:Megakaryocytes / Q000737:chemistry / Q000166:cytology*; D008666:Metalloendopeptidases / Q000494:pharmacology; D008969:Molecular Sequence Data; D009369:Neoplasms / Q000097:blood / Q000188:drug therapy / Q000473:pathology / Q000628:therapy; D009439:Neuraminidase / Q000494:pharmacology; D019007:P-Selectin; D010196:Pancreatic Elastase / Q000494:pharmacology; D015539:Platelet Activation / Q000187:drug effects; D010973:Platelet Adhesiveness / Q000187:drug effects*; D010980:Platelet Membrane Glycoproteins / Q000096:biosynthesis* / Q000235:genetics; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology; D014357:Trypsin / Q000494:pharmacology
D018952:Antigens, CD34; D000943:Antigens, Differentiation; D019007:P-Selectin; D010980:Platelet Membrane Glycoproteins; D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor; D004492:Edetic Acid; D009439:Neuraminidase; D010196:Pancreatic Elastase; D014357:Trypsin; D013917:Thrombin; D008666:Metalloendopeptidases; C070738:O-sialoglycoprotein endopeptidase; D000069585:Filgrastim