Blood, Vol. 95 No. 7 (April 1), 2000:
pp. 2452-2453
CORRESPONDENCE
 |
Letter |
To the editor:
Are MpI glycosylation defects in polycythemia vera secondary to
artifactual hypoglycemia?
In their report on altered processing of the thrombopoietin
receptor (Mpl) in patients with polycythemia vera, Moliterno and Spivak1 (Oct 15 issue) show that Mpl glycosylation is
associated with disease progression. The authors demonstrate that the
nonglycosylated Mpl isoform has reduced expression and is impaired in
its ability to transit to the cell membrane, thus rendering the cells
resistant to thrombopoietin.1,2 While the authors suggest
that these findings indicate that Mpl glycosylation defects may serve
as a marker for disease progression, an alternate hypothesis should be entertained.
Artifactual hypoglycemia has been reported to occur in polycythemia
vera and is caused by in vitro autoglycolysis due to an exaggerated
consumption of glucose by blood cells.3 In polycythemia vera, this phenomenon can occur even with only modest leukocytosis and
may be due to both red and white cell-induced enhanced
glycolysis.4,5 Serum glucose concentrations can drop
significantly within 2 hours after sample collection, and longer
intervals of time are associated with greater reductions in glucose
levels. Additionally, recent work from other laboratories has indicated
that glucose deprivation causes defects in receptor processing and
glycosylation.6-8 By depriving cells of glucose, both the
epidermal growth factor and insulin receptors were impaired in their
glycosylation and exhibited reduced surface expression, similar to the
findings of Moliterno et al with regard to Mpl. Therefore, it is
possible that there is an artifactual association between Mpl
glycosylation and disease progression due to factitious hypoglycemia.
The authors further state that this defect is a "fossil record"
of a progenitor cell because "there is no evidence that platelets
can synthesize or metabolize Mpl"; however, others have shown that
platelets can efficiently synthesize protein.9
To their credit, the authors use controls with other myeloproliferative
disorders, some of which exhibit the potential for in vitro artifactual
hypoglycemia; however, no blood cell count data is provided in this
report. Interestingly, in their previous paper,2 the
authors show that the mean white cell count in the polycythemia vera
patients is much greater than in any of their controls. Furthermore, no
other hematopoietic cytokine receptors were studied to prove
specificity of the effect. It is unclear whether artifactual
hypoglycemia has influenced the validity of the Moliterno study;
however, if this methodology is to be used to stage disease progression
then the possible effects of artifactual hypoglycemia should be considered.
Kevin G. Otto
Division of Hematology
University of Washington
Seattle,
WA
| |
References |
1.
Moliterno AR, Spivak JL.
Posttranslational processing of the thrombopoietin receptor is impaired in polycythemia vera.
Blood.
1999;94:2555-2561[Abstract/Free Full Text].
2.
Moliterno AR, Hankins WD, Spivak JL.
Impaired expression of the thrombopoietin receptor by platelets from patients with polycythemia vera.
N Engl J Med.
1988;338:572-580[Abstract/Free Full Text].
3.
Murphy S.
Polycythemia vera. In Williams WJ, Beutler E, Erslev AJ, Lichtman MA, eds. Hematology. New York, NY: McGraw-Hill.; 1990:193-202.
4.
Arem R, Jeang MK, Blevens TC, Waddell CC, Field JB.
Polycythemia rubra vera and artifactual hypoglycemia.
Arch Intern Med.
1982;142:2199-2201[Abstract/Free Full Text].
5.
Billington CJ, Casciato DA, Choquette DL, Morley JE.
Artifactual hypoglycemia associated with polycythemia vera.
JAMA.
1983;249:774-775[Abstract/Free Full Text].
6.
Cai B, Tomida A, Mikami K, Nagata K, Tsuruo T.
Downregulation of epidermal growth factor receptor-signaling pathway by binding of GRP78/BiP to the receptor under glucose-starved stress conditions.
J Cell Physiol.
1998;177:282-288[Medline]
[Order article via Infotrieve].
7.
Hwang JB, Frost SC.
Effect of alternative glycosylation on insulin receptor processing.
J Biol Chem.
1999;274:22,813-22,820[Abstract/Free Full Text].
8.
Briata P, Briata L, Gherzi R.
Glucose starvation and glycosylation inhibitors reduce insulin receptor gene expression. Characterization and potential mechanism in human cells.
Biochem Biophys Res Commun.
1990;169:397-405[Medline]
[Order article via Infotrieve].
9.
Pabla R, Weyrich AS, Dixon DA, et al.
Integrin-dependent Control of Translation: Engagement of Integrin 
IIb
3 regulates synthesis of proteins in activated human platelets.
J Cell Biol.
1999;144:175-184[Abstract/Free Full Text].
Response:
MpI glycosylation defects in polycythemia vera are not secondary
to artifactual hypoglycemia
We thank Dr Otto for his hypothesis, which is directly refuted
by our published observation1 that the Mpl abnormality
observed in polycythemia vera (PV) platelets was also present in PV
megakaryocytes as demonstrated by in situ immunohistochemical staining
of fixed bone marrow biopsy sections, an approach not subject to
confounding by artifactual leukocyte-induced in vitro hypoglycemia.
Nevertheless, given the biologic significance of our observations with
respect to the pathophysiology of PV, we think that it is important to individually address the specific contentions upon which Dr Otto's hypothesis is based.
As Dr Otto indicates, leukocyte-induced in vitro hypoglycemia in blood
samples kept at room temperature in the absence of metabolic inhibitors
can be seen in myeloproliferative disorders such as PV2,3
as well as with normal leukocytes.4 Dr Otto, however, cites
a publication demonstrating in vitro hypoglycemia induced by PV
leukocytes when the leukocyte count was less than 20 000/µL,
although not when it was normal.3 This observation is not
only unprecedented,5 but the authors also observed
artifactual hypoglycemia equivalent to that observed with PV leukocytes
in a secondary erythrocytosis patient with a normal leukocyte count. However, 4 hours of in vitro incubation were required to obtain these
results.3 Dr Otto does note that we included the
appropriate leukocytosis controls in our initial studies1
and thus he cannot have it both ways. We have never observed impaired
platelet Mpl glycosylation in patients who did not have a
myeloproliferative disorder, regardless of the height of the leukocyte
count. Conversely, we have documented impaired platelet Mpl
glycosylation in patients with PV with normal leukocyte counts (35% of
our patients) as well as in IMF patients with leukopenia. At the same
time, we have never seen impaired platelet Mpl glycosylation in
patients with secondary erythrocytosis.
Dr Otto also notes that in our second publication,6 unlike
the first, we failed to provide patient leukocyte counts. That was
because, as we stated in the paper, they were irrelevant to the
results. For example, in the patient illustrated in Figure 6 of the
paper6 showing progression of the platelet Mpl
glycosylation defect, the leukocyte count was 13 100/µL in 5/19 and
14 700/µL in 1/99, hardly a change to which even Dr. Otto would
ascribe biochemical significance. However, since the issue was raised, we wish to state for the record that the mean leukocyte count for the
"A" group in Table 1 was 13 200/µL (range 6200-22 400), for
the "A=B" group, 9500/µL (range 6600-28 100), for
the "B>A" group, 31 660/µL (range 7000-50 000), and for the
"B" group 45 300/µL (range 9800-145 437). While there is the
expected correlation between disease duration and mean leukocyte count,
it is equally clear from both the means and the ranges that there is no
correlation between the leukocyte counts and the extent of the Mpl
glycosation defect. We should also add that while we routinely process
our clinical blood samples within 2 hours of collection, we have found that the integrity of Mpl remains intact in normal platelets in whole
blood shipped unrefrigerated overnight. Thus, Dr Otto's contention
that the leukocyte count is a confounder with respect to our platelet
observations is undefendable.
The second component of Dr Otto's hypothesis with respect to blood
platelets concerns the effect of hypoglycemia on receptor processing.
However, when one examines Dr Otto's chosen examples, they fail to
recapitulate the type of defect that we observed. In one study,
glucose-deprivation caused a reduction in insulin receptor gene
transcription.7 However, the mRNA content of Mpl in PV platelets was not different than normal. In a
second study, while glucose deprivation was associated with impaired insulin receptor glycosylation,8 the improperly processed
receptor was endoglycosidase H-resistant. This is contrary to
what we observed, namely, Mpl in PV platelets was
endoglycosidase H-sensitive.6 Furthermore, where the
conditions of glucose deprivation were defined, the glucose
concentrations ranged from 0 to 5.5 mM.7,8 However, the range of normal for blood glucose is 3.3 to 5.5 mM, implying that Mpl processing should be impaired in normal circulating platelets and of course it is not. Additionally, we demonstrated both A
and B forms of Mpl in Dami cells in the absence of
hypoglycemia,6 which strongly supports our contention that
glucose deprivation is irrelevant to the PV Mpl defect.
Finally, Dr Otto's hypothesis is also based on the ability of
platelets to synthesize protein. However, in the publication he
cites,9 protein synthesis was observed only in
thrombin-activated platelets and even then required a time lag. We only
studied unstimulated platelets and have never been able to obtain
incorporation of 35S-methionine into Mpl in resting normal
platelets. Also, we did not study other hematopoietic growth factor
receptors simply because none of the relevant ones (IL-3 beta and
gp130) were immunologically detectable in platelets as clearly stated
in our paper.6 We did, however, examine 2 important
integral membrane platelet glycoproteins, gpIIb and multimerin. Neither
were abnormally glycosylated in a PV patient with markedly
impaired Mpl processing (see reference 6, figure 5), a
specificity, given the abundance of these former proteins, that would
be unusual with a nonspecific metabolic insult such as hypoglycemia.
Unfortunately, Dr Otto fails to mention these important observations
that also refute his hypothesis.
Taking everything together, it is very clear that artifactual,
leukocyte-induced, in vitro hypoglycemia cannot explain either the
specific impairment of Mpl processing in PV megakaryocytes and
platelets or its progression with disease extent and duration.
Alison R. Moliterno
Jerry L. Spivak
Division of Hematology
Johns Hopkins University
School of
Medicine
Baltimore, Maryland
| |
References |
1.
Moliterno AR, Hankins W, Spivak JL.
Impaired expression of the thrombopoietin receptor by platelets from patients with polycythemia Vera.
N Engl J Med.
1998;338:572-580.
2.
Arem R, Jeang MK, Blevens TC, Waddell CC, Field JB.
Polycythemia rubra vera and artifactual hypoglycemia.
Arch Intern Med.
1982;142:2199-2201.
3.
Billington CJ, Casciato DA, Choquette DL, Morley JE.
Artifactual hypoglycemia associated with polycythemia vera.
JAMA.
1983;249:774-775.
4.
Rawnsley HM, Bowman HM.
Autoglycolysis in leukemic and nonleukemic blood.
Am J Med Sci.
1965;249:203-210[Medline]
[Order article via Infotrieve].
5.
Field JB, Williams HE.
Artifactual hypoglycemia associated with leukemia.
N England J Med.
1966;265:946-948.
6.
Moliterno AR, Spivak JL.
Posttranslational processing of the thrombopoietin receptor is impaired in polycythemia vera.
Blood.
1999;94:2555-2561.
7.
Briata P, Briata L, Gherzi R.
Glucose starvation and glycosylation inhibitors reduce insulin receptor gene expression: characterization and potential mechanism in human cells.
Biochem Biophys Res Comm.
1990;169:397-405.
8.
Hwang JB, Frost SC.
Effect of alternative glycosylation on insulin receptor processing.
J Biol Chem.
1999;274:22,813-22,820.
9.
Pabla R, Weyrich AS, Dixon DA, Bray PF, McIntyre TM, Prescott SM, Zimmerman GA.
Intergrin-independent control of translation: engagment of integrin IIb3 regulates synthesis of proteins in activated human platelets.
J Cell Biol.
1999;144:175-184.
