Blood, Vol. 93 No. 9 (May 1), 1999:
pp. 3152-3154
CORRESPONDENCE
Where Does Platelet Factor V Originate?
 |
LETTER |
To the Editor:
I have read with interest the article by Camire et al1
proposing "endocytosis by megakaryocytes as the major mechanism by
which platelet-derived factor V is acquired. . . . ."
The approach is clever, identifying the type of factor V derived from
two patients, each heterozygous for factor V Leiden, who received
allogeneic transplantation of bone marrow and liver, respectively, from
a donor with wild-type factor V.
The only data presented in detail is a series of four Western blots
and, while the patterns exhibited are consistent with the
hypothesis, this technique does not lend itself well to quantitative evaluation. In fact, the conclusion is based in part on the failure to demonstrate platelet uptake of factor V, an experiment presented within the text and as "data not shown." Moreover, the authors did not attempt to show that megakaryocytes can endocytose factor V. Further, the possibility that the result of the transplant is a chimera
is not considered in detail.
Against this proposition are two reports from my laboratory
indicating that factor V can be biosynthesized in guinea pig
platelets2 and in human megakaryocytes3
performed by the incorporation of radiolabeled amino acids into
factor V. A third report from our group, in collaboration with Alan
Gewirtz, indicates that mature human megakaryocytes bind and
synthesize factor V and have mRNA for factor V.4 Thus, the
question is not whether there is biosynthesis, but whether uptake or
synthesis is the "major" mechanism.
In a recent study from the laboratory of Ginsburg5 using
factor V "knockout" mice transplanted with marrow progenitor
cells, different cellular origins for the biosynthesis of murine
plasma factor V and murine platelet factor V were demonstrated,
suggesting that free interchange does not take place in a situation
in which greater than 80% engraftment occurs. Because the
differences between mice, guinea pigs, and humans could be caused by
true biological variability, this study supports, but does not
prove, that platelet factor V derives from megakaryocytes.
Nevertheless, Camire et al have not excluded an important
contribution to platelet factor V from megakaryocyte biosynthesis
because they have not adequately addressed the effect of bone marrow
chimerism on their results, and have perhaps overestimated the
ability of Western blotting to quantify the concentration of factor V.
Robert W. Colman
Sol Sherry Thrombosis
Research Center
Hematology Division
Department of Medicine
Temple
University School of Medicine
Philadelphia, PA
| |
REFERENCES |
1.
Camire RM, Pollak ES, Kaushansky K, Tracy PB:
Secretable human platelet-derived factor V orginates from the plasma pool.
Blood
92:3035, 1998[Abstract/Free Full Text]
2.
Chiu HC, Schick PK, Colman RW:
Biosynthesis of factor V in isolated guinea pig megakaryocytes.
J Clin Invest
75:339, 1985
3.
Gewirtz AM, Keefer M, Doshi K, Annamalai AE, Chiu HC, Colman RW:
Biology of human megakaryocyte factor V.
Blood
67:1639, 1986[Abstract/Free Full Text]
4.
Gewirtz AM, Shapiro C, Shen YM, Boyd R, Colman RW:
Cellular and molecular regulation of factor V expression in human megakaryocytes.
J Cell Physiol
153:277, 1992[Medline]
[Order article via Infotrieve]
5.
Yang TL, Yang A, Ciu J, Ginsburg D:
Biological function of distinct platelet and plasma factor V pools in mice.
Blood
92:707a, 1998 (abstr, suppl 1)
Response
We thank Dr Colman for his interest in our study and appreciate the
opportunity to respond to his thoughtful comments. He raises three
major points. First, it should be noted that important cross-species
differences have been demonstrated in the coagulation system (eg,
unlike human platelets, which express PAR1, mouse platelets express
PAR31). In particular, Dr Colman refers to data in his
past publication2 and a recent abstract from the Ginsburg
laboratory3 regarding guinea pigs and mice, respectively,
that support megakaryocyte synthesis of factor V in those two species.
However, those observations should be contrasted to our previous work
in the bovine system where platelet-derived factor V contributes very
little to the total blood factor V pool,4 arguing against
significant levels of synthesis or uptake. Further, aortic endothelial
cell synthesis of factor V has been shown in the bovine
system,5 in contrast to the human system.6
Given this spread of results, we believe animal models must be viewed
with caution when interpreting the different mechanisms by which factor
V accumulates in any tissue bed.
Our second point addresses the quantitative assessment of Western
blots. Western blotting of factor V has been done in our laboratory7 as well as by others in our
institution,8-10 routinely and quantitatively, over the
last several years. Based on previously published data with samples
from individuals homozygous for factor VLeiden,11 we estimate our lower level of
detection for factor VLeiden, based on the appearance of
its unique activated protein C cleavage products (54- and 60-kD bands),
to be 2 to 3 ng/lane under the conditions described in our current
report.12 In fact, as noted in that paper, we overexposed
the Western blot of patient FW by fivefold to make certain we had the
opportunity to detect even small amounts of platelet factor
VLeiden which would have arisen because of megakaryocyte
synthesis. Under those conditions we also failed to detect any specific
bands at 54 and 60 kD. Thus, while we agree with Dr Colman that we
cannot entirely rule out megakaryocyte synthesis of factor V, based on our collective data, it is unlikely that such production, if present, could account for more than 5% of the secretable platelet-derived factor V.
Finally, regarding the possible chimeric nature of our patients,
polymerase chain reaction (PCR) (35 cycles) of DNA extracted from the
peripheral blood cells of JMW posttransplant failed to reveal the
presence of an FVLeiden allele due to retention of her
original marrow. Because we were concerned about total marrow ablation
pretransplant, we believed PCR analysis would be an exquisitely
sensitive method to demonstrate lack of chimerism. Furthermore, because
this transplant was male into female, marrow recovery was assessed by
XY chromosome cytogenetic display in
phytohemagglutinin-stimulated marrow cells. JMW is now
84% XY, 0.2% XX, and 15% XO. The 15% XO cells are most likely XY
(donor) that lost their Y, because additional analyses of the 55-year-old donor indicated he had lost his Y in several cells. While we cannot be certain that the XO were not in fact XX(recipient) that lost an X, this latter scenario is very unlikely, but still formally possible. Therefore, JMW is at least 84% donor blood cells,
and most likely 99% based on the argument detailed above. These data
coupled with the PCR analyses strongly argue for little, if any, chimerism.
Inadvertent bone marrow transplantation in liver transplants, without
evidence of graft-versus-host disease, has not been documented. FW
had no clinical evidence of graft-versus-host disease; hence, chimerism
is very unlikely. Furthermore, no FVLeiden protein was
detectable in her platelets and PCR analyses of DNA extracted from her
peripheral blood cells posttransplant indicated a heterozygous
FVLeiden genotype. Because of the undetectable level of
factor VLeiden in FW's platelets, if any chimerism were
present, the donor megakaryocytes would be the only ones producing
platelets. Such a scenario is highly improbable.
Consequently, we maintain that the most plausible interpretation of our
collective data is that endocytosis of plasma factor V, most likely by
the megakaryocyte, is the primary mechanism by which secretable factor
V accumulates in human platelets. In both our patients, the phenotype
of their secretable platelet factor V mirrored that of their plasma
factor V, regardless of the factor V genotype present in their
hematopoietic cells.
Paula B. Tracy
University
of Vermont College of Medicine
Burlington, VT
Rodney M. Camire
Eleanor S. Pollak
Children's Hospital of
Philadelphia
Philadelphia, PA
Kenneth Kaushansky
University of Washington School of Medicine
Seattle,
WA
| |
REFERENCES |
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Ishihara H, Connolly AJ, Dewan Z, Kahn ML, Zheng YW, Timmons C, Tram T, Coughlin SR:
Protease-activated receptor 3 is a second thrombin receptor in humans.
Nature
386:502, 1997[Medline]
[Order article via Infotrieve]
2.
Chiu HC, Schick PK, Colman RW:
Biosynthesis of factor V in isolated guinea pig megakaryocytes.
J Clin Invest
75:339, 1985
3.
Yang TL, Yang A, Ciu J, Ginsburg D:
Biological function of distinct platelet and plasma factor V pools in mice.
Blood
92:707a, 1998 (abstr, suppl 1)
4.
Tracy PB, Peterson JM, Nesheim ME, McDuffie FC, Mann KG:
Interaction of coagulation factor V and factor Va with platelets.
J Biol Chem
254:10354, 1979[Free Full Text]
5.
Cerveny TJ, Fass DN, Mann KG:
Synthesis of coagulation factor V by cultured aortic endothelium.
Blood
63:1467, 1984[Abstract/Free Full Text]
6.
Jenny FJ, Pittman DD, Toole JJ, Kriz RW, Aldape RA, Hewick RM, Kaufman RJ, Mann KG:
Complete cDNA and derived amino acid sequence of human factor V.
Proc Natl Acad Sci USA
84:4846, 1987[Abstract/Free Full Text]
7.
Tracy RP, Rubin DZ, Mann KG, Bovill EG, Rand M, Geffken D, Tracy PB:
Thrombolytic therapy and proteolysis of factor V.
J Am Coll Cardiol
30:716, 1997[Abstract]
8.
Rand MD, Lock JB, van't Veer C, Gaffney D, Mann KG:
Blood clotting in minimally altered whole blood.
Blood
88:3432, 1996[Abstract/Free Full Text]
9.
van't Veer C, Kalafatis M, Bertina RM, Simioni P, Mann KG:
Increased tissue factor-initiated prothrombin activation as a result of the Arg506 
Gln mutation in factor VLeiden.
J Biol Chem
272:20721, 1997[Abstract/Free Full Text]
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Cawthern KM, van't Veer C, Lock JB, DiLorenzo ME, Branda RF, Mann KG:
Blood coagulation in hemophilia A and hemophilia C.
Blood
91:4581, 1998[Abstract/Free Full Text]
11.
Camire RM, Kalafatis M, Simioni P, Girolami A, Tracy PB:
Platelet-derived factor Va/VaLeiden cofactor activities are sustained on the surface of activated platelets despite the presence of activated protein C.
Blood
91:2818, 1998[Abstract/Free Full Text]
12.
Camire RM, Pollak ES, Kaushansky K, Tracy PB:
Secretable human platelet-derived factor V originates from the plasma pool.
Blood
92:3035, 1998
