Full Text HL-93-03-B


NIH GUIDE, Volume 21, Number 36, October 9, 1992

RFA:  HL-93-03-B

P.T. 34

  Cell Lines 
  Tissue Culture 
  Biological Markers 
  Growth Factors 
  Biology, Molecular 

National Heart, Lung, and Blood Institute

Letter of Intent Receipt Date:  January 15, 1993
Application Receipt Date:  March 15, 1993


The Division of Blood Diseases and Resources (DBDR), of the National
Heart, Lung, and Blood Institute (NHLBI) invites applications for
studies that may contribute to better understanding of regulation of
the proliferation and maturation of megakaryocytes and the control of
platelet production.  The knowledge gained may allow manipulation of
megakaryocytopoiesis and platelet level in patient populations.


The Public Health Service (PHS) is committed to achieving the health
promotion and disease prevention objectives of "Healthy People 2000,"
a PHS-led national activity for setting priority areas.  This RFA,
Cell and Molecular Biology of Megakaryocytopoiesis, is related to the
priority area of platelet disorders and bone marrow transplantation.
Potential applicants may obtain a copy of "Healthy People 2000" (Full
Report:  Stock No. 017-001-00474-0 or Summary Report:  Stock No.
017-001-00473-1) through the Superintendent of Documents, Government
Printing Office, Washington, DC 20402-9325 (telephone 202-782-3238).


Applications may be submitted by domestic and foreign, for-profit and
non-profit organizations, public and private, such as universities,
colleges, hospitals, laboratories, units of State and local
governments, and eligible agencies of the Federal government.  Awards
in response to this RFA will be made to foreign institutions only for
research of very unusual merit, need, and promise, and in accordance
with PHS policy governing such awards.  Foreign institutions are not
eligible for First Independent Research Support and Transition
(FIRST) Awards (R29).  Applications from minority individuals and
women are encouraged.


This RFA solicits applications for the National Institutes of Health
(NIH) individual research grant (R01) and FIRST Award (R29) and is a
one-time solicitation.  Applicants, who will plan and execute their
own research programs, are requested to furnish their own estimates
of the time required to achieve the objectives of the proposed
research project.  Up to five years of support may be requested.  At
the end of the official award period, renewal applications may be
submitted for peer review and competition for support through the
regular grant program of the NHLBI.  It is anticipated that support
for the present program will begin in September 1993.  Administrative
adjustments in project period and/or amount of support may be
required at the time of the award.  All current policies and
requirements that govern the research grant programs of the NIH will
apply to grants awarded in connection with this RFA.


Although the financial plans for fiscal year 1994 include $1.5
million for this program, award of grants pursuant to this RFA is
contingent upon receipt of funds for this purpose.  It is anticipated
that about six grants will be awarded under this program.  The
specific amount to be funded will, however, depend on the merit and
scope of the applications received and on the availability of funds.
Since a variety of approaches would represent valid responses to this
announcement, it is anticipated that there will be a range of costs
among individual grants awarded.  If collaborative arrangements
involve sub-contracts with other institutions, the NHLBI Grants
Operations Branch (telephone 301-496-7257) may be consulted regarding
procedures to be followed.


The bone marrow megakaryocytes are the cells that produce the blood
platelets.  The number of platelets and their biochemical integrity
are important for normal hemostasis, and both of these parameters are
dependent upon the normal development of the parent cells, the
megakaryocytes.  The megakaryocyte is the least understood
hematopoietic cell of the bone marrow.  It possesses features unlike
those of the other hematopoietic cells:  the megakaryocyte nuclei are
highly polyploid, and several thousand platelets are formed from a
remarkable process of synthesis and organization of the cytoplasmic
compartment of each megakaryocyte.  The factors which regulate the
process of endomitosis, the extent of polyploidy, and the
ultrastructural changes which occur in the nucleus during maturation
are not understood.  Likewise, the factors which regulate cytoplasmic
development are not known.  Specific growth and maturation factors
which trigger endomitosis and cytoplasmic maturation of
megakaryocytes are not yet defined, in contrast to the considerable
knowledge that has been obtained concerning factors specific for red
and white cells.  The in vivo location of platelet production needs
to be defined; although it is generally thought that the
megakaryocytes release their platelets in the marrow, it has also
been suggested that the megakaryocytes travel to the lung and release
platelets in the lung.  Optimal methods for assessing the rate of
production of platelets have not been established.  The feedback
mechanisms by which thrombocytopenia signals an increase in
production of platelets have not been characterized.  Thus
considerable work remains to be done in understanding the
developmental regulation of megakaryocytes and the factors which lead
to platelet release.

Disorders of megakaryocytopoiesis can lead to serious clinical
problems.  The loss of normal regulation of the production of
platelets and abnormal platelet function are seen in
myeloproliferative disorders and leukemias.  In myeloproliferative
disorders, this can result paradoxically in hemorrhage in the
presence of thrombocytosis.  It is reasonable to suspect that the
biochemical development of the megakaryocyte is abnormal in these
circumstances.  However, no consistent abnormalities have been
defined in studies of proteins of platelets obtained from patients
with these disorders.  An understanding of the biochemical
abnormalities which cause the thrombopathies would be very important,
and would depend on definition of abnormalities in platelets and on
an improved understanding of factors that influence megakaryocyte

A rapidly growing clinical area in which the understanding of
megakaryocytopoiesis is critical is bone marrow transplantation,
since patients undergoing transplantation suffer from a prolonged
loss of platelet production.  Approximately 15 percent of bone marrow
transplant recipients die due to primary or secondary hemorrhagic
complications, mainly due to thrombocytopenia.  On the average, each
patient requires about 100 to 150 units of platelet transfusion until
recovery of marrow function occurs, usually after 40 to 50 days.
Some patients remain thrombocytopenic for a prolonged period and may
become refractory to platelet transfusion therapy.  An understanding
of megakaryocytopoiesis and platelet production is likely to be
beneficial for the support of bone marrow recipients.  If appropriate
growth factors could be isolated and cloned, they will find ready
application not only in these patients but also in other cases where
thrombocytopenia is a concern, e.g., patients with AIDS or cancer.

Similarly, although probably by a different mechanism, sepsis in
neonates is associated with prolonged thrombocytopenia.  Another
clinically relevant area which is poorly understood is the frequent
occurrence of megakaryoblastic leukemias in children with Down's
syndrome; this phenomenon is found as a transient leukemia in
neonates and often develops into a full-blown leukemia later in
childhood.  The characterization of the genetics and biochemistry of
this disorder may be very useful in elucidating the process of
megakaryocytopoiesis.  It is important to develop an understanding of
growth factors and other factors influencing hematopoiesis in order
to maintain normal blood cell production and thus be able to improve
treatment for the bleeding problems.

A major area of interest has been the interaction of proliferation
and growth factors with megakaryoblasts and megakaryocytes, both at
the progenitor level and at the level of cytoplasmic maturation.
Thus far, this work has largely depended on a mixed population of
progenitor cells for the starting assay, and a major obstacle to
understanding the effects of growth factors has been the inability to
isolate a pure normal megakaryoblastic cell line.  Studies at the
single cell level or with a pure cell population with newer
techniques (e.g., cell sorter or polymerase chain reaction) could be
productive.  Several cell lines derived from megakaryocytic leukemias
have been described recently, some of which contain a fraction of
cells capable of achieving a ploidy of 8N.  These cells may be useful
in elucidating some of the early stages of polyploidy.  However, they
all appear to have multiple trisomies and chromosomal rearrangements
and deletions, and the contribution of these factors to the lack of
normal development is not understood.  The chromosomal abnormalities
may restrict the usefulness of these cell lines in understanding the
normal dynamics of both development of polyploidy and cytoplasmic
maturation.  The availability of megakaryocytic cell lines which
behave in a normal manner will significantly help to answer some of
the outstanding questions in this area.  These questions include
identification of lineage-specific factors involved in stem cell
commitment to the megakaryocytic lineage, nuclear transcription
factors which may be unique to megakaryocytes, factors which
stimulate DNA and protein synthesis in megakaryocytes, and factors
which lead to cytoplasmic maturation and finally to platelet
production from the mature megakaryocyte cytoplasm.

New technology, such as the use of transgenic animals, provides the
potential for manipulating megakaryocytopoiesis in vivo.  For
example, a portion of the regulatory region of the gene for a
platelet-specific protein, platelet factor 4 (PF4) was used to insert
foreign genes into the germ cells of mice and to obtain
megakaryocyte-specific expression of the foreign proteins.  The
transgene expression occurred earliest in the 2N megakaryocytes, the
construct was expressed in a developmentally accurate fashion, and
there were no major positional effects between two cell lines with
regard to transgene expression.  The selective expression of the
transgene in bone marrow progenitors and peripheral blood cells can
be employed to devise simple bone marrow assays to monitor the
transition and commitment of hemopoietic stell cells to megakaryocyte
precursors.  This may allow the identification, isolation and cloning
of the elusive megakaryocyte maturation and growth factors.  The
transgenic mouse can be utilized to selectively target gene products
to megakaryocytes and platelets and then study their effects in vivo.

Examples of Areas of Interest

The following are only examples and prospective applicants are urged
to use their own ideas as to the area of research.

Development of Cell Lines and Culture Systems

The critical molecular events which lead to commitment of progenitor
cells to the megakaryocytic lineage and to complete maturation of
megakaryocytes to platelet production have not been identified.
These studies require the development of cell lines which can enter
and complete the normal maturation cycle.  Immortalized cell lines
could be generated which can be triggered to undergo differentiation
under restricted conditions.  Another approach would be to develop
bone marrow culture systems which allow the transition of stem cells
to megakaryocytes to be specifically monitored.  This approach could
employ precursor cells isolated from transgenic mice that possess
megakaryocyte-specific promotors coupled to reporter molecules in
conjunction with purified hematopoietic growth factors.  The role of
known oncogenes, growth factor receptors, and other signaling
proteins could be investigated in the above systems with dominant
negative suppressor techniques, antisense approaches, or injection of
specific neutralizing antibodies.  The function of previously
unidentified components might be sought by somatic cell genetic
techniques coupled to expression cloning methods.

Nuclear and Cytoplasmic Development

The biochemical steps which are responsible for the development and
extent of polyploidy and for maturation of the cytoplasm and the
formation of granule and membrane systems are not defined.  The
molecules that are specific to megakaryocytes and to different stages
of development must be defined; new markers other than those
currently used in studies of platelets should be sought.  The cis
acting regions and transacting factors of genes specifically
expressed in megakaryocytes should be determined, and the effects of
hematopoietic growth factors and cytokines on these events should be

Specific areas of study which might elucidate nuclear development
include the role of cyclins, kinases, phosphatases, and contractile
proteins in endomitosis.  Specific areas of study regarding
cytoplasmic development include stage-specific events in granule
formation and synthesis of membrane and cytoskeletal components,
including targeting of proteins to specific structures.  The
mechanism by which the cytoplasm becomes organized into the mature
platelets is unknown, and studies which could address this question
would be useful.  It would also be important to identify the specific
growth factors which control nuclear and cytoplasmic development and
platelet formation.

Sensors and Signals

Investigations are needed to clarify the sensors and molecular
signals which modulate the plasma levels of the megakaryocyte
specific growth factors and thereby regulate megakaryocytopoiesis.

Platelet Production

Newly available techniques might be used to clarify questions related
to platelet production.  For example, megakaryocytes that have been
genetically labeled with short-lived reporter molecules might serve
as in vivo markers for quantitating platelet production.  The site of
megakaryocyte fragmentation might be determined by injecting the
labeled cells and measuring the formation of the circulating

Genetic Defects in Megakaryocytopoiesis

Chromosomal abnormalities have been associated with megakaryocytic
leukemias, notably in Down syndrome and in adult leukemias in
otherwise genetically normal individuals.  Other congenital syndromes
which include defects in megakaryocytopoiesis have been identified,
such as TAR syndrome and Epstein's syndrome.  In addition, a number
of animal models of defective megakaryocytopoiesis have been
described.  Characterization of the genetic abnormalities in these
various conditions could provide useful information on the factors
regulating megakaryocytopoiesis.

Disciplines and Expertise

Among the disciplines and expertise that may be appropriate for this
program are hematology, biochemistry, cell biology, medicine, and
molecular biology.


Epidemiological studies and large-scale clinical trials are
specifically excluded from this RFA.


Upon initiation of the program, the NHLBI will sponsor annual
meetings to encourage and exchange of information among investigators
who participate in this program.  In the preparation of the budget
for the grant application, applicants must request additional travel
funds for one meeting each year to be held in Bethesda, MD.
Applicants must also include a statement in the applications
indicating their willingness to participate in such meetings.



NIH policy is that applicants for NIH clinical research grants and
cooperative agreements will be required to include minorities and
women in study populations so that research findings can be of
benefit to all persons at risk of the disease, disorder or condition
under study; special emphasis should be placed on the need for
inclusion of minorities and women in studies of diseases, disorders
and conditions which disproportionately affect them.  This policy is
intended to apply to males and females of all ages.  If women or
minorities are excluded or inadequately represented in clinical
research, particularly in proposed population-based studies, a clear
compelling rationale should be provided.

The composition of the proposed study population must be described in
terms of gender and racial/ethnic group.  In addition, gender and
racial/ethnic issues should be addressed in developing a research
design and sample size appropriate for the scientific objectives of
the study. This information should be included in the form PHS 398 in
Sections 1-4 of the Research Plan AND summarized in Section 5, Human
Subjects.  Applicants are urged to assess carefully the feasibility
of including the broadest possible representation of minority groups.
However, the NIH recognizes that it may not be feasible or
appropriate in all research projects to include representation of the
full array of United States racial/ethnic minority populations (i.e.,
Native Americans including American Indians or Alaskan Natives),
Asian/Pacific Islanders, Blacks, Hispanics).  The rationale for
studies on single minority population groups should be provided.

For the purpose of this policy, clinical research includes human
biomedical and behavioral studies of etiology, epidemiology, (and
preventive strategies), diagnosis, or treatment of diseases,
disorders or conditions, including but not limited to clinical

The usual NIH policies concerning research on human subjects also
apply.  Basic research or clinical studies in which human tissues
cannot be identified or linked to individuals are excluded.  However,
every effort should be made to include human tissues from women and
racial/ethnic minorities when it is important to apply the results of
the study broadly, and this should be addressed by applicants.

For foreign awards, the policy on inclusion of women applies fully;
since the definition of minority differs in other countries, the
applicant must discuss the relevance of research involving foreign
population groups to the United States' populations, including

If the required information is not contained within the application,
the application will be returned.

Peer reviewers will address specifically whether the research plan in
the application conforms to these policies.  If the representation of
women or minorities in a study design is inadequate to answer the
scientific question(s) addressed AND the justification for the
selected study population is inadequate, it will be considered a
scientific weakness or deficiency in the study design and will be
reflected in assigning the priority score to the application.

All applications for clinical research submitted to NIH are required
to address these policies.  NIH funding components will not award
grants or cooperative agreements that do not comply with these


The NHLBI requests that prospective applicants submit, by January 15,
1993, a letter of intent that includes a brief synopsis of the
proposed research and identification of any other participating
institutions.   Such letters are requested only for the purpose of
providing an indication of the number and scope of applications to be
received; therefore their receipt is usually not acknowledged.  A
letter of intent is not binding, and it will not enter into the
review of any application subsequently submitted, nor is it a
necessary requirement for publication.  This letter of intent is to
be sent to:

Dr. Charles L. Turbyfill
Division of Extramural Affairs
National Heart, Lung, and Blood Institute
Westwood Building, Room 553
Bethesda, MD  20892
Telephone:  (301) 496-7351
FAX:  (301) 496-7033


Applications are to be submitted on the research grant application
form PHS 398 (rev. 9/91).  This form is available in most applicant
institution's offices of sponsored research or business offices; and
from the Office of Grants Inquiries, Division of Research Grants,
National Institutes of Health, 5333 Westbard Avenue, Room 449,
Bethesda, MD 20892, telephone 301/496-7441.  Use the conventional
format for research-project grant applications and ensure that the
points identified in the Section on Review Considerations are
fulfilled.  FIRST Award applications must include at least three
sealed letters of reference attached to the face page of the original
application.  FIRST Award applications submitted without the required
number of reference letters will be considered incomplete and will be
returned without review.

To identify the application as a response to this RFA, check "YES" on
Item 2a of page 1 of the application and enter the title and number
of the RFA, Cell and Molecular Biology of Megakaryocytopoiesis, HL-

The RFA label available in application form PHS 398 must be affixed
to the bottom of the face page of the original copy of the
application.  Failure to use this label could result in delayed
processing of your application.

Send or deliver the completed application and three signed, exact
photocopies of it to:

Division of Research Grants
National Institutes of Health
Westwood Building, Room 240
Bethesda, MD  20892**

At the same time, send two additional copies of the completed PHS 398
application to Dr. Charles Turbyfill at the address listed under
LETTER OF INTENT.  It is important to send these two copies at the
same time as the original and three copies are sent to the Division
of Research Grants, otherwise the NHLBI cannot guarantee that the
application will be reviewed in competition for this RFA.

Applications must be received by March 15, 1993.  An application
received after this date will be considered ineligible.


Upon receipt, applications will be reviewed for completeness by the
DRG and for responsiveness by the NHLBI.  Incomplete applications
will be returned to the applicant without further consideration.  If
the application is not responsive to the RFA, NHLBI staff will
contact the applicant to determine whether to return the application
to the applicant or submit it for review in competition with
unsolicited applications at the next review cycle.

Applications may be triaged by an NHLBI peer review group on the
basis of relative competitiveness.  The NHLBI will withdraw from
further competition those applications judged to be non-competitive
for award and notify the applicant Principal Investigator and
institutional official.  Those applications judged to be competitive
will undergo further scientific merit review.  Those applications
that are complete and responsive will be evaluated in accordance with
the criteria stated below for scientific/technical merit by an
appropriate peer review group convened by the NHLBI.  The second
level of review will be provided by the National Advisory NHLBI

Review Criteria

The factors to be considered in the evaluation of scientific merit of
each application will be similar to those used in the review of
research grant applications, including the novelty, originality, and
feasibility of the approach; the training, experience and research
competence of the investigator(s); the adequacy of the experimental
design; the suitability of the facilities; and the appropriateness of
the requested budget to the work proposed.

The second level review will be by the National Heart, Lung, and
Blood Advisory Council.


The anticipated date of award is September 1993.

Funding decisions will be made on the basis of scientific and
technical merit as determined by peer review, program needs and
balance, and the availability of funds.

Awards in response to this RFA will be made to foreign institutions
only for research of very unusual merit, need, and promise, and in
accordance with PHS policy governing such awards.


Inquiries regarding this request for applications to:

Dr. Pankaj Ganguly
Chief, Thrombosis and Hemostasis Branch
Division of Blood Diseases and Resources
National Heart, Lung, and Blood Institute
Federal Building, Room 5C14
Bethesda, MD  20892
Telephone:  (301) 402-2237
FAX:  (301) 402-1622

Fiscal and administrative matters to:

Ms. Jane R. Davis
Chief, Blood Division Grants Management Section
Division of Extramural Affairs
National Heart, Lung, and Blood Institute
Westwood Building, Room 4A15
Bethesda, MD  20892
Telephone:  (301) 496-7257
FAX:  (301) 402-1200


The programs of the Division of Blood Diseases and Resources, NHLBI,
are described in the Catalog of Federal Domestic Assistance No.
93.839.  Awards will be made under the authority of the Public Health
Service Act, Section 301 (42 USC 241) and administered under PHS
grants policies and Federal Regulations 42 CFR Part 52 and 45 CFR
Part 74.  This program is not subject to the intergovernmental review
requirements of Executive Order 12372 or to Health Systems Agency


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