THROMBOPOIETIN, MEGAKARYOCYTOPOIESIS AND PLATELET PRODUCTION

NIH Guide, Volume 26, Number 39, December 5, 1997

PA NUMBER:  PA-98-014

P.T.

National Heart, Lung, and Blood Institute
National Institute of Diabetes, Digestive and Kidney Diseases

PURPOSE

The objective of this initiative is to understand the actions of thrombopoietin
(TPO) at the molecular level in megakaryocyte proliferation and development, in
platelet production, and in the regulation of platelet levels in vivo. The
ultimate goal is to be able to regulate platelet levels as needed in vivo in a
safe and efficacious manner.

HEALTHY PEOPLE 2000

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 PA, Thrombopoietin,
Megakaryocytopoiesis and Platelet Production, is related to the priority areas
of cancer, and heart diseases and stroke.  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 202-512-1800).

ELIGIBILITY REQUIREMENTS

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 or local governments, and eligible agencies of the
federal government.  Awards in response to this PA 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.  Racial/ethnic minority
individuals, women, and persons with disabilities are encouraged to apply.

The Mentored Clinical Scientist Development Award (K08) provides support to
develop outstanding clinician research scientists.  The candidate should hold a
clinical doctoral degree and should have initiated postgraduate clinical
training.  Candidates must be  U.S. Citizens, non-citizen nationals, or lawfully
admitted for permanent residence.  Applications may be submitted by a public or
private, non-federal organization on behalf of the applicant.  The candidate must
identify a mentor with extensive research experience and must devote 75 percent
effort to research career development activities during the period of the award. 
Former principal investigators on PHS research grants are not eligible and a
concurrent PHS award may not be held.

The National Research Service Award Individual Postdoctoral Fellowships (F32)
offers health scientist the opportunity to receive full time research training
in areas that reflect the national need for biomedical and behavioral research. 
By the award start date, applicants must have received a Ph.D., M.D., D.D.S.,
D.V.M., or equivalent degree and have arranged to work with a particular sponsor
affiliated with a private or public institution that has the staff and facilities
needed for the proposed training.  Applicants must be U.S. Citizens, non-citizen
nationals, or lawfully admitted for permanent residence. These awards are not
intended for study leading to the M.D., D.O., D.D.S., or equivalent professional
degrees nor do these awards support residency training.

The National Research Service Award Senior Postdoctoral Fellowships (F33) permit
experienced scientists to make major changes in the direction of their research
careers, or to broaden their scientific background by acquiring new research
capabilities.  The Award may be used in conjunction with a sabbatical leave. By
the award start date, an applicant must have at least 7 years of relevant
postdoctoral research or professional training, and must have a sponsor
affiliated with a public or private institution with appropriate facilities for
the proposed training.  These awards are not intended for study leading to a
professional degree nor do these awards support residency training.  Fellows must
be U.S. citizens, non-citizen nationals, or have been lawfully admitted to the
United States for permanent residence.

MECHANISM OF SUPPORT

This PA will use the research project grant (R01), the Mentored Clinical
Scientist Development Award (K08), and National Research Service Award Individual
and Senior Postdoctoral Fellowships (F32 and F33).  Applications for R01 grants
may request up to five years of support.  Responsibility for the planning,
direction, and execution of the proposed research for all  applicable mechanisms
of support will be solely that of the applicant.

RESEARCH OBJECTIVES

Background

Platelets play an essential role in hemostasis and thrombosis.  They are produced
from bone marrow megakaryocytes and arise from fragmentation of the cytoplasm. 
If the level of circulating platelets drops below a certain number
(thrombocytopenia), the patient runs the risk of catastrophic hemorrhage. 
Patients with cancer who have received chemotherapy or bone marrow transplants
usually have thrombocytopenia, and the slow recovery of platelet count in these
patients has been a concern.  The demand for platelet units for transfusion has
been steadily increasing primarily because of the need to maintain a certain
platelet level in such patients with cancer or those undergoing major cardiac
surgery.

More than three decades ago, it was reported that a humoral factor present in the
plasma of patients with severe thrombocytopenia increased the platelet count when
injected in animals and this regulator was named thrombopoietin or TPO.  The
isolation of this cytokine proved to be a difficult task because of its low
concentration in plasma and the lack of reliable assays. Meanwhile, other
hematopoietic regulators with some proliferative effect on megakaryocytes were
discovered: interleukin-3 (IL-3), IL-6, IL-11, and leukemia inhibitory factor
(LIF).  However, their action in increasing the platelet number proved to be
minimal, and each showed some undesirable, non-specific effects.

Identification of the myeloproliferative leukemia virus (MPLV) had a profound
influence on efforts to isolate TPO.  The responsible viral oncogene was cloned
in 1990, and, two years later, the corresponding cellular proto-oncogene, c-mpl,
was identified.  The amino acid sequence of the latter showed the typical
features of a cytokine receptor; in fact, it contained two "cytokine receptor
motifs."  In 1994, several groups almost simultaneously reported the isolation
and characterization of TPO using three distinct strategies.  The Mpl ligand,
TPO, was cloned, and recombinant TPO proved to be powerful in increasing the
number of platelets in vivo; however, its safety, specificity, and mechanism of
action remain to be defined.  With this magnificent discovery,
megakaryocytopoiesis became a science and new talents and tools were brought to
bear on the process.  It became equally important to identify those basic
research areas which are important but are unlikely to be supported by the
biotechnology  industry.  It is also vital to consider all effects of TPO that
might be involved in its use in humans, particularly in those patients with
malignancies of the blood system.

It is unknown whether or not thrombopoietin will be effective in raising platelet
levels in humans specifically and safely. Preclinical testing of full-length TPO
or a truncated, pegylated derivative molecule, termed megakaryocyte growth and
development factor (MGDF), produced impressive results in mice and monkeys.  The
most recent data show that either form of TPO administered before chemotherapy
resulted in a dose-dependent increase in peripheral platelet count.  The rise
occurred within 4 to 6 days after TPO administration and continued for up to 11
days.  These data suggest that TPO affects early stages of hematopoiesis and
confirms previous reports that it increases the frequency of multiple types of
hematopoietic progenitors in the marrow and in blood.  The magnitude of the
response was also impressive since a modest amount of TPO for only a single day
raised peripheral platelet count by > 200%.  No significant adverse effects were
associated with the drugs although in vitro studies indicated that TPO may prime
platelets to respond to subthreshold levels of agonists.  It should be noted,
however, that patients selected for these studies were carefully screened to
exclude pre-existing cardiovascular diseases, making the safety of these agents
in an unselected patient population still an unresolved question.

Another open issue is the effectiveness of TPO administered after cytotoxic
therapy since an optimal cellular substrate of adequate quantity and quality will
be necessary for TPO to be effective.  TPO is a unique cytokine in that it not
only promotes the differentiation and polyploidization of megakaryocytes, but it
also increases the proliferation of megakaryocyte precursors. In conjunction with
other cytokines, it is also known to increase the proliferation of CD34+ cells
and the development of other lineages.  Thus, administration of TPO to patients
with blood malignancies may have a long term effect on cellular proliferation and
on the malignant phenotype.  It then becomes essential to understand the basic
molecular mechanism by which TPO exerts each of its effects.  Indeed TPO has been
reported to upregulate the expression of specific cyclins and other cell cycle
genes, which may control the cell cycle of a variety of hematopoietic lineages. 
The results of such basic studies are essential for safe and efficacious use of
TPO in humans.

Considerable excitement has been generated over the possible use of cord blood
stem cells for transplantation. Recent data indicate that human bone marrow CD34+
CD38+ cells, which contain megakaryocyte progenitors, may be cultured in vitro
with TPO in a serum-free medium leading to the formation of proplatelets and
coagulant microparticles.  The action of TPO on cord blood stem cells needs to
be defined.

In addition to its use in patients with thrombocytopenia, TPO is likely to be
considered for administration to normal donors of platelets.  Over 7 million
units of platelet concentrates are transfused annually in the United States.
Approximately 35 to 50% of these are solicited from individual donors in whom the
use of TPO could increase platelet yields by 200 to 400%.

Both EPO or G-CSF have been used in normal donors as part of autologous blood or
stem cell collections, or as part of allogeneic granulocyte donation programs.
However, the effects of  TPO on normal individuals are unknown and need to be
investigated before such use.

In spite of significant progress, the manner of production and regulation of TPO
and the physiological sensing mechanisms remain unclear.  Megakaryocytopoiesis
is likely to involve an interplay between stem cells, stromal elements, and
growth factors.  Which of these cells play a primary role in the production of
TPO?  How is the TPO gene expression regulated?  How is the TPO level in blood
controlled?  The mechanism of signal transduction,  e.g., the structure of the
TPO receptor, kinases, phosphorylation, etc., need to be defined.  There is a
need to characterize the properties of platelets produced in the presence of TPO
and evaluate the risk of thrombosis.  The cloning of TPO was first reported in
the literature in June, 1994; clinical trials began in  1995, and the early
results are now being published.  Thrombopoietin has moved from theory to
reality, and from cloning to the clinic in three short years.  It appears to
belong to the lineage-dominant hormones, and the results of pre-clinical studies
are encouraging.  While the speed of modern biotechnology from drug discovery to
clinical application is impressive, experience with the therapeutic use of other
cytokines indicates the need for caution.  Accordingly, the focus of this
initiative is on the basic biology of TPO, megakaryocytopoiesis,  and platelet
production, areas which are unlikely to be fully investigated by the
pharmaceutical industry.  Logical extension of basic studies to animal research
and limited patient studies are encouraged.

Examples of Research Areas

The following are examples of broad areas of research interests related to
thrombopoietin. Applicants are encouraged to develop their own ideas for research
studies within the context of this program announcement.

o  Regulation of thrombopoietin expression
o  Regulation of c-mpl expression in different cell types
o  Determination of sites for binding to Mpl and signaling
o  Elucidation of mechanisms of interaction with other cytokines
o  Identification of the transcriptional targets of megakaryocyte signal
transducers and transactivators
o  Identification of genes for megakaryocyte lineage commitment and development
o  Elucidation of mechanisms underlying polyploidy
o  Definition of action of TPO in cell proliferation
o  Possible role of TPO in malignancy and thrombosis
o  Regulation of TPO level in blood
o  Developmental changes and use in neonates
o  Elucidation of effects of TPO on cord blood cells

INCLUSION OF WOMEN AND MINORITIES IN RESEARCH INVOLVING HUMAN SUBJECTS

It is the policy of the NIH that women and members of minority groups and their
subpopulations must be included in all NIH supported biomedical and behavioral
research projects involving human subjects, unless a clear and compelling
rationale and justification is provided that inclusion is inappropriate with
respect to the health of the subjects or the purpose of research.  This policy
results from the NIH Revitalization Act of 1993 (Section 492B of Public Law
103-43).  All investigators proposing research involving human subjects should
read the "NIH Guidelines for Inclusion of Women and Minorities as Subjects in
Clinical Research", which have been published in the Federal Register of March
28, 1994 (FR 59 14508-14513), and in the NIH GUIDE FOR GRANTS AND CONTRACTS of
March 18, 1994, Volume 23, Number
11.

Investigators may obtain copies from these sources or from the program staff or
contact person listed below. Program staff may also provide additional relevant
information concerning the policy.

APPLICATION PROCEDURES

Investigators who are considering preparing an application in response to this
program announcement are invited, but not required, to discuss their project with
NHLBI and NIDDK staff listed under INQUIRIES in advance of formal submission. 
Specific information on research training and career development mechanisms and
eligibility may also be accessed at the following address on the NIH Home Page
on the world wide web: http://www.nih.gov/grants/training/careerdev/RTCDINTRO.HTM

Applications for R01 and K08 grants are to be submitted on the research grant
application form, PHS 398 (rev. 5/95).  Applications for National Research
Service Awards (F32 and F33) are to be submitted on PHS 416-1.  Application kits
are available in an applicant institution's office of sponsored research and from
the Division of Extramural Outreach and Information Resources, National
Institutes of Health, 6701 Rockledge Drive, MSC 7910, Bethesda, MD 20892-7910,
telephone (301) 710-0267, email: ASKNIH@od.nih.gov.

Application receipt dates for each mechanism are listed in the application kits.

To identify the application as a response to the PA, check "YES" on Item 2 of
page 1 of the application and enter the title and PA number:  THROMBOPOIETIN,
MEGAKARYOCYTES AND PLATELET PRODUCTION, PA-98-XXX.

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

CENTER FOR SCIENTIFIC REVIEW (formerly Division of Research Grants)
NATIONAL INSTITUTES OF HEALTH
6701 ROCKLEDGE DRIVE, ROOM 1040 - MSC 7710
BETHESDA, MD  20892-7710
BETHESDA, MD  20817 (for express/courier service)

REVIEW CONSIDERATIONS

Applications will be assigned on the basis of established Public Health Service
referral guidelines.  Applications will be reviewed for scientific and technical
merit in accordance with the standard NIH peer review procedures.  Applications
that are complete will be evaluated for scientific and technical merit by an
appropriate peer review group convened in accordance with the standard NIH peer
review procedures.  As part of the initial merit review, all applications will
receive a written critique and undergo a process in which only those applications
deemed to have the highest scientific merit, generally the top half of
applications under review, will be discussed, assigned a priority score, and
receive a second level review by the appropriate national advisory council or
board.

Review Criteria

1.  Significance.  Does this study address an important problem?  If the aims of
the application are achieved, how will scientific knowledge be advanced?  What
will be the effect of these studies on the concepts or methods that drive this
field?

2.  Approach.  Are the conceptual framework, design, methods, and analyses
adequately developed, well-integrated, and appropriate to the aims of the
project?  Does the applicant acknowledge potential problem areas and consider
alternative tactics?

3.  Innovation.  Does this project employ novel concepts, approaches or methods? 
Are the aims original and innovative?  Does the project challenge existing
paradigms or develop new methodologies or technologies?

4.  Investigator.  Is the investigator appropriately trained and well suited to
carry out this work?  Is the work proposed appropriate to the experience level
of the principal investigator and other researchers  (if any)?

5.  Environment.  Does the scientific environment in which the work will be done
contribute to the probability of success?  Do the proposed experiments take
advantage of unique features of the scientific  environment or employ useful
collaborative arrangements?  Is there evidence for institutional support?

AWARD CRITERIA

Applications will compete for available funds with all other approved
applications.  The following will be considered in making funding decisions:
quality of the proposed project as determined by peer review; availability of
funds; and program priority.

INQUIRIES

Inquiries concerning this PA are encouraged.  The opportunity to clarify any
issues or questions from potential applicants is welcome.

Inquiries regarding programmatic issues may be directed to:

Dr. Pankaj Ganguly
Division of Blood Diseases and Resources
National Heart, Lung, and Blood Institute
6701 Rockledge Drive, Room 10146
Bethesda, MD  20892-7950
Telephone:  (301) 435-0070
FAX:  (301) 480-1046
Email:  gangulyp@gwgate.nhlbi.nih.gov

Dr. David G. Badman
Division of Kidney, Urologic and Hematologic Diseases
National Institute of Diabetes, Digestive and Kidney Disorders
45 Center Drive, Room 6AS-13C, MSC 6600
Bethesda, MD  20892-6600
Telephone:  (301) 594-7717
FAX:  (301) 480-3510
Email:  David_Badman@nih.gov

Direct inquiries regarding fiscal matters to:

Ms. Jane R. Davies
Grants Management Office
National Heart, Lung, and Blood Institute
6701 Rockledge Drive, MSC 7926
Bethesda, MD  20892-7926
Telephone:  (301) 435-0166
FAX:  (301) 480-3310
Email:  jane_davis@nih.gov

Ms. Aretina Perry
Division of Extramural Activities
National Institute of Diabetes, Digestive, and Kidney Diseases
45 Center Drive, Room 6N-38B, MSC 6600
Telephone:  (301) 594-8862
Email:  perrya@ep.niddk.nih.gov

AUTHORITY AND REGULATIONS

This program is described in the Catalog of Federal Domestic Assistance Nos.
93.839 and 93.849.  Awards will be made under the authority of the Public Health
Service Act, Section 301 (42 USC 241) and administered under PHS grant policies
and Federal regulations, most specifically 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 Review.

The PHS strongly encourages all grant and contract recipients to provide a smoke-
free workplace and promote the non-use of all tobacco products.  In addition,
Public Law 103-227, the Pro-Children Act of 1994, prohibits smoking in certain
facilities (or in some cases, any portion of a facility) in which regular or
routine education, library, day care, health care or early childhood development
services are provided to children.  This is consistent with the PHS mission to
protect and advance the physical and mental health of the American people.

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