Full Text HL-93-03-B CELL AND MOLECULAR BIOLOGY OF MEGAKARYOCYTOPOIESIS NIH GUIDE, Volume 21, Number 36, October 9, 1992 RFA: HL-93-03-B P.T. 34 Keywords: Cell Lines Tissue Culture Biological Markers Growth Factors Biology, Molecular Genetics National Heart, Lung, and Blood Institute Letter of Intent Receipt Date: January 15, 1993 Application Receipt Date: March 15, 1993 PURPOSE 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. 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 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). 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 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. MECHANISM OF SUPPORT 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. FUNDS AVAILABLE 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. RESEARCH OBJECTIVES 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 biochemistry. 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 assessed. 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 platelets. 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. Exclusions Epidemiological studies and large-scale clinical trials are specifically excluded from this RFA. SPECIAL REQUIREMENTS 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. STUDY POPULATIONS SPECIAL INSTRUCTIONS TO APPLICANTS REGARDING IMPLEMENTATION OF NIH POLICIES CONCERNING INCLUSION OF WOMEN AND MINORITIES IN CLINICAL RESEARCH STUDY POPULATIONS 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 trials. 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 minorities. 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 policies. LETTER OF INTENT 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 APPLICATION PROCEDURES 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- 93-03-B. 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. REVIEW CONSIDERATIONS 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 Council/Board. 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. AWARD CRITERIA 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 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 AUTHORITY AND REGULATIONS 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 review. .
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