Patients with inherited platelet function disorders and bleeding manifestations are widely encountered in clinical practice of hematology. In the vast majority of such patients, the molecular mechanisms are completely unknown. The longstanding goals of our studies have been to obtain insights into normal platelet mechanisms and the involved proteins through the study of patients with inherited platelet defects. Our studies to date have been highly successful, leading to the first descriptions of hitherto unrecognized deficiencies in three major signaling proteins, phospholipase C-2, the GTP-binding protein Gq and protein kinase C-, and into the platelet dysfunction associated with haplodeficiency of a major transcription factor, RUNX1. These patients are an invaluable repository of untapped information on normal platelet mechanisms.
Specific aim of this project is to obtain insights into the molecular basis of the platelet defects associated with mutation in transcription factor RUNX1 (Core binding factor A2, RUNX1) and into the genes regulated in platelets/ megakaryocytes by RUNX1 through studies in patients characterized with RUNX1 haplodeficiency. RUNX1 haplodeficiency is associated with familial thrombocytopenia, impaired platelet function and predisposition to acute leukemia. Our patient has mild thrombocytopenia, markedly abnormal aggregation and secretion associated with diminished agonist-stimulated phosphorylation of pleckstrin and myosin light chain (MLC), decreased PKC- and impaired activation of GPIIb-IIIa. This patient has a heterozygous mutation in RUNX1. Platelet expression profiling studies in this patient showed downregulation of specific platelet/megakaryocyte (MK) genes in association with RUNX1 haplodeficiency. To date we have established that four genes (platelet factor 4 (PF4), myosin light chain (MYL9), 12-lipoxygenase (ALOX12) and protein kinase C- (PRKCQ) are indeed transcriptional targets of RUNX1, and they provide important insights into the platelet defects in the patient. We are now proposing to study specific other genes that are shown to be downregulated in our patient platelets including a) Pallidin (PLDN): dense and alpha granule defects are a major feature of RUNX1 haplodeficiency and the mechanisms unknown. Pallid (pldn) deficiency is associated with delta-SPD in mouse models. b) Small GTPases, RAB1B and RAB31, and RAPGAP1L that are major players in vesicular trafficking, secretion and granule targeting of proteins; and c) FLNA, TUBB1, related to cytoskeletal system, and play a major role in platelet production, cell shape, adhesion and secretion. To our knowledge a human deficiency in platelet pallidin or filamin has not been reported. For each gene we will a) demonstrate a decrease in the specific protein in platelets from the patient; b) establish through molecular biology studies that the gene is a direct transcriptional target of RUNX1; c) study the effect of downregulation and overexpression of RUNX1 on the gene and associated features (protein level, functional effects, MK development and thrombopoiesis); d) perform functional and biochemical studies in patient platelets/MK based on the specific gene. In addition, we will perform selected studies on regulation of RUNX1 in MK. We will extend these studies to other patients with RUNX1 mutation. These studies will provide information on hitherto unrecognized alterations in platelets/MK in RUNX1 haplodeficiency, the genes/proteins regulated by RUNX1 and on their platelet/MK role. They will advance our understanding of the mechanisms in normal and abnormal platelet function.

Public Health Relevance

Platelets are tiny circulating blood cells that play a role in stopping bleeding following injury and in the formation of blood clots that lead to heart attacks and stroke. Our longstanding interest has been to define the molecular mechanisms in platelets through the study of patients with platelet bleeding disorders, a group of patients widely encountered but poorly understood. This project will provide new insights into the mechanisms operating in platelets in health and disease, and will form the basis for developing new therapeutic agents for cardiovascular diseases and bleeding disorders.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Special Emphasis Panel (ZRG1-VH-B (02))
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Sarkar, Rita
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Temple University
Internal Medicine/Medicine
Schools of Medicine
United States
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Jalagadugula, Gauthami; Goldfinger, Lawrence E; Mao, Guangfen et al. (2018) Defective RAB1B-related megakaryocytic ER-to-Golgi transport in RUNX1 haplodeficiency: impact on von Willebrand factor. Blood Adv 2:797-806
Songdej, Natthapol; Rao, A Koneti (2017) Hematopoietic transcription factor mutations: important players in inherited platelet defects. Blood 129:2873-2881
Songdej, Natthapol; Rao, A Koneti (2017) Inherited platelet dysfunction and hematopoietic transcription factor mutations. Platelets 28:20-26
Mao, G F; Goldfinger, L E; Fan, D C et al. (2017) Dysregulation of PLDN (pallidin) is a mechanism for platelet dense granule deficiency in RUNX1 haplodeficiency. J Thromb Haemost 15:792-801
Rao, A Koneti (2017) Editorial: Platelet Genomics and Disorders of Platelet Number and Function. Platelets 28:2
Rao, A Koneti; Songdej, Natthapol (2017) Parsing the repertoire of GPIb-IX-V disorders. Blood 129:403-404
Mao, Guangfen; Songdej, Natthapol; Voora, Deepak et al. (2017) Transcription Factor RUNX1 Regulates Platelet PCTP (Phosphatidylcholine Transfer Protein): Implications for Cardiovascular Events: Differential Effects of RUNX1 Variants. Circulation 136:927-939
Rao, A K; Poncz, M (2017) Defective acid hydrolase secretion in RUNX1 haplodeficiency: Evidence for a global platelet secretory defect. Haemophilia 23:784-792
Michael, James V; Wurtzel, Jeremy G T; Mao, Guang Fen et al. (2017) Platelet microparticles infiltrating solid tumors transfer miRNAs that suppress tumor growth. Blood 130:567-580
Voora, Deepak; Rao, A Koneti; Jalagadugula, Gauthami S et al. (2016) Systems Pharmacogenomics Finds RUNX1 Is an Aspirin-Responsive Transcription Factor Linked to Cardiovascular Disease and Colon Cancer. EBioMedicine 11:157-164

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